Compositions containing HC-HA / PTX3 complexes and methods of use thereof

Abstract

Provided herein are methods for producing natural and reconstructed hyaluronic acid (HA) complexes containing pentraxin-3 (PTX3) and heavy chain 1 (HC1) of inter-alpha inhibitor (IαI). Provided are compositions containing the complexes and methods of treatment using the complexes. Also provided are combinations and kits for implementing the methods.

Description

[0001] This application is a divisional application of Chinese patent application No. 201380047367.2, filed on July 10, 2013, entitled "Composition containing HC-HA / PTX3 complex and method of use thereof" (the corresponding PCT application was filed on July 10, 2013, and was filed with application number PCT / US2013 / 049983).

[0002] Cross-references to related applications

[0003] This application claims the benefit of U.S. Application No. 61 / 670,571, filed July 11, 2012, which is incorporated herein by reference in its entirety.

[0004] Statement regarding federally funded research or development

[0005] The work described in this article was partially funded by federal government research grants RO1 EY06819, R44 EY017497, and R43 EY021045 from the National Eye Institute and the National Institutes of Health (Bethesda, MD, USA).

[0006] The reference to the sequence list submitted via EFS-WEB as a text file is incorporated.

[0007] This application contains a sequence list, which was submitted via EFS-Web as a computer-readable text file in ASCII format and is incorporated herein by reference in its entirety. The text file was created on July 8, 2013, named 34157-732-601SEQ.txt, and is 462KB in size. Background Technology

[0008] The amnion (AM) is an avascular sac filled with amniotic fluid surrounding the fetus. Like the placenta, the AM is derived from the epiblast that forms during the development of the fertilized egg. The AM forms the innermost membrane surrounding the fetus within the amniotic cavity. In placental mammals, the umbilical cord (funiculus umbilicalis) connects the developing fetus to the placenta. The umbilical cord consists of the amnion (UCAM) and Wharton's jelly. The amnion forms the outer layer of the umbilical cord. The UCAM regulates fluid pressure within the UC. Wharton's jelly is the gel-like substance within the umbilical cord, primarily composed of mucopolysaccharides (hyaluronic acid and chondroitin sulfate). It also contains some fibroblasts and macrophages. The umbilical cord further comprises two arteries (umbilical arteries) and one vein (umbilical vein) embedded within Wharton's jelly. Summary of the Invention

[0009] This article describes methods for identifying HC-HA / PTX3 complexes in fetal tissues such as the amnion and umbilical cord. It also describes methods for isolating native HC-HA / PTX3 complexes from fetal tissues such as the amnion and umbilical cord. Furthermore, it describes methods for producing reconstituted HC-HA / PTX3 complexes. Finally, it describes methods for using the native and reconstituted HC-HA / PTX3 complexes provided herein.

[0010] In some embodiments, methods for producing HC-HA / PTX3 complexes are described herein. In some embodiments, the method includes isolating a natural HC-HA / PTX3 (nHC-HA / PTX3) complex from an extract prepared from tissues or cells. In some embodiments, the method includes generating a reconstituted HC-HA / PTX3 (rcHC-HA / PTX3) complex.

[0011] In some embodiments, this document describes a method for isolating a natural HC-HA / PTX3 (nHC-HA / PTX3) complex from amniotic tissue, such as umbilical cord or amnion. In some embodiments, the nHC-HA / PTX3 complex is isolated from isolated cells. In some embodiments, the nHC-HA / PTX3 complex is isolated from cultured cells. In some embodiments, the nHC-HA / PTX3 complex is isolated from stem cells. In some embodiments, the nHC-HA / PTX3 complex is isolated from a water-soluble fraction of an extract prepared from tissue such as umbilical cord or amnion. In some embodiments, the water-soluble fraction is extracted using an isotonic salt solution. In some embodiments, the nHC-HA / PTX3 complex is isolated from a water-insoluble fraction of an extract prepared from tissue such as umbilical cord or amnion. In some embodiments, the insoluble fraction is extracted using GnHCl.

[0012] In some embodiments, this document describes a method for producing a reconstituted HC-HA / PTX3 (rcHC-HA / PTX3) complex in vitro: (a) contacting (i) high molecular weight hyaluronic acid (HMW HA) immobilized on a solid support and (ii) pentacyclic protein 3 (PTX3) to form an immobilized PTX3 and HMW HA complex (immobilized PTX3 / HA); and (b) contacting the immobilized PTX3 / HA with a meta-α-inhibitor (IαI) protein containing heavy chain 1 (HC1) and tumor necrosis factor α-stimulated gene 6 (TSG-6) to form the immobilized rcHC-HA / PTX3 complex. In some embodiments, steps (a) and (b) of this method are performed sequentially. In some embodiments of the method, the method includes simultaneously contacting high molecular weight hyaluronic acid (HMW HA) with pentacyclic protein 3 (PTX3) protein, a meso-α-inhibitor (IαI) protein containing heavy chain 1 (HC1), and tumor necrosis factor α-stimulated gene 6 (TSG-6). In some embodiments of the method, TSG-6 catalyzes the covalent linking of IαI HC1 to HA. In some embodiments, the method further includes removing unbound PTX3 protein after step (a) and before step (b). In some embodiments, the method further includes removing unbound TSG-6 after step (b). In some embodiments, the PTX3 protein used in the method is a naturally occurring PTX3 protein isolated from a cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is an amniotic cell. In some embodiments, the cell is an umbilical cord cell. In some embodiments, the cell is an amniotic cell derived from the umbilical cord. In some embodiments, the amniotic cell is an amniotic epithelial cell. In some embodiments, the amniotic cell is an umbilical cord epithelial cell. In some embodiments, the amniotic cell is an amniotic stromal cell. In some embodiments, the amniotic cell is an umbilical cord stromal cell. In some embodiments, the PTX3 protein is a recombinant protein. In some embodiments, the PTX3 protein used in the method comprises a polypeptide having the sequence shown in SEQ ID NO:33 or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity with the polypeptide having the sequence shown in SEQ ID NO:33. In some embodiments, the PTX3 protein used in the method comprises a polypeptide having the sequence shown in any one of SEQ ID NO:32-45 or a species variant or allele variant thereof. In some embodiments, the PTX3 protein used in the method is a multimeric protein.In some embodiments, the PTX3 protein used in this method is a homopolymer (i.e., a multimeric protein composed of two or more identical components). In some embodiments, the PTX3 homopolymer is a dimer, trimer, tetramer, pentamer, hexamer, or octamer. In some embodiments, the PTX3 homopolymer is an octamer. In some embodiments, the PTX3 protein includes a modified multimerizing domain or a heteromerizing domain. In some embodiments, the TSG-6 protein used in this method is a naturally occurring TSG-6 protein isolated from a cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is an amnion cell. In some embodiments, the cell is an umbilical cord cell. In some embodiments, the cell is an amnion cell derived from the umbilical cord. In some embodiments, the amnion cell is an amniotic epithelial cell. In some embodiments, the amnion cell is an umbilical cord epithelial cell. In some embodiments, the amnion cell is an amniotic stromal cell. In some embodiments, the amniotic cell is an umbilical cord stromal cell. In some embodiments, the TSG-6 protein is a recombinant protein. In some embodiments, the TSG-6 protein comprises a polypeptide having the sequence shown in SEQ ID NO:2 or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity with the polypeptide having the sequence shown in SEQ ID NO:2. In some embodiments, the TSG-6 protein used in the method comprises a polypeptide having the sequence shown in any one of SEQ ID NO:1-31, or a species variant or allele variant thereof. In some embodiments, the HC1 used in the method comprises a polypeptide having the sequence shown in SEQ ID NO:47 or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity with the polypeptide having the sequence shown in SEQ ID NO:47. In some embodiments, the meta-α-inhibitor (IαI) protein used as the HC1 source in this method further comprises HC2 and urostatin linked by a chondroitin sulfate chain. In some embodiments, the HC1 comprises a polypeptide having the sequence shown in any one of SEQ ID NO:46-47, or a species variant or allele variant thereof. In some embodiments, the HC2 comprises a polypeptide having the sequence shown in SEQ ID NO:49, or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity with the polypeptide having the sequence shown in SEQ ID NO:49.In some embodiments, the HC2 used in the method comprises a polypeptide having the sequence shown in any one of SEQ ID NO:48-49, or a species variant or allele variant thereof. In some embodiments, urostatin comprises a polypeptide having the sequence shown in SEQ ID NO:53, or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity with the polypeptide having the sequence shown in SEQ ID NO:53. In some embodiments, the urostatin used in the method comprises a polypeptide having the sequence shown in any one of SEQ ID NO:52-53, or a species variant or allele variant thereof. In some embodiments, the IαI protein used in the method is isolated from blood, serum, plasma, amnion, chorionic villus, amniotic fluid, or a combination thereof. In some embodiments, the IαI protein used in the method is isolated from serum. In some embodiments, the IαI protein used in the method is isolated from human serum. In some embodiments, the IαI protein used in the method is produced by mammalian cells. In some embodiments, the cell is a human cell. In some embodiments, the cell is an amniotic cell. In some embodiments, the cell is an umbilical cord cell. In some embodiments, the cell is an amniotic cell derived from the umbilical cord. In some embodiments, the amniotic cell is an amniotic epithelial cell. In some embodiments, the amniotic cell is an umbilical cord epithelial cell. In some embodiments, the amniotic cell is an amniotic stromal cell. In some embodiments, the amniotic cell is an umbilical cord stromal cell. In some embodiments, the IαI and TSG-6 protein are contacted at a molar ratio of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, or 20:1. In some embodiments, the IαI and TSG-6 protein are contacted at a molar ratio of 3:1. In some embodiments of the method, the weight-average molecular weight of the HMW HA is about 500 kDa to about 10,000 kDa, about 800 kDa to about 8,500 kDa, about 1,100 kDa to about 5,000 kDa, or about 1,400 kDa to about 3,500 kDa. In some embodiments of the method, the weight-average molecular weight of the HMW HA is 3,000 kDa. In some embodiments, the HMW HA is attached to a solid support by direct attachment. In some embodiments of the method, the HMW HA is attached to a solid support by indirect attachment. In some embodiments of the method, the HMW HA is attached to a solid support by covalent attachment. In some embodiments of the method, the HMW HA is attached to a solid support by non-covalent attachment. In some embodiments of the method, the HMW HA is attached to a solid support by connection via a severable connector.In some embodiments, the linker is a chemically or enzymatically cleavable linker. In some embodiments, the method further includes dissociating the rcHC-HA / PTX3 complex from the solid support after step (b). In some embodiments, dissociation includes cleavage of the cleavable linker. In some embodiments, the method further includes purification of the dissociated rcHC-HA / PTX3 complex. In some embodiments, purification includes affinity purification, centrifugation, filtration, chromatography, or a combination thereof. In some embodiments of the method, the PTX3, IαI HC1, or TSG-6 peptide contains an affinity tag. In some embodiments, the affinity tag is selected from hemagglutinin tags, polyhistidine tags, myc tags, FLAG tags, and glutathione S-transferase (GST) tags. In some embodiments, HMW HA is immobilized by binding HMW HA to an intermediate peptide. In some embodiments, the intermediate peptide is covalently attached to the solid support. In some embodiments, the binding of HMW HA to the intermediate peptide is non-covalent. In some embodiments, the intermediate peptide is a HA-binding protein (HABP). In some embodiments, the intermediate polypeptide is selected from HAPLN1, HAPLN2, HAPLN3, HAPLN4, aggregate proteoglycans, multipotent proteoglycans, neuroproteoglycans, short proteoglycans, phosphaglucan, TSG-6, CD44, stabillin-1, stabillin-2, or HABP sufficient to bind a portion of HA. In some embodiments, the intermediate polypeptide is a multipotent proteoglycan. In some embodiments, the intermediate polypeptide includes a linker module. In some embodiments, the intermediate polypeptide includes a linker module of HAPLN1, HAPLN2, HAPLN3, HAPLN4, aggregate proteoglycans, multipotent proteoglycans, neuroproteoglycans, short proteoglycans, phosphaglucan, TSG-6, CD44, stabillin-1, or stabillin-2. In some embodiments, the intermediate polypeptide includes a linker module of a multipotent proteoglycan. In some embodiments, the intermediate polypeptide includes a polypeptide shown in any one of SEQ ID NO:54-99. In some embodiments, the intermediate polypeptide comprises a polypeptide linker. In some embodiments, the linker is attached to a solid support. In some embodiments, the method further comprises dissociating the rcHC-HA / PTX3 complex from the intermediate polypeptide after step (b). In some embodiments, dissociating the rcHC-HA / PTX3 complex from the intermediate polypeptide comprises contacting the complex with a dissociating agent. In some embodiments, the dissociating agent is guanidine hydrochloride or urea. In some embodiments, the dissociating agent is guanidine hydrochloride of about 4M to about 8M. In some embodiments, the intermediate polypeptide or linker comprises a proteolytic cleavage sequence.In some embodiments, dissociation of the rcHC-HA / PTX3 complex includes cleavage of the intermediate polypeptide or linker at a proteolytic cleavage sequence. In some embodiments, cleavage includes contacting the proteolytic cleavage sequence with a protease. In some embodiments, the protease is selected from furin, 3C protease, caspase, matrix metalloproteinase, and TEV protease.

[0013] In some embodiments, this document describes a method for producing a reconstituted HC-HA / PTX3 (rcHC-HA / PTX3) complex in vitro, comprising contacting a PTX3 / HA complex immobilized on a solid support with a meta-α-inhibitor (IαI) protein containing heavy chain 1 (HC1) and TSG-6. In some embodiments, the PTX3 / HA complex is produced by contacting high molecular weight hyaluronic acid (HMW HA) with pentacyclic protein 3 (PTX3) protein under conditions that effectively form a PTX3 and HMW HA complex (PTX3 / HA), wherein the HMW HA is immobilized on a solid support. In some embodiments, the method further comprises removing unbound PTX3 protein prior to contacting the PTX3 / HA complex with IαI and TSG-6. In some embodiments, the method further comprises removing unbound TSG-6. In some embodiments, the PTX3 protein is a recombinant protein. In some embodiments, the PTX3 protein comprises a polypeptide having the sequence shown in SEQ ID NO:33 or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity with the polypeptide having the sequence shown in SEQ ID NO:33. In some embodiments, the PTX3 protein used in this method is a multimeric protein. In some embodiments, the PTX3 protein used in this method is a homopolymer. In some embodiments, the PTX3 homopolymer is a dimer, trimer, tetramer, pentamer, hexamer, or octamer. In some embodiments, the PTX3 homopolymer is an octamer. In some embodiments, PTX3 comprises a modified multipolymerized domain or a heteropolymerized domain. In some embodiments, TSG-6 is a recombinant protein. In some embodiments, TSG-6 comprises a polypeptide having the sequence shown in SEQ ID NO:2 or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity with the polypeptide having the sequence shown in SEQ ID NO:2. In some embodiments, HC1 comprises a polypeptide having the sequence shown in SEQ ID NO:47 or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity with the polypeptide having the sequence shown in SEQ ID NO:47. In some embodiments, the IαI protein further comprises HC2. In some embodiments, HC2 comprises a polypeptide having the sequence shown in SEQ ID NO:49 or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity with the polypeptide having the sequence shown in SEQ ID NO:49.In some embodiments, the IαI protein further comprises urostatin. In some embodiments, urostatin comprises a polypeptide having the sequence shown in SEQ ID NO:53 or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity with the polypeptide having the sequence shown in SEQ ID NO:53. In some embodiments, IαI further comprises a chondroitin sulfate chain. In some embodiments, the IαI protein is a recombinant protein. In some embodiments, the IαI protein is isolated from blood, plasma, serum, amnion, chorionic villus, amniotic fluid, or combinations thereof. In some embodiments, the IαI protein is isolated from serum. In some embodiments, the IαI protein is isolated from human serum. In some embodiments, the IαI and TSG-6 protein are contacted at a molar ratio of 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, or 20:1. In some embodiments, the IαI and TSG-6 protein are contacted at a molar ratio of 3:1. In some embodiments, the weight-average molecular weight of the HMW HA is about 500 kDa to about 10,000 kDa, about 800 kDa to about 8,500 kDa, about 1100 kDa to about 5,000 kDa, or about 1400 kDa to about 3,500 kDa. In some embodiments, the weight-average molecular weight of the HMW HA is 3,000 kDa. In some embodiments, the HMW HA is immobilized on a solid support by direct attachment. In some embodiments, the HMW HA is immobilized on a solid support by indirect attachment. In some embodiments, HMW HA is immobilized to a solid support via covalent attachment. In some embodiments, HMW HA is immobilized to a solid support via non-covalent attachment. In some embodiments, the method further includes dissociating the rcHC-HA / PTX3 complex from the solid support. In some embodiments, the method further includes purifying the dissociated rcHC-HA / PTX3 complex. In some embodiments, purification includes affinity purification, centrifugation, filtration, chromatography, or a combination thereof. In some embodiments, the PTX3, IαI HC1, or TSG-6 peptide contains an affinity tag. In some embodiments, the affinity tag is selected from hemagglutinin tags, polyhistidine tags, myc tags, FLAG tags, and glutathione S-transferase (GST) tags. In some embodiments, HMW HA is immobilized by binding HMW HA to an intermediate peptide. In some embodiments, the intermediate peptide is covalently attached to the solid support. In some embodiments, the binding of HMW HA to the intermediate peptide is non-covalent. In some implementations, the intermediate polypeptide is a HA-binding protein (HABP).In some embodiments, the intermediate polypeptide is selected from HAPLN1, HAPLN2, HAPLN3, HAPLN4, aggregate proteoglycans, multipotent proteoglycans, neuroproteoglycans, short proteoglycans, phosphoproteoglycans, TSG-6, CD44, stapillin-1, stapillin-2, or HABP sufficient to bind a portion of HA. In some embodiments, the intermediate polypeptide is a multipotent proteoglycan. In some embodiments, the intermediate polypeptide includes a linker module. In some embodiments, the intermediate polypeptide includes a linker module of HAPLN1, HAPLN2, HAPLN3, HAPLN4, aggregate proteoglycans, multipotent proteoglycans, neuroproteoglycans, short proteoglycans, phosphoproteoglycans, TSG-6, CD44, stapillin-1, or stapillin-2. In some embodiments, the intermediate polypeptide includes a linker module of a multipotent proteoglycan. In some embodiments, the intermediate polypeptide includes a polypeptide represented by any one of SEQ ID NO:54-99. In some embodiments, the intermediate polypeptide includes a polypeptide linker. In some embodiments, the linker is attached to a solid support. In some embodiments, the method further includes dissociating the rcHC-HA / PTX3 complex from the intermediate polypeptide. In some embodiments, dissociating the rcHC-HA / PTX3 complex from the intermediate polypeptide includes contacting the complex with a dissociation agent. In some embodiments, the dissociation agent is guanidine hydrochloride or urea. In some embodiments, the dissociation agent is guanidine hydrochloride in a concentration of about 4 M to about 8 M. In some embodiments, the intermediate polypeptide or linker contains a proteolytic cleavage sequence. In some embodiments, dissociation includes cleaving the intermediate polypeptide or linker at the proteolytic cleavage sequence. In some embodiments, cleavage includes contacting the proteolytic cleavage sequence with a protease. In some embodiments, the protease is selected from Fehling's protease, 3C protease, caspase, matrix metalloproteinase, and TEV protease.

[0014] In some embodiments, this document describes reconstituted HC-HA (rcHC-HA / PTX3) complexes produced by any of the methods provided herein for generating rcHC-HA / PTX3 complexes.

[0015] In some embodiments, this document describes a reconstituted HC-HA / PTX3 (rcHC-HA / PTX3) complex comprising high molecular weight hyaluronic acid (HMW HA), PTX3, and IαI HC1, wherein the rcHC-HA / PTX3 complex promotes M2 polarization of macrophages. In some embodiments, this document describes a native HC-HA / PTX3 (nHC-HA / PTX3) complex comprising high molecular weight hyaluronic acid (HMW HA), PTX3, and IαI HC1, wherein the nHC-HA / PTX3 complex promotes M2 polarization of macrophages. In some embodiments, this document describes a method for inducing M2 polarization of macrophages, comprising contacting macrophages with rcHC-HA / PTX3 or a separated nHC-HA / PTX3 complex.

[0016] In some embodiments, this document describes a reconstituted HC-HA / PTX3 (rcHC-HA / PTX3) complex comprising high molecular weight hyaluronic acid (HMW HA), PTX3, and IαI HC1, wherein the rcHC-HA / PTX3 complex reduces the expression of IL-12p40 in LPS-stimulated macrophages, wherein the IL-12p40 level expressed by LPS-stimulated macrophages is lower when the rcHC-HA / PTX3 complex is applied compared to the IL-12p40 level expressed by LPS-stimulated macrophages in the absence of the rcHC-HA / PTX3 complex. In some embodiments, the level of IL-12p40 mRNA is reduced. In some embodiments, the level of IL-12p40 protein is reduced.

[0017] In some embodiments, this document describes a natural HC-HA / PTX3 (nHC-HA / PTX3) complex comprising high molecular weight hyaluronic acid (HMW HA), PTX3, and IαI HC1, wherein the nHC-HA / PTX3 complex reduces the expression of IL-12p40 in LPS-stimulated macrophages, wherein the IL-12p40 level expressed by LPS-stimulated macrophages is lower when the macrophages are exposed to the nHC-HA / PTX3 complex compared to the IL-12p40 level expressed by LPS-stimulated macrophages in the absence of the nHC-HA / PTX3 complex. In some embodiments, the level of IL-12p40 mRNA is reduced. In some embodiments, the level of IL-12p40 protein is reduced.

[0018] In some embodiments, this document describes a method for reducing the level of IL-12p40 expressed in LPS-stimulated macrophages, which includes contacting LPS-stimulated macrophages with rcHC-HA / PTX3 or a separated nHC-HA / PTX3 complex. In some embodiments, the level of IL-12p40 mRNA is reduced. In some embodiments, the level of IL-12p40 protein is reduced.

[0019] In some embodiments, this document describes a reconstituted HC-HA / PTX3 (rcHC-HA / PTX3) complex comprising high molecular weight hyaluronic acid (HMW HA), PTX3, and IαI HC1, wherein the rcHC-HA / PTX3 complex reduces the expression of IL-12p70 protein in LPS-stimulated macrophages, wherein the amount of IL-12p70 protein expressed by LPS-stimulated macrophages is lower when the LPS-stimulated macrophages are exposed to the rcHC-HA / PTX3 complex compared to the amount of IL-12p70 expressed by LPS-stimulated macrophages in the absence of the rcHC-HA / PTX3 complex.

[0020] In some embodiments, this document describes a natural HC-HA / PTX3 (nHC-HA / PTX3) complex comprising high molecular weight hyaluronic acid (HMW HA), PTX3, and IαI HC1, wherein the nHC-HA / PTX3 complex reduces the expression of IL-12p70 protein in LPS-stimulated macrophages, wherein the amount of IL-12p70 protein expressed by LPS-stimulated macrophages is lower when the LPS-stimulated macrophages are exposed to the nHC-HA / PTX3 complex compared to the amount of IL-12p70 protein expressed by LPS-stimulated macrophages in the absence of the nHC-HA / PTX3 complex.

[0021] In some embodiments, this document describes a method for reducing the level of IL-12p70 protein expressed by LPS-stimulated macrophages, including contacting LPS-stimulated macrophages with rcHC-HA / PTX3 or isolated nHC-HA / PTX3 complex.

[0022] In some embodiments, this document describes a reconstituted HC-HA / PTX3 (rcHC-HA / PTX3) complex comprising high molecular weight hyaluronic acid (HMW HA), PTX3, and IαI HC1, wherein the rcHC-HA / PTX3 complex reduces IL-23 expression in LPS-stimulated macrophages, wherein when LPS-stimulated macrophages are exposed to the rcHC-HA / PTX3 complex, the IL-23 level expressed by LPS-stimulated macrophages is lower than that expressed by LPS-stimulated macrophages in the absence of the rcHC-HA / PTX3 complex. In some embodiments, the level of IL-23 mRNA is reduced. In some embodiments, the level of IL-23 protein is reduced.

[0023] In some embodiments, this document describes a natural HC-HA / PTX3 (rcHC-HA / PTX3) complex comprising high molecular weight hyaluronic acid (HMW HA), PTX3, and IαI HC1, wherein this complex reduces IL-23 expression in LPS-stimulated macrophages, wherein when LPS-stimulated macrophages are exposed to the nHC-HA / PTX3 complex, the IL-23 level expressed by LPS-stimulated macrophages is lower than that expressed by macrophages in the absence of the nHC-HA / PTX3 complex. In some embodiments, the level of IL-23 mRNA is reduced. In some embodiments, the level of IL-23 protein is reduced.

[0024] In some embodiments, this document describes a method for reducing the level of IL-23 expressed in LPS-stimulated macrophages, which includes contacting LPS-stimulated macrophages with rcHC-HA / PTX3 or a separated nHC-HA / PTX3 complex. In some embodiments, the level of IL-23 mRNA is reduced. In some embodiments, the level of IL-23 protein is reduced.

[0025] In some embodiments, this document describes a reconstituted HC-HA / PTX3 (rcHC-HA / PTX3) complex comprising high molecular weight hyaluronic acid (HMW HA), PTX3, and IαI HC1, wherein the rcHC-HA / PTX3 complex enhances IL-10 expression in LPS-stimulated macrophages, wherein LPS-stimulated macrophages exhibit higher IL-10 levels upon contact with the rcHC-HA / PTX3 complex compared to IL-10 levels expressed by LPS-stimulated macrophages in the absence of the rcHC-HA / PTX3 complex. In some embodiments, IL-10 mRNA levels are increased. In some embodiments, IL-10 protein levels are increased.

[0026] In some embodiments, this document describes a natural HC-HA / PTX3 (nHC-HA / PTX3) complex comprising high molecular weight hyaluronic acid (HMW HA), PTX3, and IαI HC1, wherein this complex enhances IL-10 expression in LPS / IFNγ-stimulated macrophages, wherein the amount of IL-10 expressed by LPS-stimulated macrophages upon contact with the nHC-HA / PTX3 complex is higher than the amount of IL-10 expressed by LPS / IFNγ-stimulated macrophages in the absence of the nHC-HA / PTX3 complex. In some embodiments, IL-10 mRNA levels are increased. In some embodiments, IL-10 protein levels are increased.

[0027] In some embodiments, this document describes methods for increasing the level of IL-10 expressed in LPS-stimulated macrophages, including contacting LPS-stimulated macrophages with rcHC-HA / PTX3 or isolated nHC-HA / PTX3 complexes. In some embodiments, the level of IL-10 mRNA is increased. In some embodiments, the level of IL-10 protein is increased.

[0028] In some embodiments, this document describes a reconstituted HC-HA / PTX3 (rcHC-HA / PTX3) complex comprising high molecular weight hyaluronic acid (HMW HA), PTX3, and IαI HC1, wherein the rcHC-HA / PTX3 complex promotes apoptosis of LPS-stimulated neutrophils but does not promote apoptosis in resting neutrophils. In some embodiments, the reconstituted HC-HA / PTX3 (rcHC-HA / PTX3) complex comprising high molecular weight hyaluronic acid (HMW HA), PTX3, and IαI HC1 promotes apoptosis of LPS-stimulated neutrophils, wherein when an LPS-stimulated neutrophil sample is contacted with the rcHC-HA / PTX3 complex, the number of apoptotic LPS-stimulated neutrophils in the sample is higher than the number of apoptotic LPS-stimulated neutrophils in the sample without the rcHC-HA / PTX3 complex.

[0029] In some embodiments, this document describes a natural HC-HA / PTX3 (nHC-HA / PTX3) complex comprising high molecular weight hyaluronic acid (HMW HA), PTX3, and IαI HC1, wherein the nHC-HA / PTX3 complex promotes apoptosis of LPS-stimulated neutrophils but does not promote apoptosis in resting neutrophils. In some embodiments, the nHC-HA / PTX3 complex comprising high molecular weight hyaluronic acid (HMW HA), PTX3, and IαI HC1 promotes apoptosis of LPS-stimulated neutrophils, wherein when an LPS-stimulated neutrophil sample is contacted with the nHC-HA / PTX3 complex, the number of apoptotic LPS-stimulated neutrophils in the sample is higher than the number of apoptotic LPS-stimulated neutrophils in the sample in the absence of the nHC-HA / PTX3 complex.

[0030] In some embodiments, this document describes a method for inducing apoptosis in LPS-stimulated neutrophils, which includes contacting LPS-stimulated neutrophils with rcHC-HA / PTX3 or isolated nHC-HA / PTX3 complex.

[0031] In some embodiments, this document describes a reconstituted HC-HA / PTX3 (rcHC-HA / PTX3) complex comprising high molecular weight hyaluronic acid (HMW HA), PTX3, and IαI HC1, wherein the rcHC-HA / PTX3 complex promotes phagocytosis of apoptotic neutrophils, wherein when a sample containing apoptotic neutrophils and LPS-stimulated macrophages is contacted with the rcHC-HA / PTX3 complex, the number of apoptotic neutrophils phagocytosed by LPS-stimulated macrophages in the sample is higher than the number of neutrophils phagocytosed by LPS-stimulated macrophages in the absence of the rcHC-HA / PTX3 complex. In some embodiments, this document describes a reconstituted HC-HA / PTX3 (rcHC-HA / PTX3) complex comprising high molecular weight hyaluronic acid (HMW HA), PTX3, and IαI HC1, wherein the complex promotes phagocytosis of apoptotic neutrophils, wherein when a sample containing apoptotic neutrophils and resting macrophages is contacted with the rcHC-HA / PTX3 complex, the number of apoptotic neutrophils phagocytosed by resting macrophages in the sample is higher than the number of apoptotic neutrophils phagocytosed by resting macrophages in the absence of the rcHC-HA / PTX3 complex.

[0032] In some embodiments, this document describes a natural HC-HA / PTX3 (nHC-HA / PTX3) complex comprising high molecular weight hyaluronic acid (HMW HA), PTX3, and IαI HC1, wherein the nHC-HA / PTX3 complex promotes phagocytosis of apoptotic neutrophils, wherein when a sample containing apoptotic neutrophils and LPS-stimulated macrophages is contacted with the nHC-HA / PTX3 complex, the number of apoptotic neutrophils phagocytosed by LPS-stimulated macrophages in the sample is higher than the number of neutrophils phagocytosed by LPS-stimulated macrophages in the absence of the nHC-HA / PTX3 complex. In some embodiments, this document describes a natural HC-HA / PTX3 (nHC-HA / PTX3) complex comprising high molecular weight hyaluronic acid (HMW HA), PTX3, and IαI HC1, wherein the nHC-HA / PTX3 complex promotes phagocytosis of apoptotic neutrophils, wherein when a sample containing apoptotic neutrophils and LPS-stimulated macrophages is contacted with the nHC-HA / PTX3 complex, the number of apoptotic neutrophils phagocytosed by resting macrophages in the sample is higher than the number of apoptotic neutrophils phagocytosed by resting macrophages in the absence of the nHC-HA / PTX3 complex.

[0033] In some embodiments, this document describes a method for inducing phagocytosis of apoptotic neutrophils, which includes contacting a sample containing apoptotic neutrophils and LPS-stimulated or resting macrophages with rcHC-HA / PTX3 or isolated nHC-HA / PTX3 complex.

[0034] In some embodiments, this document describes a reconstituted HC-HA / PTX3 (rcHC-HA / PTX3) complex comprising high molecular weight hyaluronic acid (HMW HA), PTX3, and IαI HC1, wherein the rcHC-HA / PTX3 complex promotes the attachment of LPS-stimulated macrophages to at least the same level as a natural HC-HA / PTX3 (nHC-HA / PTX3) complex isolated from human umbilical cord or human amnion, or a combination of nHC-HA / PTX3 complexes derived from both human umbilical cord and human amnion, wherein attachment includes contacting LPS-stimulated macrophages with the rcHC-HA / PTX3 or nHC-HA / PTX3 complex fixed to a solid support. In some embodiments, the nHC-HA / PTX3 is isolated from human umbilical cord. In some embodiments, the nHC-HA / PTX3 is isolated from human amnion. In some embodiments, the nHC-HA / PTX3 is isolated from a combination of nHC-HA / PTX3 complexes derived from both human umbilical cord and human amnion.

[0035] In some embodiments, the weight-average molecular weight of the HMWHA of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is about 500 kDa to about 10,000 kDa, about 800 kDa to about 8,500 kDa, about 1,100 kDa to about 5,000 kDa, or about 1,400 kDa to about 3,500 kDa. In some embodiments, the weight-average molecular weight of the HMWHA of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is about 3,000 kDa.

[0036] In some embodiments, the HCl of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is covalently linked to HA.

[0037] In some embodiments, the PTX3 protein of the rcHC-HA / PTX3 complex is a recombinant protein. In some embodiments, the PTX3 of the rcHC-HA / PTX3 complex comprises a polypeptide having the sequence shown in SEQ ID NO:33 or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity with the polypeptide having the sequence shown in SEQ ID NO:33. In some embodiments, the PTX3 protein used in this method is a multimeric protein. In some embodiments, the PTX3 protein used in this method is a homopolymer. In some embodiments, the PTX3 homopolymer is a dimer, trimer, tetramer, pentamer, hexamer, or octamer. In some embodiments, the PTX3 homopolymer is a trimer, tetramer, or octamer. In some embodiments, the PTX3 homopolymer is an octamer.

[0038] In some embodiments, the IαI HC1 of the rcHC-HA / PTX3 complex comprises a polypeptide having the sequence shown in SEQ ID NO:47 or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity with the polypeptide having the sequence shown in SEQ ID NO:47. In some embodiments, the IαI HC1 of the rcHC-HA / PTX3 complex is a recombinant protein.

[0039] In some embodiments, the rcHC-HA / PTX3 complex comprises TSG-6. In some embodiments, the TSG-6 protein is a recombinant protein. In some embodiments, the TSG-6 protein comprises a polypeptide having the sequence shown in SEQ ID NO:2 or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity with the polypeptide having the sequence shown in SEQ ID NO:2.

[0040] In some embodiments, the PTX3, IαI HC1, or TSG-6 peptide of the rcHC-HA / PTX3 complex includes an affinity tag. In some embodiments, the affinity tag is selected from hemagglutinin tags, polyhistidine tags, myc tags, FLAG tags, and glutathione S-transferase (GST) tags.

[0041] In some embodiments, this document describes a pharmaceutical composition comprising an nHC-HA / PTX3 or rcHC-HA / PTX3 complex produced by the methods described herein or provided herein. In some embodiments, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier or excipient. In some embodiments, the pharmaceutical composition is in the form of a solution, suspension, powder, ointment, tablet, capsule, or aerosol. In some embodiments, the pharmaceutical composition is in the form of a solid, cross-linked gel, or liposome. In some embodiments, the pharmaceutical composition is in the form of a cross-linked hyaluronic acid hydrogel. In some embodiments, the pharmaceutical composition comprises a natural polymer. In some embodiments, the natural polymer comprises fibronectin, collagen, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparin sulfate, chondroitin sulfate, or combinations thereof. In some embodiments, the pharmaceutical composition further comprises an anti-inflammatory agent, an anti-scarring agent, an antitumor agent, a chemotherapeutic agent, an immunosuppressant, a cytotoxic agent, an antimicrobial agent, or a combination thereof.

[0042] In some embodiments, this document describes the use of nHC-HA / PTX3 or rcHC-HA / PTX3 complexes produced by the methods described herein or provided herein for the production of pharmaceuticals.

[0043] In some embodiments, this document describes a combination comprising: (a) an nHC-HA / PTX3 or rcHC-HA / PTX3 complex produced by the methods described herein or provided herein; and (b) an anti-inflammatory agent, an anti-scarring agent, an antitumor agent, a chemotherapeutic agent, an immunosuppressant, a cytotoxic agent, an antimicrobial agent, or a combination thereof.

[0044] In some embodiments, treatment methods described herein include administration of a pharmaceutical composition comprising an nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein or produced by the methods provided herein.

[0045] In some embodiments, methods for preventing or reversing scar formation or fibrosis in tissue are described herein, comprising administering to a subject in need an effective amount of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein or produced by the methods provided herein. In some embodiments, the method comprises contacting the tissue with an effective amount of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex. In some embodiments, the scar is a dermatitis scar, keloid, contracture scar, hypertrophic scar, or acne-related scar. In some embodiments, the use of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein or produced by the methods provided herein for reducing or preventing scar formation is described herein. In some embodiments, the application of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein or produced by the methods provided herein reduces or prevents scar formation or fibrosis by reducing or inhibiting TGF-β signaling in the tissue.

[0046] In some embodiments, methods for preventing or reducing inflammation in a subject in need are described herein, comprising administering to the subject an effective amount of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein or produced by the methods provided herein. In some embodiments, the method comprises contacting inflamed tissue with the nHC-HA / PTX3 or rcHC-HA / PTX3 complex. In some embodiments, the inflammation is acute or chronic. In some embodiments, the subject suffers from an inflammatory condition. In some embodiments, the inflammatory condition is macrophage-mediated, Th-17-mediated, or T-cell-mediated. In some embodiments, the subject suffers from an autoimmune disease, allergic reaction, leukocyte deficiency, infection, graft-versus-host disease, tissue transplant rejection, or a combination thereof. In some embodiments, the inflammatory condition is rheumatoid arthritis. In some embodiments, the inflammatory condition is an ocular inflammation. In some embodiments, the inflammatory condition is conjunctivitis, keratitis, blepharitis, palpebral conjunctivitis, scleritis, superficial scleritis, uveitis, retinitis, or choroiditis. In some embodiments, the acute inflammation is caused by myocardial infarction, stroke, endotoxic shock, or sepsis. In some embodiments, the subject has atherosclerosis. In some embodiments, the subject has cancer. In some embodiments, the subject has inflammation of a solid tumor. In some embodiments, the subject is given a combination of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex and an additional anti-inflammatory agent. In some embodiments, the additional anti-inflammatory agent is selected from anti-TGF-β antibodies, anti-TGF-β receptor blocking antibodies, anti-TNF antibodies, anti-TNF receptor blocking antibodies, anti-IL1β antibodies, anti-IL1β receptor blocking antibodies, anti-IL-2 antibodies, anti-IL-2 receptor blocking antibodies, anti-IL-6 antibodies, anti-IL-6 receptor blocking antibodies, anti-IL-12 antibodies, anti-IL-12 receptor blocking antibodies, anti-IL-17 antibodies, anti-IL-17 receptor blocking antibodies, anti-IL-23 antibodies, or anti-IL-23 receptor blocking antibodies. In some embodiments, the type 1 interferon is IFN-α or IFN-β. In some embodiments, the use of nHC-HA / PTX3 or rcHC-HA / PTX3 complexes produced by the methods described herein or provided herein for reducing or preventing inflammation is described herein.

[0047] In some embodiments, this document describes a method for treating skin trauma or ulcers in a subject in need, comprising administering to the subject an effective amount of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein or produced by the methods provided herein. In some embodiments, the method comprises contacting the skin trauma or ulcer with an effective amount of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex. In some embodiments, this document describes the use of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein or produced by the methods provided herein for the treatment of skin trauma or ulcers. In some embodiments, the skin trauma or ulcer is an unhealed ulcer.

[0048] In some embodiments, methods for promoting or inducing bone formation in a subject of interest are described herein, comprising administering to the subject an effective amount of the rcHC-HA / PTX3 complex or nHC-HA / PTX3 complex described herein or produced by the methods provided herein. In some embodiments, the subject suffers from arthritis, osteoporosis, alveolar bone degradation, Paget's disease, or a bone tumor. In some embodiments, the use of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein or produced by the methods provided herein for promoting or inducing bone formation in a subject is described herein.

[0049] In some embodiments, methods for preventing or reducing abnormal angiogenesis in subjects of need are described herein, comprising administering to the subject an effective amount of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein or produced by the methods provided herein. In some embodiments, the subject has wet age-related macular degeneration (wARMD) or diabetic proliferative retinopathy. In some embodiments, the subject has cancer. In some embodiments, the subject has a solid tumor. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is administered to the subject in combination with anticancer therapy. In some embodiments, the anticancer therapy includes the administration of an antitumor agent, a cytotoxic agent, an antiangiogenic agent, a chemotherapeutic agent, or radiotherapy. In some embodiments, the anticancer therapy is administered sequentially, simultaneously, or intermittently with the nHC-HA / PTX3 or rcHC-HA / PTX3 complex. In some embodiments, the use of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein or produced by the methods provided herein for reducing or preventing angiogenesis is described herein.

[0050] In some embodiments, methods for preventing transplant rejection in transplant recipients are described herein, comprising administering to the transplant recipient an effective amount of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein or produced by the methods provided herein. In some embodiments, the method comprises contacting the graft with the nHC-HA / PTX3 or rcHC-HA / PTX3 complex. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is administered before, after, or during a transplant procedure. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is administered in combination with an immunosuppressant. In some embodiments, the use of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein or produced by the methods provided herein for preventing transplant rejection in transplant recipients is described herein. In some embodiments, the graft is a corneal graft.

[0051] In some embodiments, this document describes a method for inducing stem cell expansion in a subject in need, comprising administering to the subject an effective amount of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein or produced by the methods provided herein. In some embodiments, the method comprises contacting stem cells with the nHC-HA / PTX3 or rcHC-HA / PTX3 complex. In some embodiments, this document describes the use of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein or produced by the methods provided herein for inducing stem cell expansion. In some embodiments, administration of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein or produced by the methods provided herein induces stem cell expansion by inhibition of TGF-β signaling and / or upregulation of the BMP signaling pathway. In some embodiments, administration of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein or produced by the methods provided herein induces stem cell expansion by reprogramming differentiated cells into stem cells (or induced progenitor cells, iPSCs).

[0052] In some embodiments, this document describes the use of nHC-HA / PTX3 or rcHC-HA / PTX3 complexes produced by the methods described herein or provided herein for cell therapy.

[0053] In some embodiments, this document describes a method of administering cell therapy to a subject in need, comprising administering to the subject a composition comprising an effective amount of an nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein or produced by the methods provided herein, combined with therapeutic cells. In some embodiments, the therapeutic cells and the nHC-HA / PTX3 or rcHC-HA / PTX3 complex are administered locally to damaged tissue. In some embodiments, the therapeutic cells and the nHC-HA / PTX3 or rcHC-HA / PTX3 complex are administered systemically. In some embodiments, the therapeutic cells are stem cells. In some embodiments, the therapeutic agent is stem cells. In some embodiments, the cell therapy includes the administration of stem cells. In some embodiments, the stem cells are mesenchymal stem cells. In some embodiments, the stem cells are induced progenitor stem cells. In some embodiments, the cell therapy includes the administration of differentiated cells. In some embodiments, the therapeutic cells are insulin-producing cells. In some embodiments, the insulin-producing cells are pancreatic islet cells. In some embodiments, the subject has type 1 diabetes.

[0054] In some embodiments, this document describes a method of administering cell therapy to a subject in need, comprising administering to the subject in combination an effective amount of an nHC-HA / PTX3 or rcHC-HA / PTX3 complex, generated by the methods described herein or provided herein, and cells contained in a cell delivery device. In some embodiments, the cells are contained in a microcapsule. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is attached to the microcapsule. In some embodiments, the microcapsule is applied topically to damaged tissue. In some embodiments, the therapeutic cells and the nHC-HA / PTX3 or rcHC-HA / PTX3 complex are administered systemically. In some embodiments, the therapeutic cells are stem cells. In some embodiments, the therapeutic agent is a stem cell. In some embodiments, the cell therapy includes the administration of stem cells. In some embodiments, the stem cells are mesenchymal stem cells. In some embodiments, the stem cells are induced progenitor stem cells. In some embodiments, the cell therapy includes the administration of differentiated cells. In some embodiments, the therapeutic cells are insulin-producing cells. In some embodiments, the insulin-producing cells are islet cells. In some implementations, the subject has type 1 diabetes.

[0055] In some embodiments, methods for preventing or reducing pain in subjects in need are described herein, comprising administering to the subject an effective amount of an nHC-HA / PTX3 or rcHC-HA / PTX3 complex produced by the methods described herein or provided herein, wherein the pain is caused by radiation from chemical burns, severe bacterial keratitis, Stevens-Johnson syndrome, toxic epidermal necrolysis, or ocular tumors. In some embodiments, the use of an nHC-HA / PTX3 or rcHC-HA / PTX3 complex produced by the methods described herein or provided herein for the relief of pain in subjects, wherein the pain is caused by radiation from chemical burns, severe bacterial keratitis, Stevens-Johnson syndrome, toxic epidermal necrolysis, or ocular tumors is described herein.

[0056] In some embodiments, methods for inducing or promoting tissue regeneration in a subject in need are described herein, comprising administering to the subject an effective amount of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein or produced by the methods provided herein. In some embodiments, the method comprises contacting damaged tissue of the subject with an effective amount of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex. In some embodiments, the tissue is bone or gingiva, corneal tissue, or conjunctival tissue. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is administered in combination with therapeutic cells, multiple therapeutic cells, or a tissue graft. In some embodiments, the tissue graft is an allogeneic graft or an autologous graft. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is administered in combination with a tissue-based therapy. In some embodiments, the use of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein or produced by the methods provided herein for inducing or promoting tissue regeneration in a subject is described herein.

[0057] In some embodiments, this document describes a method for treating fibrosis in a subject in need, comprising administering to the subject an effective amount of an nHC-HA / PTX3 or rcHC-HA / PTX3 complex produced by the methods described herein or provided herein. In some embodiments, this treatment inhibits or prevents scar formation. In some embodiments, this document describes the use of an nHC-HA / PTX3 or rcHC-HA / PTX3 complex produced by the methods described herein or provided herein for the treatment of fibrosis in a subject.

[0058] In some embodiments, this document describes a method for treating obesity or insulin resistance in a subject in need, comprising administering to the subject an effective amount of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein or produced by the methods provided herein. In some embodiments, this treatment inhibits or reduces the number of M1 adipose tissue macrophages in the subject. In some embodiments, this document describes a method for inhibiting or reducing the number of M1 adipose tissue macrophages in a subject in need by administering to the subject an effective amount of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein or produced by the methods provided herein. In some embodiments, the subject has been diagnosed with obesity or insulin resistance. In some embodiments, this document describes the use of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein or produced by the methods provided herein for treating obesity or insulin resistance in a subject.

[0059] In some embodiments, this document describes a method for treating conjunctival laxity in a subject in need, comprising administering to the subject an effective amount of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein or produced by the methods provided herein. In some embodiments, the method comprises contacting the conjunctiva of the subject with an effective amount of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex. In some embodiments, this document describes the use of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein or produced by the methods provided herein for treating conjunctival laxity in a subject.

[0060] In some embodiments, a cell culture is described herein comprising a substrate suitable for culturing cells and an rcHA / PTX3 complex or an nHC-HA / PTX3 complex produced by the methods described herein or provided herein. In some embodiments, the rcHA / PTX3 complex or nHC-HA / PTX3 complex is immobilized on the substrate.

[0061] In some embodiments, treatment methods described herein include administering to a subject an effective amount of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein or produced by the methods provided herein, along with an additional therapeutic agent. In some embodiments, the additional therapeutic agent is selected from interferon, antitumor necrosis agents, interleukin-1 (IL-1) receptor antagonists, interleukin-2 (IL-2) receptor antagonists, interleukin-6 (IL-6) receptor antagonists, interleukin-12 (IL-12) receptor antagonists, interleukin-17 (IL-17) receptor antagonists, interleukin-23 (IL-23) receptor antagonists, cytotoxic agents, antimicrobial agents, interleukins, immunomodulators, antibiotics, T-cell co-stimulation blockers, disease-relieving antirheumatic agents, and immunomodulators. Inhibitors, anti-lymphocyte antibodies, anti-angiogenic agents, chemotherapeutic agents, antitumor agents, antimetabolites, Akt inhibitors, IGF-1 inhibitors, angiotensin II antagonists, cyclooxygenase inhibitors, heparinase inhibitors, lymphokine inhibitors, cytokine inhibitors, IKK inhibitors, p38MAPK inhibitors, anti-apoptotic pathway inhibitors, apoptosis pathway agonists, PPAR agonists, Ras inhibitors, telomerase inhibitors, protease inhibitors, metalloproteinase inhibitors, aminopeptidase inhibitors, SHIP activators, and combinations thereof. In some embodiments, the antimicrobial agent is an antiviral, antibacterial, or antifungal agent.

[0062] In some embodiments, the treatment method described herein involves administering an effective amount of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein or produced by the methods provided herein, and the subject is a mammal. In some embodiments, the mammal is a human.

[0063] In some embodiments, treatment methods described herein involve attaching an nHC-HA / PTX3 or rcHC-HA / PTX3 complex to a solid surface. In some embodiments, the solid surface is the surface of nanoparticles, beads, microcapsules, or an implantable medical device, or a portion thereof.

[0064] In some embodiments, a medical device is described herein comprising a substrate coated with an nHC-HA / PTX3 or rcHC-HA / PTX3 complex produced by the methods described herein or provided herein. In some embodiments, the substrate comprises at least one of the following: a scaffold, a joint, a screw, a rod, a pin, a plate, a nail, a shunt, a clamp, a clip, a suture, a suture anchor, an electrode, a catheter, a lead, a graft, a dressing, a pacemaker, a pacemaker housing, a cardioverter, a cardioverter housing, a defibrillator, a defibrillator housing, a prosthesis, an ear drain, an ophthalmic implant, an orthopedic device, a spinal disc, a bone substitute, an anastomosis device, a perivascular wrap, a colostomy bag attachment device, a hemostatic barrier, a vascular implant, a vascular support, a tissue adhesive, a tissue sealant, a tissue scaffold, and an endoluminal device.

[0065] In some embodiments, this document describes a device comprising an nHC-HA / PTX3 or rcHC-HA / PTX3 complex fixed to a surface described herein or produced by the methods provided herein. In some embodiments, the surface is a polystyrene, polyethylene, silica, metal, or polymer surface. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is attached to a microcapsule. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is attached to nanoparticles. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is attached to beads, chips, glass slides, or filters. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is attached to a contact lens. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is attached to a surgical implant or prosthesis. In some embodiments, the implant or prosthesis is an artificial joint, bone implant, suture, or scaffold. In some embodiments, the artificial joint is an artificial hip joint, an artificial knee joint, an artificial shoulder joint, or an artificial knee. In some embodiments, the stent is a coronary artery stent, a ureteral stent, a urethral stent, a prostate stent, an esophageal stent, or a bone stent.

[0066] In some embodiments, a medical device is described herein comprising: a structure suitable for implantation in a patient, wherein a surface or a portion thereof of the structure is attached to an nHC-HA / PTX3 or rcHC-HA / PTX3 complex comprising the methods described herein or provided herein. In some embodiments, attachment comprises covalent or non-covalent attachment of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex to a surface or a portion thereof of the structure. In some embodiments, attachment comprises coating a surface or a portion thereof of the structure with a composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex. In some embodiments, the structure is a vascular stent, an artificial joint, a suture, or a microcapsule. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex inhibits bacterial biofilm formation. In some embodiments, the microcapsule contains therapeutic cells. In some embodiments, the therapeutic cells are stem cells.

[0067] In some embodiments, methods for modulating macrophage activity are described herein, comprising contacting macrophages with an amount of an nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein or produced by methods provided herein, such amount being sufficient to reduce or inhibit the expression of IL-12 or IL-23, but also promoting the expression of IL-10 in polarized macrophages from the M1 to M2 phenotypes. In some embodiments, the macrophages have been stimulated with a pro-inflammatory mediator. In some embodiments, the pro-inflammatory mediator is lipopolysaccharide (LPS), tumor necrosis factor-α (TNF-α), interferon-γ (IFNγ), or a combination thereof. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is contacted with macrophages in a subject. In some embodiments, the subject is a mammal. In some embodiments, the mammal is a human. In some embodiments, the method is performed in vitro. In some embodiments, treatment methods are described herein, comprising administering macrophages modulated by methods provided herein for modulating macrophage activity.

[0068] In some embodiments, a kit is described herein comprising an nHC-HA / PTX3 or rcHC-HA / PTX3 complex produced by the methods described herein or provided herein, an apparatus for administering the composition, and optional instructions for administration.

[0069] In some embodiments, this document describes a combination or mixture comprising: (a) a complex of PTX3 pre-bound to HMW HA (PTX3 / HA); (b) a meta-α-inhibitor (IαI) protein containing heavy chain 1 (HC1); and (c) TSG-6.

[0070] In some embodiments, this document describes a combination or mixture comprising: (a) a complex of TSG-6 pre-bound to HC-HA; and (b) PTX3.

[0071] In some embodiments, this document describes a method for producing a reconstituted HC-HA / PTX3 (rcHC-HA / PTX3) complex in vitro, comprising: (a) contacting (i) high molecular weight hyaluronic acid (HMW HA) immobilized on a solid support, (ii) a meta-α-inhibitor (IαI) protein containing heavy chain 1 (HC1), and (iii) TSG-6 to form a rcHC-HA complex pre-bound to TSG-6; and (b) contacting the rcHC-HA complex pre-bound to TSG-6 with a pentacyclic protein 3 (PTX3) protein to form the rcHC-HA / PTX3 complex.

[0072] In some embodiments, this document describes a method for producing a reconstituted HC-HA / PTX3 (rcHC-HA / PTX3) complex in vitro, comprising contacting (i) high molecular weight hyaluronic acid (HMW HA) immobilized on a solid support, (ii) pentacyclic protein 3 (PTX3) protein, (iii) a meta-α-inhibitor (IαI) protein containing heavy chain 1 (HC1), and (iv) tumor necrosis factor α-stimulated gene 6 (TSG-6) to form an immobilized rcHC-HA / PTX3 complex.

[0073] In some embodiments, this document describes a complex comprising immobilized HA bound to PTX3.

[0074] In some embodiments, this document describes a method for producing in vitro a complex comprising immobilized HA bound to PTX3, comprising contacting high molecular weight hyaluronic acid (HMW HA) with a PTX3 protein under conditions that effectively form a PTX3 / HA complex, wherein the HMW HA is immobilized on a solid support. In some embodiments, the PTX3 protein is a naturally occurring PTX3 protein isolated from a cell. In some embodiments, the cell is a mammalian cell. In some embodiments, the cell is a human cell. In some embodiments, the cell is an amniotic cell. In some embodiments, the cell is an umbilical cord cell. In some embodiments, the cell is an amniotic cell derived from the umbilical cord. In some embodiments, the amniotic cell is an amniotic epithelial cell. In some embodiments, the amniotic cell is an umbilical cord epithelial cell. In some embodiments, the amniotic cell is an amniotic stromal cell. In some embodiments, the amniotic cell is an umbilical cord stromal cell. In some embodiments, the PTX3 protein is a recombinant protein. In some embodiments, the PTX3 protein comprises a polypeptide having the sequence shown in SEQ ID NO:2 or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity with the polypeptide having the sequence shown in SEQ ID NO:2. In some embodiments, the PTX3 protein used in this method is a multimeric protein. In some embodiments, the PTX3 protein used in this method is a homopolymer. In some embodiments, the PTX3 homopolymer is a dimer, trimer, tetramer, pentamer, hexamer, or octamer. In some embodiments, the PTX3 homopolymer is a trimer, tetramer, or octamer. In some embodiments, the PTX3 homopolymer is an octamer. In some embodiments, the PTX3 comprises a modified multipolymerized domain or a heteropolymerized domain. In some embodiments, immobilizing the HMW HA comprises non-covalent attachment to a solid support. In some embodiments, immobilizing HMW HA includes binding HMW HA to an intermediate polypeptide. In some embodiments, the intermediate polypeptide is covalently attached to a solid support. In some embodiments, the binding of HMW HA to the intermediate polypeptide is non-covalent. In some embodiments, the intermediate polypeptide is an HA-binding protein (HABP). In some embodiments, the intermediate polypeptide is selected from HAPLN1, HAPLN2, HAPLN3, HAPLN4, agglutinin, pluripotent proteoglycans, neuroproteoglycans, short proteoglycans, phosphoproteoglycans, TSG-6, CD44, stapillin-1, stapillin-2, or a portion thereof sufficient to bind to HA. In some embodiments, the intermediate polypeptide is a pluripotent proteoglycan. In some embodiments, the intermediate polypeptide includes a linker module.In some embodiments, the intermediate polypeptide comprises a linker module of HAPRN1, HAPRN2, HAPRN3, HAPRN4, agglutinin, pluripotent proteoglycan, neuroproteoglycan, short proteoglycan, phosphoproteoglycan, TSG-6, CD44, stabillin-1, or stabillin-2. In some embodiments, the intermediate polypeptide comprises a linker module of a pluripotent proteoglycan. In some embodiments, the intermediate polypeptide comprises a polypeptide shown in any one of SEQ ID NO:54-99. In some embodiments, a PTX3 / HA complex produced by the foregoing method is described herein. In some embodiments, a pharmaceutical composition comprising a PTX3 / HA complex produced by the foregoing method is described herein. In some embodiments, the use of the PTX3 / HA complex for the manufacture of a medicament is described herein. In some embodiments, a treatment method comprising administering the PTX3 / HA complex for the prevention or inhibition of scar formation, inflammation, angiogenesis, cancer, diabetes, obesity, or fibrosis.

[0075] In some embodiments, methods for inducing or maintaining pluripotency in cells are described herein, including culturing cells with an nHC-HA / PTX3 complex or an rcHC-HA / PTX3 complex to induce or maintain pluripotency in the cells. In some embodiments, the cells heterologously express proteins selected from Sox2, myc, Oct4, and KLF4. In some embodiments, the cells heterologously express one, two, or three proteins selected from Sox2, myc, Oct4, and KLF4. In some embodiments, the nHC-HA / PTX3 complex or the rcHC-HA / PTX3 complex is immobilized. In some embodiments, the cells are adult differentiated cells. In some embodiments, the cells are fibroblasts. In some embodiments, the cells are human corneal fibroblasts. In some embodiments, the cells are embryonic stem cells, adult stem cells, fetal stem cells, or induced pluripotent stem cells. In some embodiments, the cells are limbic epithelial progenitor cells, limbic stromal septum cells, umbilical cord stem cells, amniotic stem cells, or adipose stem cells. In some embodiments, the nHC-HA / PTX3 complex is an amniotic nHC-HA / PTX3 complex. In some embodiments, the nHC-HA / PTX3 is an umbilical cord nHC-HA / PTX3 complex. In some embodiments, the method further includes purifying the nHC-HA / PTX3 complex by ultracentrifugation of the amniotic extract. In some embodiments, the method further includes purifying the nHC-HA / PTX3 complex by ultracentrifugation of an amniotic extract prepared in PBS buffer to produce an nHC-HA / PTX3 extract (i.e., soluble nHC-HA / PTX3). In some embodiments, the method further includes purifying the nHC-HA / PTX3 complex by ultracentrifugation of an amniotic extract prepared in GnHCl buffer to produce an nHC-HA / PTX3 extract (i.e., soluble nHC-HA / PTX3). In some embodiments, the method further includes purifying the nHC-HA / PTX3 complex by ultracentrifugation of an umbilical cord extract. In some embodiments, the umbilical cord extract comprises umbilical amnion, umbilical cord matrix, Wharton's jelly, or any combination thereof. In some embodiments, the method further comprises purifying the nHC-HA / PTX3 complex by ultracentrifugation of the umbilical cord extract prepared in PBS buffer to produce an nHC-HA / PTX3 extract (i.e., soluble nHC-HA / PTX3). In some embodiments, the method further comprises purifying the nHC-HA / PTX3 complex by ultracentrifugation of the umbilical cord extract prepared in GnHCl buffer to produce an nHC-HA / PTX3 extract (i.e., soluble nHC-HA / PTX3).In some embodiments, the method further includes performing two, three, or four rounds of ultracentrifugation. In some embodiments, the method further includes performing four rounds of ultracentrifugation. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex comprises PTX3. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex comprises small leucine-rich proteoglycan (SLRP). In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex comprises PTX3 and small leucine-rich proteoglycan (SLRP). In some embodiments, the small leucine-rich proteoglycan is selected from dermal proteoglycan, disaccharide proteoglycan, fibrinolytic proteoglycan, luminal proteoglycan, PRELP (proline-arginine-rich teloleucine-rich protein), corneal proteoglycan, osteoadhesion proteoglycan, epipycan, and osteoglobin. In some embodiments, the small leucine-rich proteoglycan is covalently attached to a glycosaminoglycan. In some embodiments, the glycosaminoglycan is keratin sulfate.

[0076] It should be understood that the foregoing general description and the following detailed description are merely exemplary and illustrative, and not intended to limit any subject matter for which protection is sought. Attached Figure Description

[0077] Figure 1 The purification of the natural HC-HA / PTX3 (nHC-HA / PTX3) complex from amniocentesis (AME) and the analysis of its protein composition and HA size are illustrated. (AB) Total protein and HA concentrations in fractions obtained by ultracentrifugation with CsCl / 4M guanidine hydrochloride. (C) HA stained with a stain-all dye on a 0.5% agarose gel. (DJ) Analysis of proteins present in nHC-HA / PTX3 by Western blotting using antibodies against HC1 (D and F), PTX3 (E and G), HC2 (H), HC3 (I), urostatin (J), TSG-6 (K), or TSP-1 (L). NaOH or N = treatment with 0.05NNaOH at 25°C for 1 hour. HAase or H = treatment with 20 units / ml of hyaluronidase at 60°C for 2 hours (FK). A bar chart of relative protein mass (M) determined by dot analysis using antibodies against various antigens, including IGFBP1-3, PF4, or TIMP-1.

[0078] Figure 2This illustrates the attachment of LPS-stimulated macrophages to immobilized nHC-HA / PTX3 cells mediated by CD44 and TLR4 receptors. (A) Cell attachment. RAW264.7 cells (100 μl of 2.5 x 10⁻⁶ cells) were attached to immobilized nHC-HA / PTX3 cells. 5 RAW264.7 cells were seeded at 1 cell / ml onto immobilized HA (2 μg / well) or nHC-HA / PTX3 (2 μg / well) (n=3) and stimulated with LPS (1 μg / ml). After 90 min of incubation, unattached cells were removed and attached cells were counted by CyQuant analysis. Scale bar represents 100 μm. Asterisk (*) indicates p-value <0.05 (HA or nHC-HA / PTX3 vs. PBS control, or nHC-HA / PTX3 vs. HA). (B) Cell viability. LPS-stimulated RAW264.7 cells were incubated for 24 h on immobilized PBS control, HA, or nHC-HA / PTX3 (n=3). Cell viability was measured by MTT analysis. No significant differences in cell viability were observed between cells on these immobilized substrates (all p-values ​​>0.05). (C) CD44 and TLR4 receptors are responsible for the attachment of LPS-stimulated macrophages to immobilized nHC-HA / PTX3. RAW264.7 cells (2.5 x 10⁻⁶) 5 Pre-incubate (n=3) on ice for 30 min with blocking antibodies against CD44, TLR2, TLR4, integrin αv, β1, β2 or β3 or RGD peptide, and an isotype control antibody or RGD control peptide. After adding LPS (1 μg / ml), incubate for 90 min and perform cell attachment analysis as described in section A. An asterisk (*) indicates a p-value < 0.05.

[0079] Figure 3 This illustrates how immobilized nHC-HA / PTX3 polarizes LPS-stimulated macrophages toward the M2 phenotype. (A) Relative mRNA expression of M1 (TNF-α, IL-12p40) or M2 (IL-10, Arg-1, LIGHT, and SPHK1) markers in macrophages bound to PBS control or immobilized HA or nHC-HA / PTX3, as determined by quantitative PCR. (B) Relative TNF-α protein levels, as determined by ELISA. (C) Western blot (left) and cell localization (right) in macrophages bound to PBS control or immobilized nHC-HA / PTX3, stained with IRF5 (a marker for M1). (D) Apoptosis of resting, fMLP-stimulated, or LPS-stimulated neutrophils after incubation with immobilized nHC-HA / PTX3. An asterisk (*) indicates p < 0.05. (E) Phagocytosis of apoptotic neutrophils by resting or LPS-stimulated macrophages. An asterisk (*) indicates p < 0.05.

[0080] Figure 4 The role of CD44 in maintaining M2 macrophage polarization on immobilized nHC-HA / PTX3 is illustrated. (A) Relative mRNA expression of macrophage markers M1 (IL-12p40) and M2 (IL-10, LIGHT, and SPHK1) after binding to nHC-HA / PTX3, as determined by qPCR, after macrophages pre-incubated with PBS or antibodies against CD44, TLR4, or CD44 / TLR4 binding. An asterisk (*) indicates p < 0.05 compared to the antibody-free treatment (none) in the same group. (B) Amounts of IL-12 and IL-10 proteins as determined by ELISA. An asterisk (*) indicates p < 0.05 compared to the antibody-free treatment (none) in the same group.

[0081] Figure 5 nHC-HA / PTX3 (4th) promoted cell aggregation, but both nHC-HA / PTX3 (2nd) and nHC-HA / PTX3 (4th) inhibited the production of IL-12p40 and IL-23 proteins in IFN-γ / LPS-stimulated macrophages. RAW264.7 cells (2.5 x 10⁻⁶) 5 Cells were cultured on immobilized substrates (PBS as a control) and stimulated with IFN-γ / LPS for 4 hours (A) or 24 hours (B and C). (A) Cell morphology 4 hours post-seeding. Alternatively, cells were stimulated with LPS for 24 hours, and the protein levels of IL-12p40 and IL-23 in the cell culture supernatant were measured by their respective ELISA (B and C). p-values ​​are shown in B and C.

[0082] Figure 6 Examples of HMW HA and nHC-HA / PTX3 with 96-hole CovaLink are shown. TM Dose-dependent and covalent coupling on the plate surface. (A) HA ELISA of bound HMW HA and nHC-HA / PTX3 after removal of unbound HMW HA and nHC-HA / PTX3. (B) HA ELISA of bound and unbound HA from HMW HA and nHC-HA / PTX3.

[0083] Figure 7 The dose-dependent binding of TSG-6 to immobilized HA (iHA) and its resistance to various dissociating and reducing agents are illustrated. (A) TSG-6 bound to iHA as measured by TSG-6 ELISA. (B) TSG-6 bound to iHA as measured by TSG-6 ELISA after treatment with 6M guanidine hydrochloride, 8M guanidine hydrochloride, 2% SDS, 100mM DTT, or 25mM NaOH.

[0084] Figure 8 The dose-dependent binding of PTX3 to immobilized HA (iHA) and its resistance to various dissociating and reducing agents are illustrated. (A) PTX3 bound to iHA as measured by PTX3 ELISA. (B) PTX3 bound to iHA as measured by PTX3 ELISA after treatment with 6M guanidine hydrochloride, 8M guanidine hydrochloride, 2% SDS, 100mM DTT, or 25mM NaOH.

[0085] Figure 9 Examples show no competitive or synergistic effect between TSG-6 and PTX3 on iHA binding. For bound TSG-6 or PTX3, relative absorbance measured by ELISA is shown for each factor individually or in combination with iHA. No statistically significant difference was observed between individual and combination binding of TSG-6 or PTX3 to iHA (p>0.05).

[0086] Figure 10 This illustrates partial inhibition of PTX3 binding to iHA pre-bound with TSG-6, and no inhibition of TSG-6 binding to iHA pre-bound with PTX3. The figure shows the TSG-6 and PTX3 ELISA results for subsequent binding of TSG-6 or PTX3 to pre-bound TSG-6 / iHA (A) or pre-bound PTX3 / iHA (B). p-values ​​are shown in (A), and no statistical significance was observed between groups in (B).

[0087] Figure 11 Examples illustrate the attachment of LPS-stimulated RAW264.7 macrophages to PBS (control), HA (iHA), nHC-HA / PTX3, TSG-6 / iHA, or PTX3 / iHA. Cells were photographed 24 hours after incubation.

[0088] Figure 12 The relative gene expression in RAW264.7 macrophages after incubation on PBS (control), HA (iHA), nHC-HA / PTX3, TSG-6 / iHA, or PTX3 / iHA is illustrated. Total RNA was isolated, and the mRNA expression of IL-12p40(A) and IL-10(D) was measured by quantitative PCR. Alternatively, cells were stimulated with LPS (B and E) or IFN-γ / LPS (C) for 24 hours, and the protein expression of IL-12p70(B), IL-23(C), and IL-10(E) in cell culture medium was measured using their respective ELISA. An asterisk (*) indicates p < 0.05 compared to control.

[0089] Figure 13Examples illustrate the efficiency of free TSG-6 in solution for transferring HC1 and HC2 from IαI to iHA compared to bound TSG-6. (A, B) Relative bound HC1 (A) or IαI (B) as determined by their respective ELISA after simultaneous or sequential addition of TSG-6 and IαI to iHA. An asterisk (*) indicates p < 0.05 at the same TSG-6 concentration when added simultaneously and sequentially. (C) Western blot of the sample from A digested with hyaluronidase (HAase) and analyzed with anti-TSG-6 antibody. (D) Relative bound HC1 and PTX3 to iHA as determined by ELISA after simultaneous incubation of PTX3 and IαI with iHA.

[0090] Figure 14 Examples show the complexes formed in solution after simultaneous incubation of IαI and TSG-6 with PTX3 or without incubation with PTX3. (AD) Western blots obtained with antibodies against HC1 (A), HC2 (B), TSG-6 (C), or urostatin (D). HAase = treatment with hyaluronidase. (E) Schematic diagram of the interaction between TSG-6 and IαI. (F) Schematic diagram of the inhibition of HC2·TSG-6 formation by PTX3. (G) Western blot obtained with anti-IαI antibody.

[0091] Figure 15 Examples of complexes formed on iHA after simultaneous incubation of IαI and TSG-6 with PTX3 or without incubation with PTX3 are shown. After washing with 8M GnHCl and PBS, the binding of HCl, TSG-6, and PTX3 (A, D, F) was determined by their respective ELISA. An asterisk (*) indicates p < 0.05 compared to 1 μg / ml PTX3. The complex was washed again with 8M GnHCl and PBS, and the bound fraction was digested with 1 unit / ml hyaluronidase for 2 hours. The digested samples were analyzed by Western blotting using antibodies against HC1 (B), HC2 (C), TSG-6 (E), and PTX3 (G).

[0092] Figure 16 The complex formed on iHA after the sequential addition of IαI and TSG-6, followed by the addition of PTX3, is illustrated. The binding of HCl, TSG-6, and PTX3 (A, D, F) was determined by their respective ELISA. The complex was washed again with 8M GnHCl and PBS, and the bound fraction was digested with 1 unit / ml of hyaluronidase for 2 hours. The digested samples were analyzed by Western blotting using antibodies against HC1 (B), HC2 (C), TSG-6 (E), and PTX3 (G).

[0093] Figure 17 The complex formed on iHA after the sequential addition of PTX3 followed by the addition of IαI and TSG-6 is illustrated. The binding of HCl, TSG-6, and PTX3 (A, C, E) was determined by their respective ELISA. The complex was washed again with 8M GnHCl and PBS, and the bound fraction was digested with 1 unit / ml of hyaluronidase for 2 hours. The digested samples were analyzed by Western blotting using antibodies against PTX3 (B), TSG-6 (D), HC1 (F), and HC2 (G).

[0094] Figure 18 Examples illustrate the attachment of LPS-stimulated RAW264.7 macrophages to PBS (control), HA (iHA), nHC-HA / PTX3, (IαI / TSG-6 / PTX3) / iHA (IαI, TSG-6, or PTX3 simultaneously bind to iHA), (IαI / TSG-6) / PTX3 / iHA (PTX3 is sequentially added to iHA pre-incubated with IαI and TSG-6), or (PTX3) / IαI / TSG-6 / iHA (IαI and TSG-6 are sequentially added to iHA pre-incubated with PTX3). Cells were photographed 24 hours after incubation.

[0095] Figure 19 Gene expression in RAW264.7 macrophages cultured on immobilized substrates and stimulated with LPS is illustrated. Total RNA was isolated, and the expression of IL-10 and IL-12p40 mRNA was determined by quantitative PCR (A and C). IL-10 and IL-12p70 proteins in cell culture supernatants were determined by their respective ELISA results (B and D). (E) IL-23 protein in cell culture supernatants of resting RAW264.7 cells (without) or stimulated for 24 hours with IFN-γ (200 units / ml), LPS (1 μg / ml), IFN-γ / LPS, LPS containing immune complexes (LPS / IC), or IL-4 (10 ng / ml) was determined by IL-23 ELISA. (F) IL-23 in cell culture supernatants of RAW264.7 cells cultured on immobilized substrates and stimulated for 24 hours with IFN-γ / LPS was determined by IL-23 ELISA. An asterisk (*) indicates that p < 0.05.

[0096] Figure 20Immunostaining for HA, PTX3, TSG-6, HC, and urinary astatin in human umbilical cord (A) or amnion (B) is illustrated. Frozen sections of human umbilical cord were probed with biotinylated HABP, with or without hyaluronidase digestion, using antibodies against PTX3 and TSG-6 and chain-specific antibodies against the illustrated IαI and PαI fractions. Nuclei were counterstained with Hoechst 33342 (blue). Epi, epithelium. Strips represent 100 μm.

[0097] Figure 21 Comparison of PTX3 levels in sequential PBS and GnHCl extracts from AM, CH, and UC is illustrated. A, lanes 2 and 3 each contained 2 μg HA, while lanes 4–11 each contained 20 μg total protein. B, lanes 3–10 each contained 40 μg total protein.

[0098] Figure 22 The comparison of HCl, urinary acetaminophen, and IαI in sequential PBS and GnHCl extracts from AM, CH, and UC is illustrated. In A and C, each lane contained 20 μg of total protein, while in B, excluding the positive control, each lane contained 40 μg of total protein.

[0099] Figure 23 A comparison of TSG-6 in AM and UC GnHCl extracts is illustrated. Each lane contained 40 μg of total protein, excluding the positive TSG-6 control.

[0100] Figure 24 Western blot analysis of PTX3(A), HC1(B), HC2(C), and TSG-6(D) in the AM-HC·HA complexes 1 through 4 is illustrated. Each lane contained 4 μg of HA, except for the positive control.

[0101] Figure 25 Western blot analysis of TSP-1 in AME, AM GnHCl, and HC-HA complexes 1 through 4 is illustrated. Lanes 3, 4, 10, and 11 each contain 30 μg of total protein. Lanes 5–8 and 12–15 each contain 4 μg of HA.

[0102] Figure 26 Western blot analysis of PTX3(A), HC1(B), HC2(C), HC3(D), and TSG-6(E) in the 4UC HC-HA complex is illustrated. Each lane contained 4 μg of HA, except for the positive control.

[0103] Figure 27A comparison of HC1(A) and PTX3(B) in the 4HC·HA complex from PBS and GnHCl extracts is illustrated. Each lane contained 4 μg of HA, except for the positive control.

[0104] Figure 28 The comparison of the 4HC-HA complex from PBS and GnHCl in agarose gels is illustrated. Each lane contained 15 μg of HA, except for the positive HA control.

[0105] Figure 29 Examples of Coomassie blue staining of SDS-PAGE gels containing GnHCl HC-HA and PBS HC-HA are shown. A, AMPBS and GnHCl HC-HA. B, UC PBS and GnHCl HC-HA. Each lane contains 30 μg of HA.

[0106] Figure 30 This illustrates the presence of keratin sulfate and osteoadhesive proteoglycan in AM GnHCl HC-HA, but not in PBS HC-HA. A, Coomassie blue staining. Each lane contains 30 μg HA. B and C, Western blotting of keratin sulfate (B) and osteoadhesive proteoglycan (C). Each lane contains 4 μg HA. D, Immunostaining of keratin sulfate in AM.

[0107] Figure 31 Deglycosylation of AM GnHCl HC-HA and analysis by SDS-PAGE or Western blot stained with CB (Coomassie blue) are illustrated. A, Coomassie blue staining. Each lane contains 30 μg HA, except for lane 6 which contains 5 μg HA. BH, Western blots of osteoadhesive proteoglycan (B), keratin proteoglycan (C, D), disaccharide proteoglycan (E, F), keratin sulfate (G), and PTX3 (H). H: hyaluronidase; C: chondroitinase (Cabc); K: keratinase (keratin sulfate endopeptidase-β-galactosidase); T: TFMSA (trifluoromethanesulfonic acid). Each lane contains 4 μg HA.

[0108] Figure 32Examples show that keratin and disaccharide proteoglycans are abundant in UC GnHCl HC-HA, but not in PBS HC-HA. Keratin sulfate, osteoadhesive proteoglycans, and urostatin are also present in UC GnHCl HC-HA, but not in PBS HC-HA (except for keratin sulfate). A, Coomassie blue staining. Each lane contains 30 μg HA, except for lane 6 which contains 5 μg HA. BH, Western blots of keratin (B), disaccharide proteoglycans (C), urostatin (D), PTX3 (E), keratin sulfate (F), and osteoadhesive proteoglycans (G). H: hyaluronidase; C: chondroitinase (Cabc); K: keratinase (keratin sulfate endopeptidase-β-galactosidase). Each lane contains 4 μg HA.

[0109] Figure 33 Immunolocalization of PTX3 in human amnion membranes is illustrated. Frozen sections of human fetal membranes were explored with anti-PTX3, biotinylated HABP, with or without hyaluronidase digestion and using a chain-specific antibody against the IαI fraction. Cell nuclei were counterstained with Hoechst 33342 (blue). AM, amnion; Epi, epithelium; CH, chorion. Bars represent 100 μm.

[0110] Figure 34 PTX3 was demonstrated in the AM soluble extract and the purified HC-HA complex. The purified PTX3, AM extract (AME), and AM HC-HA complex were treated with 50 mM NaOH at 25°C for 1 hour, or with hyaluronidase (HAase) at 37°C for 1 hour, followed by Western blot analysis using anti-PTX3 (A) and anti-HC1 (B) antibodies, and analyzed on 0.5% agarose gel electrophoresis, then stained with a full-dye dye (C). PTX3 and its multimeric forms were found in the AM soluble extract and the purified HC-HA complex. M, protein step molecular weight standard.

[0111] Figure 35 PTX3 mRNA and protein expression in AMEC and AMSCs are illustrated. RNA and protein were extracted from human skin fibroblasts (SkinFib.) and both AMEC and AMSCs. The expression of PTX3 mRNA (A) and protein in supernatant and cell lysate (B) were compared. PTX3 siRNA transfection was performed to confirm PTX3 expression in AMEC and AMSCs (C).

[0112] Figure 36Morphological changes of human skin fibroblasts (HSF), AMSCs, and AMECs after agarose covering are illustrated. HSF, AMSCs, and AMECs were cultured under serum-free and serum-containing conditions with or without 3% agarose covering for five days, and cell morphology was photographed. Scale bar, 50 µm.

[0113] Figure 37 Agarose covering was demonstrated to reduce the release of HA into the culture medium from HSF, AMSC, and AMEC cultures. HA concentrations were determined by ELISA in media from HSF, AMSC, and AMEC under serum-free and serum-containing conditions, with or without agarose covering.

[0114] Figure 38 Immunolocalization of PTX3, HA, and HC1 in agarose-covered cell cultures is illustrated. HSF, AMSC, and AMEC were cultured with or without TNF treatment and covered with agarose, and hyaluronic acid (red), PTX3 (green, AF; red, JL), and HC1 (green) (nucleus in blue) were examined. Colocalization of HC1 with HA was observed in all cultures, but colocalization of PTX3 with either HA or HC1 was observed only in AMSC and AMEC. Scale bar, 50 μm.

[0115] Figure 39 An example is shown of the HC-HA / PTX3 complex in AMSCs but not HSFs under agarose covering. GnHCl extracts from cell layers of agarose-covered HSF and AMSC cultures were analyzed for PTX3 using Western blot analysis with and without NaOH treatment. M represents the protein ladder molecular weight standard.

[0116] Figure 40 The reconstruction of the HC-HA / PTX3 complex on immobilized HA in vitro is illustrated. iHA (~14 μg / ml), IαI (5 μg / ml), and TSG-6 (12 μg / ml) were incubated simultaneously with or without PTX3 (1, 5, or 20 μg / ml) at 37 °C for 2 h. For sequential incubation, iHA (~14 μg / ml), IαI (12 μg / ml), and TSG-6 (12 μg / ml) were incubated in reaction buffer at 37 °C for 2 h, followed by the addition of PTX3 (1, 5, or 20 μg / ml) and incubation at 37 °C for another 2 h. After washing with 8 M GnHCl and PBS, the iHA with bound components was digested with 1 unit / ml of hyaluronidase at 60 °C for 2 h. The samples were analyzed by Western blotting using antibodies against IαI (A), TSG-6 (B), and PTX3 (C).

[0117] Figure 41 Examples of cell morphology of human corneal fibroblasts cultured for 2 days in DMEM / 10% FBS (A) or 2 days in DMEM / 10% FBS ± 3 days in TGF-β1 (B) up to day 3 are shown.

[0118] Figure 42 This study demonstrates that soluble HC-HA (PBS) inhibits TGFβ1 but activates TGFβ3 signaling, while insoluble HC-HA (GnHCl) activates both TGFβ1 and TGFβ3 signaling under serum-free conditions and is further enhanced upon TGFβ1 stimulation.

[0119] Figure 43 Examples show that both soluble HC-HA (PBS) and insoluble HC-HA (GnHCl) inhibit the expression of TGFβR2 and TGFβR3 upon TGFβ1 stimulation. A, TGFβR mRNA expression. B, TGFβR protein expression.

[0120] Figure 44 An example is given of the inhibition of nuclear translocation of pSMAD2 / 3 signaling by inhibiting TGFβ1 signaling via HC-HA.

[0121] Figure 45 An example of HC-HA inhibition of α-smooth muscle actin formation is shown.

[0122] Figure 46 This illustrates the activation of BMP6 transcripts by HA and soluble / insoluble HC-HA. The addition of TGFβ1 activated BMP6 transcript expression on plastics, but significantly activated BMP4 / 6 mRNA expression in HCF on both HA and soluble / insoluble HC-HA.

[0123] Figure 47 This illustrates that HC-HA, rather than HA, activates the transcript expression of BMPR1A in HCF stimulated with TGFβ1, while additional TGFβ1 nonspecifically activates the mRNA expression of BMPR1B and BMPR2 in HCF.

[0124] Figure 48 Examples show that soluble HC-HA (PBS) and insoluble HC-HA (GnHCl) activate BMP4 / 6 signaling via pSMAD1 / 5 / 8.

[0125] Figure 49 Activation of SMAD / 1 / 5 / 8 leads to upregulation of its downstream genes—DNA binding inhibitors 1, 3, and 4 (ID1, ID3, and ID4), downstream targets of BMP signaling.

[0126] Figure 50Examples show that HC-HA (PBS) and HC-HA (GnHCl) promoted corneal proteoglycan mRNA expression by 14-fold and 16-fold, respectively, which was significantly downregulated by additional TGFβ1.

[0127] Figure 51 Examples show that HC-HA (PBS) and HC-HA (GnHCl) promote the protein expression of corneal proteoglycans.

[0128] Figure 52 Examples show that HCF expresses more ESC markers on 4X HC-HA (PBS) and insoluble HC-HA (GnHCl) than on plastic and when cells are stimulated by the addition of TGFβ1.

[0129] Figure 53 Cell viability of MC3T3-E1 cells as measured by MTT assay is illustrated.

[0130] Figure 54 Mineralization of MC3T3-E1 cells as measured by alizarin red staining is illustrated.

[0131] Figure 55 The morphology of MC3T3-E1 cells treated with HC-HA (A) or AMP (B) on day 13 of induction is illustrated.

[0132] Figure 56 Alizarin Red staining of MC3T3-E1 cells treated with HC-HA (A) or AMP (B) on day 13 of induction is illustrated.

[0133] Figure 57 Quantitative analysis of ARS staining in MC3T3-E1 cells treated with HC-HA (A) or AMP (B) on day 13 of induction is illustrated.

[0134] Figure 58 The ALP activity (IU / L) of cells treated with HC-HA (A) and AMP (B) on day 13 of induction is illustrated.

[0135] Figure 59 Phase-contrast microscopy of morphological changes in MC3T3-E1 cells after induction from day 3 is illustrated. (A) Uninduced cells were cultured for 7 days in αMEM containing 10% FBS. (B) MC3T3-E1 cells were cultured to confluence in flat-bottomed 96-well plates one day after seeding (day 0). Cells were then induced with ascorbic acid and β-glycerophosphate.

[0136] Figure 60 Phase contrast microscopy examination of morphological changes in MC3T3-E1 cells induced by treatment with HC-HA (A) or AMP (B) from day 1 to day 7 is illustrated.

[0137] Figure 61 A phase-contrast microscopy examination of spindle loop formation in induced MC3T3-E1 cells is shown on day 3 of induction.

[0138] Figure 62 Phase contrast microscopy of spindle loop formation in MC3T3-E1 cells induced by HC-HA (A) and AMP (B) treatment is illustrated (days 0 to 6).

[0139] Figure 63 ARS staining and extraction using GnHCl are illustrated in MC3T3-E1 cells induced by HC-HA and AMP treatment.

[0140] Figure 64 The extraction and quantification of ARS from MC3T3-E1 cells using acetic acid and 10% ammonium hydroxide are illustrated. The ARS extract obtained by acetic acid treatment was neutralized with 10% ammonium hydroxide and then added to a 96-well clear-bottom plate (A) for reading on a spectrophotometer (B). * indicates statistical significance.

[0141] Figure 65 The extraction and quantification of ARS from MC3T3-E1 cells using GnHCl is illustrated. The ARS extract obtained by GnHCl treatment was added to a 96-well clear-bottom plate (A) for reading on a spectrophotometer (B). * indicates statistical significance.

[0142] Figure 66 ARS staining (A) and quantification (B) of HC-HA-treated MC3T3-E1 cells on day 19 (day 18 of induction) are illustrated. ARS staining was performed on day 19 of culture (day 18 of induction).

[0143] Figure 67 ARS staining (A) and quantification (B) of AMP-treated MC3T3-E1 cells are illustrated. ARS staining was performed on day 19 of culture (day 18 of induction).

[0144] Figure 68 A spindle-like loop observed in induced MC3T3-E1 cells on day 14 of induction is illustrated.

[0145] Figure 69 Phase contrast microscopy of cell morphology of induced MC3T3-E1 cells (A) without Transwell or (B) with Transwell, after 14 days of induction, is shown.

[0146] Figure 70An example is shown of ARS staining in induced MC3T3-E1 cells on day 14 of induction.

[0147] Figure 71 The quantitative analysis of ARS staining in induced MC3T3-E1 cells on day 14 of induction is illustrated.

[0148] Figure 72 ARS staining and quantification of MC3T3-E1 cells treated with AMP are illustrated. (A) Phase contrast photographs and ARS staining images of MC3T3-E1 cells taken on day 21 of culture (day 20 of induction). (B) Quantification of ARS staining on day 21 of culture (day 20 of induction). * indicates statistical significance of p < 0.05.

[0149] Figure 73 The illustration shows images of different differentiations of progenitor cells into osteoblasts, with common factors added to the culture medium shown.

[0150] Figure 74 ARS staining and quantification in MC3T3-E1 cells are illustrated. (A) Phase-contrast micrographs of HUVEC, hBMMSC, and hAM mesenchymal stem cells with and without ARS staining from day 4 to day 21. (B) Quantification of ARS staining. * indicates statistical significance of p < 0.05 compared to the negative control.

[0151] Figure 75 The figure illustrates the timeline of bone formation in MC3T3-E1 cells.

[0152] Figure 76 ARS staining and quantification in MC3T3-E1 cells are illustrated. (A) Cell morphology and ARS staining of MC3T3-E1 cells treated with AMP (days 1, 2, 7, and 10). (B) ARS quantification of MC3T3-E1 cells treated with AMP (days 1, 2, 7, and 10). * indicates statistical significance of p < 0.05.

[0153] Figure 77 The timeline of cell viability and proliferation as determined by MTT assay is illustrated. (A) MTT assay of MC3T3-E1 cell viability and metabolic activity on days 1, 2, and 4. * indicates statistical significance relative to day 1, p < 0.05. (B) BrdU assay of MC3T3-E1 cell proliferation on days 1, 2, and 16. * indicates statistical significance relative to day 1, p < 0.05.

[0154] Figure 78Examples of mRNA expression of multiple genes as determined by qPCR after differentiation induction with or without AMP treatment are shown. (A) hMSC and (B) MC3T3-E1 cell expression.

[0155] Figure 79 Phase contrast microscopy and quantification of ARS analysis are illustrated. MC3T3-E1 cells deposited and mineralized during the duration of the experiment (8 days). ARS was then used for qualitative analysis. (A) CovaLink-NH 96-well plates immobilized with PBS, HA, and HC-HA. (B) Conventional 96-well plates with negative controls and AGM (inducer) and AMP as positive controls. (C) Quantification of ARS analysis. * indicates statistical significance of p < 0.05 relative to day 1.

[0156] Figure 80 The following illustrations show the mRNA expression of several genes tested after differentiation induction with or without HC-HA / PTX3 treatment, as determined by qPCR. (A) Runx2 and Sox9, the main transcription factors for osteogenic and chondrogenic processes, on days 1, 7, and 14. (B) Bone morphogenetic protein (BMP) expression on day 14. (C) The chondrogenic marker collagen 2 (COL2) and the osteogenic marker alkaline phosphatase (ALPL) expressed on days 7 and 14. (D) The hypertrophy markers collagen 10 (COL10) and MMP13 expressed on day 14. (E) The osteogenic markers collagen 1 (COL1), Osterix (OSX), and bone salivary protein (BSP) on day 14.

[0157] Figure 81 The following are examples of phase microscopy (A) and quantification (B) of ARS analysis after differentiation induction with or without HC-HA / PTX3 (soluble or insoluble) treatment on day 14.

[0158] Figure 82 CD4 is an example. + T cell activation and differentiation. Immature CD4 cells, under different stimuli... + T helper cells (Th) differentiate into Th1, Th2, Th17, or Treg and secrete different cytokines. Th1 cytokines (e.g., IFN-γ and IL-2) tend to produce pro-inflammatory responses.

[0159] Figure 83The procedure for measuring cell proliferation and cytokine production is illustrated. Spleen cells were isolated from OT-II mice expressing ovalbumin (OVA)-specific transgenic TCRs and stimulated with OVA for up to 4 days. Cell proliferation was measured by BrdU labeling, and the expression of cytokines (IFN-γ and IL-2) was measured by their respective ELISA.

[0160] Figure 84 An example of nHC-HA / PTX3 on CD4 is given. + Inhibition of T cell proliferation. Spleen cells isolated from Ova T cell receptor transgenic mice were stimulated with OVA (0-10 μM) for 4 days. AM extract (AME, 25 μg / ml) and nHC-HA / PTX3 (25 μg / ml) inhibited the clonal growth of activated T cells induced by increased OVA concentration (top). Proliferation of spleen cells treated with HA, AME, or nHC-HA / PTX3 and labeled with CFSE for 4 days was measured by flow cytometry. Both AME and nHC-HA / PTX3 inhibited cell proliferation in a dose-dependent manner (middle). 25 μg / ml nHC-HA / PTX3 inhibited CD4+ labeled with BrdU. + T cell proliferation (bottom) (*, p<0.05 compared to control).

[0161] Figure 85 This study illustrates the inhibitory effect of nHC-HA / PTX3 on Th1 cytokines IFN-γ and IL-2. Spleen cells treated with PBS, 25 μg / ml HA, 25 μg / ml AME, or 25 μg / ml nHC-HA / PTX3 were stimulated with 10 μM OVA for 4 days. IFN-γ and IL-2 levels in the culture supernatant were measured by their respective ELISA assays. Both AME and nHC-HA / PTX3 inhibited the production of IFN-γ and IL-2 (*p<0.05 compared to control).

[0162] Figure 86 This study illustrates how nHC-HA / PTX3 reduces the influx of macrophages (labeled by enhanced green fluorescent protein or EGFP). LPS (2 μl, 2 μg / ml) was injected into the corneal stroma of Mafia mice. Immediately afterwards, 5 μl of PBS or nHC-HA / PTX3 (1 mg / ml) was injected through the subconjunctival tissue into each quadrant of a cornea from the same mouse. EGFP was monitored using in vivo biomicroscopy on days 1, 2, 3, and 6 following LPS treatment. +Macrophage influx (top). Alternatively, mouse corneas were treated with LPS simultaneously with PBS or nHC-HA / PTX3 (pretreatment (-) or three days prior to LPS treatment (pretreatment (+)). On day 4 post-LPS treatment, cells from the cornea were isolated by collagenase digestion and sorted for EGFP by FACS. - or EGFP + (Macrophages) (* and ** compared with the control, p<0.05 and p<0.01, respectively).

[0163] Figure 87 This illustrates how nHC-HA / PTX3 polarizes macrophages to the M2 phenotype. The polarization was demonstrated by qPCR in LPS-induced macrophage (EGFP) infiltration into the mouse cornea. + The mRNA expression of M2 markers (Arg-1 and IL-10) and M1 markers (IL-12p40 and IL-12p35) in the study was quantified (* and ** compared with the control, p<0.05 and p<0.01, respectively).

[0164] Figure 88 This illustrates the improvement in corneal allogeneic graft survival achieved by nHC-HA / PTX3. Rat corneal allogeneic graft survival was significantly improved by injection of nHC-HA / PTX3 (10 μg / dose, twice weekly, top left image) at a single quadrant subconjunctival site, but even more significantly by injection of nHC-HA / PTX3 (20 μg / dose, twice weekly, top right image) at four quadrant subconjunctival sites. The image below shows photographs of corneal allogeneic grafts treated with PBS (day 21 post-surgery, left) or nHC-HA / PTX3 (day 40 post-surgery, right).

[0165] Figure 89 Examples include classical M1 activation of macrophages (e.g., induced by IFN-γ and / or TLR ligands such as LPS) to express high levels of pro-inflammatory cytokines (such as TNF-α, IL-12, and IL23), which activate Th1 and Th17 lymphocytes, leading to many chronic inflammatory diseases.

[0166] Figure 90This illustrates LPS-induced macrophage infiltration into the mouse cornea after treatment with PBS (control), HC-HA / PTX3, or AMP. Both eyes of Mafia mice (macrophages were EGFP+) were intramedullary injected with LPS matrix (5 μg). OS was treated with PBS, and OD was treated with HC-HA (2 injection sites) (A), HC-HA (4 injection sites) (B), AMP (2 injection sites) (C), and AMP (4 injection sites) (D), with each injection being 5 μl. Treatment was performed immediately after LPS injection. Images were taken on days 1, 2, 3, and 6. Cell counts were based on the intensity of green fluorescence.

[0167] Figure 91 Examples illustrate LPS-induced macrophage infiltration of the mouse cornea and their subtypes (M1 and M2) after treatment or pretreatment with PBS (control), nHC-HA / PTX3, or AMP. Pretreatment and treatment in Mafia mice are compared with... Figure 86 The procedure was the same as described above. On day 4, corneal flaps were cut and digested with collagenase at 37°C for 2 hours. EGFP-positive / negative cells were sorted by FACS. The ratio of EGFP-positive macrophages to EGFP-negative cells was calculated and used as an arbitrary unit to determine the degree of macrophage infiltration (A). Total RNA was extracted from the sorted EGFP-positive macrophages and converted into cDNA. The expression of Arg-1, IL-10, IL-12p40, and IL-12p35 was determined by quantitative PCR (B).

[0168] Figure 92 This is an illustration of the injection site. The injection location will be subconjunctival, near the fornix. nHC-HA / PTX3 or AMP can reduce DS-induced ALKC in a mouse model of experimental dry eye.

[0169] Figure 93 This study illustrates how HC-HA activates IGF1-HIF1α-VEGF signaling to promote angiogenesis. When cells were at rest, HC-HA induced a 2- to 6-fold increase in IGF1 mRNA and a 2-fold increase in VEGF mRNA. When cells were stimulated with TGFβ (10 ng / ml), HC-HA induced a 5- to 12-fold increase in IGF1 mRNA and a 5- to 9-fold increase in VEGF mRNA. n = 4, *p < 0.05, **p < 0.01. IGF1, insulin-like growth factor 1; HIF1α, hypoxia-inducible factor 1-α; VEGF, vascular endothelial growth factor. Detailed Implementation

[0170] Specific terms

[0171] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the claimed subject matter pertains. Unless otherwise stated, all patents, patent applications, published applications and publications, GENBANK sequences, websites and other published materials referenced herein are incorporated herein by reference in their entirety. Where multiple definitions exist for terms herein, the definition in this section shall prevail. When references are made to URLs or other such identifiers or addresses, it should be understood that such identifiers may vary and specific information on the Internet may vary, but equivalent information is known and readily accessible, such as by searching the Internet and / or appropriate databases. References attest to the availability and public dissemination of this information.

[0172] As used herein, ranges and quantities can be expressed as “about” a specific value or range. “About” also includes the exact quantity. Therefore, “about 5 μg” means “about 5 μg” and also “5 μg”. Typically, the term “about” includes the quantity expected to be within experimental error.

[0173] As used herein, the reconstructed HC-HA / PTX3 (rcHC-HA / PTX3) complex is formed by assembling the component molecules of the complex in vitro. Methods for assembling rcHC-HA / PTX3 include: reconstruction using purified natural proteins or molecules from biological sources, generation of recombinant proteins via recombinant methods, or synthesis of the molecules via in vitro synthesis. In some cases, the purified natural protein used to assemble rcHC-HA / PTX3 is a protein in a complex formed with other proteins (i.e., a multimer, a multi-chain protein, or other complex). In some cases, PTX3 is purified from cells as a multimer (e.g., a homomultimer) and used to assemble the rcHC-HA / PTX3 complex.

[0174] As used herein, purified natural HC-HA / PTX3 (nHC-HA / PTX3) complexes refer to HC-HA / PTX3 complexes purified from biological sources, such as cells, tissues, or biological fluids. Such complexes are typically assembled in vivo in a subject or in vitro in cells, tissues, or biological fluids derived from a subject, including humans or other animals.

[0175] As used herein, the PTX3 / HA complex refers to an intermediate complex formed by contacting PTX3 with immobilized HA. In the method provided herein, the PTX3 / HA complex is generated prior to the addition of hydrochloric acid to the HA.

[0176] As used herein, “hyaluronan,” “hyaluronic acid,” or “hyaluronate” (HA) are used interchangeably to refer to a substantially nonsulfated linear glycosaminoglycan (GAG) having repeating disaccharide units of D-glucuronic acid and N-acetylglucosamine (D-glucuronic acid-N-acetylglucosamine).

[0177] As used herein, the term "high molecular weight" or "HMW," such as in high molecular weight hyaluronic acid (HMW HA), means a weight-average molecular weight greater than about 500 kilodaltons (kDa), for example, about 500 kDa to about 10,000 kDa, about 800 kDa to about 8,500 kDa, about 1,100 kDa to about 5,000 kDa, or about 1,400 kDa to about 3,500 kDa. In some embodiments, HMW HA has a weight-average molecular weight of 3,000 kDa or greater. In some embodiments, HMW HA has a weight-average molecular weight of 3,000 kDa. In some embodiments, HMW HA is a product with a weight-average molecular weight of about 3,000 kDa. In some embodiments, HMW HA has a molecular weight of about 500 kDa to about 10,000 kDa. In some embodiments, HMW HA has a molecular weight of about 800 kDa to about 8,500 kDa. In some embodiments, HMW HA has a molecular weight of about 3,000 kDa.

[0178] As used herein, the term "low molecular weight" or "LMW," as in low molecular weight hyaluronic acid (LMW HA), means a weight-average molecular weight of less than 500 kDa, for example, less than about 400 kDa, less than about 300 kDa, less than about 200 kDa, or HA of about 200-300 kDa or about 1-300 kDa.

[0179] As used herein, pentacyclic protein 3 or PTX3 protein or polypeptide refers to any PTX3 protein, including but not limited to recombinant proteins, synthetic proteins, native PTX3 proteins, and PTX3 proteins extracted from cells or tissues. PTX3 includes multimeric forms of PTX3 (e.g., homopolymers), including but not limited to dimers, trimers, tetramers, pentamers, hexamers, tetramers, octamers, and other naturally or artificially produced multimeric forms.

[0180] As used herein, tumor necrosis factor-stimulated gene-6 (TSG-6) refers to any TSG-6 protein or polypeptide, including but not limited to recombinant proteins, synthetic proteins, native TSG-6 proteins, and TSG-6 proteins extracted from cells or tissues.

[0181] As used herein, meso-α-inhibitors (IαI) refer to IαI proteins composed of a light chain (i.e., urostatin) covalently linked by a chondroitin sulfate chain and one or two heavy chains of type HC1 or HC2. In some embodiments, IαI is derived from serum or from IαI-producing cells, such as hepatocytes, amniotic epithelial cells, stromal cells, or umbilical cord epithelial cells, upon constitutive stimulation by pro-inflammatory cytokines such as IL-1 or TNF-α.

[0182] As used in this article, "hyaluronic acid-binding protein," "HA-binding protein," or "HABP" refers to any protein that specifically binds to HA.

[0183] As used in this article, "connecting module" refers to the hyaluronic acid binding domain.

[0184] As used herein, “bioactivity” refers to the in vivo activity or physiological response of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex or a composition or mixture thereof when administered in vivo. Therefore, bioactivity includes the therapeutic and pharmaceutical activities of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex and its compositions and mixtures.

[0185] As used herein, the terms “subject,” “individual,” and “patient” are used interchangeably. None of these terms should be construed as requiring supervision by a medical professional (such as a physician, nurse, physician assistant, caregiver, or hospice staff). As used herein, a subject is any animal, including mammals (e.g., humans or non-human animals) and non-mammals. In one embodiment of the methods and compositions provided herein, the mammal is a human.

[0186] As used herein, the terms “treat,” “treating,” or “treatment” and other grammatical equivalents include alleviating, mitigating, or improving one or more symptoms of a disease or condition; improving, preventing, or reducing the occurrence, severity, or frequency of one or more additional symptoms of a disease or condition; improving or preventing the underlying metabolic cause of one or more symptoms of a disease or condition; inhibiting a disease or condition, for example, preventing its development; alleviating a disease or condition; causing a disease or condition to subside; alleviating symptoms caused by a disease or condition; or inhibiting the symptoms of a disease or condition in prevention and / or treatment. In a non-limiting example, to obtain a beneficial preventive effect, the rcHC-HA / PTX3 complex or composition disclosed herein is applied to an individual who is at risk of having a particular condition, is at risk of having a particular condition, or is applied to an individual who reports having one or more physiological symptoms of a condition.

[0187] As used in this article, the "placenta" refers to the organ that connects the developing fetus to the mother's uterine wall, allowing for nutrient absorption, waste excretion, and gas exchange via the mother's blood supply. The placenta consists of three layers. The innermost layer surrounding the fetus is called the amnion. The allantoic membrane is the middle layer of the placenta (derived from the embryonic hindgut); blood vessels originating from the umbilicus pass through this membrane. The outermost layer of the placenta, the chorion, is in contact with the uterine lining. The chorion and allantoic membrane fuse to form the chorioallantoic membrane.

[0188] As used herein, the "chorion" refers to the membrane formed by the extraembryonic mesoderm and two trophoblastic layers. The chorion consists of two layers: an outer layer formed by the trophoblast and an inner layer formed by the body wall mesoderm; the amnion is in contact with the latter. The trophoblast consists of a cubic or prismatic inner layer of cells, a cytotrophoblast or Langhans layer, and a nucleus-rich outer layer of protoplasm lacking cell boundaries (i.e., the syncytiotrophoblast). The avascular amnion is attached to the inner layer of the chorion.

[0189] As used herein, “amnion-chorion” refers to a product comprising the amnion and the chorion. In some embodiments, the amnion and chorion are not separated (i.e., the amnion is naturally attached to the inner layer of the chorion). In some embodiments, the amnion is initially separated from the chorion and subsequently merged back into the chorion during processing.

[0190] As used in this article, the "umbilical cord" refers to the organ that connects the developing fetus to the placenta. The umbilical cord is composed of Wharton's jelly, a gelatinous substance primarily made of mucopolysaccharides. It contains one vein (which carries oxygenated, nutrient-rich blood to the fetus) and two arteries (which carry away deoxygenated, nutrient-depleted blood).

[0191] As used herein, “placental amnion” (PAM) refers to the amnion derived from the placenta. In some implementations, the PAM is substantially separated.

[0192] As used herein, “umbilical cord amnion” (UCAM) refers to the amnion derived from the umbilical cord. The UCAM is a translucent membrane. The UCAM has multiple layers: an epithelial layer (basement membrane); a compact layer; a fibroblast layer; and a spongy layer. It lacks blood vessels or a direct blood supply. In some embodiments, the UCAM includes Wharton's jelly. In some embodiments, the UCAM includes blood vessels and / or arteries. In some embodiments, the UCAM includes Wharton's jelly and blood vessels and / or arteries.

[0193] As used herein, the terms “purified” and “isolated” mean material that is substantially or substantially free of components that are normally associated with it in its native state (e.g., nHC-HA / PTX3 complex). In some embodiments, “purified” or “isolated” means free of components that are normally associated with it in its native state in a proportion of about 50% or higher, for example, material that is free of components that are normally associated with it in its native state in a proportion of about 50%, about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% (e.g., nHC-HA / PTX3 complex).

[0194] Overview: nHC-HA / PTX3 and rcHC-HA / PTX3 complexes

[0195] Hyaluronic acid (HA) is a essentially non-sulfated linear glycosaminoglycan (GAG) composed of repeating disaccharide subunits of D-glucuronic acid and N-acetyl-D-glucosamine linked by GlcUA-β1,3-GlcNAc-β1,4- bonds. HA is synthesized by HA synthases (e.g., HAS1, HAS2, and HAS3) and deposited into the extracellular matrix, where it contributes to tissue structural integrity and also regulates many cellular processes through interactions with proteins, including cell surface receptors. The molecular weight of HA typically ranges from about 200 to about 10,000 kDa. Normal levels of HA in tissues are maintained through a balance between biosynthesis by HAS enzymes and catabolism by hyaluronidases such as Hyal1.

[0196] High molecular weight hyaluronic acid (HMWHA), typically greater than 500 kDa, promotes cell quiescence and structural integrity in tissues such as cartilage and vitreous humor (body fluids) in the eye, and is associated with wound healing in scarless fetuses. In some cases, HMWHA inhibits gene expression of pro-inflammatory mediators and angiogenesis.

[0197] Under certain pathogenic conditions, HMW HA is degraded into smaller fragments and oligosaccharides (e.g., by hyaluronidase or free radical oxidation). LMW HA fragments stimulate vascular endothelial cell proliferation, migration, collagen synthesis, bud formation, and angiogenesis in rat skin, myocardial infarction, and cryodamaged skin graft models by promoting gene expression of pro-inflammatory and pro-angiogenic mediators.

[0198] The biological function of HA is mediated through the interaction of HA with HA-binding proteins (HABPs) (also known as hyaluronans). Such proteins include, but are not limited to, tumor necrosis factor-α stimulated gene 6 (TSG-6), aggregate proteoglycans, multipotent proteoglycans, neuroproteoglycans, short proteoglycans, LYVE-1, CD44, and inter-α-inhibitors (IαI). In some cases, HABPs contain a linker module domain that binds to HA. TSG-6, aggregate proteoglycans, multipotent proteoglycans, neuroproteoglycans, short proteoglycans, LYVE-1, and CD44 are exemplary HABPs containing a linker module.

[0199] IαI comprises two heavy chains (HC1 and HC2) linked by ester bonds to a chondroitin sulfate chain attached to a light chain (i.e., urostatin). In some cases, HA forms a covalent complex (hereinafter referred to as "HC-HA") with one or both HCs of IαI via covalent linkage to the IαI heavy chain. In some cases, IαI is found in serum and / or obtained from IαI-producing cells, such as hepatocytes, amniotic epithelial or stromal cells, or umbilical cord epithelial or stromal cells, upon constitutive stimulation by pro-inflammatory cytokines such as IL-1 or TNF-α.

[0200] In some cases, TSG-6 promotes, catalyzes, and / or transfers HC1 and HC2 of IαI to HA. TSG-6 forms a stable complex (TSG-6·HA) with immobilized HA, leading to the transfer of HC1 and HC2 to HA, thereby forming an HC-HA complex and releasing TSG-6 from this complex. TSG-6 expression is typically induced by inflammatory mediators such as TNF-α and interleukin-1 during inflammatory processes such as ovulation and cervical ripening.

[0201] Amniotic membrane (AM) regulates wound healing in adults and promotes tissue regeneration. In some cases, AM promotes epithelial formation while suppressing stromal inflammation, angiogenesis, and scar formation. AM has been successfully used as a surgical graft or temporary biological patch for treating ophthalmic conditions requiring corneal and conjunctival surface reconstruction, including but not limited to persistent epithelial defects, deep corneal ulcers, infectious keratitis, symptomatic bullous keratopathy, acute Stevens-Johnson syndrome / toxic epidermal necrolysis (SJS / TEN), limbic stem cell defects, pterygium, conjunctival macular degeneration, conjunctival laxity, symblepharon, fornix reconstruction, and conjunctival tumors.

[0202] The avascular stromal matrix of AM contains high levels of HA and constitutively expresses IαI (Zhang et al. (2012), J. Biol. Chem. 287(15):12433-44). HMW HA in AM forms an nHC-HA complex (He et al. (2009), J. Biol. Chem. 284(30):20136-20146). As shown herein, in the examples provided, this nHC-HA complex also contains pentacyclic protein 3, PTX3 ( Figure 1 This compound, referred to herein as the "nHC-HA / PTX3 complex," was derived from AM. The natural HC-HA / PTX3 complex extracted from AM exhibited inhibition of TGF-β promoter activity, promotion of macrophage death, and inhibition of angiogenesis. Therefore, the nHC-HA / PTX3 complex from AM plays a positive role in the anti-inflammatory, anti-scarring, and anti-angiogenic effects of AM.

[0203] As described herein, nHC-HA / PTX3 complexes are also found in the umbilical cord (UC). These UC HC-HA / PTX3 complexes differ in their biochemical composition regarding HA content and the presence and / or relative abundance of various components of the complex, including proteoglycans such as small leucine-rich proteoglycans (SLRP). In some embodiments, SLRP is gypsum proteoglycan, disaccharide proteoglycan, and / or osteoadhesion proteoglycan. As described herein, these complexes also differ in the content of specific sulfated glycosaminoglycans such as keratin sulfate. Furthermore, as described herein, complexes isolated from AM or UC using different extraction methods (e.g., PBS extraction versus GnHCl extraction) result in complexes with different biochemical compositions and biological properties. In some cases, complexes isolated from insoluble fractions obtained from umbilical cord tissue by GnHCl extraction have been found to exhibit improved properties.

[0204] PTX3 is a multimeric protein that has been shown to interact directly with TSG-6 and IαI HC. PTX3 is upregulated in response to inflammatory modulators and has been shown to play a crucial role in HA organization in the extracellular matrix of the cumulous oophorus during oocyte maturation. As demonstrated in this paper, PTX3 is also found within the nHC-HA complex (i.e., nHC-HA / PTX3) in the amnion and umbilical cord and plays a key role in M2 macrophage polarization.

[0205] M1 macrophages, or classically activated pro-inflammatory macrophages, are induced by interferon (IFN) alone or in combination with lipopolysaccharide (LPS) or tumor necrosis factor (TNF) α. M1 macrophages are characterized by high expression of interleukin-12 (IL-12) and IL-23, and low levels of IL-10. In contrast, M2 macrophages, or “alternatively activated” macrophages, exhibit wound healing and tissue regeneration properties and are characterized by low IL-12 / IL-23 and high or approximately equal IL-10 ratios. In some cases, M2 macrophages also show high expression of TGF-β.

[0206] The embodiments provided herein demonstrate that PTX3 binds directly to immobilized HA, as demonstrated by resistance to dissociation agents. This document confirms that in vitro reconstituted complexes of HA bound to PTX3 exhibit different properties than those of in vitro reconstituted complexes of HA bound to TSG-6. For example, in some embodiments, the PTX3 / HA complex promotes attachment of LPS-stimulated macrophages without aggregation and induces IL-10 expression in LPS-stimulated macrophages. In some embodiments, the PTX3 / HA complex disclosed herein increases IL-10 expression in LPS-stimulated macrophages by approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to IL-10 expression in the absence of the PTX3 / HA complex. In contrast, in some embodiments, the TSG-6 / HA complex reduces cell attachment and promotes aggregation of LPS-stimulated macrophages without inducing IL-10 expression in LPS-stimulated macrophages. Furthermore, in some embodiments, TSG-6 pre-bound to HA inhibits subsequent binding of PTX3 to the complex. In some embodiments, both the TSG-6 / HA complex and the PTX3 / HA complex reduce IL-12 expression in LPS-stimulated macrophages. In some embodiments, the PTX3 / HA complex or TSG-6 / HA complex disclosed herein reduces or inhibits IL-12 expression in LPS-stimulated macrophages by approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the absence of the PTX3 / HA complex or the TSG-6 / HA complex. In some embodiments, both the TSG-6 / HA complex and the PTX3 / HA complex enhance IL-23 expression in LPS / IFNγ-stimulated macrophages. In some embodiments, the PTX3 / HA complex or TSG-6 / HA complex disclosed herein reduces or inhibits IL-23 expression in LPS / IFNγ-stimulated macrophages by approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the absence of the PTX3 / HA complex or the TSG-6 / HA complex.

[0207] Furthermore, this paper demonstrates that the in vitro reconstituted rcHC-HA / PTX3 complex exhibits different biological activities depending on whether the following steps are employed to form the rcHC-HA / PTX3 complex: prebinding HA to TSG-6 in the presence of IαI, followed by the addition of PTX3 or HA prebinding to PTX3, and then the addition of TSG-6 with IαI. This paper provides exemplary methods for reconstructing the rcHC-HA / PTX3 complex using HA prebinding to TSG-6 or HA prebinding to PTX3. In some embodiments, the rcHC-HA / PTX3 complex formed using immobilized HA prebinding to TSG-6 leads to the aggregation of LPS-stimulated macrophages. In some embodiments, the rcHC-HA / PTX3 complex formed using immobilized HA prebinding to PTX3 promotes the attachment of LPS-stimulated macrophages without aggregation.

[0208] In some embodiments, the rcHC-HA / PTX3 complex formed using immobilized HA pre-bound to PTX3 reduces or inhibits the expression of M1 macrophage markers such as IL-12 and IL-23. In some embodiments, the rcHC-HA / PTX3 complex formed using immobilized HA pre-bound to PTX3 reduces IL-12 expression in LPS-stimulated macrophages compared to the absence of the rcHC-HA / PTX3 complex. In some embodiments, the rcHC-HA / PTX3 complex disclosed herein reduces or inhibits IL-12 expression in LPS-stimulated macrophages by approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to the absence of the rcHC-HA / PTX3 complex. In some embodiments, the rcHC-HA / PTX3 complex formed using immobilized HA pre-bound to PTX3 reduced or inhibited IL-23 expression in LPS / IFNγ-stimulated macrophages compared to IL-12 expression in the absence of the rcHC-HA / PTX3 complex. In some embodiments, the rcHC-HA / PTX3 complex formed using immobilized HA pre-bound to PTX3 reduced or inhibited IL-23 expression in LPS / IFNγ-stimulated macrophages by approximately 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100% compared to IL-23 expression in the absence of the rcHC-HA / PTX3 complex. In some embodiments, the activity of the nHC-HA / PTX3 complex isolated from the amnion is replicated by using an immobilized HA pre-bound to PTX3 to form an rcHC-HA / PTX3 complex.

[0209] In some embodiments, the rcHC-HA / PTX3 complex formed with immobilized HA pre-bound to TSG-6 reduced or inhibited the expression of M1 macrophage markers such as IL-12, but increased the expression of IL-23. In some embodiments, the rcHC-HA / PTX3 complex formed with immobilized HA pre-bound to TSG-6 reduced or inhibited IL-12 expression. In some embodiments, the rcHC-HA / PTX3 complex formed with immobilized HA pre-bound to TSG-6 increased IL-23 expression.

[0210] This document provides a method for preparing reconstituted HC-HA / PTX3 complexes using immobilized HA pre-bound to PTX3 and their uses. This document also provides a method for preparing reconstituted HC-HA / PTX3 complexes using immobilized HA pre-bound to TSG-6 and their uses. In some embodiments, the reconstituted HC-HA / PTX3 complexes provided herein are applied to treat a variety of diseases or conditions, including but not limited to treating, reducing, preventing, or mitigating inflammation, immune responses leading to autoimmunity or immune rejection, adhesions, scarring, angiogenesis, conditions requiring cell or tissue regeneration, tissue reperfusion injury due to ischemia (including myocardial infarction and stroke), and the risk of symptoms arising therefrom. In some embodiments, the reconstituted HC-HA / PTX3 complexes provided herein are applied to treat inflammation. In some embodiments, the reconstituted HC-HA / PTX3 complexes provided herein are applied to treat scarring. In some embodiments, the reconstituted HC-HA / PTX3 complexes provided herein are applied to treat angiogenesis. In some embodiments, the reconstituted HC-HA / PTX3 complex provided herein is applied to treat immune responses leading to autoimmunity or immune rejection. In some embodiments, the reconstituted HC-HA / PTX3 complex provided herein is applied to treat conditions requiring inhibition of cell adhesion. In some embodiments, the reconstituted HC-HA / PTX3 complex provided herein is applied to treat conditions requiring cell or tissue regeneration.

[0211] Furthermore, the examples presented herein demonstrate the ability of the HC-HA / PTX3 complex to maintain stem cells in an undifferentiated state and to induce adult fibroblasts to differentiate into younger progenitor cells in a human corneal fibroblast model. These human corneal fibroblasts are derived from corneal cells differentiated by the addition of exogenous TGF-β1, and they further differentiate into scar-forming myofibroblasts. The data presented herein show that culturing cells in the presence of HC-HA prevents cells from differentiating into myofibroblasts upon TGF-β1 stimulation. In the absence of TGF-β1, the HC-HA / PTX3 complex restores human corneal fibroblasts to corneal cells expressing corneal proteoglycans and CD34. In the presence of TGF-β1, human corneal fibroblasts are further reprogrammed into younger progenitor cells that lack corneal proteoglycan expression but express multiple neural crest cell markers (such as Osr2, FGF10, and Sox9) and embryonic stem cell markers (such as c-myc, KLF4, Nanog, nestin, Oct4, Rex-1, Sox-2, and SSEA-4).

[0212] Transcription factors Sox2, Oct4, c-Myc, and KLF4 are known to play important roles in the induction of adult differentiated cells into iPSCs. Therefore, in some embodiments, the HC-HA / PTX3 complex described herein is used to reprogram adult differentiated cells into iPSCs. In some embodiments, induction of iPSCs using the HC-HA / PTX3 complex in combination with one or more of Sox2, Oct4, c-Myc, and KLF4 is significantly more efficient than conventional methods using these four transcription factors without HC-HA / PTX3. In some embodiments, the addition of the HC-HA / PTX3 complex promotes stem cell induction by blocking TGF-β signaling to prevent differentiation and by promoting reprogramming into younger progenitor cells such as iPSCs by turning on BMP signaling. In some embodiments, the addition of the HC-HA / PTX3 complex promotes stem cell induction by reprogramming cells into younger progenitor cells and inducing stem cell markers. In some embodiments, the addition of the HC-HA / PTX3 complex helps maintain the characteristics of stem cells during in vitro expansion, thereby eliminating the need for a feeder layer composed of mouse embryonic fibroblasts. Therefore, in some implementations, the HC-HA / PTX3 complex is used as a carrier or scaffold to help deliver in vitro expanded stem cells to human patients, thereby enhancing the efficacy of the stem cell therapy.

[0213] Method for preparing isolated nHC-HA / PTX3 complex

[0214] This article discloses a method for producing isolated natural HC-HA / PTX3 complexes (nHC-HA / PTX3).

[0215] In some embodiments, the isolated nHC-HA / PTX3 complex is isolated from amniotic tissue. In some embodiments, the isolated nHC-HA / PTX3 complex is isolated from the amnion or umbilical cord. In some embodiments, the isolated nHC-HA / PTX3 complex is isolated from fresh, frozen, or previously frozen placental amnion (PAM), fresh, frozen, or previously frozen umbilical cord amnion (UCAM), fresh, frozen, or previously frozen placenta, fresh, frozen, or previously frozen umbilical cord, fresh, frozen, or previously frozen chorion, fresh, frozen, or previously frozen amniochorion, or any combination thereof. Such tissue can be obtained from any mammal, such as, but not limited to, humans, non-human primates, cattle, or pigs.

[0216] In some implementations, nHC-HA / PTX3 is purified by any suitable method. In some embodiments, the nHC-HA / PTX3 complex is purified by centrifugation (e.g., ultracentrifugation, gradient centrifugation), chromatography (e.g., ion exchange chromatography, affinity chromatography, size exclusion chromatography, and hydroxyapatite chromatography), gel filtration or differential dissolution, ethanol precipitation, or any other feasible technique for protein purification (see, for example, Scopes, Protein Purification Principles and Practice, 2nd edition, Springer-Verlag, New York, 1987; Higgins, SJ and Hames, BD, eds., Protein Expression: A Practical Approach, Oxford Univ Press, 1999; and Deutscher, MP, Simon, MI, Abelson, JN, eds., Guide to Protein Purification: Methods in Enzymology (Methods in Enzymology Series, Vol. 182), Academic Press, 1997, all of which are incorporated herein by reference).

[0217] In some embodiments, nHC-HA / PTX3 is isolated from the extract. In some embodiments, the extract is prepared from an amnion extract. In some embodiments, the extract is prepared from an umbilical cord extract. In some embodiments, the umbilical cord extract comprises umbilical cord matrix and / or Wharton's jelly. In some embodiments, the nHC-HA / PTX3 complex is contained in an extract prepared by ultracentrifugation. In some embodiments, the nHC-HA / PTX3 complex is contained in an extract prepared by ultracentrifugation using a CsCl / 4-6M guanidine hydrochloride gradient. In some embodiments, the extract is prepared by at least two rounds of ultracentrifugation. In some embodiments, the extract is prepared by more than two rounds of ultracentrifugation (i.e., nHC-HA / PTX3, second). In some embodiments, the extract is prepared by at least four rounds of ultracentrifugation (i.e., nHC-HA / PTX3, fourth). In some embodiments, the nHC-HA / PTX3 complex comprises small leucine-rich proteoglycans. In some implementations, the nHC-HA / PTX3 complex comprises HCl, HA, PTX3, and / or a small leucine-rich proteoglycan.

[0218] In some embodiments, the extract prepared by extraction is ultracentrifuged in an isotonic solution. In some embodiments, the isotonic solution is PBS. For example, in some embodiments, tissue is homogenized in PBS to prepare a homogenized sample. The homogenized sample is then separated into soluble and insoluble fractions by centrifugation. In some embodiments, the soluble fraction of the tissue extracted with PBS is ultracentrifuged. In such embodiments, nHC-HA / PTX3 purified by ultracentrifugation of tissue extracted with PBS is referred to as the nHC-HA / PTX3 soluble complex. In some embodiments, the nHC-HA soluble complex comprises a small leucine-rich proteoglycan. In some embodiments, the nHC-HA / PTX3 soluble complex comprises HCl, HA, PTX3, and / or a small leucine-rich proteoglycan.

[0219] In some embodiments, the extract prepared by direct guanidine hydrochloride extraction (e.g., 4-6 MgnHCl) of amnion and / or umbilical cord tissue is ultracentrifuged. In some embodiments, the tissue of the GnHCl extract is subsequently centrifuged to produce GnHCl-soluble and GnHCl-insoluble fractions. In some embodiments, the GnHCl-soluble fraction is ultracentrifuged. In such embodiments, nHC-HA / PTX3 purified by ultracentrifugation of the tissue extracted with guanidine hydrochloride is referred to as the nHC-HA / PTX3 insoluble complex. In some embodiments, the nHC-HA insoluble complex comprises a small leucine-rich proteoglycan. In some embodiments, the nHC-HA / PTX3 insoluble complex contains HCl, HA, PTX3, and / or a small leucine-rich proteoglycan.

[0220] In some embodiments, the extract prepared by further guanidine hydrochloride extraction of the insoluble fraction of tissue extracted with PBS is subjected to ultracentrifugation. For example, in some embodiments, the tissue is homogenized in PBS to prepare a homogenate sample. The homogenate sample is then separated into soluble and insoluble fractions by centrifugation. The insoluble fraction is then further extracted in guanidine hydrochloride (e.g., 4-6 M GnHCl) and centrifuged to produce guanidine hydrochloride soluble and insoluble fractions. In some embodiments, the guanidine hydrochloride soluble fraction is ultracentrifuged. In such embodiments, nHC-HA / PTX3 purified by ultracentrifugation of tissue extracted with guanidine hydrochloride is referred to as the nHC-HA / PTX3 insoluble complex. In some embodiments, the nHC-HA insoluble complex comprises a small leucine-rich proteoglycan. In some embodiments, the nHC-HA / PTX3 insoluble complex comprises HCl, HA, PTX3, and / or a small leucine-rich proteoglycan.

[0221] In some embodiments, the method for purifying the isolated nHC-HA / PTX3 extract includes: (a) dissolving the isolated extract (e.g., prepared by the soluble or insoluble methods described herein) in CsCl / 4-6M guanidine hydrochloride with an initial density of 1.35 g / ml to produce a CsCl mixture; (b) centrifuging the cesium chloride mixture at 125,000 x g for 48 h at 15 °C to produce a first purified extract; and (c) extracting the first purified extract and dialyzing it relative to distilled water to remove cesium chloride and guanidine hydrochloride, thereby producing a dialysate. In some embodiments, the method for purifying the isolated extract further includes: (d) mixing dialysate with three volumes of 95% (v / v) ethanol containing 1.3% (w / v) potassium acetate for 1 h at 0 °C to produce a first dialysate / ethanol mixture; (e) centrifuging the first dialysate / ethanol mixture at 15,000 x g to produce a second purified extract; and (f) extracting the second purified extract. In some embodiments, the method for purifying the isolated extract further includes: (g) washing the second purified extract with ethanol (e.g., 70% ethanol) to produce a second purified extract / ethanol mixture; (h) centrifuging the second purified extract / ethanol mixture to produce a third purified extract; and (i) extracting the third purified extract. In some embodiments, the method for purifying the isolated extract further includes: (j) washing the third purified extract with ethanol (e.g., 70% ethanol) to produce a third purified extract / ethanol mixture; (k) centrifuging the third purified extract / ethanol mixture to produce a fourth purified extract; and (l) extracting the fourth purified extract. In some implementations, the purified extract contains an nHC-HA / PTX3 complex.

[0222] In some embodiments, the nHC-HA / PTX3 complex is purified by immunoaffinity chromatography. In some embodiments, an anti-HC1 antibody, an anti-HC2 antibody, or both, is generated and attached to a fixation support. In some embodiments, the unpurified HC-HA complex (i.e., the mobile phase) is passed through the support. In some cases, the HC-HA complex binds to an antibody (e.g., by the interaction of (a) anti-HC1 antibody and HCl, (b) anti-HC2 antibody and HC2, (c) anti-PTX antibody and PTX3, (d) anti-SLRP antibody and SLRP, or (e) any combination thereof). In some embodiments, the support is washed (e.g., with PBS) to remove any unbound or loosely bound molecules. In some embodiments, the support is subsequently washed with a solution capable of eluting the nHC-HA / PTX3 complex from the support (e.g., 1% SDS, 6M guanidine hydrochloride, or 8M urea).

[0223] In some embodiments, the nHC-HA / PTX3 complex is purified by affinity chromatography. In some embodiments, HABP is generated and attached to a stationary support. In some embodiments, the unpurified nHC-HA / PTX3 complex (i.e., the mobile phase) is passed through the support. In some cases, the nHC-HA / PTX3 complex binds to HABP. In some embodiments, the support is washed (e.g., with PBS) to remove any unbound or loosely bound molecules. In some embodiments, the support is subsequently washed with a solution capable of eluting the HC-HA complex from the support.

[0224] In some embodiments, the nHC-HA / PTX3 complex is purified by HABP affinity chromatography and immunoaffinity chromatography using an anti-HC1 antibody, an anti-HC2 antibody, an anti-PTX3 antibody, an antibody against SLRP or a combination of SLRP, or any combination of antibodies against the latter.

[0225] In some embodiments, the nHC-HA / PTX3 complex is purified from the insoluble fraction as described herein using one or more antibodies. In some embodiments, the nHC-HA / PTX3 complex is purified from the insoluble fraction as described herein using an anti-SLRP antibody.

[0226] In some embodiments, the nHC-HA / PTX3 complex is purified from the soluble fraction as described herein. In some embodiments, an anti-PTX3 antibody is used to purify the nHC-HA / PTX3 complex from the soluble fraction as described herein.

[0227] In some embodiments, the nHC-HA / PTX3 complex comprises small leucine-rich proteoglycans (SLRPs). In some embodiments, the nHC-HA / PTX3 complex comprises class I, II, or III SLRPs. In some embodiments, the small leucine-rich proteoglycan is selected from class I SLRPs, such as dermal proteoglycans and disaccharide proteoglycans. In some embodiments, the small leucine-rich proteoglycan is selected from class II SLRPs, such as fibrinolytic proteoglycans, lumican, PRELP (proline-arginine-rich terminal leucine-rich protein), corneal proteoglycans, and osteoadhesion proteoglycans. In some embodiments, the small leucine-rich proteoglycan is selected from class III SLRPs, such as epiphyseal proteoglycans and osteoglobin proteoglycans. In some embodiments, the small leucine-rich proteoglycan is selected from urostatin, dermal proteoglycans, disaccharide proteoglycans, and osteoadhesion proteoglycans. In some embodiments, the small leucine-rich protein comprises glycosaminoglycans. In some embodiments, the small leucine-rich proteoglycan comprises keratin sulfate.

[0228] Method for producing rcHC-HA / PTX3 complex

[0229] This paper discloses a method for generating reconstituted HC-HA / PTX3 complexes (rcHC-HA / PTX3) with or without SLRP. Intermediate combinations of the rcHC-HA / PTX3 complexes and components generated by this method are also disclosed.

[0230] In some embodiments, a method for generating a reconstituted HC-HA / PTX3 complex includes: (a) contacting immobilized high molecular weight hyaluronic acid (HMW HA) with pentacyclic protein 3 (PTX3) under appropriate conditions to form a PTX3 / HA complex, and (b) contacting the PTX3 / HA complex with IαI and tumor necrosis factor-stimulated gene 6 (TSG-6). This document provides an rcHC-HA / PTX3 complex produced by this method. In some embodiments, TSG-6 catalyzes the transfer of the heavy chain 1 (HCl) of the meta-α-inhibitor (IαI) to HA. In some embodiments, the HCl of IαI forms a covalent bond with HA. In some embodiments, steps (a) and (b) of the method are performed sequentially.

[0231] In some embodiments, the method for generating the reconstituted HC-HA / PTX3 complex includes contacting the PTX3 / HA complex with IαI and TSG-6. In some embodiments, TSG-6 catalyzes the transfer of the heavy chain 1 (HCl) of the meta-α-inhibitor (IαI) to HA. This document provides the rcHC-HA / PTX3 complex generated by this method. In some embodiments, the HCl of IαI forms a covalent bond with HA.

[0232] In some embodiments, methods for generating a PTX3-bound HA complex include contacting immobilized high molecular weight hyaluronic acid (HMW HA) with pentacyclic protein 3 (PTX3) under appropriate conditions to form a PTX3 / HA complex. This document provides a PTX3 / HA complex generated by this method.

[0233] In some embodiments, a method for generating a reconstituted HC-HA / PTX3 complex includes (a) contacting immobilized high molecular weight hyaluronic acid (HMW HA) with IαI and TSG-6 targeting HA to form a pre-bound HC-HA complex to TSG-6, and (b) contacting the HC-HA complex with pentacyclic protein 3 (PTX3) under appropriate conditions to form an rcHC-HA / PTX3 complex. This document provides an rcHC-HA / PTX3 complex generated by this method. In some embodiments, HCl of IαI forms a covalent bond with HA. In some embodiments, steps (a) and (b) of the method are performed sequentially. In some embodiments, the method includes contacting the pre-bound HC-HA complex to TSG-6 with PTX3.

[0234] In some embodiments, the method includes: first contacting high molecular weight hyaluronic acid (HMW HA) with pentacyclic protein 3 (PTX3) under appropriate conditions to form a PTX3 / HA complex, and then contacting the PTX3 / HA complex with IαI and TSG-6.

[0235] In some embodiments, the IαI protein is contacted with the TSG-6 protein at a molar ratio of about 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, 10:1, 15:1, or 20:1 (IαI:TSG-6) to form a complex. In some embodiments, the IαI:TSG-6 ratio ranges from about 1:1 to about 20:1, for example, from about 1:1 to about 10:1, for example, from about 1:1 to about 5:1, such as from about 1:1 to about 3:1. In some embodiments, the IαI:TSG-6 ratio ranges from 3:1 or higher. In some embodiments, the IαI:TSG-6 ratio is 3:1.

[0236] In some embodiments, steps (a) and (b) of the method are performed sequentially. In some embodiments, the method includes contacting the PTX3 / HA complex with IαI and TSG-6.

[0237] In some cases, TSG-6 interacts with IαI and forms covalent complexes with HCl and HC2 of IαI (i.e., HCl·TSG-6 and HC2·TSG-6). In other cases, HC is transferred to HA in the presence of HA to form rcHC-HA. In some embodiments, the TSG-6·HCl complex is added to a pre-bound PTX3 / HA complex to catalyze the transfer of HCl to HA. In some embodiments, the method includes: first contacting immobilized high molecular weight hyaluronic acid (HMW HA) with pentacyclic protein 3 (PTX3) under appropriate conditions to form a PTX3 / HA complex, and then contacting the PTX3 / HA complex with the HC1·TSG-6 complex. In some embodiments, a combination of the HC1·TSG-6 complex and the HC2·TSG-6 complex is added to the PTX3 / HA complex.

[0238] In some embodiments, the step of contacting PTX3 with immobilized HMW HA is performed for at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, or at least 24 hours or longer. In some embodiments, the step of contacting PTX3 with immobilized HMW HA is performed for at least 2 hours. In some embodiments, the step of contacting PTX3 with immobilized HMW HA is performed at 37°C. In some embodiments, the step of contacting PTX3 with immobilized HMW HA is performed in 5 mM MgCl2 in PBS.

[0239] In some embodiments, the step of contacting the PTX3 / HA complex with IαI and TSG-6 against HA is performed for at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, or at least 24 hours or longer. In some embodiments, the step of contacting the PTX3 / HA complex with the HC1·TSG-6 complex and / or the HC2·TSG-6 complex is performed for at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, or at least 24 hours or longer. In some embodiments, the step of contacting the PTX3 / HA complex with the HC1·TSG-6 complex and / or the HC2·TSG-6 complex is performed for at least 2 hours or longer. In some embodiments, the step of contacting the PTX3 / HA complex with the HC1·TSG-6 complex and / or the HC2·TSG-6 complex is performed for at least 2 hours. In some embodiments, the step of contacting the PTX3 / HA complex with the HCl·TSG-6 complex and / or the HCl·TSG-6 complex is performed at 37°C. In some embodiments, the step of contacting the PTX3 / HA complex with the HCl·TSG-6 complex and / or the HCl·TSG-6 complex is performed in 5 mM MgCl2 in PBS.

[0240] In some embodiments, the method includes simultaneously contacting high molecular weight hyaluronic acid (HMW HA) with pentacyclic protein 3 (PTX3) protein, a meta-α-inhibitor (IαI) protein containing heavy chain 1 (HCl), and tumor necrosis factor α-stimulated gene 6 (TSG-6) under appropriate conditions to form an HC-HA / PTX3 complex. In some embodiments, the contacting of HMW HA with PTX3, IαI, and TSG-6 is performed for at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, or at least 24 hours or longer. In some embodiments, the contacting step of HMW HA, PTX3, IαI, and TSG-6 is performed at 37°C. In some embodiments, the contacting step of HMW HA, PTX3, IαI, and TSG-6 is performed in 5 mM MgCl2 in PBS.

[0241] In some embodiments, the method includes sequentially contacting high molecular weight hyaluronic acid (HMW HA) with pentacyclic protein 3 (PTX3) protein, a meta-α-inhibitor (IαI) protein containing heavy chain 1 (HCl), and tumor necrosis factor α-stimulated gene 6 (TSG-6) under appropriate conditions in any order to form an HC-HA / PTX3 complex. In some embodiments, the contacting of HMW HA with PTX3, IαI, and TSG-6 is performed for at least 10 minutes, at least 30 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 6 hours, at least 12 hours, or at least 24 hours or longer. In some embodiments, the contacting step of HMW HA, PTX3, IαI, and TSG-6 is performed at 37°C. In some embodiments, the contacting step of HMW HA, PTX3, IαI, and TSG-6 is performed in 5 mM MgCl2 in PBS.

[0242] In some embodiments, the method for producing the rcHC-HA / PTX3 complex further includes adding one or more small leucine-rich proteoglycans (SLRPs). In some embodiments, the method for producing the reconstituted HC-HA / PTX3 complex includes: (a) contacting immobilized high molecular weight hyaluronic acid (HMW HA) with pentacyclic protein 3 (PTX3) under appropriate conditions to form a PTX3 / HA complex, (b) contacting the PTX3 / HA complex with IαI and tumor necrosis factor-stimulated gene 6 (TSG-6), and (c) contacting the PTX3 / HA complex with one or more SLRPs. The rcHC-HA / PTX3 complex produced by this method is provided herein. In some embodiments, TSG-6 catalyzes the transfer of the heavy chain 1 (HCl) of the meta-α-inhibitor (IαI) to HA. In some embodiments, the HCl of IαI forms a covalent bond with HA. In some embodiments, steps (a), (b), and (c) of the method are performed sequentially. In some embodiments, steps (a), (b), and (c) of the method are performed simultaneously. In some embodiments, step (a) of the method is performed, followed by steps (b) and (c) of the method in sequence. In some embodiments, step (a) of the method is performed, followed by steps (b) and (c) of the method simultaneously.

[0243] In some embodiments, a method for generating a reconstituted HC-HA / PTX3 complex includes: (a) contacting immobilized high molecular weight hyaluronic acid (HMW HA) with IαI and TSG-6 targeting HA to form a pre-bound HC-HA complex to TSG-6; (b) contacting the HC-HA complex with pentacyclic protein 3 (PTX3); and (c) contacting the HC-HA complex with one or more SLRPs under appropriate conditions to form an rcHC-HA / PTX3 complex. This document provides an rcHC-HA / PTX3 complex generated by this method. In some embodiments, HCl of IαI forms a covalent bond with HA. In some embodiments, the method includes contacting the pre-bound HC-HA complex to TSG-6 with PTX3. In some embodiments, steps (a), (b), and (c) of the method are performed sequentially. In some embodiments, steps (a), (b), and (c) of the method are performed simultaneously. In some embodiments, step (a) of the method is performed, followed by steps (b) and (c) of the method sequentially. In some implementations, step (a) of the method is performed, and then steps (b) and (c) of the method are performed simultaneously.

[0244] In some embodiments, the SLRP is selected from Class I, Class II, or Class II SLRPs. In some embodiments, the SLRP is selected from Class I SLRPs, such as dermal proteoglycans and disaccharide proteoglycans. In some embodiments, the small leucine-rich proteoglycan is selected from Class II SLRPs, such as fibrinolytic proteoglycans, luminal proteoglycans, PRELP (proline-arginine-rich, terminal leucine-rich protein), corneal proteoglycans, and osteoadhesion proteoglycans. In some embodiments, the small leucine-rich proteoglycan is selected from Class III SLRPs, such as epiphyseal proteoglycans and osteoglobin proteoglycans. In some embodiments, the small leucine-rich proteoglycan is selected from urostatin, dermal proteoglycans, disaccharide proteoglycans, and osteoadhesion proteoglycans. In some embodiments, the small leucine-rich protein comprises glycosaminoglycans. In some embodiments, the small leucine-rich proteoglycan comprises keratin sulfate.

[0245] PTX3

[0246] In some embodiments, the PTX3 used in the method is isolated from one or more cells (e.g., tissue extracts). Exemplary cells suitable for expressing PTX3 include, but are not limited to, animal cells (including, but not limited to, mammalian cells, primate cells, human cells, rodent cells, insect cells, bacteria, and yeast) and plant cells (including, but not limited to, algae, angiosperms, gymnosperms, ferns, and bryophytes). In some embodiments, the PTX3 used in the method is isolated from human cells. In some embodiments, the PTX3 used in the method is isolated from cells whose PTX3 expression is upregulated by stimulation with one or more pro-inflammatory cytokines. In some embodiments, the pro-inflammatory cytokines are IL-1 or TNF-α.

[0247] In some embodiments, the PTX3 used in the method is isolated from amniotic cells. In some embodiments, the PTX3 used in the method is isolated from amniotic cells derived from the umbilical cord. In some embodiments, the amniotic cells are stimulated with one or more pro-inflammatory cytokines to upregulate PTX3 expression. In some embodiments, the pro-inflammatory cytokines are IL-1 or TNF-α.

[0248] In some embodiments, the PTX3 used in the method is isolated from umbilical cord cells. In some embodiments, the umbilical cord cells are stimulated with one or more pro-inflammatory cytokines to upregulate PTX3 expression. In some embodiments, the pro-inflammatory cytokines are IL-1 or TNF-α.

[0249] In some embodiments, the PTX3 used in the method is isolated from amniotic epithelial cells. In some embodiments, the PTX3 used in the method is isolated from umbilical cord epithelial cells. In some embodiments, the amniotic epithelial cells or umbilical cord epithelial cells are stimulated with one or more pro-inflammatory cytokines to upregulate PTX3 expression. In some embodiments, the pro-inflammatory cytokines are IL-1 or TNF-α.

[0250] In some embodiments, the PTX3 used in the method is isolated from amniotic stromal cells. In some embodiments, the PTX3 used in the method is isolated from umbilical cord stromal cells. In some embodiments, the amniotic stromal cells or umbilical cord stromal cells are stimulated with one or more pro-inflammatory cytokines to upregulate PTX3 expression. In some embodiments, the pro-inflammatory cytokines are IL-1 or TNF-α.

[0251] In some embodiments, the PTX3 used in the method is a naturally occurring PTX3 protein isolated from cells. In some embodiments, the cells are stimulated with one or more pro-inflammatory cytokines to upregulate PTX3 expression. In some embodiments, the pro-inflammatory cytokines are IL-1 or TNF-α.

[0252] In some embodiments, PTX3 is prepared using recombinant technology. In some embodiments, PTX3 is expressed from a recombinant expression vector. In some embodiments, the nucleic acid encoding PTX3 is operatively linked to a constitutive promoter. In some embodiments, the nucleic acid encoding PTX3 is operatively linked to an inducible promoter. In some embodiments, PTX3 is expressed in transgenic animals. In some embodiments, PTX3 is a recombinant protein. In some embodiments, PTX3 is a recombinant protein isolated from cells. In some embodiments, PTX3 is a recombinant protein produced from cell-free extracts.

[0253] In some embodiments, PTX3 is purified from the amnion, umbilical cord, umbilical cord-amnion, chorion, amniotic fluid, or combinations thereof. In some embodiments, PTX3 is purified from amniotic cells. In some embodiments, the amniotic cells are amniotic epithelial cells. In some embodiments, the amniotic cells are umbilical cord epithelial cells. In some embodiments, the amniotic cells are amniotic stromal cells. In some embodiments, the amniotic cells are umbilical cord stromal cells. In some embodiments, the amniotic cells are stimulated with one or more pro-inflammatory cytokines to upregulate PTX3 expression. In some embodiments, the pro-inflammatory cytokines are IL-1 or TNF-α.

[0254] In some implementations, PTX3 is not isolated from one or more cells (e.g., tissue extracts).

[0255] In some embodiments, PTX3 comprises a polypeptide having the sequence shown in SEQ ID NO:33 or a variant thereof, the variant having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity with the polypeptide having the sequence shown in SEQ ID NO:33. Exemplary variants include, for example, species variants, allelic variants, and variants containing conserved and non-conserved amino acid mutations. In some embodiments, PTX3 comprises a PTX3 fragment sufficient to bind to HA and promote the formation of the rcHC-HA / PTX3 complex. In some embodiments, PTX3 comprises Glu18 to Ser277 of human PTX3. Variants of PTX3 used in the provided methods include variants having amino acid modifications (such as amino acid substitutions, deletions, or insertions). In some embodiments, the modification improves one or more properties of the PTX3 peptide, such as improving one or more therapeutic properties of the rcHC-HA / PTX3 complex (e.g., anti-inflammatory, anti-immune, anti-angiogenic, anti-scarring, anti-adhesion, regenerative, or other therapeutic activities as described herein).

[0256] In some implementations, the PTX3 protein is obtained from a commercial source. Exemplary commercial sources of PTX3 are, but are not limited to, PTX3 (catalog number 1826-TS; R&D Systems, Minneapolis, MN).

[0257] In some embodiments, the PTX3 protein used in the method is a multimeric protein. In some embodiments, the PTX3 protein used in the method is a homopolymer. In some embodiments, the homopolymer is a dimer, trimer, tetramer, hexamer, pentamer, or octamer. In some embodiments, the PTX3 homopolymer is a trimer, tetramer, or octamer. In a specific embodiment, the PTX3 homopolymer is an octamer. In some embodiments, the multimerizing domain is modified to improve the multimerization of the PTX3 protein. In some embodiments, a heteropolymerizing domain (e.g., an Fc multimerizing domain or a leucine zipper) that improves the multimerization of PTX3 when fused with PTX3 is used instead of the multimerizing domain.

[0258] TSG-6

[0259] In some embodiments, the TSG-6 used in the method is isolated from one or more cells (e.g., tissue extracts). Exemplary cells suitable for expressing TSG-6 include, but are not limited to, animal cells (including, but not limited to, mammalian cells, primate cells, human cells, rodent cells, insect cells, bacteria, and yeast) and plant cells (including, but not limited to, algae, angiosperms, gymnosperms, ferns, and bryophytes). In some embodiments, the TSG-6 used in the method is isolated from human cells. In some embodiments, the TSG-6 used in the method is isolated from cells whose TSG-6 expression is upregulated by stimulation with one or more pro-inflammatory cytokines. In some embodiments, the pro-inflammatory cytokines are IL-1 or TNF-α.

[0260] In some embodiments, the TSG-6 used in the method is isolated from amniotic cells. In some embodiments, the TSG-6 used in the method is isolated from amniotic cells derived from the umbilical cord. In some embodiments, the TSG-6 used in the method is isolated from amniotic cells whose TSG-6 expression has been upregulated by stimulation with one or more pro-inflammatory cytokines. In some embodiments, the pro-inflammatory cytokines are IL-1 or TNF-α.

[0261] In some embodiments, the TSG-6 used in the method is isolated from umbilical cord cells. In some embodiments, the TSG-6 used in the method is isolated from umbilical cord cells stimulated with one or more pro-inflammatory cytokines to upregulate TSG-6 expression. In some embodiments, the pro-inflammatory cytokines are IL-1 or TNF-α.

[0262] In some embodiments, TSG-6 used in the method is isolated from amniotic epithelial cells. In some embodiments, TSG-6 used in the method is isolated from umbilical cord epithelial cells. In some embodiments, TSG-6 used in the method is isolated from amniotic epithelial cells or umbilical cord epithelial cells whose TSG-6 expression is upregulated by stimulation with one or more pro-inflammatory cytokines. In some embodiments, the pro-inflammatory cytokines are IL-1 or TNF-α.

[0263] In some embodiments, TSG-6 used in the method is isolated from amniotic stromal cells. In some embodiments, TSG-6 used in the method is isolated from umbilical cord stromal cells. In some embodiments, TSG-6 used in the method is isolated from amniotic stromal cells or umbilical cord stromal cells whose TSG-6 expression is upregulated by stimulation with one or more pro-inflammatory cytokines. In some embodiments, the pro-inflammatory cytokines are IL-1 or TNF-α.

[0264] In some embodiments, the TSG-6 used in the method is a naturally occurring TSG-6 protein isolated from cells. In some embodiments, the cells are stimulated with one or more pro-inflammatory cytokines to upregulate TSG-6 expression. In some embodiments, the pro-inflammatory cytokines are IL-1 or TNF-α.

[0265] In some embodiments, TSG-6 is prepared using recombinant technology. In some embodiments, TSG-6 is expressed from a recombinant expression vector. In some embodiments, the nucleic acid encoding TSG-6 is operatively linked to a constitutive promoter. In some embodiments, the nucleic acid encoding TSG-6 is operatively linked to an inducible promoter. In some embodiments, TSG-6 is expressed in transgenic animals. In some embodiments, TSG-6 is a recombinant protein. In some embodiments, TSG-6 is a recombinant protein isolated from cells. In some embodiments, TSG-6 is a recombinant protein produced from cell-free extracts.

[0266] In some embodiments, TSG-6 is purified from the amnion, amnion membrane, chorion, amniotic fluid, or combinations thereof. In some embodiments, PTX3 is purified from amniotic cells. In some embodiments, the amniotic cells are amniotic epithelial cells. In some embodiments, the amniotic epithelial cells are umbilical cord epithelial cells. In some embodiments, the amniotic cells are amniotic stromal cells. In some embodiments, the amniotic cells are umbilical cord stromal cells. In some embodiments, the amniotic cells are stimulated with one or more pro-inflammatory cytokines to upregulate TSG-6 expression. In some embodiments, the pro-inflammatory cytokines are IL-1 or TNF-α.

[0267] In some implementations, TSG-6 is not isolated from one or more cells (e.g., tissue extracts).

[0268] In some embodiments, TSG-6 comprises a TSG-6 fragment sufficient to promote or catalyze the transfer of HC1 to HA from IαI. In some embodiments, TSG-6 comprises a linker module of TSG-6. In some embodiments, TSG-6 comprises amino acids Trp18 to Leu277 of TSG-6. In some embodiments, TSG-6 comprises a polypeptide having the sequence shown in SEQ ID NO:2 or a variant thereof, the variant having at least 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity with the polypeptide having the sequence shown in SEQ ID NO:2. Exemplary variants include, for example, species variants, allelic variants, and variants containing conserved and non-conserved amino acid mutations. Natural allelic variants of human TSG-6 include, for example, TSG-6 containing the amino acid substitution Q144R. Variants of TSG-6 used in the provided methods or HA-binding fragments thereof comprise variants having amino acid modifications (substitution, deletion, or insertion). In some embodiments, the modification improves one or more properties of the TSG-6 peptide, such as improving the transfer of HC1 from IαI to HA or improving the release of the TSG-6 peptide from the rcHC-HA / PTX3 complex after the transfer of HC1 from IαI to HA.

[0269] In some embodiments, TSG-6 includes an affinity tag. Exemplary affinity tags include, but are not limited to, hemagglutinin tags, polyhistidine tags, myc tags, FLAG tags, and glutathione S-transferase (GST) tags. Such affinity tags are known in the art for purification. In some embodiments, such affinity tags are introduced into the TSG-6 peptide as a fusion protein or via a chemical linker. In some embodiments, TSG-6 includes an affinity tag, and unbound TSG-6 is removed from the rcHC-HA / PTX3 complex by affinity purification.

[0270] In some implementations, the TSG-6 protein is obtained from a commercial source. Exemplary commercial sources of TSG-6 are, but are not limited to, TSG-6 (catalog number 2104-TS R&D Systems, Minneapolis, MN).

[0271] IαI

[0272] In some embodiments, the IαI comprises an HC1 chain. In some embodiments, the IαI comprises HC1 and HC2 chains. In some embodiments, the IαI comprises HC1 and urostatin. In some embodiments, the IαI comprises HCl and HC2 chains, as well as urostatin. In some embodiments, the IαI comprises HCl and HC2 chains linked by chondroitin sulfate chains, as well as urostatin.

[0273] In some embodiments, IαI is isolated from a biological sample. In some embodiments, the biological sample is a biological sample from a mammal. In some embodiments, the mammal is a human. In some embodiments, the biological sample is a blood, serum, plasma, liver, amnion, chorionic villus, or amniotic fluid sample. In some embodiments, the biological sample is a blood, serum, or plasma sample. In some embodiments, the biological sample is a blood sample. In some embodiments, the biological sample is a serum sample. In some embodiments, the biological sample is a plasma sample. In some embodiments, the IαI is purified from human blood, plasma, or serum. In some embodiments, IαI is isolated from human serum. In some embodiments, IαI is not isolated from serum. In some embodiments, the IαI used in the method is produced in amniotic cells. In some embodiments, the IαI used in the method is produced in umbilical cord cells. In some embodiments, the IαI used in the method is produced in amniotic cells derived from the umbilical cord. In some embodiments, the IαI used in the method is produced in amniotic epithelial cells. In some embodiments, the IαI used in the method is produced in umbilical cord epithelial cells. In some embodiments, the IαI used in the method is produced in amnion stromal cells. In some embodiments, the IαI used in the method is produced in umbilical cord stromal cells. In some embodiments, the IαI used in the method is produced in hepatocytes. In some embodiments, IαI is prepared using recombinant technology.

[0274] In some embodiments, HC1 of IαI is isolated from a biological sample. In some embodiments, the biological sample is a biological sample from a mammal. In some embodiments, the mammal is a human. In some embodiments, the biological sample is a blood, serum, plasma, liver, amnion, chorionic villus, or amniotic fluid sample. In some embodiments, the biological sample is a blood, serum, or plasma sample. In some embodiments, the biological sample is a blood sample. In some embodiments, the biological sample is a serum sample. In some embodiments, the biological sample is a plasma sample. In some embodiments, HC1 of IαI is purified from human blood, plasma, or serum. In some embodiments, IαI is isolated from human serum. In some embodiments, HC1 of IαI is not purified from serum. In some embodiments, HC1 of IαI is prepared using recombinant technology. In some embodiments, HC1 of IαI is purified from hepatocytes. In some embodiments, HC1 of IαI is purified from amniotic cells. In some embodiments, HC1 of IαI is purified from amniotic epithelial cells or umbilical cord epithelial cells. In some implementations, HC1 of IαI is purified from amniotic stromal cells or umbilical cord stromal cells.

[0275] In some embodiments, HC1 comprises a polypeptide having the sequence shown in SEQ ID NO:47 or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity with the polypeptide having the sequence shown in SEQ ID NO:47.

[0276] In some embodiments, the HC2 of IαI is isolated from a biological sample. In some embodiments, the biological sample is a biological sample from a mammal. In some embodiments, the mammal is a human. In some embodiments, the biological sample is a blood, serum, plasma, liver, amnion, chorionic villus, or amniotic fluid sample. In some embodiments, the biological sample is a blood, serum, or plasma sample. In some embodiments, the biological sample is a blood sample. In some embodiments, the biological sample is a serum sample. In some embodiments, the biological sample is a plasma sample. In some embodiments, the HC2 of IαI is purified from human blood, plasma, or serum. In some embodiments, the HC2 of IαI is isolated from human serum. In some embodiments, the HC2 of IαI is isolated from human serum. In some embodiments, the HC2 of IαI is not isolated from serum. In some embodiments, the HC2 of IαI is prepared using recombinant technology. In some embodiments, the HC2 of IαI is purified from hepatocytes. In some embodiments, the HC2 of IαI is purified from amniotic cells. In some embodiments, the HC2 of IαI is purified from amniotic epithelial cells or umbilical cord epithelial cells. In some embodiments, the HC2 of IαI is purified from amniotic stromal cells or umbilical cord stromal cells.

[0277] In some embodiments, HC2 comprises a polypeptide having the sequence shown in SEQ ID NO:49 or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity with the polypeptide having the sequence shown in SEQ ID NO:49.

[0278] In some embodiments, IαI comprises urostatin. In some embodiments, urostatin comprises a polypeptide having the sequence shown in SEQ ID NO:53 or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity with the polypeptide having the sequence shown in SEQ ID NO:53. In some embodiments, IαI comprises a chondroitin sulfate chain.

[0279] HA

[0280] In some embodiments, HA is purified from cell, tissue, or fluid samples. In some embodiments, HA is obtained from a commercial supplier (e.g., Sigma Aldrich or Advanced Medical Optics, Irvine, CA (e.g., Healon)). In some embodiments, HA is obtained from a commercial supplier as a powder. In some embodiments, HA is expressed in cells. Exemplary cells suitable for expressing HA include, but are not limited to, animal cells (including, but not limited to, mammalian cells, primate cells, human cells, rodent cells, insect cells, bacteria, and yeast) and plant cells (including, but not limited to, algae, angiosperms, gymnosperms, ferns, and bryophytes). In some embodiments, HA is expressed in human cells. In some embodiments, HA is expressed in transgenic animals. In some embodiments, HA is obtained from cells expressing hyaluronic acid synthases (e.g., HAS1, HAS2, and HAS3). In some embodiments, the cells contain a recombinant expression vector expressing HA synthases. In some cases, HA synthase prolongs hyaluronic acid production by repeatedly adding glucuronic acid and N-acetylglucosamine to the nascent polysaccharide as it is squeezed through the cell membrane into the extracellular space.

[0281] The HA used in the method is typically a high molecular weight (HMW) HA. In some embodiments, the weight-average molecular weight of the HMW HA is greater than about 500 kilodaltons (kDa), for example, about 500 kDa to about 10,000 kDa, about 800 kDa to about 8,500 kDa, about 1,100 kDa to about 5,000 kDa, or about 1,400 kDa to about 3,500 kDa. In some embodiments, the weight-average molecular weight of the HMW HA is about 3,000 kDa.

[0282] Additional components

[0283] In some embodiments, one or more additional components are added to generate the rcHC-HA / PTX3 complex. In some embodiments, small leucine-rich proteoglycans (SLRPs) are added to generate the rcHC-HA / PTX3 complex. In some embodiments, the SLRP is a class I, class II, or class III SLRP. In some embodiments, the SLRP is selected from class I SLRPs, such as dermal proteoglycans and disaccharide proteoglycans. In some embodiments, the SLRP is selected from class II SLRPs, such as fibrinolytic proteoglycans, luminal proteoglycans, PRELP (proline-arginine-rich, terminal leucine-rich protein), corneal proteoglycans, and osteoadhesion proteoglycans. In some embodiments, the SLRP is selected from class III SLRPs, such as epiphyseal proteoglycans and osteoglobin proteoglycans. In some embodiments, the SLRP is selected from urostatin, dermal proteoglycans, disaccharide proteoglycans, and osteoadhesion proteoglycans. In some embodiments, the SLRP contains glycosaminoglycans. In some embodiments, the SLRP contains keratin sulfate.

[0284] HA immobilization

[0285] In some embodiments, HMW HA is immobilized by any suitable method. In some embodiments, HMW HA is immobilized on a solid support, such as a petri dish, beads, column, or other suitable surface, for example, the surface of an implantable medical device or a portion thereof, or on a surface subsequently attached or incorporated with an implantable medical device as described herein. In some embodiments, HMW HA is directly immobilized on the solid support, for example, by chemical bonding. In some embodiments, HMW HA is indirectly attached to the solid support via a linker or intermediate protein. Numerous heterobifunctional crosslinking agents for forming covalent bonds between amino and thiol groups and introducing thiol groups into proteins are known to those skilled in the art. In some embodiments, HMW HA is directly immobilized on the solid support by crosslinking with the solid support. In some embodiments, HMW HA is directly immobilized on the solid support without crosslinking with the solid support. In some embodiments, HMW HA is directly immobilized on the solid support as a coating. In some embodiments, HMW HA is immobilized on Covalink. TM -NH surface. In some embodiments, HMW HA is directly fixed to a solid support as a coating. In some embodiments, HMW HA is fixed to Covalink at 4°C. TM -NH surface for about 16 hours.

[0286] In some embodiments, the method includes solidifying HMW HA on a solid surface by direct attachment to a solid support (i.e., without intermediate proteins). In some embodiments, the solid support is washed to remove unbound HMW HA before contacting the immobilized HA with PTX3. In some embodiments, the solid support is washed with a washing solution of 8M GnHCl and PBS to remove unbound HMW HA before contacting the immobilized HA with PTX3.

[0287] In some embodiments, the method includes immobilizing HA onto a solid surface via an intermediate protein or linker. In some embodiments, the linker is a peptide linker. In some embodiments, the intermediate protein is an HA-binding protein (HABP). In some embodiments, the HABP is first attached to a solid support (e.g., by cross-linking, chemical linking, or via a chemical linker). In some embodiments, the solid support containing the HABP is subsequently contacted with HA (e.g., HMW HA) to immobilize HA onto the solid support by binding the HABP to the HA. In some embodiments, the solid support is washed to remove unbound HMW HA before contacting the immobilized HMW HA with PTX3. In some embodiments, the solid support is washed with a washing solution of 8M GnHCl and PBS before contacting the immobilized HA with PTX3 to remove unbound HMW HA.

[0288] In some embodiments, the method includes immobilizing the HA on a solid surface by attaching a peptide linker to a solid support and attaching the HA to the peptide linker. In some embodiments, the peptide linker includes a protease cleavage site.

[0289] In some embodiments, the method includes fixing the HA to a solid surface by attaching a cuttable chemical linker (e.g., but not limited to a disulfide chemical linker).

[0290] In some embodiments, the HABP selected for the method is a HABP that has dissociated from HA after the formation of the rcHC-HA / PTX3 complex. In some embodiments, the HABP is non-covalently bound to HA. In some embodiments, the method further includes dissociating the rcHC-HA / PTX3 complex from the HABP using one or more dissociating agents. Dissociating agents for disrupting non-covalent interactions (e.g., guanidine hydrochloride, urea, and various detergents, such as SDS) are known in the art. In some embodiments, the dissociating agent is urea. In some embodiments, the dissociating agent is guanidine hydrochloride. In some embodiments, the dissociating agent is guanidine hydrochloride at about 4M to about 8M. In some embodiments, the dissociating agent is guanidine hydrochloride at about 4M, about 5M, about 6M, about 7M, or about 8M. In some embodiments, the dissociating agent is guanidine hydrochloride at pH 7.5 at about 4M to about 8M in PBS.

[0291] In some embodiments, such a dissociating agent is used to dissociate the rcHC-HA / PTX3 complex from the intermediate HABP. The HABP used in the method is selected such that its binding affinity for HA is strong enough to allow the assembly of the rcHC-HA / PTX3 complex, but a suitable dissociating agent can be used to dissociate it from the rcHC-HA / PTX3 complex. In some embodiments, the dissociating agent is guanidine hydrochloride.

[0292] Exemplary HABPs for use with the methods provided herein include, but are not limited to, HAPLN1, HAPLN2, HAPLN3, HAPLN4, aggregate proteoglycans, multipotent proteoglycans, neuroproteoglycans, short proteoglycans, phosphoproteoglycans, TSG-6, CD44, stabylin-1, stabylin-2, or a portion thereof sufficient to bind HA (e.g., its linker module). In some embodiments, the HABP comprises a polypeptide having the sequence shown in any one of SEQ ID NO: 54-99 or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity with a polypeptide having the sequence shown in any one of SEQ ID NO: 54-99. In some embodiments, the HABP is a multipotent proteoglycan. In some embodiments, the HABP is a recombinant protein. In some embodiments, the HABP is a recombinant mammalian protein. In some embodiments, the HABP is a recombinant human protein. In some embodiments, HABP is a recombinant pluripotent proteoglycan protein or a portion thereof sufficient to bind to HA. In some embodiments, HABP is a recombinant aggregated proteoglycan protein or a portion thereof sufficient to bind to HA. In some embodiments, HABP is a natural HABP or a portion thereof sufficient to bind to HA. In some embodiments, natural HABP is isolated from mammalian tissues or cells. In some embodiments, HABP is isolated from bovine nasal cartilage (e.g., HABP from Seikagaku containing an HA-binding domain of aggregated proteoglycan and a linker protein).

[0293] In some embodiments, the HABP includes a linker module comprising HAPLN1, HAPLN2, HAPLN3, HAPLN4, agglutinin, pluripotent proteoglycan, neuroproteoglycan, short proteoglycan, phosphoproteoglycan, TSG-6, CD44, stabillin-1, or stabillin-2. In some embodiments, the HABP containing the linker module comprises a polypeptide having the sequence shown in any of the linker domains of SEQ ID NO:54-99, or a polypeptide having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence amino acid identity with a polypeptide having the sequence shown in any of the linker domains of SEQ ID NO:54-99. In some embodiments, the HABP comprises a linker module of pluripotent proteoglycan. In some embodiments, the HABP containing the linker module is a recombinant protein. In some embodiments, the HABP containing the linker module of pluripotent proteoglycan is a recombinant protein.

[0294] In some embodiments, the intermediate protein, such as HABP, comprises a protease cleavage sequence that is recognized and hydrolyzed by a site-specific protease (such as Fehling's protease, 3C protease, caspase, matrix metalloproteinase, or TEV protease). In such embodiments, the assembled rcHC-HA / PTX3 complex is released from a solid support by contacting the immobilized complex with a protease that cleaves the specific cleavage sequence.

[0295] In some embodiments, the rcHC-HA / PTX3 complex is purified. In some embodiments, the rcHC-HA / PTX3 complex is purified by any suitable method or a combination of these methods. The embodiments described below are not exclusive and are merely exemplary.

[0296] In some embodiments, the rcHC-HA / PTX3 complex is purified by chromatography (e.g., ion exchange chromatography, affinity chromatography, size exclusion chromatography, and hydroxyapatite chromatography), gel filtration, centrifugation (e.g., gradient centrifugation), differential dissolution, ethanol precipitation, or any other feasible technique for protein purification.

[0297] In some embodiments, the rcHC-HA / PTX3 complex is purified by immunoaffinity chromatography. In some embodiments, antibodies against components of the rcHC-HA / PTX3 complex (e.g., antibodies against HCl, PTX, one or more SLRPs of the rcHC-HA / PTX3 complex, such as anti-urostatin, anti-glucan, anti-disaccharide proteoglycan, or anti-osteoadhesion proteoglycan) are generated and attached to a solid support. In some embodiments, the unpurified rcHC-HA / PTX3 complex (i.e., the mobile phase) is passed through the support. In some cases, the rcHC-HA / PTX3 complex binds to the antibody. In some embodiments, the support is washed (e.g., with PBS) to remove any unbound or loosely bound molecules. In some embodiments, the support is subsequently washed with a solution capable of eluting the rcHC-HA / PTX3 complex from the support (e.g., 1% SDS, 6M guanidine hydrochloride, or 8M urea). In some embodiments, the dissociating agent is removed from the dissociated rcHC-HA / PTX3 complex. In some embodiments, the dissociating agent is removed from the dissociated rcHC-HA / PTX3 complex by methods including but not limited to ion exchange chromatography, dialysis, gel filtration chromatography, ultrafiltration, or percolation.

[0298] In some embodiments, the rcHC-HA / PTX3 complex is purified by affinity chromatography. In some embodiments, HABP is used to bind to the rcHC-HA / PTX3 complex to purify the complex and attach it to a fixed support. In some embodiments, the unpurified rcHC-HA / PTX3 complex (i.e., the mobile phase) is passed through the support. In some cases, the rcHC-HA / PTX3 complex is bound to HABP. In some embodiments, the support is washed (e.g., with PBS) to remove any unbound or loosely bound molecules. In some embodiments, the support is subsequently washed with a solution (e.g., a dissociating agent) capable of eluting the rcHC-HA / PTX3 complex from the support. In some embodiments, the dissociating agent is removed from the dissociated rcHC-HA / PTX3 complex by methods including, but not limited to, ion exchange chromatography, dialysis, gel filtration chromatography, ultrafiltration, or percolation.

[0299] In some embodiments, the rcHC-HA / PTX3 complex is purified by a combination of HABP affinity chromatography and immunoaffinity chromatography using antibodies against one or more components of the rcHC-HA / PTX3 complex.

[0300] In some embodiments, one or more components of the rcHC-HA / PTX3 complex disclosed herein contain an affinity tag (e.g., a fusion protein of PTX3 or HC1 with an affinity tag). In some embodiments, exemplary affinity tags introduced into one or more components of the rcHC-HA / PTX3 complex include, but are not limited to, hemagglutinin tags, polyhistidine, myc tags, FLAG tags, or glutathione S-transferase sequences. In some embodiments, a ligand for the affinity tag is attached to a solid support. In some embodiments, the unpurified rcHC-HA / PTX3 complex is passed through the support. In some cases, the rcHC-HA / PTX3 complex binds to the ligand. In some embodiments, the support is washed (e.g., with PBS) to remove any unbound or loosely bound molecules. In some embodiments, the support is subsequently washed with a solution capable of eluting the rcHC-HA / PTX3 complex disclosed herein from the support. In some embodiments, the eluent is removed from the dissociated rcHC-HA / PTX3 complex by methods including but not limited to ion exchange chromatography, dialysis, gel filtration chromatography, ultrafiltration, or percolation.

[0301] In some embodiments, PTX3, TSG-6, and / or HC1 are coupled to a label. "Label" refers to a compound or composition that is directly or indirectly coupled to a polypeptide to produce a labeled polypeptide. In some embodiments, the label is self-detectable (e.g., radioisotope labeling or fluorescent labeling), or, in the case of enzyme labeling, catalyzes a chemical change in the composition of a detectable substrate compound. Non-limiting examples of labels include fluorescent moieties, dyes, fluorescent tags, green fluorescent protein, or luciferase.

[0302] Methods for evaluating the activity of nHC-HA / PTX3 and rcHC-HA / PTX3 complexes Assess the properties of the nHC-HA / PTX3 and rcHC-HA / PTX3 complexes provided herein using any suitable method (including in vitro and in vivo methods). Exemplary in vitro methods are provided herein and include, but are not limited to, cell culture methods for assessing the ability of the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes to promote macrophage attachment to immobilized nHC-HA / PTX3 or rcHC-HA / PTX3 complexes, inhibit or reduce macrophage aggregation, promote neutrophil apoptosis, macrophage phagocytosis of apoptotic neutrophils, and the ability of stimulated macrophages to undergo M2 polarization. In some embodiments, macrophages used in the experiments are stimulated, for example, by exposure to LPS or IFN-γ. In some embodiments, gene or protein expression in stimulated macrophages is assessed after contact with the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes. In such methods for assessing the activity of the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes, suitable controls are used for comparison. In some implementations, the control group lacks treatment using the nHC-HA / PTX3 or rcHC-HA / PTX3 complex (i.e., negative control).

[0303] In some embodiments, the activity of the rcHC-HA / PTX3 complex is compared with the activity of the natural HC-HA / PTX3 complex. In some embodiments, natural HC-HA / PTX3 is isolated from the amnion.

[0304] In some implementations, gene expression in treated macrophages is assessed using PCR, RT-PCR, Northern blotting, Western blotting, dot blot, immunohistochemistry, chromatography, or other suitable methods for detecting proteins or nucleic acids. In some implementations, the expression levels of IL-10, IL-12, IL23, LIGHT, and SPHK1 are assessed.

[0305] Exemplary in vitro methods for evaluating the activity of the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes provided herein include, but are not limited to, animal models of various diseases and conditions. A variety of animal models for diseases and conditions are available and well-known in the art, including, but not limited to, animal models for various inflammatory and autoimmune diseases and conditions, including, but not limited to: ischemia-reperfusion injury, type 1 and type 2 diabetes, inflammatory diseases, collagen-induced arthritis, rheumatoid arthritis, antigen-induced autoimmune diseases such as collagen-induced arthritis and myelin peptide-induced experimental allergic encephalomyelitis, inflammatory bowel disease (IBD) / ulcerative colitis, multiple sclerosis, surgery-induced osteoarthritis and nephritis, psoriasis, inflammatory dermatitis, LPS-induced endotoxin shock, LPS-induced lung injury, allergic rhinitis, liver injury, chronic stress, asthma (e.g., rodent and primate models), and xenograft and allogeneic transplantation models for various cancers.

[0306] In some embodiments, the animal model is an inflammatory model, such as chronic graft-versus-host disease (cGVHD), HSV1-induced necrotizing stromal keratitis, or a rodent model of high-risk corneal transplantation. In some embodiments, the reduction in inflammation induced by nHC-HA / PTX3 or rcHC-HA / PTX3 treatment is assessed by measuring T cell proliferation and activation, as well as the production of immune cytokines such as IL-1α, IL-2, IL-6, IFN-γ, and TNF-α. In some embodiments, the animal model is a scarring model, such as a rodent model of laser-assisted refractive keratotomy (PRK). Exemplary methods for such animal models are provided in the embodiments provided herein.

[0307] In some embodiments, the animal model is a genetic model of an inflammatory and autoimmune disease or condition that includes one or more genetic modifications that cause the disease or condition. In some embodiments, such models are obtained from commercial sources. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex provided herein is administered to an animal model of a specific disease or condition, and the ability of the rcHC-HA / PTX3 complex to inhibit or reduce one or more symptoms of the disease or condition is evaluated.

[0308] Pharmaceutical Composition

[0309] In some embodiments, pharmaceutical compositions comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes described herein are disclosed herein. In some embodiments, pharmaceutical compositions comprising nHC-HA / PTX3 or rcHC-HA / PTX3 complexes produced by the methods provided herein are disclosed herein. In some embodiments, the pharmaceutical compositions are formulated in a conventional manner using one or more physiologically acceptable carriers (including excipients and adjuvants that facilitate the processing of the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes into formulations suitable for pharmaceutical use). Appropriate formulations depend on the chosen route of administration. Any known techniques, carriers, and excipients may be used as suitable techniques, carriers, and excipients as understood in the art.

[0310] In some embodiments, pharmaceutical compositions comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are disclosed. In some embodiments, the pharmaceutical composition further comprises at least one pharmaceutically acceptable carrier. In some embodiments, the pharmaceutical composition also comprises adjuvants, excipients, preservatives, agents for delayed absorption, fillers, binders, adsorbents, buffers, and / or solubilizers. Exemplary pharmaceutical compositions formulated to comprise the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes provided herein include, but are not limited to, solutions, suspensions, emulsions, syrups, granules, powders, ointments, tablets, capsules, pills, or aerosols.

[0311] Dosage form

[0312] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are applied as an aqueous suspension. In some embodiments, the aqueous suspension comprises water, Ringer's solution, and / or isotonic sodium chloride solution. In some embodiments, the aqueous suspension comprises a sweetener or flavoring agent, a coloring agent or dye, and, if necessary, an emulsifier or suspending agent, and includes diluents such as water, ethanol, propylene glycol, glycerin, or combinations thereof. In some embodiments, the aqueous suspension comprises a suspending agent. In some embodiments, the aqueous suspension comprises sodium carboxymethyl cellulose, methylcellulose, hydroxypropyl methylcellulose, sodium alginate, polyvinylpyrrolidone, tragacanth gum, and / or gum arabic. In some embodiments, the aqueous suspension comprises a dispersant or wetting agent. In some embodiments, the aqueous suspension comprises naturally occurring phospholipids (e.g., lecithin), or condensation products of alkyl epoxides and fatty acids (e.g., polyoxyethylene stearate), or condensation products of ethylene oxide and long-chain aliphatic alcohols (e.g., heptadecaethylene-oxycetanol), or condensation products of ethylene oxide and esters derived from fatty acids and hexitols (e.g., polyoxyethylene sorbitan monooleate), or condensation products of ethylene oxide and esters derived from fatty acids and hexitol anhydrides (e.g., polyethylene dehydrated sorbitan monooleate). In some embodiments, the aqueous suspension comprises a preservative. In some embodiments, the aqueous suspension comprises ethylparaben or n-propylparaben. In some embodiments, the aqueous suspension comprises a sweetener. In some embodiments, the aqueous suspension comprises sucrose, saccharin, or aspartame.

[0313] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are applied as an oily suspension. In some embodiments, the oily suspension is formulated by suspending the active ingredient in a vegetable oil (e.g., peanut oil, olive oil, sesame oil, or coconut oil) or a mineral oil (e.g., liquid paraffin). In some embodiments, the oily suspension contains a thickener (e.g., beeswax, hard paraffin, or cetyl alcohol). In some embodiments, the oily suspension contains a sweetener (e.g., those listed above). In some embodiments, the oily suspension contains an antioxidant (e.g., butylated hydroxyanisole or α-tocopherol).

[0314] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated for parenteral injection (e.g., by injection or infusion, including intra-arterial, intracardiac, intradermal, intraduodenal, intramedullary, intramuscular, intraosseous, intraperitoneal, intrathecal, intravascular, intravenous, intravitreal, epidural, and / or subcutaneous). In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are administered as a sterile solution, suspension, or emulsion.

[0315] In some embodiments, the formulation for parenteral administration includes aqueous and / or non-aqueous (oil-like) sterile injectable solutions of the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein (which, in some embodiments, contain antioxidants, buffers, antibacterial agents, and / or solutes that make the formulation isotonic with the blood of the intended recipient); and / or aqueous and / or non-aqueous sterile suspensions (which, in some embodiments, include suspending agents and / or thickeners). In some embodiments, the formulation for parenteral administration includes suitable stabilizers or reagents that improve the solubility of the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein to allow for the preparation of high-concentration solutions.

[0316] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are applied as oil-in-water microemulsions (wherein the active ingredient is dissolved in the oil phase). In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are dissolved in fatty oils (e.g., sesame oil or synthetic fatty acid esters (e.g., ethyl oleate or triglycerides)) or liposomes. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are dissolved in a mixture of soybean oil and / or lecithin. In some embodiments, the oil solution is introduced into a mixture of water and glycerol and treated to form a microemulsion.

[0317] In some embodiments, the composition for parenteral administration is formulated as a single bolus injection. In some embodiments, the composition for parenteral administration is formulated via a continuous intravenous delivery device (e.g., Deltec CADD-PLUS). TM Administer via a 5400 intravenous pump.

[0318] In some embodiments, the formulation for injection is presented in unit dose form, for example, in ampoules or multi-dose containers, and is fortified with preservatives. In some embodiments, the formulation for injection is stored in powder form or under lyophilized (freeze-dried) conditions, so that a sterile liquid carrier (e.g., saline or sterile pyrogen-free water) only needs to be added immediately before use.

[0319] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated for topical application. Topical formulations include, but are not limited to, ointments, creams, lotions, solutions, pastes, gels, films, rods, liposomes, and nanoparticles. In some embodiments, the topical formulation is applied using patches, bandages, or wound dressings.

[0320] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated into compositions in the form of solids, cross-linked gels, or liposomes. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated into insoluble cross-linked hydrogels.

[0321] In some embodiments, the topical formulation comprises a gelling (or thickening) agent. Suitable gelling agents include, but are not limited to, cellulose, cellulose derivatives, cellulose ethers (e.g., carboxymethyl cellulose, ethyl cellulose, hydroxyethyl cellulose, hydroxymethyl cellulose, hydroxypropyl methyl cellulose, hydroxypropyl cellulose, methyl cellulose), guar gum, xanthan gum, locust bean gum, alginate (e.g., alginic acid), silicates, starch, tragacanth gum, carboxyvinyl polymer, carrageenan, paraffin wax, petrolatum, gum arabic, agar, magnesium aluminum silicate, sodium alginate, sodium stearate, fucus vesiculosus, bentonite, carbomer, carrageenan, carbopol, xanthan gum, cellulose, microcrystalline cellulose (MCC), carob gum, chondrus, glucose, furcelellaran, gelatin, ghatina var. aizoon. Guar gum, lithium montmorillonite, lactose, sucrose, maltodextrin, mannitol, sorbitol, honey, corn starch, wheat starch, rice starch, potato starch, gelatin, sterculia gum, polyethylene glycol (e.g., PEG 200-4500), tragacanth gum, ethyl cellulose, ethyl hydroxyethyl cellulose, ethyl methyl cellulose, methyl cellulose, hydroxyethyl cellulose, hydroxyethyl methyl cellulose, hydroxypropyl cellulose, poly(hydroxyethyl methacrylate), oxidized polygelatin, pectin, polygelatin peptides, povidone, propylene carbonate, methyl vinyl ether / maleic anhydride copolymer (PVM / MA), poly(methoxyethyl methacrylate), poly(methoxyethoxyethyl methacrylate), hydroxypropyl cellulose, hydroxypropyl methyl cellulose (HPMC), sodium carboxymethyl cellulose (CMC), silica, polyvinylpyrrolidone (PVP: povidone), or combinations thereof.

[0322] In some embodiments, the topical formulations disclosed herein include a softening agent. Softening agents include, but are not limited to, castor oil esters, cocoa butter esters, safflower oil esters, cottonseed oil esters, corn oil esters, olive oil esters, cod liver oil esters, almond oil esters, avocado oil esters, palm oil esters, sesame oil esters, squalene esters, kikui oil esters, soybean oil esters, acetylated monoglycerides, ethoxylated monostearate, hexyl laurate, hexyl isolaurate, isohexpalmitate, isopropyl palmitate, methyl palmitate, decyl oleate, isodecyl oleate, decyl hexadecyl stearate, isopropyl isostearate, methyl isostearate, diisopropyl adipaldehyde, diisohexyl adipate, dihexyl decyl adipate, and disaccharide. Isopropyl ester, dodecyl lactate, tetradecyl lactate and hexadecyl lactate, myristic acid oleyl alcohol, stearic acid oleyl alcohol and oleic acid oleyl ester, nonanoic acid, lauric acid, myristic acid, palmitic acid, stearic acid, isostearic acid, hydroxystearic acid, oleic acid, linoleic acid, ricinoleic acid, arachidic acid, benzyl acid, erucic acid, lauryl alcohol, myristyl alcohol, cetyl alcohol, hexadecyl alcohol, stearyl alcohol, isostearyl alcohol, hydroxystearyl alcohol, oleyl alcohol, ricinoleyl alcohol, benzyl alcohol, guarana alcohol, 2-octyldodecyl alcohol, lanolin and lanolin derivatives, beeswax, cetyl wax, myristate, stearate, carnauba wax, candelilla wax, lecithin and cholesterol.

[0323] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is formulated with one or more natural polymers. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is formulated with a natural polymer (which is fibronectin, collagen, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparan sulfate, chondroitin sulfate). In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is formulated with a polymer gel derived from a natural polymer. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is formulated with a polymer gel derived from a natural polymer, such as, but not limited to, fibronectin, collagen, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparan sulfate, chondroitin sulfate, and combinations thereof. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated with a crosslinked polymer. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated with a non-crosslinked polymer. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated with both a non-crosslinked polymer and a crosslinked polymer. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated with a crosslinked hyaluronic acid gel. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated with an insoluble crosslinked HA gel. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated with a non-crosslinked hyaluronic acid gel. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated with a collagen matrix. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated with a fibrin matrix. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated with a fibrin / collagen matrix.

[0324] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated for application to the eye or associated tissues. Formulations suitable for ocular application include, but are not limited to, solutions, suspensions (e.g., aqueous suspensions), ointments, gels, creams, liposomes, vesicles, pharmacosomes, nanoparticles, or combinations thereof. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are applied topically to the eye via spray, wash, or a combination thereof. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are applied to the eye via an injectable reservoir formulation.

[0325] As used herein, a "reservoir formulation" is a controlled-release formulation implanted into the eye or associated tissues (e.g., the sclera) (e.g., subcutaneously, intramuscularly, intravitreally, or subconjunctivally). In some embodiments, the reservoir formulation is formulated by forming a microcapsule encapsulation matrix (also called a microcapsule matrix) within a biodegradable polymer to form the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein. In some embodiments, the reservoir formulation is formulated by encapsulating the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein in liposomes or microemulsions.

[0326] Formulations for ophthalmic application have ophthalmologically acceptable osmoticity. In some cases, tears have an isotonicity equivalent to a 0.9% sodium chloride solution. In some embodiments, an isotonicity of about 0.6% to about 1.8% sodium chloride equivalent is suitable for topical ophthalmic application. In some embodiments, the ophthalmic formulations disclosed herein have a molar osmolality of about 200 to about 600 mOsm / L. In some embodiments, the ophthalmic formulations disclosed herein are hypotonic, thus requiring the addition of any suitable additives to achieve an appropriate osmotic range. Ophthalmologically acceptable substances for adjusting osmoticity include, but are not limited to, sodium chloride, potassium chloride, sodium thiosulfate, sodium bisulfite, and ammonium sulfate.

[0327] Formulations intended for application to the eye have ophthalmologically acceptable clarity. Examples of ophthalmologically acceptable clarifying agents include, but are not limited to, polysorbate 20, polysorbate 80, or combinations thereof.

[0328] In some embodiments, the formulation for application to the eye comprises an ophthalmologically acceptable viscosity enhancer. In some embodiments, the viscosity enhancer increases the time the formulation disclosed herein remains in the eye. In some embodiments, increasing the time the formulation disclosed herein remains in the eye allows for better drug absorption and efficacy. Non-limiting examples of mucosal adhesion polymers include carboxymethyl cellulose, carbomer (acrylic polymer), poly(methylisobutylene acid), polyacrylamide, polycarbofil, acrylate / butyl acrylate copolymer, sodium alginate, and dextran.

[0329] In some embodiments, the formulation for application to the eye is administered or delivered to the posterior segment of the eye (e.g., the retina, choroid, vitreous body, and optic nerve). In some embodiments, the topical formulations disclosed herein for application to the eye or for delivery to the posterior segment of the eye contain a solubilizer, such as dextran sulfate and / or cyclodextrin. Dextran sulfate used in some embodiments includes, but is not limited to, dextran sulfate, cyclodextrin sulfate, and β-1,3-glucan sulfate (natural and their derivatives), or any compound temporarily bound to and retained in tissue, containing fibroblast growth factor (FGF), which improves the stability and / or solubility of the drug, and / or enhances the penetration and ocular absorption of the topical formulations disclosed herein for application to the eye. Cyclodextrin derivatives used as solubilizers in some embodiments include, but are not limited to, α-cyclodextrin, β-cyclodextrin, γ-cyclodextrin, hydroxyethyl β-cyclodextrin, hydroxypropyl γ-cyclodextrin, hydroxypropyl β-cyclodextrin, sulfated α-cyclodextrin, sulfated β-cyclodextrin, and sulfobutyl ether β-cyclodextrin.

[0330] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are formulated for rectal or vaginal administration. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are administered as suppositories. In some embodiments, compositions suitable for rectal administration are prepared by mixing the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein with a suitable non-irritating excipient (which is solid at room temperature but liquid at rectal temperature and thus will melt in the rectum to release the drug). In some embodiments, compositions suitable for rectal administration are prepared by mixing the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein with cocoa butter, glycerin gelatin, hydrogenated vegetable oil, mixtures of polyethylene glycols of various molecular weights, or fatty acid esters of polyethylene glycol.

[0331] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are optionally blended with controlled-release particles, lipid complexes, liposomes, nanoparticles, microspheres, microparticles, nanocapsules, or other agents that enhance or facilitate local delivery to the skin. Examples of conventional microencapsulation processes for preparing pharmaceutical formulations are described in U.S. Patent No. 3,737,337, which is incorporated herein by reference.

[0332] dose

[0333] The amount of the pharmaceutical composition administered depends in part on the individual being treated. When the pharmaceutical composition is administered to a human subject, the daily dose is typically determined by the prescribing physician, and the dose generally varies based on age, sex, diet, weight, general health condition and individual response, severity of individual symptoms, the exact disease or condition being treated, the severity of the disease or condition being treated, time of administration, route of administration, disposal of the composition, rate of excretion, combination of drugs, and the prescribing physician's judgment.

[0334] In some embodiments, the dose of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is from about 0.001 to about 1000 mg / kg body weight / day. In some embodiments, the amount of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is from about 0.5 to about 50 mg / kg / day. In some embodiments, the amount of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is from about 0.001 to about 7 g / day. In some embodiments, the amount of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is from about 0.01 to about 7 g / day. In some embodiments, the amount of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is from about 0.02 to about 5 g / day. In some embodiments, the amount of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is from about 0.05 to about 2.5 g / day. In some embodiments, the amount of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is from about 0.1 to about 1 g / day.

[0335] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is administered before, during, or after the onset of a disease or condition. In some embodiments, combination therapy is administered before, during, or after the onset of a disease or condition. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is administered in combination with therapy before, during, or after the onset of a disease or condition. In some embodiments, the timing of administration of a composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 disclosed herein varies. Therefore, in some instances, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is administered as a prophylactic agent and continuously to subjects prone to developing a condition or disease to prevent its occurrence. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is administered to the subject during or as soon as possible after the onset of symptoms. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is introduced within 48 hours before the onset of symptoms, preferably within 48 hours before symptom onset, more preferably within 6 hours before symptom onset, and most preferably within 3 hours of symptom onset. In some embodiments, initial administration is by any practical route, such as, for example, intravenous injection, a single rapid injection, an infusion of about 5 minutes to about 5 hours, pills, capsules, transdermal patches, oral delivery, or a combination thereof. The nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is preferably administered as soon as possible after the detection or suspicion of disease or condition, and is continued for the desired duration of treatment, such as, for example, about 1 month to about 3 months. In some embodiments, the duration of treatment varies from subject to subject, and the duration is determined using known criteria. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein, or a formulation containing the complex, is administered for at least 2 weeks, preferably about 1 month to about 5 years, more preferably about 1 month to about 3 years.

[0336] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is administered as a single dose once daily. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is administered in multiple doses more than once daily. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is administered twice daily. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is administered three times daily. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is administered four times daily. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is administered more than four times daily.

[0337] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is administered for prophylactic and / or therapeutic treatment. In therapeutic applications, in some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is administered to an individual already suffering from the disease or condition in an amount sufficient to cure or at least partially stop the symptoms of the disease or condition. The effective amount for this purpose depends on the severity and duration of the disease or condition, prior treatment, the individual's health status, weight, and response to the drug, as well as the judgment of the treating physician.

[0338] In prophylactic applications, in some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is administered to an individual at risk for a specific disease. Such a quantity is defined as a “prophylactically effective quantity or dose.” In such uses, the precise quantity also depends on the individual’s health status, weight, and other physical parameters.

[0339] If an individual's condition does not improve, based on the physician's judgment, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed in this article may be used for a prolonged period of time, including throughout the individual's entire lifespan, to improve or otherwise control or limit the symptoms of the individual's disease or condition.

[0340] In some implementations, if an individual's condition does improve, the physician may, based on their judgment, continue administering the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein, or temporarily reduce the dosage of the medication or suspend it for a certain period of time (i.e., a "withdrawal period"). In some implementations, the length of the withdrawal period varies from 2 days to 1 year, including, for example, 2 days, 3 days, 4 days, 5 days, 6 days, 7 days, 10 days, 12 days, 15 days, 20 days, 28 days, 35 days, 50 days, 70 days, 100 days, 120 days, 150 days, 180 days, 200 days, 250 days, 280 days, 300 days, 320 days, 350 days, or 365 days. In some implementations, the dose reduction during the withdrawal period is between 10% and 100%, by way of example only, including 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 100%.

[0341] Once an individual's condition improves, a maintenance dose is administered if necessary. In some implementations, the dose or frequency, or both, is subsequently reduced as a function of symptoms to maintain the level of improvement in the disease, disorder, or condition. In some implementations, long-term intermittent treatment is required should any recurrence of symptoms occur.

[0342] In some embodiments, the pharmaceutical compositions disclosed herein are in a single-dose unit dose suitable for precise dosing. In unit dosage forms, the formulation is divided into unit doses containing an appropriate amount of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein. In some embodiments, the unit dose is in the form of a package containing a separated amount of the formulation. Non-limiting examples are packaged tablets or capsules, and powders in vials or ampoules. In some embodiments, aqueous suspension compositions are packaged in single-dose, non-resealable containers. In some embodiments, multi-dose, resealable containers are used, in which case the inclusion of a preservative in the composition is typical. In some embodiments, the formulation for parenteral injection is present in unit dose form, wherein the unit dose form includes, but is not limited to, ampoules, or, in multi-dose containers, the addition of a preservative.

[0343] Suitable daily doses for the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are, for example, about 0.01 to 2.5 mg / kg per body weight. Daily doses indicated in larger mammals (including, but not limited to, humans) range from about 0.5 mg to about 100 mg, conveniently administered in fractionated doses, including, but not limited to, up to four times a day or in an extended-release form. Suitable unit doses for oral administration comprise about 1 to 50 mg of the active ingredient. The above ranges are merely recommendations, as the number of variables relating to individual treatment regimens is vast, and considerable deviations from these recommendations are not uncommon. In some embodiments, the dose varies depending on many variables, not limited to the activity of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex used, the disease or condition to be treated, the route of administration, the individual subject's requirements, the severity of the disease or condition being treated, and the physician's judgment.

[0344] In some implementations, the toxicity and therapeutic efficacy of such treatment regimens are determined through standard pharmaceutical procedures in cell cultures or laboratory animals, including but not limited to, LD50. 50 (50% lethal dose in the population) and ED 50 The determination of the dose (the effective dose in 50% of the population). In some implementations, the dose ratio between toxicity and therapeutic effect is the therapeutic index, which can be expressed as LD50. 50 and ED 50 The ratio between them. nHC-HA / PTX3 or rcHC-HA / PTX3 complexes exhibit a preferred high therapeutic index. In some embodiments, data obtained from cell culture assays and animal studies were used to formulate dosage ranges for human use. The dosages of the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are preferably within the range of ED with minimal toxicity. 50 Within the cyclic concentration range. In some embodiments, the dose varying within this range depends on the dosage form used and the route of administration employed.

[0345] In some embodiments, the pharmaceutical composition of nHC-HA / PTX3 or rcHC-HA / PTX3 complex is packaged into a manufactured article comprising packaging material, a pharmaceutical composition effective in preventing and / or treating a disease or condition, and a label indicating that the pharmaceutical composition is used to treat the disease or condition. In some embodiments, the pharmaceutical composition is packaged in a unit dosage form containing an amount of the pharmaceutical composition for a single dose or multiple doses. In some embodiments, the packaged composition comprises a lyophilized powder pharmaceutical composition reconstituted prior to administration (e.g., with water or saline).

[0346] Medical devices and biomaterial compositions

[0347] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are directly assembled onto a surface or formulated as a coating for implantable medical devices. Methods for covalently attaching hyaluronic acid to surfaces (such as, but not limited to, metallic, polymeric, ceramic, silica, and composite surfaces) are well known in the art, and in some embodiments, they are used in conjunction with methods for assembling the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes provided herein onto such surfaces (see, for example, U.S. Patents 5,356,433, 5,336,518, 4,613,665, 4,810,784, 5,037,677, and 8,093,365). In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes are directly assembled onto the surface of an implantable medical device or a portion thereof. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex produced according to the methods provided herein is purified and then directly attached to the surface of an implantable medical device or a portion thereof. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex produced according to the methods provided herein is purified and then formulated into a coating for attachment to a medical device or a portion thereof. In some embodiments, the coating is applied directly to a surface or to a pretreated or coated surface, wherein the pretreatment or coating is designed to facilitate adhesion of the coating to a substrate. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex produced according to the methods provided herein is purified and then attached to a medical device or a portion thereof that has been coated with a substance capable of promoting attachment of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex. For example, in some embodiments, the medical device or a portion thereof is coated with an adhesive polymer that provides functional groups of hyaluronic acid on its surface for covalent attachment of the nHC-HA / PTX3 or rcHC-HA / PTX3 complex. In some embodiments, a coupling agent, such as, but not limited to, carbodiimide, is used to attach the nHC-HA / PTX3 or rcHC-HA / PTX3 complex to the polymer coating. In some embodiments, photoimmobilization is used to covalently attach the nHC-HA / PTX3 or rcHC-HA / PTX3 complex generated according to the methods provided herein to a medical device or a portion thereof. In some embodiments, a spacer molecule is used to attach the nHC-HA / PTX3 or rcHC-HA / PTX3 complex generated according to the methods provided herein to a medical device or a portion thereof, the spacer molecule containing photochemical or thermochemical reactive groups.

[0348] In some embodiments, a coating formulation comprising an nHC-HA / PTX3 or rcHC-HA / PTX3 complex is applied to a substrate by, for example, dip coating. Other methods of application include, but are not limited to, spraying, washing, vapor deposition, brushing, roller coating, curtain coating, spin coating, and other methods known in the art.

[0349] Exemplary implantable medical devices include, but are not limited to, artificial joints, orthopedic devices, bone implants, contact lenses, sutures, surgical staples, surgical clips, catheters, angioplasty balloons, sensors, surgical devices, electrodes, needles, syringes, wound drainage tubes, shunts, urethral inserts, metal or plastic implants, heart valves, artificial organs, sutured band rings, valve repair rings, guidewires, Kirschner wires or Denham pins, stents, stent grafts, vascular grafts, pacemakers, pills, chips, medical catheters, infusion cannulas, implantable defibrillators, neurostimulators, glucose sensors, cerebrospinal fluid shunts, implantable drug pumps, spinal supports, artificial spinal discs, eye implants, cochlear implants, breast implants, nucleus pulposus replacement devices, ear tubes, intraocular lenses, drug delivery systems, microparticles, nanoparticles, and microcapsules.

[0350] In specific embodiments, the implantable medical device is an implant or prosthesis comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein. In specific embodiments, the prosthesis is an artificial joint. In some embodiments, the prosthesis is an artificial hip joint, an artificial knee, an artificial glenohumeral joint, or an artificial ankle.

[0351] In specific implementations, the implant is a stent. In specific implementations, the implant is a coronary artery stent, ureteral stent, urethral stent, prostate stent, bone stent, or esophageal stent. In specific implementations, the implant is a bone implant, such as, but not limited to, osseointegrated implants or craniofacial prostheses (e.g., artificial ears, orbital prostheses, nasal prostheses).

[0352] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are directly assembled onto microparticles or nanoparticles for delivering the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes to a subject (see, for example, WO 03 / 015755 and US2004 / 0241248).

[0353] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex provided herein is attached to, assembled on, or provided as a coating on a surface or as part of any such implantable medical device provided herein or known in the art. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is eluted from the coating and enters the surrounding tissue after implantation.

[0354] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are directly assembled onto scaffolds, microparticles, microcapsules, or microcarriers for delivering biomaterials, such as stem cells or insulin-producing cells. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are directly attached to or assembled onto microcapsules. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes are combined with materials for forming microcapsules, resulting in microcapsules containing the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes are used to coat the inner surface of microcapsules. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes are used to coat the outer surface of microcapsules. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes are used to coat both the inner and outer surfaces of microcapsules.

[0355] Exemplary materials for encapsulating cells include, but are not limited to, thermosetting hydrogels such as agarose, alginate, and synthetic polymers such as poly(NiPAAm-co-AAC), poly(ethylene glycol) (PEG), and PEG derivatives such as PEG diacrylate and oligo(poly(ethylene glycol)) fumarate. Methods for culturing and microcapsulating stem cells in some embodiments are known in the art and are used to produce microcapsules containing the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes provided herein.

[0356] In some embodiments, the microcapsule comprises cells, multiple cells, or other biological material. In some embodiments, the cells or multiple cells are stem cells, such as, but not limited to, mesenchymal stem cells. In some embodiments, the cells or multiple cells are differentiated cells, such as, but not limited to, insulin-producing cells. In some embodiments, the cells or multiple cells are autologous cells (i.e., recipient cells derived from or derived from cells). In some embodiments, the cells or multiple cells are allogeneic cells (i.e., recipient cells not derived from or derived from cells). In some embodiments, the microcapsule comprises cells, multiple cells, or other biological material, and the inner surface of the microcapsule is coated with the nHC-HA / PTX3 or rcHC-HA / PTX3 complex provided herein. In some embodiments, the microcapsule comprises cells, multiple cells, or other biological material, and the outer surface of the microcapsule is coated with the nHC-HA / PTX3 or rcHC-HA / PTX3 complex provided herein. In some embodiments, the microcapsule comprises cells, multiple cells, or other biological material, and both the outer and inner surfaces of the microcapsule are coated with the nHC-HA / PTX3 or rcHC-HA / PTX3 complex provided herein. In some implementations, microcapsules are administered to treat a disease or condition. Exemplary diseases and conditions, as well as treatments in which microcapsules can be administered, are described elsewhere herein, including but not limited to inflammatory and immune-related diseases.

[0357] Treatment

[0358] In some embodiments, methods of treating an individual in need disclosed herein include administering the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes described herein to the individual. In some embodiments, methods of treating an individual in need disclosed herein include administering the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes produced by the methods described herein to the individual. Examples of non-limiting treatment methods include administering the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are used to inhibit at least one of the following: scar formation, inflammation, immune responses leading to autoimmune diseases or immune rejection, adhesion, angiogenesis, and conditions requiring cell or tissue regeneration. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are used to promote wound healing. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are used to promote stem cell expansion. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are used to promote tissue regeneration.

[0359] In some embodiments, methods of treating an individual in need include administering the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein to that individual by any suitable method. In some embodiments, methods of treating an individual in need include administering the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein to that individual by any suitable route. The suitable method for administration will depend on the disease or condition to be treated. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is applied topically to the treatment site. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is administered systemically. Exemplary methods of administering the nHC-HA / PTX3 or rcHC-HA / PTX3 complex provided herein include, but are not limited to, parenteral, enteral, subcutaneous, percutaneous, transdermal, intradermal, intravenous, local, inhalation, or implantation.

[0360] Scar formation

[0361] In some embodiments, methods for preventing, reducing, or reversing scar formation in subjects in need are described herein, comprising administering to the subject a composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein.

[0362] As used herein, “scar formation” refers to the formation of a scar. In one aspect, the scar is a hypertrophic scar, a keloid scar, or a scar caused by acne. As used herein, a scar is a region of fibrous tissue caused by excessive collagen production. In some cases, wound healing involves the migration of fibroblasts to the site of injury. In some cases, fibroblasts deposit collagen. In some cases, fibroblasts deposit excessive collagen at the site of injury, resulting in a scar.

[0363] In some cases, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein prevent or inhibit TGF-β signaling. In some cases, TGF-β regulates the extracellular matrix by stimulating cellulose proliferation and collagen deposition and inhibiting extracellular matrix degradation (by upregulating the synthesis of protease inhibitors). In some cases, prevention or inhibition of TGF-β expression leads to prevention of reduced scar intensity. In some embodiments, application of the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein prevents or reduces scar formation.

[0364] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein inhibit or prevent the ability of fibroblasts to differentiate into myofibroblasts. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein reverse the differentiation of myofibroblasts into fibroblasts.

[0365] In some embodiments, the methods disclosed herein are used to prevent, reduce, or reverse scar formation. In some embodiments, the methods disclosed herein include administering the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein to an individual suffering from a condition leading to scar formation (e.g., dermatitis scars, keloids, contracture scars, hypertrophic scars, or acne-related scars). In some embodiments, the methods disclosed herein include administering the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein to an individual in need before or after trauma. In some embodiments, the methods disclosed herein include administering the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein to an individual in need before or after surgery.

[0366] In some embodiments, the methods disclosed herein are used to prevent or reduce scarring on the eye or surrounding tissues. In some embodiments, the methods disclosed herein include administering the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein to an individual suffering from a condition caused by scarring of the eye or surrounding tissues (e.g., retinopathy of prematurity). In some embodiments, the methods disclosed herein include administering the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein to an individual in need before or after trauma to the eye or surrounding tissues. In some embodiments, the methods disclosed herein include administering the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein to an individual in need before or after surgery on the eye or surrounding tissues.

[0367] inflammation

[0368] In some embodiments, this document describes methods for preventing or reducing inflammation in subjects in need, comprising administering to the subject a composition disclosed herein containing an nHC-HA / PTX3 or an rcHC-HA / PTX3 complex. As used herein, “inflammation” means a physiological response resulting from the migration of plasma and / or leukocytes (e.g., lymphocytes, macrophages, granulocytes, and neutrophils) to a site of infection or trauma (e.g., blunt force trauma, penetrating trauma, or surgery).

[0369] In some cases, leukocytes secrete cytokines upon contact with antigens. As used herein, "cytokine" refers to a signaling protein or glycoprotein. In some cases, cytokines bind to cell surface receptors. In some cases, cytokines induce chemotaxis of leukocytes to the site of infection. In some cases, cell surface receptors on leukocytes detect the chemical gradient of cytokines. In some cases, leukocytes follow a gradient to the site of infection. In some cases, the binding of cytokines to cell surface receptors leads to the upregulation or downregulation of certain genes and their transcription factors. In some cases, changes in gene expression lead to cytokine production, increased cytokine yield, or increased presence of cell surface receptors.

[0370] As non-limiting examples, cytokines include interleukins IL-1, IL-6, IL-8, MCP-1 (also known as CCL2), and TNF-α. Interleukin 1 exists in the body in two isoforms: IL-1α and IL-1β. In some cases, the presence of IL-1 increases the expression of adhesion factors on endothelial cells. This, in turn, enables leukocytes to migrate to sites of infection. In some cases, IL-8 induces leukocyte chemotaxis. In some cases, TNF-α induces leukocyte chemotaxis. In some cases, MCP-1 recruits leukocytes to sites of tissue damage and infection.

[0371] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein inhibit the production and / or activity of cytokines. In certain cases, the reduction in cytokine concentrations reduces or prevents inflammation by decreasing the number of leukocytes and / or the rate at which leukocytes migrate to the site of injury.

[0372] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein induce apoptosis of leukocytes (e.g., macrophages, neutrophils, or lymphocytes). In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein reduce the number of activated leukocytes or the rate at which such leukocytes are activated. In certain cases, the reduction in leukocyte concentration reduces or prevents inflammation by decreasing the number of cells migrating to the site of injury (e.g., by promoting the death of such cells through apoptosis).

[0373] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein inhibit macrophage polarization toward an inflammatory phenotype (i.e., in the M1 type). In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein reduce or inhibit the expression of IL-12 or IL-23 in stimulated macrophages. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein promote the polarization of stimulated macrophages toward a regulator or damage-healing M2 phenotype. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein inhibit or reduce inflammation in a subject. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein inhibit or reduce tissue damage caused by a condition or disease that induces inflammation in a subject.

[0374] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is administered to a subject suffering from inflammation. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is administered to a subject suffering from acute inflammation. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is administered to a subject suffering from chronic inflammation. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is administered to a subject suffering from an inflammatory condition. In some embodiments, the inflammatory condition is macrophage-mediated. In some embodiments, the inflammatory condition is T-cell-mediated. In some embodiments, the inflammatory condition is a Th-17-mediated immune condition.

[0375] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is administered to a subject suffering from an acute inflammatory response. In some embodiments, the acute inflammatory response is caused by, for example, allergic reactions, sepsis, endotoxin shock, or local ischemia, such as, but not limited to, myocardial infarction and stroke. In some embodiments, the acute inflammatory response is caused by bacterial infection, protozoan infection, viral infection, fungal infection, or a combination thereof. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex inhibits or reduces acute inflammation. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex inhibits or reduces tissue damage caused by acute inflammation. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex inhibits or reduces tissue reperfusion injury caused by local ischemia, including myocardial infarction and stroke.

[0376] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are administered to subjects with chronic inflammation associated with adaptive immunity and lymphocyte activation. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are administered to subjects with a Th1 response. In some embodiments, the Th1 response leads to immune rejection of the biological transplant. In some embodiments, the transplant is an allogeneic transplant. In some embodiments, the graft is an autologous transplant. In some embodiments, the inflammatory condition is graft-versus-host disease or tissue transplant rejection. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes suppress or reduce chronic inflammation in the subject.

[0377] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are administered to subjects with chronic inflammation associated with Th17 cell immune responses related to inflammatory conditions. In some embodiments, the inflammatory condition is an autoimmune disease or a leukocyte deficiency.

[0378] In some implementations, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are administered to subjects suffering from inflammatory conditions such as acute disseminated encephalomyelitis, Addison's disease, ankylosing spondylitis, antiphospholipid syndrome, autoimmune hemolytic anemia, autoimmune hepatitis, autoimmune inner ear disease, bullous pemphigoid, Chagas disease, chronic obstructive pulmonary disease, celiac disease, dermatomyositis, type 1 diabetes, type 2 diabetes, endometriosis, pulmonary hemorrhage nephritis syndrome, Graves' disease, Guillain-Barré syndrome, Hashimoto's disease, idiopathic thrombocytopenic purpura, interstitial cystitis, systemic lupus erythematosus (SLE), metabolic syndrome, multiple sclerosis, myasthenia gravis, myocarditis, narcolepsy, obesity, pemphigus vulgaris, pernicious anemia, polymyositis, primary biliary cirrhosis, rheumatoid arthritis, schizophrenia, and scleroderma. Behcet's syndrome, vasculitis, vitiligo, Wegener's granulomatosis, allergic rhinitis, prostate cancer, non-small cell lung cancer, ovarian cancer, breast cancer, melanoma, gastric cancer, colorectal cancer, brain cancer, metastatic bone disease, pancreatic cancer, lymphoma, nasal polyps, gastrointestinal tumors, ulcerative colitis, Crohn's disease, collagenous colitis, lymphocytic colitis, ischemic colitis, shunt colitis, Behcet's syndrome Syndrome), infectious enteritis, indeterminate colitis, inflammatory liver disease, ischemia, myocardial infarction, stroke, endotoxic shock, septic shock, rheumatoid spondylitis, ankylosing spondylitis, gouty arthritis, polymyalgia rheumatica, Alzheimer's disease, Parkinson's disease, epilepsy, AIDS dementia, asthma, adult respiratory distress syndrome, bronchitis, cystic fibrosis, acute leukocyte-mediated lung injury, distal proctitis, Wegener's granulomatosis, fibromyalgia, uveitis, conjunctivitis, psoriasis, eczema, dermatitis, smooth muscle hyperplasia, meningitis, herpes zoster, encephalitis, nephritis, tuberculosis, retinitis, atopic dermatitis, pancreatitis, periodontal gingivitis, coagulative necrosis, liquefactive necrosis, fibrinoid necrosis, neointimal hyperplasia or combinations thereof.

[0379] In some embodiments, the inflammatory condition is an inflammatory condition of the eye or surrounding tissues. In some embodiments, the inflammatory condition is conjunctivitis. In some cases, conjunctivitis is caused by exposure to an allergen. In some cases, conjunctivitis is caused by a bacterial infection. In some embodiments, the inflammatory condition is keratitis. As used herein, "keratitis" is a condition characterized by inflammation of the cornea. In some embodiments, the inflammatory condition is keratoconjunctivitis (i.e., a combination of conjunctivitis and keratitis (i.e., corneal inflammation)). In some embodiments, the inflammatory condition is blepharitis. As used herein, "blepharitis" is an ophthalmic disease characterized by inflammation of the eyelid margin. In some embodiments, the inflammatory condition is palpebral conjunctivitis (i.e., a combination of conjunctivitis and blepharitis (i.e., blepharitis)). In some embodiments, the inflammatory condition is scleritis. As used herein, "scleritis" is a condition characterized by inflammation of the sclera. In some embodiments, the inflammatory condition is episcleritis. As used herein, “epistymitis” is an inflammatory condition of the outer layer of the sclera characterized by congestion and bulbar conjunctival edema. In some embodiments, the inflammatory condition is uveitis. As used herein, “uveitis” is an inflammatory condition characterized by the uvea. In some embodiments, the condition is retinitis. As used herein, “retinitis” is an inflammatory condition of the retina. In some embodiments, the condition is choroiditis. As used herein, “choroiditis” is an inflammatory condition of the uvea, ciliary body, and choroid.

[0380] Abnormal angiogenesis

[0381] In some embodiments, this document discloses methods for preventing or reducing angiogenesis in subjects with this need, the method comprising administering to the subject a composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein. As used herein, "angiogenesis" refers to the formation of new blood vessels. In some cases, angiogenesis promotes tumor growth and metastasis. Furthermore, in some cases, abnormal angiogenesis is the basis of wet age-related macular degeneration (wARMD) and diabetic proliferative retinopathy. In some cases, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein prevents or reduces angiogenesis.

[0382] In some cases, the binding of the ligand to VEGF receptor-2 (VEGFR-2) initiates a tyrosine kinase signaling cascade that stimulates the production of factors that differently promote vascular permeability (eNOS, producing NO), proliferation / survival (bFGF), migration (ICAM / VCAM / MMP), and ultimately differentiation into mature blood vessels. In other cases, after VEGFR-2 binds to its ligand, endothelial cells form capillary-like tubular structures.

[0383] As used in this article, “wet age-related macular degeneration,” “wARMD,” or “wet ARMD” refers to an ocular condition characterized by the proliferation of blood vessels originating from the choroid. In some cases, wet ARMD leads to vision loss due to leakage of blood and proteins under the macula. In other cases, without treatment, bleeding, leakage, and scarring from these vessels can cause irreversible damage to photoreceptors and rapid vision loss.

[0384] As used in this article, "diabetic proliferative retinopathy" refers to an eye condition characterized by incompetence of the vascular walls. In some cases, oxygen deficiency in the retina leads to blood vessel formation along the retina and in the vitreous humor. In other cases, new blood vessels bleed, obstruct vision, and damage the retina.

[0385] In some cases, capillary proliferation provides nutrients to the tumor, allowing it to proliferate. In others, it rapidly removes cellular waste, thus facilitating tumor growth. In still others, angiogenesis promotes metastasis. And in yet another context, capillary proliferation increases the likelihood that cancer cells will be able to enter blood vessels and thus form new tumors at novel sites.

[0386] In some implementations, exemplary cancer types treated with the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein include, but are not limited to, acute lymphoblastic leukemia, acute myeloid leukemia, adrenocortical carcinoma, HIV-related cancers, HIV-related lymphomas, anal cancer, astrocytoma, basal cell carcinoma, cholangiocarcinoma, bladder cancer, bone cancer, brainstem glioma, brain tumors, breast cancer, bronchial adenoma, Burkitt's lymphoma, carcinoid tumors, carcinomas, central nervous system lymphomas, cerebellar astrocytomas, etc. Cervical cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disorder, colon cancer, colorectal cancer, cutaneous T-cell lymphoma, endometrial cancer, ependymoma, esophageal cancer, extragonadal germ cell tumors, eye cancer, intraocular melanoma, retinoblastoma, gallbladder cancer, gastrointestinal carcinoid tumors, gastrointestinal stromal tumors (GIST), germ cell tumors (extracranial), germ cell tumors (extragonadal), germ cell tumors (ovarian), gestational trophoblastic tumors, glioma, hairy cell leukemia, head and neck cancer, hepatocellular carcinoma, Hodgkin's lymphoma. Lymphoma, hypopharyngeal cancer, hypothalamic and visual pathway glioma, intraocular melanoma, islet cell carcinoma (endocrine pancreas), Kaposi's sarcoma, kidney (renal cell) carcinoma, laryngeal cancer, leukemia (acute lymphoblastic), leukemia (acute myeloid leukemia), leukemia (chronic lymphocytic leukemia), leukemia (chronic myeloid leukemia), lip and oral cancer, liver cancer, lung cancer (non-small cell), lung cancer (small cell), lymphoma (skin T-cell), lymphoma (non-Hodgkin's), malignant fibrous histiocytoma / osteosarcoma of bone, medulloblastoma, melanoma, Markel cell carcinoma, mesothelioma, occult primary metastatic squamous neck cancer. Primary, multiple endocrine adenoma syndrome, multiple myeloma / plasma cell tumor, mycosis fungoides, myelodysplastic syndrome, myelodysplastic / myeloproliferative disorders, myelogenous leukemia, myeloid leukemia, myeloproliferative disorders, nasal cavity and sinus carcinoma, nasopharyngeal carcinoma, neuroblastoma, oral cancer, oropharyngeal cancer, osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, ovarian epithelial carcinoma, ovarian germ cell tumor, low-grade potential malignant tumor of the ovary, pancreatic cancer, parathyroid carcinoma, penile cancer, pheochromocytoma, pineal blastoma, and supratentorial primary neuroectodermal tumor.Tumors, pituitary adenoma, plasma cell tumor / multiple myeloma, pleural pulmonary blastoma, prostate cancer, rectal cancer, retinoblastoma, rhabdomyosarcoma, salivary gland cancer, sarcoma (Kaposi's), sarcoma (uterus), Sezary syndrome, skin cancer (non-melanoma), skin cancer (melanoma), skin cancer (Merkel cell), small intestine cancer, soft tissue sarcoma, squamous cell carcinoma, gastric (stomach) cancer, T-cell lymphoma, testicular cancer, thymoma, thyroid cancer, trophoblastic tumor, pregnancy, urethral cancer, uterine cancer, endometrial cancer, uterine sarcoma, vaginal cancer, visual pathway and hypothalamic glioma, vulvar cancer, Warren's macroglobulinemia ( Macroglobulinemia and Wilms' Tumor.

[0387] Wound repair and tissue regeneration

[0388] In some embodiments, pharmaceutical compositions comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are used as wound dressings or to promote wound healing. In some embodiments, tissue is damaged, impaired, or lost due to injury (e.g., burns; surgical incisions; necrotic areas caused by infection, trauma, or toxins; lacerations). In some embodiments, tissue is damaged, impaired, or lost due to burns. In some embodiments, tissue is damaged, impaired, or lost due to trauma (e.g., incisions, lacerations, abrasions). In some embodiments, tissue is damaged, impaired, or lost due to necrosis. In some embodiments, tissue is damaged, impaired, or lost due to ulceration.

[0389] burn

[0390] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to burns. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to first-degree burns. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to second-degree burns. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to third-degree burns. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is applied to a substrate before being placed on the burn site.

[0391] trauma

[0392] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a skin wound (e.g., an incision, laceration, abrasion, ulcer, puncture, infiltration). In some embodiments, the wound is an ischemic wound. In some embodiments, the pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is applied to a substrate before being placed on the wound. In some embodiments, the pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein treats the wound.

[0393] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to an incision site of an organ (e.g., skin, brain, stomach, kidney, liver, intestine, lung, bladder, trachea, esophagus, vagina, ureter, and blood vessel wall). In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a surgical incision site. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a colectomy site. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a gastrectomy site. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a breast surgery site (e.g., breast reduction surgery, breast augmentation surgery, and mastectomy). In some embodiments, a pharmaceutical composition comprising nHC-HA / PTX3 or rcHC-HA / PTX3 complex is applied to the base before being placed on the wound.

[0394] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is used as a covering for skin incisions (e.g., incisions in the epidermis, dermis, and / or subcutaneous tissue). In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is used to repair or repair skin after hemorrhoid surgery. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is applied to a base before being placed on a wound. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is used to treat a wound.

[0395] Necrosis

[0396] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is used as a protective graft on an area of ​​necrotic tissue (e.g., from an infection). In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is used as a protective graft on an area of ​​necrotic skin. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is placed on an area of ​​necrotic tissue. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is applied to a substrate prior to placement on the necrotic tissue. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is used to treat necrotic tissue.

[0397] ulcer

[0398] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is used as a protective covering on an ulcer. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein treats an ulcer. In some embodiments, the ulcer is a diabetic ulcer, such as a diabetic foot or leg ulcer. In some embodiments, the ulcer is an ischemic wound. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is applied to a base before being placed on the ulcer. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein treats an ulcer. In some embodiments, the ulcer is an unhealed ulcer. For example, in some embodiments, the unhealed ulcer is a wound or ulcer on the skin that has persisted for approximately 3-4 weeks without healing.

[0399] In some embodiments, the ulcer is a foot ulcer (e.g., a diabetic foot ulcer or an arterial insufficiency ulcer). In some embodiments, treating a foot ulcer includes: (a) preparing the wound (e.g., clearing the wound); and (b) placing a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein onto the wound. In some embodiments, treating a foot ulcer includes: (a) preparing the wound (e.g., clearing the wound); (b) placing a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein onto the wound; and (c) covering the pharmaceutical composition with a protective barrier (e.g., a silver cell dressing, metipel, gauze, or bandage). In some embodiments, the pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is applied to a base prior to being placed on the ulcer. In some embodiments, pharmaceutical compositions comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are used to treat ulcers.

[0400] In some embodiments, the ulcer is a venous stasis (VS) ulcer. In some embodiments, treating a VS ulcer includes: (a) preparing the wound (e.g., clearing the wound); and (b) placing a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein onto the wound. In some embodiments, treating a VS ulcer includes: (a) preparing the wound (e.g., clearing the wound); (b) placing a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein onto the wound; and (c) covering the pharmaceutical composition with a protective barrier (e.g., wound gauze, antimicrobial dressing, gauze, or bandage). In some embodiments, the pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is applied to a substrate prior to placement on the wound. In some embodiments, the pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein treats the ulcer.

[0401] In some embodiments, the ulcer is a corneal ulcer (i.e., ulcerative keratitis). In some embodiments, treating a corneal ulcer includes: (a) preparing a wound (e.g., clearing the wound); and (b) placing a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein onto the wound. In some embodiments, treating a corneal ulcer includes: (a) preparing a wound (e.g., clearing the wound); (b) placing a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein onto the wound; and (c) covering the pharmaceutical composition with a protective barrier (e.g., a contact lens or bandage). In some embodiments, the pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is applied to a substrate prior to placement on the wound. In some embodiments, the pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein treats the ulcer.

[0402] Therapeutic cell therapy

[0403] In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are used in combination with cell therapy. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are co-administered with therapeutic cells. Therapeutic cells include any cells exhibiting therapeutic properties for treating a disease or condition. In some embodiments, the therapeutic cells are recombinant cells that heterologously express one or more therapeutic gene products. In some embodiments, the therapeutic cells are transplanted cells. In some embodiments, the therapeutic cells are stem cells. In some embodiments, the therapeutic cells are cells expressing one or more stem cell markers (e.g., Oct-3 / 4 (Pou5f1), Sox2, c-Myc, and Klf4).

[0404] In some embodiments, the cell therapy is stem cell transplantation. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is administered to promote the expansion of transplanted and tissue regeneration stem cells. In some instances, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is used to reduce or inhibit inflammation, scarring, and abnormal angiogenesis induced by stem cell transplantation. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is used to maintain the properties of stem cells during in vitro expansion by replacing the feeder layer. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is used to reprogram differentiated cells into stem cells. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is used for in vitro expansion and culture of stem cells for subsequent transplantation into a subject.

[0405] In some embodiments, the stem cell therapy is embryonic stem cell therapy. In some embodiments, the stem cell therapy is adult stem cell therapy. In some embodiments, the stem cell therapy is mesenchymal stem cell therapy. In some embodiments, the stem cell therapy is administered to treat diseases or conditions, such as, but not limited to, cardiovascular disease, cancer, diabetes, spinal cord injury, neurodegenerative diseases, Alzheimer's disease, Parkinson's disease, multiple sclerosis, amyotrophic lateral sclerosis, Duchenne muscular dystrophy, muscle injury or malnutrition, stroke, burns, lung disease, retinopathy, kidney disease, osteoarthritis, and rheumatoid arthritis.

[0406] In some implementations, stem cells are used to treat diabetes. Type 1 diabetes is caused by autoimmune-mediated destruction of insulin-secreting β cells in the islets of Langerhans of the pancreas. Type 2 diabetes is caused by systemic insulin resistance and reduced insulin secretion from pancreatic β cells. Stem cells have been shown to differentiate into insulin-producing cells in vitro (see, for example, Schuldiner et al. (2000) Proc. Natl. Acad. Sci. USA. 97:11307–11312; Guo et al., (2009) Endocr Rev 30:214-227). Therefore, in some implementations, stem cells, including ESCs and ASCs and their derivatives, such as partially differentiated stem cells, are used in stem cell therapy for the regeneration of pancreatic β cells.

[0407] In some embodiments, stem cells or differentiated cells for therapeutic use are encapsulated in a microcapsule device. In some embodiments, the microcapsule comprises the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is covalently attached to the microcapsule. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is assembled onto the surface of the microcapsule, e.g., the inner or outer surface, or both. In some embodiments, nHC-HA / PTX3 or rcHC-HA / PTX3 is formulated to encapsulate the microcapsule. In some embodiments, the microcapsule includes pores to allow nutrients to enter the microcapsule through cells and / or to allow proteins and molecules (e.g., insulin) secreted by the encapsulated cells to flow out of the microcapsule. In some embodiments, the cells are first immobilized on a microcarrier (e.g., using...). The cells are coated onto beads and then encapsulated within microcapsules. Methods for encapsulating cells, such as stem cells, are known in the art and described, for example, in Serra et al., (2011) PLoS ONE 6(8):e23212. In some embodiments, any method for encapsulating cells is used in combination with the methods provided herein.

[0408] In some embodiments, allogeneic therapeutic stem cells (e.g., insulin-producing islet cells) are encapsulated in a microcapsule device for insulin production. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex promotes stem cell expansion. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex inhibits or reduces inflammatory responses to stem cell-containing microcapsules for diabetes treatment. In some embodiments, the microcapsules contain the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein. In some embodiments, the nHC-HA / PTX3 or rcHC-HA / PTX3 complex inhibits or reduces inflammatory responses to stem cell-containing microcapsules.

[0409] Soft tissue applications

[0410] In some embodiments, this document discloses the use of the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein for repairing, rebuilding, replacing, or supplementing damaged, impaired, or missing soft tissues (e.g., tendons). In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein is applied directly to a tissue. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein is administered in combination with a cell- or tissue-based therapy. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein is mixed with a single cell, multiple cells, or tissue and administered as part of a tissue-based therapy. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein is used to coat a single cell, multiple cells, or tissue and administered as part of a tissue-based therapy.

[0411] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is used as a covering for soft tissue incisions (e.g., eyelids forming a tissue plane between different soft tissue layers). In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is applied to a base and subsequently used as a covering for soft tissue incisions (e.g., eyelids forming a tissue plane between different soft tissue layers).

[0412] In some embodiments, pharmaceutical compositions comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein are used as structural (constructive) supports for soft tissues. In some embodiments, pharmaceutical compositions comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein prevent adhesions during joint or tendon repair.

[0413] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is used for tendon or joint repair (e.g., rotator cuff repair, hand tendon repair). In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is used to strengthen tendons or joints. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is used to prevent adhesions between the healing tendon and surrounding tissues, tendons, or joints. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is used to prevent the formation of scar tissue on the tendon.

[0414] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a base, and the base / nHC-HA / PTX3 or base / rcHC-HA / PTX3 complex is used to enlarge smaller tendons and ligaments of the foot and ankle, including the posterior tibialis tendon, peroneal tendon, flexor tendon, and extensor tendon, as well as the ligaments of the lateral malleolus complex. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a base, and the base / nHC-HA / PTX3 or base / rcHC-HA / PTX3 complex is used to strengthen the primary repair of the quadriceps and patellar tendon around the knee. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a base, and the base / nHC-HA / PTX3 or base / rcHC-HA / PTX3 complex is used as a periosteal patch for bone grafts in joint replacement. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 is used to enlarge the cystic tissue of the under-repaired hip and knee joints after the entire joint repair surgery.

[0415] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is used for the repair of a torn rotator cuff. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is used as a patch on a rotator cuff muscle or tendon (e.g., supraspinatus tendon). In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is used for the reconstruction of a rotator cuff muscle or tendon (e.g., supraspinatus tendon). In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is used to enlarge the rotator cuff muscles or tendons (e.g., the supraspinatus tendon). In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is used to strengthen the rotator cuff muscles or tendons (e.g., the supraspinatus tendon). In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is used to prevent soft tissue adhesions to the rotator cuff muscles or tendons (e.g., the supraspinatus tendon).

[0416] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is used to repair gingiva. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is used to repair gingival recession. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate and used as a patch on the gingiva. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate and used as a patch on an exposed root surface. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is used to reconstruct gingiva. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is used to enlarge gingiva. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is used to strengthen the gingiva. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is used to prevent soft tissue adhesions to the gingiva.

[0417] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is used as a protective graft for incisions or tears in the fascia. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is used as a structural (constructive) support for the fascia. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is used as a substitute or supplement for the fascia. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a base, and the base / nHC-HA / PTX3 or base / rcHC-HA / PTX3 complex is used to repair a hernia (e.g., for fascia repair). In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a base, and the base / nHC-HA / PTX3 or base / rcHC-HA / PTX3 complex is used to repair an inguinal hernia. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a base, and the base / nHC-HA / PTX3 or base / rcHC-HA / PTX3 complex is used to repair a femoral hernia. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a basement membrane, and the basement / nHC-HA / PTX3 or basement / rcHC-HA / PTX3 complex is used to repair an umbilical hernia. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a basement membrane, and the basement / nHC-HA / PTX3 or basement / rcHC-HA / PTX3 complex is used to repair an incisional hernia. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a basement membrane, and the basement / nHC-HA / PTX3 or basement / rcHC-HA / PTX3 complex is used to repair a diaphragmatic hernia.In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is used to repair Cooper's hernia, epigastric hernia, hiatal hernia, Littre hernia, lumbar hernia, Maydl hernia, obturator hernia, pantaloon hernia, paraesophageal hernia, paraumbilical hernia, perineal hernia, properitoneal hernia, Richter hernia, sliding hernia, ischial hernia, semilunar hernia, motor hernia, Velpeau hernia, or Amyand hernia.

[0418] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to the acupoint, and the acupoint / nHC-HA / PTX3 or acupoint / rcHC-HA / PTX3 complex is used to repair a herniated disc. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to the acupoint, and the acupoint / nHC-HA / PTX3 or acupoint / rcHC-HA / PTX3 complex is used as a protective graft on an incision or tear of the intervertebral disc. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to the acupoint, and the acupoint / nHC-HA / PTX3 or acupoint / rcHC-HA / PTX3 complex is used as a protective graft on an incision or tear of the annulus fibrosus. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to the submucosa, and the submucosa / nHC-HA / PTX3 or submucosa / rcHC-HA / PTX3 complex serves as a structural support for the intervertebral disc. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to the submucosa, and the submucosa / nHC-HA / PTX3 or submucosa / rcHC-HA / PTX3 complex serves as a structural support for the annulus fibrosus. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to the submucosa, and the submucosa / nHC-HA / PTX3 or submucosa / rcHC-HA / PTX3 complex serves as a substitute or supplement for the intervertebral disc. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to the submucosa, and the submucosa / nHC-HA / PTX3 or submucosa / rcHC-HA / PTX3 complex serves as a structural (constructive) support for the intervertebral disc. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to the submucosa, and the submucosa / nHC-HA / PTX3 or submucosa / rcHC-HA / PTX3 complex serves as a substitute or supplement for the annulus fibrosus.

[0419] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is applied to an incision in the brain or one (or all) of the meninges (i.e., the dura mater, pia mater, and / or arachnoid mater). In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex serves as a structural (constructive) support for one (or all) of the meninges (i.e., the dura mater, pia mater, and / or arachnoid mater). In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to the substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is used as a substitute for one (or all) meninges (i.e., dura mater, pia mater, and / or arachnoid mater).

[0420] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is applied to an incision in the lung or pleura. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is used as a structural (or constructive) support for the pleura. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is used as a substitute for the pleura.

[0421] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is applied to an incision in the tympanic membrane. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex serves as a structural (constructive) support for the tympanic membrane. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex serves as a substitute for the tympanic membrane.

[0422] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is used as a protective graft on an incision site in the heart or pericardium. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is used as a structural (constructive) support for the pericardium. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is used as a substitute for the pericardium.

[0423] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is used as a protective graft on a peritoneal incision. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is used as a structural (constructive) support for the peritoneum. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is used as a substitute for the peritoneum.

[0424] Ophthalmic applications

[0425] In some embodiments, this document discloses the use of pharmaceutical compositions comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein for the repair, reconstruction, replacement or replenishment of damaged, impaired or missing ocular tissues containing receptors.

[0426] Glaucoma treatment

[0427] As used in this article, "glaucoma" refers to a condition characterized by the loss of retinal ganglion cells in the optic nerve. In some cases, glaucoma is caused partly or entirely by an increase in intraocular pressure in the anterior chamber (AC). Intraocular pressure varies according to the fluid produced by the ciliary processes of the eye, aqueous humor, and its drainage through the trabecular meshwork.

[0428] A glaucoma drainage device (GDD) is a medical device implanted in the eye to reduce intraocular pressure by providing a bypass pathway for the drainage of aqueous humor. If the GDD tube is not covered, it will corrode and make the eye susceptible to intraocular infections. Therefore, the GDD tube needs to be covered. Currently, the patches used to cover the GDD tube are made of the pericardium, sclera, and cornea. These patches are approximately 400-550 micrometers thick. These thin patches cause them to melt by 25% within two years, potentially exposing the shunt tube again.

[0429] In some embodiments, the pharmaceutical composition disclosed herein comprising an nHC-HA / PTX3 or rcHC-HA / PTX3 complex is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is used to cover the GDD tube. In some embodiments, the substrate / nHC-HA / PTX3 or rcHC-HA / PTX3 complex is 300-600 micrometers thick. In some embodiments, the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex does not melt by 25% within 2 years.

[0430] Treatment of eye ulcers

[0431] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is used to cover persistent epithelial defects and / or ulcers of ophthalmic nitrile.

[0432] In some embodiments, a surgical sponge is used to remove the base of the ulcer and remove poorly adhesive epithelium adjacent to the ulcer margin (e.g., to obtain the portion of the eye where epithelial adhesion becomes very high). In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to the base, and the base / nHC-HA / PTX3 or base / rcHC-HA / PTX3 complex is transferred to the recipient eye. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to the base, and the base / nHC-HA / PTX3 or base / rcHC-HA / PTX3 complex is subsequently secured to the eye with sutures (e.g., interrupted 10-0 nylon sutures or continuous 10-0 nylon sutures) and the suture knot is buried. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is fixed to the eye using fibrin glue. In some embodiments, a protective layer is applied to the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex or the entire eye (e.g., a contact lens). In some embodiments, the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex further comprises an antibiotic (e.g., neomycin, polymyxin B sulfate, and dexamethasone).

[0433] Reconstruction of the conjunctiva, sclera, eyelids and orbital rim surfaces

[0434] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is used for the reconstruction of the conjunctival, scleral, eyelid, and orbital rim surfaces. In some embodiments, damage to the conjunctival surface is caused by the dissolution of symblepharon cells; surgical removal of tumors, lesions, and / or scar tissue; excimer laser refractive keratomy and therapeutic keratomy, or a combination thereof.

[0435] Coronary artery applications

[0436] In some embodiments, this document discloses the use of pharmaceutical compositions comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein for the repair, reconstruction, replacement or replenishment of damaged, impaired or missing coronary artery tissue.

[0437] Prevention of ischemia-reperfusion injury

[0438] This document discloses the use of pharmaceutical compositions comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes described herein for the inhibition or reduction of tissue damage resulting from acute inflammation caused by ischemia (e.g., myocardial infarction or stroke). In some embodiments, the pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein is administered to a subject suffering from an ischemic condition (e.g., but not limited to, myocardial infarction or stroke).

[0439] Coronary artery bypass

[0440] This document discloses the use of pharmaceutical compositions comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes described herein for coronary artery bypass surgery. In some embodiments, the pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complexes disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is transplanted onto the coronary artery to bypass the arterial portion characterized by atherosclerosis.

[0441] Heart valves

[0442] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is applied to a heart valve. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is used as a structural (constructive) support for the heart valve. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is used as a substitute for a heart valve.

[0443] veins and arteries

[0444] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is applied to a vein or artery. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is used as a structural (constructive) support for a vein or artery.

[0445] Neural Applications

[0446] In some embodiments, this document discloses the use of pharmaceutical compositions comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein for the repair, reconstruction, replacement or supplementation of damaged, impaired or missing nerve tissue.

[0447] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is used as a covering on a nerve (e.g., a peripheral nerve). In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is used as a covering on a nerve graft, nerve transfer, or repaired nerve. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is used as a covering on an incision in a nerve (e.g., a peripheral nerve). In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex serves as a structural (constructive) support for nerves (e.g., peripheral nerves). In some embodiments, the pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein prevents adhesions during nerve repair.

[0448] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex serves as a non-constricting encasement for the injured nerve. In some embodiments, the pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein prevents or minimizes scar formation, nerve encapsulation, chronic compression, entrapment, and nerve entrapment. In some embodiments, the pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein prevents or minimizes neuroma formation. In some embodiments, the pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein prevents or minimizes the migration of endogenous growth factors (nerve growth factors) during nerve repair.

[0449] Spinal Applications

[0450] In some embodiments, this document discloses the use of pharmaceutical compositions comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein in spinal surgery.

[0451] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein is used in laminectomy. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein is used to reduce or prevent epidural fibrosis and / or scar adhesions following spinal surgery (e.g., laminectomy). In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein is implanted between the dura mater and overlying tissue after spinal surgery (e.g., laminectomy). In some embodiments, implantation of a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein between the dura mater and overlying tissue after spinal surgery (e.g., laminectomy) reduces or prevents fibroblast migration to the dura mater and collagen deposition on the dura mater.

[0452] In some embodiments, pharmaceutical compositions comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein are used to reduce or prevent the growth of proliferative scars following spinal surgery (e.g., laminectomy). In some embodiments, pharmaceutical compositions comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein are used to reduce or prevent the growth of epidural / epidural / perineural scars following surgery (e.g., after laminectomy). In some embodiments, pharmaceutical compositions comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein are used to reduce or prevent the growth of proliferative scars following spinal surgery (e.g., laminectomy). In some embodiments, pharmaceutical compositions comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein are used to reduce or prevent the growth of membranes after laminectomy.

[0453] In some embodiments, pharmaceutical compositions comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein are used to reduce or prevent the development of epidural compression or dural restraint following spinal surgery (e.g., laminectomy). In some embodiments, pharmaceutical compositions comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein are used to reduce or prevent the growth of restrained nerve roots following spinal surgery (e.g., laminectomy). In some embodiments, pharmaceutical compositions comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex described herein are used to reduce or prevent the development of arachnoiditis following spinal surgery (e.g., laminectomy).

[0454] In some embodiments, the pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein further comprises granular bone tissue. In some embodiments, the pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein and comprising granular bone tissue is used during spinal fusion. In some embodiments, implanting the pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein and comprising granular bone tissue between adjacent vertebrae. In some embodiments, implantation of the pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein and comprising granular bone tissue between two adjacent vertebrae promotes vertebral fusion.

[0455] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is used as a protective graft over an incision in the dura mater. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is used as a structural (constructive) support for the dura mater. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a substrate, and the substrate / nHC-HA / PTX3 or substrate / rcHC-HA / PTX3 complex is used as a substitute for the dura mater.

[0456] Multiple Applications of nHC-HA / PTX3 or rcHC-HA / PTX3 Complexes

[0457] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to a patch or wound dressing.

[0458] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is used as a dermal filler. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is injected into subcutaneous facial tissue. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is injected below wrinkles and lines of aging on the face (e.g., nasolabial folds, marionette lines, crow's feet, and forehead wrinkles). In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is used for lip augmentation. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is injected into the lips.

[0459] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is used to treat arthritis (e.g., osteoarthritis, rheumatoid arthritis, septic arthritis, ankylosing spondylitis, vertebral stiffness). In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is injected into a joint suffering from arthritis (e.g., the knee).

[0460] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is used to treat foot arthritis. In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is used to treat arthritis of the first metatarsophalangeal (MTP) joint (e.g., thumb stiffness). In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is applied to an MTP joint after a dorsal lip resection. In some embodiments, administration of a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein reduces one or more adverse symptoms (e.g., scarring, joint stiffness, swelling, inflammation, and pain) associated with thumb stiffness or a dorsal lip resection procedure.

[0461] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is used to treat one or more symptoms associated with bone spurs (e.g., scarring, joint stiffness, swelling, inflammation, and pain).

[0462] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is used to inhibit bone resorption in an individual with this need. In some embodiments, the individual suffers from arthritis, osteoporosis, alveolar bone degeneration, Paget's disease, or a bone tumor. In some embodiments, the pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is injected into a joint. In some embodiments, the pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is brought into contact with bone (e.g., by using a wound dressing or bandage). In some embodiments, the pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex coats a bone scaffold, bone implant, or bone prosthesis (e.g., an osseointegrated implant). As used herein, an "osseointegrated implant" means a three-dimensional implant containing pores through which osteoblasts and supporting connective tissue migration enter. In some embodiments, the bone scaffold is inserted into the intramedullary canal of bone. In some embodiments, the bone scaffold is placed in the tarsal sinus. In some embodiments, the bone scaffold is placed in the knee or joint. In some embodiments, the bone scaffold is placed at the site of a fracture. In some embodiments, the bone scaffold is expandable or shrinkable.

[0463] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is used to promote or induce bone formation in an individual in need. In some embodiments, the individual suffers from arthritis, osteoporosis, alveolar bone degeneration, Paget's disease, or a bone tumor. In some embodiments, the pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is injected into a joint. In some embodiments, the pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is brought into contact with bone (e.g., by using a wound dressing or bandage). In some embodiments, the pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex covers a bone scaffold, bone implant, or bone prosthesis (e.g., an osseointegrated implant). As used herein, an "osseointegrated implant" means a three-dimensional implant containing pores through which osteoblasts and supporting connective tissue migration enter. In some embodiments, the bone scaffold is inserted into the intramedullary canal of bone. In some embodiments, the bone scaffold is placed in the tarsal sinus. In some embodiments, the bone scaffold is placed in the knee or joint. In some embodiments, the bone scaffold is placed at the site of a fracture. In some embodiments, the bone scaffold is expandable or shrinkable.

[0464] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is used to inhibit osteoclast differentiation. In some embodiments, the individual suffers from arthritis, osteoporosis, alveolar bone degeneration, Paget's disease, or a bone tumor. In some embodiments, the pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is injected into a joint. In some embodiments, the pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is brought into contact with bone (e.g., by using a wound dressing or bandage). In some embodiments, the pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex covers a bone scaffold, bone implant, or bone prosthesis (e.g., an osseointegrated implant). As used herein, an "osseointegrated implant" means a three-dimensional implant containing pores through which osteoblasts and supporting connective tissue migration enter. In some embodiments, the bone scaffold is inserted into the intramedullary canal of bone. In some embodiments, the bone scaffold is placed in a tarsal sinus. In some embodiments, the bone scaffold is placed in the knee or joint. In some embodiments, the bone scaffold is placed at the site of a fracture. In some embodiments, the bone scaffold is expandable or collapsible.

[0465] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is used to promote mineralization by osteoblasts in an individual in need. In some embodiments, the individual suffers from arthritis, osteoporosis, alveolar bone degeneration, Paget's disease, or a bone tumor. In some embodiments, the pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is injected into a joint. In some embodiments, the pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is brought into contact with bone (e.g., by using a wound dressing or bandage). In some embodiments, the pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex covers a bone scaffold, bone implant, or bone prosthesis (e.g., an osseointegrated implant). As used herein, an "osseointegrated implant" means a three-dimensional implant containing pores that support the migration of osteoblasts and connective tissue. In some embodiments, the bone scaffold is inserted into the intramedullary canal of bone. In some embodiments, the bone scaffold is placed in the tarsal sinus. In some embodiments, the bone scaffold is placed in the knee or joint. In some embodiments, the bone scaffold is placed at the fracture site. In some embodiments, the bone scaffold is expandable or collapsible.

[0466] In some embodiments, a pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex disclosed herein is used to balance bone resorption and bone formation in an individual in need. In some embodiments, the individual suffers from arthritis, osteoporosis, alveolar bone degeneration, Paget's disease, or a bone tumor. In some embodiments, the pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is injected into a joint. In some embodiments, the pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-HA / PTX3 complex is brought into contact with bone (e.g., by using a wound dressing or bandage). In some embodiments, the pharmaceutical composition comprising the nHC-HA / PTX3 or rcHC-H...

Claims

1. A method for producing reconstituted HC-HA / PTX3 (rcHC-HA / PTX3) complexes in vitro ， It includes (a) contacting (i) high molecular weight hyaluronic acid (HMW HA) immobilized on a solid support with (ii) pentraxin 3 (PTX3) protein to form a complex of immobilized PTX3 and HMW HA, and contacting the complex of immobilized PTX3 and HMW HA with (iii) inter-α-inhibitor (IαI) protein comprising heavy chain 1 (HC1) and heavy chain 2 (HC2), and (iv) tumor necrosis factor α-stimulated gene 6 (TSG-6) to form an immobilized rcHC-HA / PTX3 complex.

2. A reconstituted HC-HA / PTX3 (rcHC-HA / PTX3) complex produced by the method of claim 1.

3. The rcHC-HA / PTX3 of claim 2, which is immobilized on a solid support.

4. A pharmaceutical composition comprising the rcHC-HA / PTX3 complex of claim 2 and a pharmaceutically acceptable excipient.

5. The pharmaceutical composition of claim 4, further comprising fibronectin, collagen, laminin, keratin, fibrin, fibrinogen, hyaluronic acid, heparan sulfate, chondroitin sulfate or any combination thereof.

6. The pharmaceutical composition of claim 4 or claim 5, further comprising an anti-inflammatory agent, an anti-scarring agent or any combination thereof.

7. The pharmaceutical composition of claim 4 or claim 5, wherein the composition is a solution, suspension, gel, powder, ointment, tablet, capsule or aerosol.

8. The pharmaceutical composition of claim 7, wherein the gel is a cross-linked gel or a liposome.

9. The pharmaceutical composition of claim 7, wherein the gel is a cross-linked hyaluronic acid hydrogel.

10. A cell culture comprising a substrate suitable for culturing cells and the rcHC-HA / PTX3 complex of claim 2.

11. The cell culture of claim 10, wherein the rcHC-HA / PTX3 complex is immobilized to the substrate.

12. A medical device comprising a substrate coated with the rcHC-HA / PTX3 complex of claim 2.

13. The medical device of claim 12, wherein the substrate comprises at least one of a stent, a joint, a rod, a pin, a plate, a staple, a shunt, a clamp, a clip, a suture, a suture anchor, an electrode, a catheter, a lead, a graft, a dressing, a pacemaker, a pacemaker housing, a cardioverter, a cardioverter housing, a defibrillator, a defibrillator housing, a prosthesis, an ear drain, an orthopedic device, a spinal disc, an anastomotic device, a perivascular wrap, a colostomy bag attachment device, a hemostatic barrier, a vascular support, a tissue adhesive, and a tissue sealant.

14. The medical device of claim 12, wherein the substrate is a microcapsule, a nanoparticle, a bead, or any combination thereof.

15. The medical device of claim 14, wherein the microcapsules contain stem cells.

16. The medical device of claim 12, wherein the substrate is a contact lens.

17. The medical device of claim 12, wherein the substrate is a surgical implant.

18. The medical device of claim 17, wherein the implant is an artificial joint, a suture, a stent, or any combination thereof.

19. The medical device of claim 18, wherein the artificial joint is an artificial hip joint, an artificial knee joint, an artificial shoulder joint, or any combination thereof.

20. The medical device of claim 12, wherein the substrate is a screw.

21. The medical device of claim 12, wherein the substrate is a bone implant.

22. The medical device of claim 12, wherein the substrate is an artificial knee.

23. The medical device of claim 13, wherein the stent is a coronary stent, a ureteral stent, a urethral stent, a prostate stent, an esophageal stent, a bone stent, or any combination thereof.

24. The medical device of claim 12, wherein the substrate is an ophthalmic implant, a bone substitute, or a vascular implant.

25. The medical device of claim 12, wherein the substrate is an intraluminal device.

26. The medical device of claim 12, wherein the substrate is a tissue scaffold.

27. A kit comprising the rcHC-HA / PTX3 complex of claim 2, a device for administering the complex, and optional instructions for administration.

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