A protein matrix composition for hard and soft tissue matrix repair, reconstruction, and / or augmentation

Abstract

The current invention involves a method of mixing POP with a tissue matrix to obtain a matrix composition for soft and hard tissue matrix repair, reconstruction, and / or augmentation. POP is a partially ordered polypeptide and the tissue matrix is a decellularized tissue matrix derived from a source tissue. The invention involves mixing these compositions in a matrix composition and implanting or injecting the matrix composition into a subject to repair, augment, or reconstruct a tissue structure.

Description

ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 A PROTEIN MATRIX COMPOSITION FOR HARD AND SOFT TISSUE MATRIX REPAIR, RECONSTRUCTION, AND / OR AUGEMENTATION CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 686,319, filed on August 23, 2024, the content of which is herein incorporated by reference in its entirety. REFERENCE TO AN ELECTRONIC SEQUENCE LISTING

[0002] The contents of the electronic sequence listing (INSO_008_01WO_SeqList_ST26.xml; Size: 175,270 bytes; and Date of Creation: August 20, 2025) are herein incorporated by reference in its entirety. TECHNICAL FIELD

[0003] The present disclosure relates to the field of soft and hard tissue matrix reconstructive procedures. BACKGROUND

[0004] Existing tissue matrices used in hard and soft tissue matrix repair, reconstruction, and / or augmentation have several limitations as they often require extensive processing, are difficult to acquire and lose their strength and shape over time. Given the growing popularity of tissue matrices in repair, reconstruction, and / or augmentation procedures, there is a need to improve upon commonly used tissue matrix products to improve their structural and biological properties as well as to expand the potential use of these products in plastic and reconstructive procedures. BRIEF SUMMARY

[0005] The present disclosure relates to a protein matrix composition for soft and hard tissue matrix repair, reconstruction, and / or augmentation.

[0006] According to embodiments, the present disclosure further relates to a matrix composition for replacement of tissue, comprising: a partially ordered polypeptide, and an allograft tissue matrix that is not derived from an adipose tissue, wherein the composition comprises from about 10% v / v to about 90% v / v partially ordered polypeptide.ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035

[0007] According to embodiments, the present disclosure further relates to a method of hard or soft tissue matrix repair, reconstruction, or augmentation in a subject, the method comprising mixing a volume of a partially ordered polypeptide and a volume of allograft tissue matrix that is not derived from an adipose tissue to form a matrix composition and administering a volume of the matrix composition to the subject.

[0008] According to embodiments, the present disclosure further relates to a use of a volume of a partially ordered polypeptide and a volume of allograft tissue matrix to replace hard or soft tissue in a subject.

[0009] According to embodiments, the present disclosure further relates to a matrix composition, comprising a polypeptide comprising a plurality of disordered domains and a plurality of ordered domains, wherein each disordered domain independently comprises a PG or GP motif and each ordered domain independently comprises a polyalanine motif and an allograft tissue matrix that is not derived from an adipose tissue.

[0010] According to embodiments, the present disclosure further relates to a method of hard or soft tissue matrix repair, reconstruction, or augmentation in a subject, the method comprising administering a volume of a partially ordered polypeptide to the subject, administering a volume of allograft tissue matrix that is not derived from an adipose tissue to the subject, wherein the administered volume of the allograft tissue matrix is co-located with the administered volume of the partially ordered polypeptide, thereby forming a matrix composition. BRIEF DESCRIPTION OF THE DRAWINGS

[0011] The accompanying figures, which are incorporated herein and form a part of the specification, illustrate some, but not the only or exclusive, example embodiments and / or features. It is intended that the embodiments and figures disclosed herein are to be considered illustrative rather than limiting.

[0012] FIG. 1 is a flow diagram of a method of preparing a matrix composition to be administered to a subject for tissue matrix repair, reconstruction, and / or augmentation.

[0013] FIG.2 is a flow diagram of a sub process of a method of preparing a matrix composition to be administered to a subject for tissue matrix repair, reconstruction, and / or augmentation.

[0014] FIGS. 3A-3C are images depicting the combination of allograft tissue matrices and partially ordered polypeptide (Fractomer™). FIG. 3A depicts syringe preparations of bone- derived matrix (BDM) and intestinal submucosa matrix (ISM), as exemplary allograft tissue matrices, and saline, as a control. BDM-based allograft tissue matrices were prepared byATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 dissolving X grams of bone matrix in a solvent. ISM-based allograft tissue matrices were prepared by dicing intestinal submucosa sheets into portions and suspending the diced portions within a solution. FIG. 3B depicts the addition of Fractomer™ to the syringe preparations of the allograft tissue matrices via a second syringe. The BDM preparation, the ISM preparation, and the saline preparation are shown from top to bottom. FIG.3C depicts the final mixtures of each preparation, yielding an injectable Fractomer™ / allograft tissue matrix.

[0015] FIG. 4 is a visualization of varying matrix compositions after aggregation of the FractomerTM. Fractomer™ was combined with allograft tissue matrices (BDM, ISM) are ratios of 50:50, 70:30, and 90:10 (FractomerTM:allograft tissue matrix). A 100% FractomerTMcomposition is provided for comparison. Prior to aggregation, each of the matrix compositions was extruded into cylindrical molds. The mold-bound matrix compositions were then incubated at body temperature for 10 minutes to permit FractomerTMaggregation. Upon removal of the BDM-based and ISM-based matrix compositions from their respective molds, visual analysis of the aggregated matrix compositions indicated good shape retention at each of the mixing ratios. Mixtures are shown as A:B where A is percentage of Fractomer™ and B is percentage of allograft by volume. A Fractomer™-only control is shown on the right.

[0016] FIG.5 depicts histological images of the 50:50 matrix compositions shown in FIG.4. Hematoxylin and eosin staining of the matrix compositions show that the Fractomer™ architecture is supplemented with allograft tissue matrices throughout the bulk material. A FractomerTMonly control is provided for reference. The bottom row of images are magnified portions of respective ones of the top row of images.

[0017] FIG. 6A and FIG. 6B depicts mechanical stiffness of each of the BDM-based matrix composition and the ISM-based matrix composition after aggregation. Adjusting the ratio of Fractomer™ to allograft tissue matrix can augment mechanical stiffness of the matrix composition. In each figure, the x-axis shows the percentage of allograft tissue matrix present relative to Fractomer™. As shown in FIG. 6A, ISM-based matrix compositions show increasing mechanical stiffness with decreasing Fractomer™ concentration, as measured by Youngs Modulus. As shown in FIG. 6B, BDM-based matrix compositions show decreasing mechanical stiffness with decreasing Fractomer™ concentration, as measured by Youngs Modulus. DETAILED DESCRIPTION

[0018] All publications, patents and patent applications, including any drawings and appendices, are herein incorporated by reference to the same extent as if each individualATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 publication or patent application was specifically and individually indicated to be incorporated by reference.

[0019] The following description includes information that may be useful in understanding the present disclosure. It is not an admission that any of the information provided herein is prior art or relevant to the presently claimed disclosures, or that any publication specifically or implicitly referenced is prior art. Definitions

[0020] 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. For example, any nomenclatures used in connection with, and techniques of, cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are well known and commonly used in the art. In case of conflict, the present disclosure, including definitions, will control. Exemplary methods and materials are described below, although methods and materials similar or equivalent to those described herein can be used in practice or testing of the embodiments and aspects described herein.

[0021] As used herein, the terms "amino acid," "nucleotide," "polynucleotide," "vector," 30 "polypeptide," and "protein" have their common meanings as would be understood by a biochemist of ordinary skill in the art. Standard single letter nucleotides (A, C, G, T, U) and standard single letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein.

[0022] As used herein, the terms "amino acid," "nucleotide," "polynucleotide," "vector," 30 "polypeptide," and "protein" have their common meanings as would be understood by a biochemist of ordinary skill in the art. Standard single letter nucleotides (A, C, G, T, U) and standard single letter amino acids (A, C, D, E, F, G, H, I, K, L, M, N, P, Q, R, S, T, V, W, or Y) are used herein.

[0023] As used herein, the term "a," "an," "the" and similar terms used in the context of the disclosure (especially in the context of the claims) are to be construed to cover both the singular and plural unless otherwise indicated herein or clearly contradicted by the context. In addition, "a," "an," or "the" means "one or more" unless otherwise specified.

[0024] As used herein, the term "about" or "approximately" as applied to one or more values of interest, refers to a value that is similar to a stated reference value, or within an acceptable error range for the particular value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, such as the limitations of theATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 measurement system. In one aspect, the term "about" refers to any values, including both integers and fractional components that are within a variation of up to ± 10% of the value modified by the term "about." As used herein, the symbolmeans "about" or "approximately."

[0025] All ranges disclosed herein include both end points as discrete values as well as all integers and fractions specified within the range. For example, a range of 0.1-2.0 includes 0.1, 0.2, 0.3, 0.4 ... 2.0. If the end points are modified by the term "about," the range specified is expanded by a variation of up to ±10% of any value within the range or within 3 or more standard deviations, including the end points.

[0026] As used herein, the terms "control," or "reference" are used herein interchangeably. A "reference" or "control" level may be a predetermined value or range, which is employed as a baseline or benchmark against which to assess a measured result. "Control" also refers to control experiments or control cells.

[0027] “Decellularization”, as used herein in all of its grammatical forms, is any process, including those known to the skilled artisan, by which cells and cellular components (including DNA) are removed from a tissue, thereby leaving the extracellular matrix (ECM) essentially free of such cells and cellular components.

[0028] “Delipidization” and “delipidation,” as used interchangeably herein in all of their grammatical forms, are any processes, including those known to the skilled artisan, by which lipids are removed from a tissue.

[0029] As used herein, the term "dose" denotes any form of an active ingredient formulation or composition, including cells, that contains an amount sufficient to initiate or produce a therapeutic effect with at least one or more administrations. "Formulation" and "composition" are used interchangeably herein.

[0030] The term “endogenous” as used herein refers to that which is naturally occurring in a tissue prior to its excision, explantation, or, generally, removal from a subject (e.g., a donor).

[0031] The term “exogenous” as used herein refers to that which is added to a matrix composition prior to or during injection or implantation into a subject (e.g., a patient).

[0032] The term “extracellular matrix” as used herein refers to a scaffold in a cell's external environment with which the cell interacts via specific cell surface receptors. The extracellular matrix serves many functions, including, but not limited to, providing support and anchorage for cells, segregating one tissue from another tissue, and regulating intracellular communication.ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035

[0033] As used herein, the terms "effective amount" or "therapeutically effective amount," refers to a substantially non-toxic, but sufficient amount of an agent, composition, or cell(s) being administered to a subject that will prevent, treat, or ameliorate to some extent one or more of the symptoms of the disease or condition being experienced or that the subject is susceptible to contracting. The result can be the reduction or alleviation of the signs, symptoms, or causes of a disease, or any other desired alteration of a biological system.

[0034] The term “growth factor” as used herein refers to extracellular polypeptide molecules that bind to a cell-surface receptor triggering an intracellular signaling pathway, leading to proliferation, differentiation, or other cellular response. Growth factors include, but are not limited to, cytokines and hormones.

[0035] The term “growth induction” as used herein refers to a process by which primitive, undifferentiated and tissuegenic cells are stimulated to develop into an ensemble of cells, not necessarily identical, that together carry out a specific function.

[0036] The term “growth-inductive matrix” as used herein refers to a matrix containing a substance or substances capable of recruiting or stimulating local tissuegenic cells so that the cells are induced (meaning to cause, bring about, bring about, or trigger) to differentiate and / or produce a tissue.

[0037] The terms “growth-inductive components” or “growth-inductive factors” or “tissuegenic factors” are used interchangeably to refer to the plethora of mediators associated with tissue development and repair.

[0038] The term “hematopoietic stem cell” refers to a cell isolated from the blood or from the bone marrow that can renew itself, differentiate to a variety of specialized cells, mobilize out of the bone marrow into the circulating blood, and undergo programmed cell death (apoptosis).

[0039] The term “mesenchymal stem cells (MSCs)” as used herein refers to non-blood adult stem cells found in a variety of tissues. They are characterized by their spindle-shape morphologically; by the expression of specific markers on their cell surface; and by their ability under appropriate conditions, to differentiate along a minimum of three lineages (osteogenic, chondrogenic and adipogenic).

[0040] As used herein, the term "subject" refers to an animal. Typically, the subject is a mammal. A subject also refers to primates (e.g., humans, male or female; infant, adolescent, or adult), non- human primates, rats, mice, rabbits, pigs, cows, sheep, goats, horses, dogs, cats, fish, birds, and the like. In one embodiment, the subject is a primate. In one embodiment, the subject is a human. As used herein, a subject is "in need of treatment" if such subject would benefit biologically, medically, or in quality of life from such treatment.ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035

[0041] As used herein, "treatment," "therapy" and / or "therapy regimen" refer to the clinical intervention made in response to a disease, disorder or physiological condition manifested by a patient or to which a patient may be susceptible. The aim of treatment includes the alleviation or prevention of symptoms, slowing or stopping the progression or worsening of a disease, disorder, or condition and / or the remission of the disease, disorder, or condition.

[0042] As used herein, "treatment" or "treating" refers to prophylaxis of, preventing, suppressing, repressing, reversing, alleviating, ameliorating, or inhibiting the progress of biological process including a disorder or disease, or completely eliminating a disease.

[0043] "Amino acid" as used herein refers to naturally occurring and non-natural synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code. Amino acids can be referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Amino acids include the side chain and polypeptide backbone portions.

[0044] The term "host cell" is a cell that is susceptible to transformation, transfection, transduction, conjugation, and the like with a nucleic acid construct or expression vector.

[0045] Host cells can be derived from plants, bacteria, yeast, fungi, insects, animals, etc. In some embodiments, the host cell includes Escherichia coli.

[0046] Polynucleotide" as used herein can be single stranded or double stranded or can contain portions of both double stranded and single stranded sequence. The polynucleotide can be nucleic acid, natural or synthetic, DNA, genomic DNA, cDNA, RNA, or a hybrid, where the polynucleotide can contain combinations of deoxyribo- and ribo-nucleotides, and combinations of bases including uracil, adenine, thymine, cytosine, guanine, inosine, xanthine hypoxanthine, isocytosine, and isoguanine. Polynucleotides can be obtained by chemical synthesis methods or by recombinant methods.

[0047] A "peptide" or "polypeptide" is a linked sequence of two or more amino acids linked by peptide bonds. The polypeptide can be natural, synthetic, or a modification or combination of natural and synthetic. Peptides and polypeptides include proteins such as binding proteins, receptors, and antibodies. The terms "polypeptide," "protein," and "peptide" are used interchangeably herein. "Primary structure" refers to the amino acid sequence of a particular peptide. "Secondary structure" refers to locally ordered, three dimensional structures within a polypeptide. These structures are commonly known as domains, e.g., enzymatic domains, extracellular domains, transmembrane domains, pore domains, and cytoplasmic tall domains,ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 "Domains" are portions of a polypeptide that form a compact unit of the polypeptide and are typically 15 to 350 amino acids long. Exemplary domains include domains with enzymatic activity or ligand binding activity. Typical domains are made up of sections of lesser organization such as stretches of beta-sheet and alpha-helices. "Tertiary structure" refers to the complete three- dimensional structure of a polypeptide monomer. "Quaternary structure" refers to the three- dimensional structure formed by the noncovalent association of independent tertiary units. A "motif" is a portion of a polypeptide sequence and includes at least two amino acids. A motif may be 2 to 20, 2 to 15, or 2 to 10 amino acids in length, in some embodiments, a motif includes 3, 4, 5, 6, or 7 sequential amino acids. A domain may be comprised of a series of motifs, which may be similar or different.

[0048] "Recombinant" when used with reference, e.g., to a cell, or nucleic acid, protein, or vector, indicates that the cell, nucleic acid, protein, or vector, has been modified by the introduction of a heterologous nucleic acid or protein or the alteration of a native nucleic acid or protein, or that the cell is derived from a cell so modified. Thus, for example, recombinant cells express genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise abnormally expressed, under expressed, or not expressed at all.

[0049] "Sample" or "test sample" as used herein can mean any sample in which the presence and / or level of a target is to be detected or determined or any sample comprising an agent, cell, or partially ordered polypeptide (POP) as described herein. Samples may include liquids, solutions, emulsions, or suspensions. Samples may include a medical sample. Samples may include any biological fluid or tissue, such as blood, whole blood, fractions of blood such as plasma and serum, muscle, interstitial fluid, sweat, saliva, urine, tears, synovial fluid, bone marrow, cerebrospinal fluid, nasal secretions, sputum, amniotic fluid, bronchoalveolar lavage fluid, gastric lavage, emesis, fecal matter, lung tissue, peripheral blood mononuclear cells, total white blood cells, lymph node cells, spleen cells, tonsil cells, cancer cells, tumor cells, bile, digestive fluid, skin, or combinations thereof. In some embodiments, the sample comprises an aliquot. In other embodiments, the sample comprises a biological fluid. Samples can be obtained by any means known in the art. The sample can be used directly as obtained from a patient or can be pre-treated, such as by filtration, distillation, extraction, concentration, centrifugation, inactivation of interfering components, addition of reagents, and the like, to modify the character of the sample in some manner as discussed herein or otherwise as is known in the art.ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035

[0050] The term “soft tissues” refers generally to non-calcified tissues from the mammalian body.

[0051] "Substantially identical" can mean that a first and second amino acid sequence are at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% over a region of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1100 amino acids.

[0052] "Variant" as used herein with respect to a polynucleotide means (i) a portion or fragment of a referenced nucleotide sequence; (ii) the complement of a referenced nucleotide sequence or portion thereof; (iii) a polynucleotide that is substantially identical to a referenced polynucleotide or the complement thereof; or (iv) a polynucleotide that hybridizes under stringent conditions to the referenced polynucleotide, complement thereof, or a sequences substantially identical thereto.

[0053] A "variant" can further be defined as a peptide or polypeptide that differs in amino acid sequence by the insertion, deletion, or conservative substitution of amino acids, but retain at least one biological activity. Representative examples of "biological activity" include the ability to be bound by a specific antibody or polypeptide or to promote an immune response. Variant can mean a substantially identical sequence. Variant can mean a functional fragment thereof. Variant can also mean multiple copies of a polypeptide. The multiple copies can be in tandem or separated by a linker. Variant can also mean a polypeptide with an amino acid sequence that is substantially identical to a referenced polypeptide with an amino acid sequence that retains at least one biological activity.

[0054] A variant can be a polynucleotide sequence that is substantially identical over the full length of the full gene sequence or a fragment thereof. The polynucleotide sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 88%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the gene sequence or a fragment thereof. A variant can be an amino acid sequence that is substantially identical over the full length of the amino acid sequence or fragment thereof. The amino acid sequence can be 80%, 81%, 82%, 83%, 84%, 85%, 88%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100% identical over the full length of the amino acid sequence or a fragment thereof.

[0055] As used herein, "shapeable" refers to the ability of the POP and tissue matrix composition to be shaped or molded into various two- or three-dimensional shapes, areas, or volumes and the ability to maintain this shape, area, or volume over a prolonged period of time.ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 Specific exemplary shapes or volumes include breasts, buttocks, hands, knees, 2-dimensional layers for placing under skin or skin grafts, or other irregular or indefinite shapes or volumes present in a subject's body. Matrix Composition

[0056] Embodiments described herein are directed to a matrix composition for tissue matrix repair, reconstruction, and / or augmentation. The matrix composition may be administered by injection or implantation, depending on the use case. In embodiments, the tissue matrix may be derived from allograft tissue, autograft tissue, or xenograft tissue. For example, the tissue matrix may be derived from allograft tissue and referred to as allograft tissue matrix. In embodiments, the matrix composition comprises a recombinant partially ordered polypeptide (POP) or "FractomerTM" and an allograft tissue matrix. The allograft tissue matrix is combined with POP to provide various allograft tissue matrix:POP ratios as described herein to provide a matrix composition. As an example, 900 μL of allograft tissue matrix combined with 100 μL of POP solution would be a 9:1 ratio by volume or 90% allograft tissue matrix by volume. In embodiments, the matrix composition improves the properties of the matrix composition compared to injections of the allograft tissue matrix alone, including, but not limited to volume and / or shape.

[0057] Described herein is a matrix composition for replacement of tissue, comprising a partially ordered polypeptide and an allograft tissue matrix, wherein the composition comprises from about 10% v / v to about 90% v / v POP. In embodiments, the matrix composition comprises about 50% v / v to about 90% v / v partially ordered polypeptide. Partially Ordered Polypeptide

[0058] The term "FractomerTM", “partially ordered polypeptide” and “POP” are used interchangeably herein to refer to the class of recombinant, artificial proteins that are designed to mimic native elastin. POP are thermally responsive, allowing them to be injected as a liquid, yet rapidly form a porous, solid network at body temperature. Alternatively, a POP can be prepared as a liquid, formed into a desired shape, and then heated to form a solid which is then implanted. Examples of POPs are described in International Patent Application Publication No. WO2019006374A1, which is incorporated by reference herein in its entirety.

[0059] Each POP may include a plurality of disordered domains, and a plurality of structured domains. The POP may exhibit phase transition behavior by changing solubility and aggregate dissolution / formation with temperature.ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 i. Disordered Domains

[0060] The POP may include a plurality of disordered domains. The disordered domain may comprise any polypeptide that has minimal or no secondary structure as observed by CD and have phase transition behavior. The disordered domain may include an amino acid sequence of repeated amino acids, non-repeated amino acids, or a combination thereof.

[0061] The disordered domains and the structured domains of the POP can be arranged in any number of possible ways, in some embodiments, one or more disordered domains are positioned between at least two adjacent structured domains of the POP. In some embodiments, the POP includes a plurality of structured domains repeated in tandem and a plurality of disordered domains repeated in tandem, in some embodiments, the plurality of structured domains repeated in tandem are positioned C-terminal to the plurality of disordered domains repeated in tandem, in some embodiments, the plurality of structured domains repeated in tandem are positioned N-terminal to the plurality of disordered domains repeated in tandem. In some embodiments, the POP is arranged as [disordered domain]q- [structured domain]r - [disordered domain]s -[structured domain]t, wherein q, r, s, and t are independently an integer from 0 to 100, such as from 1 to 100, from 2 to 100, from 1 to 50 or from 2 to 50. In some embodiments, the POP is arranged as [disordered domain]q-[structured domain]r, wherein q and r are independently an integer from 1 to 100. in some embodiments, q, r, s, and t are independently an integer from 0 to 10, from 0 to 20, from 0 to 30, from 0 to 40, from 0 to 50, from 0 to 60, from 0 to 70, from 0 to 80, from 0 to 90, from 0 to 100, from 1 to 10, from 1 to 20, from 1 to 30, from 1 to 40, from 1 to 150, from 1 to 60, from 1 to 70, from 1 to 80, from 1 to 90 or from 1 to 100.

[0062] In some embodiments, the disordered domain independently comprises a PG or GP motif. In some embodiments, each disordered domain comprises an amino acid sequence of (GXGVP)n(SEQ ID NO: 1), wherein X is any amino acid except proline and n is an integer greater than or equal to 1, and each ordered domain comprises a polyalanine motif. In some embodiments, each disordered domain comprises an amino acid sequence of (GXGVP)n (SEQ ID NO: 2), wherein each X is independently Val or Ala, and wherein n is an integer from 1 to 50. In some embodiments, each disordered domain comprises an amino acid sequence of (GXGVP)n (SEQ ID NO: 27 or SEQ ID NO: 28), wherein each X is independently Val or Ala, and wherein n is an integer from 1 to 50, wherein a ratio of Ala to Val in the amino acid sequence of (GXGVP)n ranges from 10:1 to 1:10. In some embodiments, each disordered domain comprises an amino acid sequence of (GXGVP)n (e.g., SEQ ID NO: 27 or SEQ ID NO: 28), wherein X is any amino acid except proline and n is an integer greater than or equal to 1,ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 and each ordered domain comprises a polyalanine motif, wherein the polyalanine motif comprises (A)m (SEQ ID NO: 29) wherein m is an integer from 5 to 50. In some embodiments, the polyalanine motif comprises one or more of (A)n (SEQ ID NO: 3), K(A)nK (SEQ ID NO: 4), D(A)nK (SEQ ID NO: 5), GD(An)K (SEQ ID NO: 6), or GK(An)K (SEQ ID NO: 7), wherein n is an integer from 2 to 100. In some embodiments, the polyalanine motif comprises one or more of (A)25(SEQ ID NO: 8), K(A)25K (SEQ ID NO: 9), D(A)25K (SEQ ID NO: 10), GD(A25)K (SEQ ID NO: 11); or GK(A25)K (SEQ ID NO: 12).

[0063] A POP comprising structured domain of oligoalanine amino acids (from 5 to 500, but typically A25) that form a-helices and are periodically inserted into an unstructured elastin-like polypeptide (ELP) that is composed of typically 80-120 total repeats of a (GXGVP)n pentapeptide motif (~30-50 kDa) (SEQ ID NO: 1), where X is any standard amino acid except proline. In some embodiments, the unstructured polypeptide or “disordered domain’’ is a (GXGVP)n motif (SEQ ID NO: 2), wherein X is Val (SEQ ID NO: 25), or Ala (SEQ ID NO: 26), or mixture of Ala and Val, as in SEQ ID NOs: 27 and 28, and wherein n is an integer from 1 to 50. In some embodiments, X is an alternating iteration of Ala and Val in a ratio from 10:1 to 1:10 (Ala:Val). In some embodiments, X is an alternating iteration of Ala and Val in a ratio of 1:1 (SEQ ID NO: 27) or 1:4 (SEQ ID NO: 28). POPs are recombinantly synthesized in E. coli by overexpression of a plasmid-borne gene that encodes the POP.

[0064] In some embodiments, about 20% to about 99%, such as about 25% to about 97%, about 35% to about 95% or about 50% to about 94% of the POP comprises disordered domains. At least about 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the POP may comprise disordered domains.

[0065] In some embodiments, the disordered domain comprises an amino acid sequence of (GXGVP)n(SEQ ID NO: 1), wherein X is any amino acid and n is an integer greater than or equal to 1. In some embodiments, mis an integer from 1 to 500. In some embodiments, is at least, at most, or exactly 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145, 150, 155, 160, 165, 170, 175, 180, 185, 190, 195, 200, 205, 210, 215, 220, 225, 230, 235, 240, 245, 250, 255, 260, 265, 270, 275, 280, 285, 290, 295, 300, 305, 310, 315, 320, 325, 330, 335, 340, 345, 350, 355, 360, 365, 370, 375, 380, 385, 390, 395, 400, 405, 410, 415, 420, 425, 430, 435, 440, 445, 450, 455, 460, 465, 470, 475, 480, 485, 490, 495, or 500. In some embodiments, may be less than 500, less than 400, less than 300, less than 200, or less than 100. In some embodiments, mis from 1 to 500, from 1 to 400, from 1 to 300, from 1 to 200, or from 60 to 180. In some embodiments, m is 60, 120, or 180. In some embodiments, X is anyATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 amino acid except praline. In some embodiments, X is Val, or Ala, or an alternating iteration of Ala and Val. In some embodiments, X is Val. In some embodiments, X is Ala. in some embodiments, X is an alternating iteration of Ala and Val. In some embodiments, X is an alternating iteration of Ala and Val in a ratio of 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, 1:10, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1. In some embodiments, X is a mixture of Ala and Val in a ratio of 1:1 or 1:4. In some embodiments, X is an alternating iteration of Ala and Val in a ratio from 10:1 to 1:10 (Ala:Val), such as from 5:1 to 1:5 or from 1:1 to 1:4. ii. Structured Domains

[0066] The POP may include a plurality of structured domains. The structured domain may have a secondary structure as observed by CD, such as, for example, an alpha helix. The structured domain may comprise at least one of a polyproline domain and a polyalanine domain, in some embodiments, the POP comprises alternating disordered domains and structured domains. In some embodiments, the structured domain comprises only polyalanine domains. In some embodiments, the structured domain comprises only polyproline domains.

[0067] In some embodiments, about 4% to about 75%, such as about 5% to about 70%, about 6% to about 60% or about 7% to about 50% of the POP comprises structured domains. At least about 2%, 3%, 4%, 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the POP may comprise structured domains.

[0068] In some embodiments, the structured domain comprises a polyalanine domain. Each polyalanine domain may include at least 4 or at least 5 alanine residues (e.g., 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 150, 200, 250, 300, 350, 400, 450, 500, including all values and ranges therein). Each polyalanine domain may have at least about 60% of the amino acids in an alpha-helical conformation. In some embodiments, the structured domain comprises a polyalanine domain (A)m(SEQ ID NO: 29) wherein m is an integer from 5 to 500. In some embodiments, the polyalanine domain comprises of one or more of: (A)25 (SEQ ID NO: 8); K(A)25K (SEQ ID NO: 9); D(A)25K (SEQ ID NO: 10); GD(A25)K (SEQ ID NO: 11); or GK(A25)K (SEQ ID NO: 12).

[0069] In one embodiment, the POP comprises a plurality of disordered domains, each comprising a PG motif comprising an amino acid sequence selected from PG, P(X)nG (SEQ ID NO: 21), and (B)mP(X)nG(Z)p (SEQ ID NO: 22), or a combination thereof, wherein m, n, and p are independently an integer from 1 to 15, and wherein B, X, and Z are independently any amino acid, and a plurality of structured domains, each comprising a polyalanine domain, each polyalanine domain comprising at least 5 alanine residues and having at least about 50% of the amino acids in an a-helical conformation, wherein the POP exhibits phase transitionATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 behavior. In one embodiment, at least one disordered domain comprises an amino acid sequence of (GXGVP)n (SEQ ID NO:1), wherein X is any amino acid except proline and n is an integer greater than or equal to 1. In another embodiments, at least about 60% of the amino acids in each polyalanine domain are in an a-helical conformation. In another embodiment, each polyalanine domain comprises an amino acid sequence of [Bp(A)qZr]n(SEQ ID NO: 23) or [(BAs)tZr]n(SEQ ID NO: 24), wherein B is Lys, Arg, Asp, or Glu; A is Ala; Z is Lys, Arg, Asp, or Glu; n is an integer from 1 to 50; p is an integer from 0 to 2; q is an integer from 1 to 50; r is an integer from 0 to 2; s is an integer from 1 to 5; and t is an integer from 1 to 50. In another embodiment, the structured domain comprises one or more of (A)25(SEQ ID NO: 8); K(A)25K (SEQ ID NO: 9); D(A)25K (SEQ ID NO: 10); GD(A25)K (SEQ ID NO: 11); or GK(A25)K (SEQ ID NO: 12). In another embodiment, about 4% to about 75% of the POP comprises structured domains. In embodiments, each ordered domain comprises a polyalanine motif comprising at least 4 alanine residues. In embodiments, the at least 4 alanine residues are consecutive. In embodiments, the at least about 50% of the amino acids in each polyalanine motif are in an alpha-helical conformation. In embodiments, the at least about 90% of the amino acids in each polyalanine motif are in an alpha-helical conformation. In embodiments, the at least about 50% of the amino acids in the polyalanine motif are alanine residues.

[0070] In another embodiment, the FractomerTMcomprises a plurality of disordered domains, and a plurality of structured domains. In one embodiment, the FractomerTMhas the general structure of [(SEQ ID NO: 1)n-a-helix]m(SEQ ID NO: 30), where n is an integer equal to or greater than 1, m is an integer equal to or greater than 1, and a-helix is any polyalanine based a-helix having about 5 to 50 alanine residues. In another embodiment, the FractomerTMhas the structure [(SEQ ID NO: 2)n-GX1(A)25X1]m(SEQ ID NO: 31), where X1is K or D; n is an integer from 10 to 20; and m is an integer from 4 to 8 (e.g., [(SEQ ID NO: 2)n-(SEQ ID NO: 11 or 12)]m). In another embodiment, the FractomerTMcomprises one or more of the following structures:

[0071] M[(GVGVP)15-GD(A25)K]6-GWP (SEQ ID NO: 13);

[0072] M[(GVGVP)15-GD(A25)K]4-GWP (SEQ ID NO: 14);

[0073] M[(GVGVP)15-GK(A25)K]6-GWP (SEQ ID NO: 15);

[0074] M[(GVGVP)15-GK(A25)K]4-GWP (SEQ ID NO: 16);

[0075] M[(G[A1:V1]GVP)16-GD(A25)K]6-GWP (SEQ ID NO: 17);

[0076] M[(G[A1:V1]GVP)16-GD(A25)K]4-GWP (SEQ ID NO: 18);

[0077] M[(G[V4:A1]GVP)15-GD(A25)K]6-GWP (SEQ ID NO: 19); or

[0078] M[(G[V4:A1]GVP)15-GD(A25)K]4-GWP (SEQ ID NO: 20).ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035

[0079] In one embodiment, the FractomerTMcomprises the following structures:

[0080] M[(GVGVP)15-GD(A25)K]6-GWP (SEQ ID NO: 13); or

[0081] M[(G[V4:A1]GVP)15-GD(A25)K]6-GWP (SEQ ID NO: 19).

[0082] A complete listing of exemplary sequences, sequence motifs, and POP constructs is provided herein.

[0083] The POP may also include amino acid derivatives that are not naturally occurring, such as a UV crosslinkable amino acid derivative. The non-native amino acid derivative can be used to introduce covalent crosslinks between different POPs and within the same POP. For example, POPs that include the UV crosslinkable amino acid derivative can be exposed to UV light, which can result in covalent crosslinks being formed between the amino acid derivative and a side chain of an amino acid of another POP or with a side chain of an amino acid of the same POP (having the amino acid derivative). The UV crosslinkable amino acid derivative may be any amino acid that has been functionalized with an azide group. In some embodiments, the amino acid derivative is para-azidophenylalanine.

[0084] The UV crosslinkable amino acid derivative may be included at varying amounts without affecting the POP's ability to transition at different temperatures. For example, the UV crosslinkable amino acid derivative may be included within the POP from about 0.1% to about 20% (of the POP), such as from about 0.5% to about 15% or from about 1% to about 10% (of the POP).

[0085] The POP alone, without the presence of tissue matrix, may form aggregates, a three- dimensional matrix, a semi-solid or a gel. The following discussion of “aggregate” refers to the aggregate formed by POP alone.

[0086] The POP may demonstrate phase transition behavior by changing solubility and aggregate formation with temperature. The phase transition behavior of the POP may derive from the phase transition behavior of the disordered domains of the POP. "Phase transition" or "transition" may refer to the aggregation of a polypeptide, which occurs sharply at a specific temperature. The phase transition may be reversible, although the specific temperature of dissolution may be the same or different from the specific temperature of aggregation.

[0087] In some embodiments, the POP is soluble below a lower critical solution temperature (LCST). LCST is the temperature below which the polypeptide is miscible.

[0088] A transition temperature (Tt) is a temperature at which the POP changes from one state to another. States may include, for example, soluble polypeptides, gels, and aggregates of varying sizes and dimensions. The POP may have a transition temperature of heating (Tt- heating) and a transition temperature of cooling (Tt-cooling). In some embodiments, theATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 transition temperature heating (Tt-heating) is concentration-dependent. In some embodiments, the transition temperature cooling (Tt-cooling) is concentration-independent. The Tt-heating may be primarily determined by the disordered domains. The Tt-cooling may be primarily determined by the structured domains.

[0089] Below the transition temperature (LCST or Tt), the POP may be highly soluble. Upon heating above the transition temperature, the POP may hydrophobically collapse and aggregate, forming a separate phase.

[0090] In some embodiments, the POP is soluble below a lower critical solution temperature (LCST). In some embodiments, the transition temperature of heating (Tt-heating) and transition temperature of cooling (Tt-cooling) are identical, or the transition temperature of heating (Tt- heating) is greater than the transition temperature of cooling (Tt-cooling). In some embodiments, the Tt-heating may be dependent on the concentration of the POP in the composition. The Tt-heating and the Tt-cooling may range from about 10 °C to about 45 °C. In some embodiments the Tt-heating and the Tt-cooling may range from about 10 °C to about 40 °C, about 15 °C to about 35 °C, and / or about 20 °C to about 30 °C. In some embodiments, the partially ordered polypeptide forms a solid aggregate above the Tt-heating.

[0091] The POP may phase transition at a variety of temperatures. The POP may have a transition temperature (Tt) from about 0 °C to about 100 °C, from about 10 °C to about 50 °C, or from about 20 °C to about 42 °C. The transition temperature of heating (Tt-heating) and transition temperature of cooling (Tt-cooling) may be identical. As used herein, temperatures may be "identical" when the temperatures are within 2.0 °C, 1.0 °C, 0.5 °C, or 0.1 °C of each other. In some embodiments, the transition temperature of heating (Tt-heating) is greater than the transition temperature of cooling (Tt-cooling). In embodiments where the POP has a Tt- heating greater than the Tt-cooling, the difference between the two transition temperatures may be referred to as a hysteresis, in some embodiments, the POP has a hysteresis of about 5 °C to about 70 °C, such as about 5 °C to about 60 °C or about 10 °C to about 50 °C.

[0092] The phase transition behavior of the POP may be utilized in purification of the POP according to a method referred to as "inverse transition cycling," in which the POP's reversible phase transition behavior is used to cycle the solution through soluble and insoluble phases, thereby removing contaminants. Phase transition may also be triggered using kosmotropic salts, such as, for example, ammonium sulfate or sodium chloride. The kosmotropic salt may be added to a solution comprising the POP, with the kosmotropic salt being added until the POP forms aggregates or is precipitated out of solution. The aggregates may be pelleted by centrifugation and resuspended in a second solution or buffer. Aggregates of the POP may re-ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 solubilize into solution once cooled below their Tt or when the kosmotropic salt is removed from the solution. In some embodiments, the POP is purified without any chromatographic purification. In some embodiments, the POP is generated recombinantly and purified from bacterial culture, such as, for example, from E. coli.

[0093] In some embodiments, the POP may form an aggregate when the temperature is greater than the Tt-heating. The aggregate may resolubilize when cooled to below a temperature less than the Tt-cooling.

[0094] The aggregate formed from a plurality of POPs may be a variety of sizes and dimensions. In some embodiments, the aggregate is a stable three-dimensional matrix. In some embodiments, the aggregate is fractal-like, in some embodiments, the aggregate is gel-like, in some embodiments, the aggregate is porous with a void volume, e.g., the nonprotein rich phase of the aggregate. In some embodiments, the void volume is tunable. For example, the aggregate may have a void volume from about 60% to about 90% (of the volume of the aggregate), in addition, the aggregate may comprise pores having a diameter of about 1 μm to about 100 μm, such as about 1 μm to about 10 μm, about 3 μm to about 5 μm, about 25 μm to about 60 μm, about 30 μm to about 50 μm, or about 3 μm to about 50 μm. In some embodiments, the aggregate may comprise pores having a diameter of about 1 pm to about 100 pm, such as about 1 pm to about 10 pm, about 3 pm to about 5 pm, about 25 pm to about 60 pm, about 30 pm to about 50 pm, or about 3 pm to about 50 pm.

[0095] In some embodiments, the solid aggregate is a stable three-dimensional matrix. This three-dimensional matrix may encapsulate the tissue matrix. In some embodiments, the solid aggregate comprises a plurality of micropores. In some embodiments, the composition comprises between about 200 μM and about 2.5 mM of the POP. In some embodiments, the composition comprises between about 300 μM and about 2.4 mM, about 400 μM and about 2.3 mM, about 500 μM and about 2.2 mM, about 600 μM and about 2.1 mM, about 700 μM and about 2.0 mM, about 800 μM and about 1.9 mM, about 900 μM and about 1.8 mM, about 1.0 mM and about 1.7 mM, about 1.1 mM and about 1.6 mM, about 1.2 mM and about 1.5 mM, or about 1.3 mM and about 1.4 mM.

[0096] The aggregate formed by a plurality of POPs may have advantageous properties that can arise from the structure of the POPs. For example, the aggregate may have physical, non- covalent crosslinks. These physical, non-covalent crosslinks may arise from helical bundling of the structured domain(s) interacting with each other. The aggregate may also have covalent crosslinks (e.g., chemical crosslinks) in addition to physical, non-covalent crosslinks. Covalent crosslinks can be included in the aggregate in order to increase their mechanical stabilityATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 without altering their porous architecture, in some embodiments, the aggregate can be formed from a plurality of POPs and can then be further stabilized by covalent crosslinking (after the formation of the aggregate). Covalent crosslinks can be introduced via a UV crosslinkable amino acid derivative having an azide functionality as described herein. Further examples of crosslinks that can be incorporated into the aggregate include, but are not limited to, small molecule crosslinks and cysteine disulfide bridges. An example of a chemical, small molecule crosslink is tetrakis(hydroxymethyl)phosphonium chloride (TMPC), which can crosslink lysines within POPs.

[0097] In addition, the aggregate formed by a plurality of POPs may have solid-like properties that distinguish it from liquid-like coacervate structures. For example, the aggregate may have a storage modulus (C) that is greater than its loss modulus (G"), such as having a G' 2* greater, 5x greater, 10x greater, 15x greater, 20x greater, 25x greater, 30xgreater, 35x greater, 50x greater or 100x greater than its G”. In some embodiments, the aggregate has a G' from 2x greater to 100x greater than its G", such as from 10x greater to 50x greater or from 20x greater to 35x greater than its G”.

[0098] Further provided are polynucleotides encoding the POPs described herein. A vector may include the polynucleotide encoding the POPs detailed herein. To obtain expression of a polypeptide, one may subclone the polynucleotide encoding the polypeptide into an expression vector that contains a promoter to direct transcription, a transcription / translation terminator, and if for a nucleic acid encoding a protein, a ribosome binding site for translational initiation. An example of a vector is pET24. Suitable bacterial promoters are well known in the art. Further provided is a host cell transformed or transfected with an expression vector comprising a polynucleotide encoding a POP as described herein. Bacterial expression systems for expressing the protein are available in, e.g., E. coli, Bacillus species., and Salmonella. See Paiva et al., Gene 22: 229-235 (1983); Mosbach et al., Nature 302: 543-545 (1983). Kits for such expression systems are commercially available. Eukaryotic expression systems for mammalian cells, yeast, and insect cells are well known in the art and are also commercially available. Retroviral expression systems can be used in the present invention.

[0099] The POP may be expressed recombinantly in a host cell according to one of skill in the art. The POP may be purified by any means known to one of skill in the art. For example, the POP may be purified using chromatography, such as liquid chromatography, size exclusion chromatography, or affinity chromatography, or a combination thereof, in some embodiments, the POP is purified without chromatography, in some embodiments, the POP is purified using inverse transition cycling.ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035

[0100] In other embodiments, the present disclosure further provides herein a scaffold comprising a plurality of POPs. The scaffold may be formed at a temperature greater than the transition temperature of the POP, such that the polypeptide forms an aggregate. The scaffold may be injectable.

[0101] Further provided in accordance with one embodiment is a cellular scaffold. A cellular scaffold includes the scaffold and a plurality of cells. The cells may include a variety of types. In some embodiments, the cells comprise stem cells, bacterial cells, or human tissue cells, or a combination thereof.

[0102] The scaffold may have low immunogenicity or low antigenicity or both. The scaffold may promote at least one of cell growth, cell recruitment, and cell differentiation, or a combination thereof. The scaffold, or cellular scaffold, may be suitable for cell transplantation, tissue regeneration, cell culture, and cell-based in vitro assays. In addition, the scaffold and / or cellular scaffold may promote the formation of vasculature, wound healing, or a combination thereof.

[0103] Further provided in accordance with one embodiment is a drug delivery composition. The drug delivery composition may include a plurality of POPs as detailed herein, self- assembled into an aggregate above the Tt-heating, and an agent encapsulated within the aggregate. In some embodiments, the POPs could have different structures.

[0104] In embodiments, the tissue matrix compositions described herein comprise the POP in a volume of about 10% v / v to about 100% v / v (e.g. 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% v / v, including any values or ranges therein). In embodiments, the POP is present at a volume comprising about 10% v / v to about 100% v / v, about 20% v / v to about 100% v / v, about 30% v / v to about 100% v / v, about 40% v / v to about 100% v / v, about 50% v / v to about 100% v / v, about 60% v / v to about 100% v / v, about 70% v / v to about 100% v / v, about 80% v / v to about 100% v / v, about 90% v / v to about 100% v / v, about 10% v / v to about 80% v / v, about 20% v / v to about 80% v / v, about 30% v / v to about 80% v / v, about 40% v / v to about 80% v / v, about 50% v / v to about 80% v / v, about 60% v / v to about 80% v / v, about 70% v / v to about 80% v / v, about 10% v / v to about 60% v / v, about 20% v / v to about 60% v / v, about 30% v / v to about 60% v / v, about 40% v / v to about 60% v / v, about 50% v / v to about 60% v / v, about 10% v / v to about 40% v / v, about 20% v / v to about 40% v / v, about 30% v / v to about 40% v / v, or about 10% v / v to about 20% v / v, including all ranges and values therein. In embodiments, the tissue matrix compositions described herein comprise the POP in a volume of about 10% v / v to about 90% v / v. In embodiments, the tissue matrix compositions described herein comprise the POP in a volume of about 50% v / v to about 90% v / v.ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 Extracellular Matrix

[0105] In multicellular organisms, cells that are specialized to perform common functions are usually organized into cooperative assemblies embedded in a complex network of secreted extracellular macromolecules (i.e., the extracellular matrix (ECM)), to form specialized tissue compartments. Individual cells in such tissue compartments are in contact with ECM macromolecules. The ECM helps hold the cells and compartments together and provides an organized lattice or scaffold within which cells can migrate and interact with one another.

[0106] The ECM is an intricate network of secreted extracellular macromolecules that largely fills the extracellular space in the tissue compartments and comprises large polymeric complexes of glycosaminoglycans (GAGs) and proteoglycans. GAGs are negatively charged unbranched polysaccharide chains comprising repeating disaccharide units. Each repeating disaccharide unit of a GAG chain contains an amino sugar (e.g., N-acetyl glucosamine), which in most cases is sulfated, and an -uronic acid (e.g., glucuronic or iduronic acid). Four main types of GAG molecules are distinguished based on sugar residues, type of linkage, number and location of sulfate groups: (1) hyaluronan; (2) chondroitan sulfate and dermatan sulfate; (3) heparan sulfate and heparin; and (4) keratin sulfate.

[0107] GAG chains are inflexible and tend to adopt extended conformations occupying a huge volume relative to their mass, forming gels even at low concentrations. Their high density of negative charges attracts cations, such as Na+, that are effective in osmotic absorption of large amounts of water into the matrix. This creates high turgor enabling the ECM to withstand compressive forces.

[0108] Hyaluronan (also termed hyaluronic acid or hyaluronate) (HA), which comprises a regular repeating sequence of up to 25,000 nonsulfated disaccharide units, serves many functions, many of which depend on the binding of HA-binding proteins and proteoglycans, which are either themselves constituents of the ECM or are integral constituents of cell surfaces. For example, HA resists compressive forces in joints as a major constituent of joint fluid serving as a lubricant; serves as a space filler during embryonic development; creates a cell-free space in the epithelial compartment to allow cell migration during the formation of heart, cornea, and other organs; and plays a role in wound repair. Excess HA is usually degraded by hyaluronidase.

[0109] All GAGs, except for HA, are covalently linked to proteins in the form of proteoglycans. During their synthesis, the polypeptide chain of proteoglycans is synthesized on membrane-bound ribosomes and threaded into the lumen of endoplasmic reticulum, from which they are sorted in the Golgi apparatus, and assembled with polysaccharide chains. WhileATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 still in the Golgi, proteoglycans undergo a series of sequential and coordinated sulfation and epimerization reactions to produce sulfated proteoglycans. Sulfated and nonsulfated proteoglycans then travel through the Golgi network and are ultimately secreted into the ECM by exocytosis with the help of secretory vesicles.

[0110] Proteoglycans are heterogenous molecules, with core proteins ranging in molecular weight from 10 kD to about 600 kD and with attached GAG chains varying in number and type, further modified by a complex variable pattern of sulfate groups. At least one of the proteoglycan sugar side chains is a GAG; the core protein is usually a glycoprotein, but may comprise up to 95% carbohydrate by weight, mostly as long unbranched GAG chains up to at least 80 sugar residues long.

[0111] Proteoglycans along with their attached GAG chains regulate the activities of secreted macromolecules. They can serve as selective molecular sieves regulating a size-based trafficking of molecules and cells, and play a role in cell-cell signaling. Proteoglycans modulate the activities of secreted factors, such as growth factors and cytokines, by binding to them For example, binding of fibroblast growth factor (FGF) to heparan sulfate chains of proteoglycans is required for FGF activation of its cell surface receptors. On the other hand, for example, binding of a ubiquitous growth regulatory factor, such as transforming growth factor (TGF- ) to core proteins of several ECM proteoglycans, such as decorin, results in inhibition of TGF- activity. Proteoglycans also bind and regulate the activities of other types of secreted proteins, such as proteases and protease inhibitors. Cell-surface proteoglycans also may act as co- receptors: for example, syndecan binds to FGF and presents it to the FGF-receptor. Similarly,betaglycan binds to TGF- and presents it to TGF- receptors.

[0112] Collagens and elastin are the major fibrous proteins of the ECM. Collagens comprise a family of highly characteristic fibrous proteins and are a major component of skin and bone. Collagen fibers consist of globular units of the collagen subunit tropocollagen. Each tropocollagen subunit molecule comprises three polypeptide chains, called a chains, each exhibiting a left-handed helical conformation that are wrapped around each other in a right- handed coiled coil structure, also called a triple helix or super helix. A characteristic feature of collagen is a repeating tripeptide unit comprising Glycine-Proline-X or Glycine-X- Hydroxyproline, where X may be any amino acid. The presence of glycine at every third position in a collagen unit is critical for maintaining the coiled coil structure, since each repeating glycine residue sits on the interior axis of the helix, which sterically hinders bulkier side chains. Prolines and hydroxyprolines help stabilize the triple helix. Collagen is secreted as procollagen molecules, which undergo proteolytic processing and subsequent assembly to formATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 collagenous fibrils. Collagens are highly glycosylated during protein trafficking through intracellular secretory pathways.

[0113] Collagens are classified into various types depending on the nature of their -chains. A lists types of collagen, composition, class and distribution can be found in Shoulders and Raines, Annu. Rev. Biochem. 2009, 78: 929-958 and Bailey's Textbook of Microscopic Anatomy, Kelly et al., Williams and Wilkins, 18thedition, 1984.

[0114] A network of elastic fibers in the ECM offers resilience and elasticity so that organs are able to recoil following transient stretch. Elastic fibers primarily comprise the fibrous protein elastin, a highly hydrophobic protein about 750 amino acids in length that is rich in proline and glycine, is not glycosylated and is low in hydroxyproline and hyroxylysine. Elastin molecules are secreted into the ECM and assemble into elastic fibers close to the plasma membrane. Upon secretion, elastin molecules become highly cross-linked to form an extensive network of fibers and sheets.

[0115] The ECM also comprises many non-collagen adhesive proteins, usually with multiple domains containing binding sites of other macromolecules and for cell-surface receptors. One such adhesive protein, fibronectin, is a large glycoprotein comprising two subunits joined by a pair of disulfide bonds near the carboxy termini. Each subunit is folded into a series of rod-like domains interspersed by regions of flexible polypeptide chains. Each domain further comprises repeating modules of various types. One major type of fibronectin repeating module, called type III fibronectin repeat, is about 90 amino acids in length and occurs at least 15 times in each subunit. Fibronectin type III repeats have characteristic Arg-Gly-Asp (RGD) tripeptide repeats that function as binding sites for other proteins such as collagen, heparin or cell surface receptors. Fibronectin not only plays an important role in cell adhesion to the ECM, but also in guiding cell migration in vertebrate embryos.

[0116] Laminin, another adhesive glycoprotein of the ECM, is a major constituent (along with type IV collagen and another glycoprotein, nidogen / entactin) of the basal lamina, a tough sheet of ECM formed at the base of epithelial cells. Laminin is a large flexible complex, about 850 kD in molecular weight, with three very long polypeptide chains arranged in the form of an asymmetric cross held together with disulfide bonds. Laminin contains numerous functional domains, e.g., one binds to type IV collagen, one to heparan sulfate, one to entactin, and two or more to laminin receptor proteins on the cell surface. Stem Cells

[0117] In embodiments, stem cells can be added to the matrix composition.ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035

[0118] In embodiments, the stem cells comprise Embryonic stem cells (EmSC). EmScs are stem cells derived from an embryo that are pluripotent (i.e., they are able to differentiate in vitro into endodermal, mesodermal and ectodermal cell types).

[0119] In embodiments, the stem cells comprise Hematopoietic stem cells. Hematopoietic stem cells (also known as the colony-forming unit of the myeloid and lymphoid cells (CFU-M,L), or CD34+ cells) are rare pluripotential cells within the blood-forming organs that are responsible for the continued production of blood cells during life. While there is no single cell surface marker exclusively expressed by hematopoietic stem cells, it generally has been accepted that human HSCs have the following antigenic profile: CD 34+, CD59+,Thy1+(CD90), CD38low / , C-kit / low and, lin . CD45 is also a common marker of HSCs,except platelets and red blood cells. HSCs can generate a variety of cell types, including erythrocytes, neutrophils, basophils, eosinophils, platelets, mast cells, monocytes, tissue macrophages, osteoclasts, and the T and B lymphocytes. The regulation of hematopoietic stem cells is a complex process involving self-renewal, survival and proliferation, lineage commitment and differentiation and is coordinated by diverse mechanisms including intrinsic cellular programming and external stimuli, such as adhesive interactions with the micro- environmental stroma and the actions of cytokines.

[0120] In embodiments, the stem cells comprise Mesenchymal stem cells (MSCs). MSCs comprise bone marrow stromal stem cells or skeletal stem cells. In embodiments, the MSCs are non-blood adult stem cells found in a variety of tissues. Exemplary cell surface markers for MSCs include but are not limited to CD105, CD166, CD90, and CD44 and that MSCs are negative for typical hematopoietic antigens, such as CD45, CD34, and CD14.

[0121] In embodiments, the stem cells are skin stem cells. In embodiments, the stem cells are from skin tissue. In embodiments, skin stem cells comprise skin-derived stem cells. In embodiments, skin stem cells comprises pluripotent stem cells, MSCs, skin progenitors and stroma obtained from skin tissue. In embodiments, the stem cells are bone stem cells. In embodiments, the stem cells are from bone tissue. In embodiments, bone stem cells comprise bone-derived stem cells. In embodiments, bone stem cells comprise pluripotent stem cells, MSCs, bone progenitors and stroma obtained from bone tissue. In embodiments, the stem cells are cartilage stem cells. In embodiments, the stem cells are from cartilage tissue. In embodiments, cartilage stem cells comprise cartilage-derived stem cells. In embodiments, cartilage stem cells comprise pluripotent stem cells, MSCs, cartilage progenitors and stroma obtained from cartilage tissue. In embodiments, the stem cells are nerve stem cells. In embodiments, the stem cells are from nerve tissue. In embodiments, nerve stem cells compriseATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 nerve-derived stem cells. In embodiments, nerve stem cells comprise pluripotent stem cells, MSCs, nerve progenitors and stroma obtained from nerve tissue. In embodiments, the stem cells are from muscle tissue. In embodiments, muscle stem cells comprise muscle-derived stem cells. In embodiments, muscle stem cells comprise pluripotent stem cells, MSCs, muscle progenitors and stroma obtained from muscle tissue. In embodiments, the stem cells are from ligament tissue. In embodiments, ligament stem cells comprise ligament-derived stem cells. In embodiments, ligament stem cells comprise pluripotent stem cells, MSCs, ligament progenitors and stroma obtained from ligament tissue. In embodiments, the stem cells are from vascular tissue. In embodiments, muscle stem cells comprise vascular-derived stem cells. In embodiments, vascular stem cells comprise pluripotent stem cells, MSCs, vascular progenitors and stroma obtained from vascular tissue. Growth Factors

[0122] In embodiments, the matrix composition comprises at least one exogenous growth- inductive substance. In embodiments, the at least one exogenous growth-inductive substance comprises extracellular polypeptide molecules.

[0123] Growth factors comprise extracellular polypeptide molecules that bind to a cell-surface receptor triggering an intracellular signaling pathway, leading to proliferation, differentiation, or other cellular response. These pathways stimulate the accumulation of proteins and other macromolecules, and they can do so by both increasing their rate of synthesis and decreasing their rate of degradation. One intracellular signaling pathway activated by growth factor receptors involves the enzyme PI 3-kinase, which adds a phosphate from ATP to the 3 position of inositol phospholipids in the plasma membrane. The activation of PI 3-kinase leads to the activation of several protein kinases, including S6 kinase. The S6 kinase phosphorylates ribosomal protein S6, increasing the ability of ribosomes to translate a subset of mRNAs, most of which encode ribosomal components, as a result of which, protein synthesis increases. When the gene encoding S6 kinase is inactivated in Drosophila, cell numbers are normal, but cell size is abnormally small, and the mutant flies are small. Growth factors also activate a translation initiation factor called elF4E, further increasing protein synthesis and cell growth.

[0124] Growth factor stimulation also leads to increased production of the gene regulatory protein Myc, which plays a part in signaling by mitogens. Myc increases the transcription of a number of genes that encode proteins involved in cell metabolism and macromolecular synthesis. In this way, it stimulates both cell metabolism and cell growth.

[0125] Some extracellular signal proteins, including platelet-derived growth factor (PDGF), can act as both growth factors and mitogens, stimulating both cell growth and cell-cycleATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 progression. This functional overlap is achieved in part by overlaps in the intracellular signaling pathways that control these two processes. The signaling protein Ras, for example, is activated by both growth factors and mitogens. It can stimulate the PI3-kinase pathway to promote cell growth and the MAP-kinase pathway to trigger cell-cycle progression. Similarly, Myc stimulates both cell growth and cell-cycle progression. Extracellular factors that act as both growth factors and mitogens help ensure that cells maintain their appropriate size as they proliferate.

[0126] Since many mitogens, growth factors, and survival factors are positive regulators of cell-cycle progression, cell growth, and cell survival, they tend to increase the size of organs and organisms. In some tissues, however, cell and tissue size also is influenced by inhibitory extracellular signal proteins that oppose the positive regulators and thereby inhibit organgrowth. The best-understood inhibitory signal proteins are TGF- and its relatives. TGF-inhibits the proliferation of several cell types, either by blocking cell-cycle progression in G1or by stimulating apoptosis. TGF- binds to cell-surface receptors and initiates an intracellularsignaling pathway that leads to changes in the activities of gene regulatory proteins called Smads. This results in complex changes in the transcription of genes encoding regulators of cell division and cell death.

[0127] Bone morphogenetic protein (BMP), a TGF- family member, helps trigger theapoptosis that removes the tissue between the developing digits in the mouse paw. Like TGF- , BMP stimulates changes in the transcription of genes that regulate cell death. Fibroblast Growth Factor (FGF)

[0128] The fibroblast growth factor (FGF) family currently has over a dozen structurally related members. FGF1 is also known as acidic FGF; FGF2 is sometimes called basic FGF (bFGF); and FGF7 sometimes goes by the name keratinocyte growth factor. Over a dozen distinct FGF genes are known in vertebrates; they can generate hundreds of protein isoforms by varying their RNA splicing or initiation codons in different tissues. FGFs can activate a set of receptor tyrosine kinases called the fibroblast growth factor receptors (FGFRs). Receptor tyrosine kinases are proteins that extend through the cell membrane. The portion of the protein that binds the paracrine factor is on the extracellular side, while a dormant tyrosine kinase (i.e., a protein that can phosphorylate another protein by splitting ATP) is on the intracellular side. When the FGF receptor binds an FGF (and only when it binds an FGF), the dormant kinase is activated, and phosphorylates certain proteins within the responding cell, activating those proteins.ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035

[0129] FGFs are associated with several developmental functions, including angiogenesis (blood vessel formation), mesoderm formation, and axon extension. While FGFs often can substitute for one another, their expression patterns give them separate functions, FGF2 is especially important in angiogenesis, whereas FGF8 is involved in the development of the midbrain and limbs.

[0130] The expression levels of angiogenic factors, such as VEGF, IGF, PDGF, HGF, FGF, TGFm Angiopoeitin-1, and stem cell factor (SCF) have been found to differ amongst bone- derived-, and cartilage-derived MSCs. (Peng et al., 2008, Stems Cells and Development, 17: 761-774). Insulin-Like Growth Factor (IGF-1)

[0131] IGF-1, a hormone similar in molecular structure to insulin, has growth-promoting effects on almost every cell in the body, especially skeletal muscle, cartilage, bone, liver, kidney, nerves, skin, hematopoietic cell, and lungs. It plays an important role in childhood growth and continues to have anabolic effects in adults. IGF-1 is produced primarily by the liver as an endocrine hormone as well as in target tissues in a paracrineiautocrine fashion. Production is stimulated by growth hormone (GH) and can be retarded by undernutrition, growth hormone insensitivity, lack of growth hormone receptors, or failures of the downstream signaling molecules, including SHP2 and STAT5B. Its primary action is mediated by binding to its specific receptor, the Insulin-like growth factor 1 receptor (IGF1R), present on many cell types in many tissues. Binding to the IGF1R, a receptor tyrosine kinase, initiates intracellular signaling; IGF-1 is one of the most potent natural activators of the AKT signaling pathway, a stimulator of cell growth and proliferation, and a potent inhibitor of programmed cell death. IGF-1 is a primary mediator of the effects of growth hormone (GH). Growth hormone is made in the pituitary gland, released into the blood stream, and then stimulates the liver to produce IGF-1. IGF-1 then stimulates systemic body growth. In addition to its insulin-like effects, IGF- 1 also can regulate cell growth and development, especially in nerve cells, as well as cellular DNA synthesis.Transforming Growth Factor Beta (TGF- )

[0132] There are over 30 structurally related members of the TGF- superfamily, and theyregulate some of the most important interactions in development. The proteins encoded byTGF- superfamily genes are processed such that the carboxy-terminal region contains themature peptide. These peptides are dimerized into homodimers (with themselves) orheterodimers (with other TGF- peptides) and are secreted from the cell. The TGF-superfamily includes the TGF- family, the activin family, the bone morphogenetic proteinsATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 (BMPs), the Vg-1 family, and other proteins, including glial-derived neurotrophic factor (GDNF, necessary for kidney and enteric neuron differentiation) and Müllerian inhibitoryfactor, which is involved in mammalian sex determination. TGF- family members TGF- 1,2, 3, and 5 are important in regulating the formation of the extracellular matrix between cellsand for regulating cell division (both positively and negatively). TGF- 1 increases the amountof extracellular matrix epithelial cells make both by stimulating collagen and fibronectinsynthesis and by inhibiting matrix degradation. TGF- s may be critical in controlling whereand when epithelia can branch to form the ducts of kidneys, lungs, and salivary glands.

[0133] The members of the BMP family were originally discovered by their ability to induce bone formation. Bone formation, however, is only one of their many functions, and they have been found to regulate cell division, apoptosis (programmed cell death), cell migration, anddifferentiation. BMPs can be distinguished from other members of the TGF- superfamily bytheir having seven, rather than nine, conserved cysteines in the mature polypeptide. The BMPs include proteins such as Nodal (responsible for left-right axis formation) and BMP4 (important in neural tube polarity, eye development, and cell death). Neural Epidermal Growth-Factor-Like 1 (NELL1)

[0134] Neural epidermal growth-factor-like 1 (NEL-like 1, NELL1) is a gene that encodes an 810-amino acid polypeptide, which trimerizes to form a mature protein involved in the regulation of cell growth and differentiation. The neural epidermal growth-factor-like (nel) gene first was detected in neural tissue from an embryonic chicken cDNA library, and its human orthologue NELL1 was discovered later in B-cells. Studies have reported the presence of NELL in various fetal and adult organs, including, but not limited to, the brain, kidneys, colon, thymus, lung, and small intestine. a. NELL1—General Structure

[0135] Generally, the arrangement of the functional domains of the 810 amino acid NELL1 protein bears resemblance to thrombospondin-1 (“THBS1”) and consists of a thrombospondin N-terminal domain (“TSPN”) and several von Willebrand factor, type C (“VWC”), and epidermal growth-factor (“EGF”) domains.

[0136] Additional studies have shown that there are two transcript variants encoding different isoforms. The nel-like 1 isoform 1 precursor transcript variant represents the longer transcript and encodes the longer isoform 1.

[0137] The conserved domains of the nel-like 1 isoform 1 precursor transcript reside in seven regions of the isoform 1 peptide and include: (1) a TSPN domain / Laminin G superfamilyATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 domain; (2) a VWC domain; (3) an EGF-like domain; (4) an EGF-like domain; (5) an EGF- like domain; (6) an EGF-like domain and (7) a VWC domain.

[0138] The first conserved domain region comprises amino acids (amino acids 29 to 213) that are most similar to a thrombospondin N-terminal-like domain. Thrombospondins are a family of related, adhesive glycoproteins, which are synthesized, secreted and incorporated into the extracellular matrix of a variety of cells, including alpha granules of platelets following thrombin activation and endothelial cells. They interact with a number of blood coagulation factors and anticoagulant factors, and are involved in cell adhesion, platelet aggregation, cell proliferation, angiogenesis, tumor metastasis, vascular smooth muscle growth and tissue repair. The first conserved domain also comprises amino acids (amino acids 82 to 206; amino acids 98 to 209) that are similar to a Laminin G-like domain. Laminin G-like (LamG) domains usually are Ca2+mediated receptors that can have binding sites for steroids, 131-integrins, heparin, sulfatides, fibulin-1, and -dystroglycans. Proteins that contain LamG domains serve a variety of purposes, including signal transduction via cell-surface steroid receptors, adhesion, migration and differentiation through mediation of cell adhesion molecules.

[0139] Much of what is known about NELL1 concerns its role in bone development. It generally is believed that during osteogenic differentiation, NELL1 signaling may involve an integrin-related molecule and tyrosine kinases that are triggered by NELL1 binding to a NELL1 specific receptor and a subsequent formation of an extracellular complex. As thus far understood, in human NELL1 (hNELL1), the laminin G domain comprises about 128 amino acid residues that show a high degree of similarity to the laminin G domain of extracellular matrix (“ECM”) proteins, such as human laminin 3 chain (hLAMA3), mouse laminin 3 chain (mLAMA3), human collagen 11 3 chain (hCOLA1), and human thrombospondin-1 (hTSP1). This complex facilitates either activation of Tyr-kinases, inactivation of Tyr phosphatases, or intracellular recruitment of Tyr-phosphorylated proteins. The ligand bound integrin (cell surface receptors that interact with ECM proteins such as, for example, laminin 5, fibronectin, vitronectin, TSP1 / 2) transduces the signals through activation of the focal adhesion kinase (FAK) followed by indirect activation of the Ras-MAPK cascade, and then leads to osteogenic differentiation through Runx2; the laminin G domain is believed to play a role in the interaction between integrins and a 67 kDa laminin receptor.

[0140] The second conserved domain (amino acids 273 to 331) and seventh conserved domain (amino acids 701 to 749; amino acids 703 to 749) are similar to von Willebrand factor type C (VWC) domains, also known as chordin-like repeats. VWC domains occur in numerousATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 proteins of diverse functions. It is thought that these domains may be involved in protein oligomerization.

[0141] The third conserved domain (amino acids 434 to 471; amino acids 434 to 466); fourth conserved domain (amino acids 478 to 512), fifth conserved domain (amino acids 549 to 586; amino acids 549 to 582), and sixth conserved domain (amino acids 596 to 627; amino acids 596 to 634) are similar to a calcium-binding EGF-like domain. Calcium-binding EGF-like domains are present in a large number of membrane-bound and extracellular (mostly animal) proteins. Many of these proteins require calcium for their biological function. Calcium-binding sites have been found to be located at the N-terminus of particular EGF-like domains, suggesting that calcium-binding may be crucial for numerous protein-protein interactions. Six conserved core cysteines form three disulfide bridges as in non-calcium-binding EGF domains whose structures are very similar.

[0142] The nel-like 1 isoform 2 precursor transcript variant lacks an alternate in-frame exon compared to variant 1. The resulting isoform 2, which has the same N- and C-termini as isoform 1 but is shorter compared to isoform 1, has six conserved regions including a TSPN domain / LamG superfamily domain (amino acids 29 to 313); VWC domains (amino acids 273 to 331; amino acids 654 to 702); and calcium-binding EGF-like domains (amino acids 478 to 512; amino acids 434 to 471; amino acids 549 to 580).

[0143] NELL1 and its orthologs are found across several species including Homo sapiens (man), Mus musculus (mouse), Rattus norvegicus (rat), Pan troglodytes (chimpanzee), Xenopus (Silurana) tropicalis (frog), Canis lupus familiaris (dog), Culex quinquefasciatus (mosquito) Pediculus humanus corporis (head louse), Aedes aegypti (mosquito), Ixodes scapularis (tick), Strongylocentrotus purpuratus (purple sea urchin), and Acyrthosiphon pisum (pea aphid). b. NELL1 is Variable

[0144] NELL1 comprises several regions susceptible to increased recombination. Studies have indicated that susceptibilities to certain diseases may be associated with genetic variations within these regions, suggesting the existence of more than one causal variant in the NELL1 gene. For example; in patients suffering irritable bowel syndrome (“IBS”), six different single nucleotide polymorphisms (SNPs) within NELL1 have been identified, with most of theseSNPs near the 5 end of the gene and fewer at the 3 end. These include R136S and A153T(which reside in the TSPN) and R354W (which resides in a VWC domain). Additional studies have identified at least 26 variants comprising some of at least 263 SNPs within the NELL1 region.ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 c. NELL1-Function

[0145] The NELL1 protein is a secreted cytoplasmic heterotrimeric protein. The complete role NELL1 plays in vivo remains unknown.

[0146] Several studies have indicated that NELL1 may play a role in bone formation, inflammatory bowel disease, and esophageal adenocarcinoma, among others. Tissue Matrices

[0147] The connective tissue compartment contains cells that primarily function to elaborate and maintain ECM structure. The character of the extracellular matrix is region-specific and is determined by the amount of the extracellular materials.

[0148] Common cell types of connective tissue compartments include: fibroblasts, macrophages, mast cells, and plasma cells. Specialized connective tissue compartments, such as cartilage, bone, and the vasculature, and those with special properties, such as tendons, ligaments, etc., have specialized cells to perform specialized functions. a. Bone (Osseous) Tissue Matrix

[0149] Osseous tissue is a rigid form of connective tissue normally organized into definite structures, the bones. These form the skeleton, serve for the attachment and protection of the soft parts, and, by their attachment to the muscles, act as levers that bring about body motion. Bone is also a storage place for calcium that can be withdrawn when needed to maintain a normal level of calcium in the blood.

[0150] Bones can be classified according to their shape. Examples of bone types include: long bones whose length is greater than their widths (e.g., femur (thigh bone), humerus (long bone of the upper limb), tibia (shin bone), fibula (calf bone), radius (the outer of the two bones of the forearm), and ulna (inner of two bones of the forearm)), short bones whose length and width is approximately equal (e.g., carpals bones (wrist bones in the hand)), flat bones (e.g., cranium (skull bones surrounding the brain), scapula (shoulder blade), and ilia (the uppermost and largest bone of the pelvis)), irregular bones (e.g., vertebra), and sesamoid bones, small bones present in the joints to protect tendons (fibrous connective tissues that connect muscles to the bones, e.g., patella bones (knee cap)).

[0151] Grossly, two types of bone may be distinguished: cancellous, trabecular or spongy bone, and cortical, compact, or dense bone.

[0152] Cortical bone, also referred to as compact bone or dense bone, is the tissue of the hard outer layer of bones, so-called due to its minimal gaps and spaces. This tissue gives bones their smooth, white, and solid appearance. Cortical bone consists of haversian sites (the canalsATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 through which blood vessels and connective tissue pass in bone) and osteons (the basic units of structure of cortical bone comprising a haversian canal and its concentrically arranged lamellae), so that in cortical bone, bone surrounds the blood supply. Cortical bone has a porosity of about 5% to about 30% and accounts for about 80% of the total bone mass of an adult skeleton. i. Cancellous Bone (Trabecular or Spongy Bone)

[0153] Cancellous bone tissue, an open, cell-porous network also called trabecular or spongy bone, fills the interior of bone and is composed of a network of rod- and plate-like elements that make the overall structure lighter and allows room for blood vessels and marrow so that the blood supply surrounds bone. Cancellous bone accounts for the remaining 20% of total bone mass but has nearly ten times the surface area of cortical bone. It does not contain haversian sites and osteons and has a porosity of about 30% to about 90%.

[0154] The head of a bone, termed the epiphysis, has a spongy appearance and consists of slender irregular bone trabeculae, or bars, which anastomose to form a lattice work, the interstices of which contain the marrow, while the thin outer shell appears dense. The irregular marrow spaces of the epiphysis become continuous with the central medullary cavity of the bone shaft, termed the diaphysis, whose wall is formed by a thin plate of cortical bone.

[0155] Both cancellous and cortical bone have the same types of cells and intercellular substance, but they differ from each other in the arrangement of their components and in the ratio of marrow space to bone substance. In cancellous bone, the marrow spaces are relatively large and irregularly arranged, and the bone substance is in the form of slender anastomosing trabeculae and pointed spicules. In cortical bone, the spaces or channels are narrow and the bone substance is densely packed.

[0156] With very few exceptions, the cortical and cancellous forms are both present in every bone, but the amount and distribution of each type vary considerably. The diaphyses of the long bones consist mainly of cortical tissue; only the innermost layer immediately surrounding the medullary cavity is cancellous bone. The tabular bones of the head are composed of two plates of cortical bone enclosing marrow space bridged by irregular bars of cancellous bone. The epiphyses of the long bones and most of the short bones consist of cancellous bone covered by a thin outer shell of cortical bone.

[0157] Each bone, except at its articular end, is surrounded by a vascular fibroelastic coat, the periosteum. The so-called endosteum, or inner periosteum of the marrow cavity and marrow spaces, is not a well-demarcated layer; it consists of a variable concentration of medullaryATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 reticular connective tissue that contains osteogenic cells that are in immediate contact with the bone tissue. ii. Components of Bone

[0158] Bone is composed of cells and an intercellular matrix of organic and inorganic substances.

[0159] The organic fraction consists of collagen, glycosaminoglycans, proteoglycans, and glycoproteins. The protein matrix of bone largely is composed of collagen, a family of fibrous proteins that have the ability to form insoluble and rigid fibers. The main collagen in bone is type I collagen.

[0160] The inorganic component of bone, which is responsible for its rigidity and may constitute up to two-thirds of its fat-free dry weight, is composed chiefly of calcium phosphate and calcium carbonate, in the form of calcium hydroxyapatite, with small amounts of magnesium hydroxide, fluoride, and sulfate. The composition varies with age and with a number of dietary factors. The bone minerals form long fine crystals that add strength and rigidity to the collagen fibers; the process by which it is laid down is termed mineralization. iii. Bone Cells

[0161] Four cell types in bone are involved in its formation and maintenance. These are 1) osteoprogenitor cells, 2) osteoblasts, 3) osteocytes, and 4) osteoclasts. Osteoprogenitor Cells

[0162] Osteoprogenitor cells arise from mesenchymal cells, and occur in the inner portion of the periosteum and in the endosteum of mature bone. They are found in regions of the embryonic mesenchymal compartment where bone formation is beginning and in areas near the surfaces of growing bones. Structurally, osteoprogenitor cells differ from the mesenchymal cells from which they have arisen. They are irregularly shaped and elongated cells having pale- staining cytoplasm and pale-staining nuclei. Osteoprogenitor cells, which multiply by mitosis, are identified chiefly by their location and by their association with osteoblasts. Some osteoprogenitor cells differentiate into osteocytes. While osteoblasts and osteocytes are no longer mitotic, it has been shown that a population of osteoprogenitor cells persists throughout life. Osteoblasts

[0163] Osteoblasts, which are located on the surface of osteoid seams (the narrow region on the surface of a bone of newly formed organic matrix not yet mineralized), are derived from osteoprogenitor cells. They are immature, mononucleate, bone-forming cells that synthesize collagen and control mineralization. Osteoblasts can be distinguished from osteoprogenitorATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 cells morphologically; generally they are larger than osteoprogenitor cells, and have a more rounded nucleus, a more prominent nucleolus, and cytoplasm that is much more basophilic. Osteoblasts make a protein mixture known as osteoid, primarily composed of type I collagen, which mineralizes to become bone. Osteoblasts also manufacture hormones, such as prostaglandins, alkaline phosphatase, an enzyme that has a role in the mineralization of bone, and matrix proteins. Osteocytes

[0164] Osteocytes, star-shaped mature bone cells derived from osteoblasts and the most abundant cell found in compact bone, maintain the structure of bone. Osteocytes, like osteoblasts, are not capable of mitotic division. They are actively involved in the routine turnover of bony matrix and reside in small spaces, cavities, gaps or depressions in the bone matrix called lacuna. Osteocytes maintain the bone matrix, regulate calcium homeostasis, and are thought to be part of the cellular feedback mechanism that directs bone to form in places where it is most needed. Bone adapts to applied forces by growing stronger in order to withstand them; osteocytes may detect mechanical deformation and mediate bone-formation by osteoblasts. Osteoclasts

[0165] Osteoclasts, which are derived from a monocyte stem cell lineage and possess phagocytic-like mechanisms similar to macrophages, often are found in depressions in the bone referred to as Howship's lacunae. They are large multinucleated cells specialized in bone resorption. During resorption, osteoclasts seal off an area of bone surface; then, when activated, they pump out hydrogen ions to produce a very acid environment, which dissolves the hydroxyapatite component. The number and activity of osteoclasts increase when calcium resorption is stimulated by injection of parathyroid hormone (PTH), while osteoclastic activity is suppressed by injection of calcitonin, a hormone produced by thyroid parafollicular cells. iv. Bone Matrix

[0166] The bone matrix accounts for about 90% of the total weight of compact bone and is composed of microcrystalline calcium phosphate resembling hydroxyapatite (60%) and fibrillar type I collagen (27%). The remaining 3% consists of minor collagen types and other proteins including osteocalcin, osteonectin, osteopontin, bone sialoprotein, as well as proteoglycans, glycosaminoglycans, and lipids.

[0167] Bone matrix is also a major source of biological information that skeletal cells can receive and act upon. For example, extracellular matrix glycoproteins and proteoglycans in bone bind a variety of growth factors and cytokines, and serve as a repository of stored signalsATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 that act on osteoblasts and osteoclasts. Examples of growth factors and cytokines found in bone matrix include, but are not limited to, Bone Morphogenic Proteins (BMPs), Epidermal Growth Factors (EGFs), Fibroblast Growth Factors (FGFs), Platelet-Derived Growth Factors (PDGFs), Insulin-like Growth Factor-1 (IGF-1), Transforming Growth Factors (TGFs), Bone-Derived Growth Factors (BDGFs), Cartilage-Derived Growth Factor (CDGF), Skeletal Growth Factor (hSGF), Interleukin-1 (IL-1), and macrophage-derived factors.

[0168] There is an emerging understanding that extracellular matrix molecules themselves can serve regulatory roles, providing both direct biological effects on cells as well as key spatial and contextual information. v. The Periosteum and Endosteum

[0169] The periosteum is a fibrous connective tissue investment of bone, except at the bone's articular surface. Its adherence to the bone varies by location and age. In young bone, the periosteum is stripped off easily. In adult bone, it is more firmly adherent, especially so at the insertion of tendons and ligaments, where more periosteal fibers penetrate into the bone as the perforating fibers of Sharpey (bundles of collagenous fibers that pass into the outer circumferential lamellae of bone). The periosteum consists of two layers, the outer of which is composed of coarse, fibrous connective tissue containing few cells but numerous blood vessels and nerves. The inner layer, which is less vascular but more cellular, contains many elastic fibers. During growth, an osteogenic layer of primitive connective tissue forms the inner layer of the periosteum. In the adult, this is represented only by a row of scattered, flattened cells closely applied to the bone. The periosteum serves as a supporting bed for the blood vessels and nerves going to the bone and for the anchorage of tendons and ligaments. The osteogenic layer; which is considered a part of the periosteum; is known to furnish osteoblasts for growth and repair, and acts as an important limiting layer controlling and restricting the extend of bone formation. Because both the periosteum and its contained bone are regions of the connective tissue compartment, they are not separated from each other or from other connective tissues by basal laminar material or basement membranes. Perosteal stem cells have been shown to be important in bone regeneration and repair. (Zhang et al., 2005, J. Musculoskelet. Neuronal. Interact.5(4): 360-362).

[0170] The endosteum lines the surface of cavities within a bone (marrow cavity and central canals) and also the surface of trabeculae in the marrow cavity. In growing bone; it consists of a delicate striatum of myelogenous reticular connective tissue; beneath which is a layer of osteoblasts. In the adult, the osteogenic cells become flattened and are indistinguishable as aATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 separate layer. They are capable of transforming into osteogenic cells when there is a stimulus to bone formation, as after a fracture. vi. Marrow

[0171] The marrow is a soft connective tissue that occupies the medullary cavity of the long bones, the larger central canals, and all of the spaces between the trabeculae of spongy bone. It consists of a delicate reticular connective tissue, in the meshes of which lie various kinds of cells. Two varieties of marrow are recognized: red and yellow. Red marrow is the only type found in fetal and young bones, but in the adult it is restricted to the vertebrae, sternum, ribs, cranial bones, and epiphyses of long bones. It is the chief site for the genesis of blood cells in the adult body. Yellow marrow consists primarily of fat cells that gradually have replaced the other marrow elements. Under certain conditions, the yellow marrow of old or emaciated persons loses most of its fat and assumes a reddish color and gelatinous consistency, known as gelatinous marrow. With adequate stimulus, yellow marrow may resume the character of red marrow and play an active part in the process of blood development. vii. Osteogenesis or Ossification

[0172] Osteogenesis or ossification is a process by which the bones are formed. There are three distinct lineages that generate the skeleton. The somites generate the axial skeleton, the lateral plate mesoderm generates the limb skeleton, and the cranial neural crest gives rise to the branchial arch, craniofacial bones, and cartilage. There are two major modes of bone formation, or osteogenesis, and both involve the transformation of a preexisting mesenchymal tissue into bone tissue. The direct conversion of mesenchymal tissue into bone is called intramembranous ossification. This process occurs primarily in the bones of the skull. In other cases, mesenchymal cells differentiate into cartilage, which is later replaced by bone. The process by which a cartilage intermediate is formed and replaced by bone cells is called endochondral ossification. viii. Intramembranous Ossification

[0173] Intramembraneous ossification is the characteristic way in which the flat bones of the scapula, the skull and the turtle shell are formed. In intramembraneous ossification, bones develop sheets of fibrous connective tissue. During intramembranous ossification in the skull, neural crest-derived mesenchymal cells proliferate and condense into compact nodules. Some of these cells develop into capillaries; others change their shape to become osteoblasts, committed bone precursor cells. The osteoblasts secrete a collagen-proteoglycan matrix that is able to bind calcium salts. Through this binding, the prebone (osteoid) matrix becomes calcified. In most cases, osteoblasts are separated from the region of calcification by a layer ofATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 the osteoid matrix they secrete. Occasionally, osteoblasts become trapped in the calcified matrix and become osteocytes. As calcification proceeds, bony spicules radiate out from the region where ossification began, the entire region of calcified spicules becomes surrounded by compact mesenchymal cells that form the periosteum, and the cells on the inner surface of the periosteum also become osteoblasts and deposit osteoid matrix parallel to that of the existing spicules. In this manner, many layers of bone are formed.

[0174] Intramembraneous ossification is characterized by invasion of capillaries into the mesenchymal zone, and the emergence and differentiation of mesenchymal cells into mature osteoblasts, which constitutively deposit bone matrix leading to the formation of bone spicules, which grow and develop, eventually fusing with other spicules to form trabeculae. As the trabeculae increase in size and number they become interconnected forming woven bone (a disorganized weak structure with a high proportion of osteocytes), which eventually is replaced by more organized, stronger, lamellar bone.

[0175] The molecular mechanism of intramembranous ossification involves bone morphogenetic proteins (BMPs) and the activation of a transcription factor called CBFA1. Bone morphogenetic proteins, for example, BMP2, BMP4, and BMP7, from the head epidermis are thought to instruct the neural crest-derived mesenchymal cells to become bone cells directly. BMPs activate the Cbfa1 gene in mesenchymal cells. The CBFA1 transcription factor is known to transform mesenchymal cells into osteoblasts. Studies have shown that the mRNA for mouse CBFA1 is largely restricted to the mesenchymal condensations that form bone, and is limited to the osteoblast lineage. CBFA1 is known to activate the genes for osteocalcin, osteopontin, and other bone-specific extracellular matrix proteins. Bone Remodeling

[0176] Bone constantly is broken down by osteoclasts and re-formed by osteoblasts in the adult. It has been reported that as much as 18% of bone is recycled each year through the process of renewal, known as bone remodeling, which maintains bone's rigidity. The balance in this dynamic process shifts as people grow older: in youth, it favors the formation of bone, but in old age, it favors resorption.

[0177] As new bone material is added peripherally from the internal surface of the periosteum, there is a hollowing out of the internal region to form the bone marrow cavity. This destruction of bone tissue is due to osteoclasts that enter the bone through the blood vessels. Osteoclasts dissolve both the inorganic and the protein portions of the bone matrix. Each osteoclast extends numerous cellular processes into the matrix and pumps out hydrogen ions onto the surroundingATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 material, thereby acidifying and solubilizing it. The blood vessels also import the blood- forming cells that will reside in the marrow for the duration of the organism's life.

[0178] The number and activity of osteoclasts must be tightly regulated. If there are too many active osteoclasts, too much bone will be dissolved, and osteoporosis will result. Conversely, if not enough osteoclasts are produced, the bones are not hollowed out for the marrow, and osteopetrosis (known as stone bone disease, a disorder whereby the bones harden and become denser) will result. x. Bone Regeneration and Fracture Repair

[0179] A fracture, like any traumatic injury, causes hemorrhage and tissue destruction. The first reparative changes thus are characteristic of those occurring in any injury of soft tissue. Proliferating fibroblasts and capillary sprouts grow into the blood clot and injured area, thus forming granulation tissue. The area also is invaded by polymorphonuclear leukocytes and later by macrophages that phagocytize the tissue debris. The granulation tissue gradually becomes denser, and in parts of it, cartilage is formed. This newly formed connective tissue and cartilage is designated as a callus. It serves temporarily in stabilizing and binding together the fractured bone. As this process is taking place, the dormant osteogenic cells of the periosteum enlarge and become active osteoblasts. On the outside of the fractured bone, at first at some distance from the fracture, osseous tissue is deposited. This formation of new bone continues toward the fractured ends of the bone and finally forms a sheath-like layer of bone over the fibrocartilaginous callus. As the amount of bone increases, osteogenic buds invade the fibrous and cartilaginous callus and replace it with a bony one. The cartilage undergoes calcification and absorption in the replacement of the fibrocartilaginous callus and intramembraneous bone formation also takes place. The newly formed bone is at first a spongy and not a compact type, and the callus becomes reduced in diameter. At the time when this subperiosteal bone formation is taking place, bone also forms in the marrow cavity. The medullary bone growing centripetally from each side of the fracture unites, thus aiding the bony union.

[0180] The process of repair is, in general, an orderly process, but it varies greatly with the displacement of the fractured ends of the bone and the degree of trauma inflicted. Uneven or protruding surfaces gradually are removed, and the healed bone, especially, in young individuals, assumes its original contour. xi. Osteogenesis and Angiogenesis

[0181] Skeletal development and fracture repair includes the coordination of multiple events such as migration, differentiation, and activation of multiple cell types and tissues. The development of a microvasculature and microcirculation is important for the homeostasis andATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 regeneration of living bone, without which the tissue would degenerate and die. Recent developments using in vitro and in vivo models of osteogenesis and fracture repair have provided a better understanding of the recruitment nature of the vasculature in skeletal development and repair.

[0182] The vasculature transports oxygen, nutrients, soluble factors and numerous cell types to all tissues in the body. The growth and development of a mature vascular structure is one of the earliest events in organogenesis. In mammalian embryonic development, the nascent vascular networks develop by aggregation of de novo forming angioblasts into a primitive vascular plexus (vasculogenesis). This undergoes a complex remodeling process in which sprouting, bridging and growth from existing vessels (angiogenesis) leads to the onset of a functional circulatory system.

[0183] The factors and events that lead to the normal development of the embryonic vasculature are recapitulated during situations of neoangiogenesis in the adult. There are a number of factors involved in neoangiogenesis; these include, but are not limited to, Vascular Endothelial Growth Factor (VEGF), basic Fibroblast Growth Factor (bFGF), various membersof the Transforming Growth factor beta (TGF ) family and Hypoxia-Inducible TranscriptionFactor (HIF). Other factors that have angiogenic properties include the Angiopoietins, (Ang- 1); Hepatocyte Growth Factor (HGF); Platelet-Derived Growth Factor (PDGF); Insulin-like Growth Factor family (IGF-1, IGF-2) and the Neurotrophins (NGF).

[0184] The VEGFs and their corresponding receptors are key regulators in a cascade of molecular and cellular events that ultimately lead to the development of the vascular system, either by vasculogenesis, angiogenesis, or in the formation of the lymphatic vascular system. Although VEGF is a critical regulator in physiological angiogenesis, it also plays a significant role in skeletal growth and repair.

[0185] In the mature established vasculature, the endothelium plays an important role in the maintenance of homeostasis of the surrounding tissue by providing the communicative network to neighboring tissues to respond to requirements as needed. Furthermore, the vasculature provides growth factors, hormones, cytokines, chemokines and metabolites, and the like, needed by the surrounding tissue and acts as a barrier to limit the movement of molecules and cells. Signals and attractant factors expressed on the bone endothelium help recruit circulating cells, particularly hematopoietic cells, to the bone marrow and coordinate with metastatic cells to target them to skeletal regions. Thus, any alteration in the vascular supply to bone tissue can lead to skeletal pathologies, such as osteonecrosis (bone death caused by reduced blood flow to bones), osteomyelitis (infection of the bone or bone marrow by microorganism), andATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 osteoporosis (loss of bone density). A number of factors have been found to have a prominent effect on the pathology of the vasculature and skeleton, including Osteoprotegerin (OPG),which inhibits Receptor Activator of NF- B Ligand (RANKL)-induced osteoclastogenic boneresorption.

[0186] Both intramembraneous and endochondral bone ossification occur in close proximity to vascular ingrowth. In endochondral ossification, the coupling of chondrogenesis and osteogenesis to determine the rate of bone ossification is dependent on the level of vascularization of the growth plate. For example, vascular endothelial growth (VEGF) factor isoforms are essential in coordinating metaphyseal and epiphyseal vascularization, cartilage formation, and ossification during endochondral bone development. HIF-1 stimulates transcription of the VEGF gene (and of other genes whose products are needed when oxygen is in short supply). The VEGF protein is secreted, diffuses through the tissue, and acts on nearby endothelial cells.

[0187] The response of the endothelial cells includes at least four components. First, the cells produce proteases to digest their way through the basal lamina of the parent capillary or venule. Second, the endothelial cells migrate toward the source of the signal. Third, the cells proliferate. Fourth, the cells form tubes and differentiate. VEGF acts on endothelial cells selectively to stimulate this entire set of effects. Other growth factors, including some members of the fibroblast growth factor family, also can stimulate angiogenesis, but they influence other cell types besides endothelial cells. As the new vessels form, bringing blood to the tissue, the oxygen concentration rises, HIF-1 activity declines, VEGF production is shut off, and angiogenesis ceases.

[0188] The vascularization of cartilage regions in long bones occurs at different stages of development. In early embryonic development, blood vessels that originate from the perichondrium invaginate into the cartilage structures. During elevated postnatal growth, capillaries invade the growth plate of long bones. In adulthood, angiogenesis periodically can be switched on during bone remodeling in response to bone trauma or pathophysiological conditions such as rheumatoid arthritis (RA) and osteoarthritis (OA). b. Cartilaginous Tissue Matrix

[0189] Cartilaginous tissue compartments are specialized connective tissue compartments comprising cartilage cells, known as chondrocytes, cartilage fibers and ground substance constituting the cartilage matrix, that collectively contribute to characteristic elastic firmness rendering cartilage capable of withstanding high levels of pressure or sheer. Cartilage isATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 histologically classified into three types depending on its molecular composition: hyaline cartilage; fibrocartilage and elastic cartilage.

[0190] Hyaline cartilage is the predominant form of cartilage comprising an amorphous matrix surrounding chondrocytes embedded within spaces, known as lacunae. Hyaline cartilage, which is commonly associated with the skeletal system and found in the nose, trachea, bronchi and larynx, predominantly functions to provide support. Hyaline cartilage associated with the articular portions of bone, forming the major component of synovial joints, is termed articular cartilage. Hyaline cartilage is usually avascular except where vessels may pass through to supply other tissues and in ossification centers involved in intracartilaginous bone development.

[0191] Fibrocartilage, which is commonly found in intervertebral discs and pubic symphysis and functions to provide tensile strength and in shock absorption, is less firm than hyaline cartilage. It comprises a combination of dense collagenous fibers with cartilage cells and a scant cartilage matrix. Fibrocartilage is not usually circumscribed by a perichondrium. Proportions of cells, fibers and ECM components in fribrocartilage are variable.

[0192] Elastic cartilage, which is found in the external ear, the Eustachian tube, epiglottis and some of the lanryngeal cartilages, is characterized by a large number of elastic fibers that branch and course in all directions to form a dense network of anastomising and interlacing fibers. i. Articular Cartilage Matrix

[0193] The chondrocytes in articular cartilage are surrounded by a narrow region of connective tissue ECM, termed the pericellular matrix (PCM), which together with the chondrocyte, is termed chondron. The PCM, which is very rich in fibronectin, proteoglycans (e.g., aggrecan, hyaluron and decorin) and collagen (types II, VI and IX), is particularly characterized by a high concentration of type VI collagen as compared to the surrounding ECM. In normal articular cartilage, type VI collagen is restricted to the chondrons, but in osteoarthritic cartilage, it is upregulated and found throughout the ECM. A proteomic analysis of articular cartilage revealed the presence of collagen 1 (II) C-propeptide, collagen 1(XI) C-propeptide, collagen 2(XI) C-propeptide, collagen 1(VI), collagen 2(VI), link protein, biglycan, decorin, osteonectin, matrillin-1, annexin-V, lactadherin, and binding immunoglobulin protein (BIP), in addition to metabolic proteins. (Wilson et. al., 2008, Methods, 48: 22-31).ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 ii. Chondrocyte Differentiation

[0194] The specific structure of articular cartilage, with endogenous chondrocytes forming adult joints, is the result of endochondral ossification, as described above under the heading, Osseous Tissue Compartments Formation.

[0195] Chondrocyte differentiation and maintenance in articular cartilage is governed by interaction of multiple factors. Key players include, but are not limited to, ions (e.g., calcium); steroids (e.g., estrogens); terpenoids (e.g., retinoic acid); peptides (e.g., Parathyroid hormone(PTH), parathyroid hormone-related peptide (PTHrP)), insulin growth factors (e.g., TGFhormones, including, without limitation, BMPs, IGF-1, VEGF, PDGF, FGF); transcription factors (e.g., Wnt, SOX-9); eicosanoids (e.g., prostaglandins); catabolic interleukins (e.g., IL- 1); and anabolic interleukins (e.g., IL-6, IL-4 and IL-10). (Gaissmaier et al., 2008, Int. J. Care Injured, 39S1: S88-S96). iii. Growth Plate

[0196] The epiphyseal plates or growth plates are a hyaline cartilage plate located in the metaphysis at the end of long bones. Whereas endochondral ossification is responsible for the formation of cartilage in utero and in infants, the growth plates are responsible for the longitudinal growth of long bones via a cartilage template. The ongoing developmental processes of proliferation and differentiation within the growth plates are mediated by a number of hormonal and paracrine factors secreted by the growth plate chondrocytes. The growth plate is a highly organized structure comprising a large number of chondrocytes in various stages of differentiation and proliferation embedded in a scaffold of ECM components. iv. Stem Cells of Cartilaginous Tissue Compartments

[0197] Multipotent mesenchymal progenitor cells with osteogenic and chondrogenic potential,and that are CD105+ / CD166+ (corresponding to TGF- type III receptor (endoglin) andALCAM, respectively), have been identified in articular cartilage. (Asalameh et al., Arthritis & Rheumatism, 2004, 50(5): 1522-1532). The presence ofCD34 / CD45 / CD44+ / CD73+ / CD90+ mesenchymal stem cells with chondrogenic andosteogenic potential also has been shown. (Peng et al., Stem Cells and Development (2008), 17: 761-774). Similar to bone-derived MSCs, articular-derived MSCs are positive for surface expression of Notch-1. (Hiraoka et al., Biorheology, 2006, 43: 447-454). A potential MSC niche positive for Stro-1, Jagged-1 and BMPr1a has also been identified in the perichondrial zone of Ranvier on the growth plate. (Karlsson et al., 2009, J. Anat.215(3): 355-63).

[0198] Differential expression of Notch-1, Stro-1 and VCAM-1 / CD106 markers has been observed in normal articular cartilage versus osteoarthritic (OA) cartilage. In normal cartilage,ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 expression of these markers is higher in the superficial zone (SZ) as compared to the middle zone (MZ) and deep zone (DZ). On the other hand, OA cartilage SZ has reduced Notch-1 and Sox-9 while MZ has increased Notch-1, Stro-1 and VCAM-1 positive cells. (Grogan et al., Arthritis Res. Ther.2009, 11(3): R85-R97). v. Intervertebral Disc Fibrocartilage Tissue Compartments

[0199] The intervertebral discs (IVD) predominantly are comprised of fibrocartilage. The IVD fibrocartilage is continuous both with and below the articular cartilage of adjacent vertebrae as well as peripherally with spinal ligaments. The IVD is a unique structure containing annulus fibrosus (AF) and nucleus pulposus (NP), a gelatinous ellipsoidal remnant of the embryonic notochord, and is sandwiched between two adjacent cartilaginous endplates (EP). IVD rupture and herniation of the nucleus pulposus into the spinal cord may cause severe pain and other neurological symptoms. The NP and AF synergistically function to achieve the primary role of IVD in transferring load, dissipating energy and facilitating in joint mobility.

[0200] The adult IVD is essentially avascular; hence, endogenous cells survive in a low- nutrient and low-oxygen microenvironment. The major ECM components of IVD include but are not limited to aggrecan, collagen (e.g., types I, II and IX), leucine rich repeat (LRR) proteins and proteoglycans (e.g., fibromodulin, decorin, lumican), cartilage oligomatrix protein, and collagen VI beaded filament network. (Feng et al., 2006, J. Bone Joint Surg. Am. 88: 25-29). The water content, GAG content, aggrecan levels and levels of type II collagen are significantly lower in older discs demonstrating the effects of IVD degeneration with age. (Murakami et al., 2010, Med. Biol. Eng. Comput.48: 469-474).

[0201] The central nucleus pulposus (NP) is rich in aggrecan and hyaluron. The developing NP is characterized by the presence of highly vacuolated chondrocytes and small chondroblasts inherited from the notochord. Primarily functioning as a primitive axial support, the integrity of the notochord is maintained by a proteoglycan (PG-) and laminin-rich sheath. As NP matures, the cellular composition becomes predominantly chondrocytic. Mature NP cells are small and have an aggrecan rich matrix, which is essential in maintaining requisite hydration levels for mechanical function. Their gene expression profile and metabolic activity are distinct from the chondrocytes of articular cartilage. The ECM of immature NP has high aggrecan levels and primarily contains type II collagen, with the type IIA isoform expressed by progenitor cells during chondrogenesis, not by mature chondrocytes. (Hsieh A. H. and Tworney J. D., J. Biomech., 2010, 43(1): 137-156).

[0202] The AF surrounds the NP with layers of unidirectional sheets of collagen parallel to the circumference of a disc to form collagen lamellae. Alternating bidirectional collagen fibersATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 intersperse the AF collagen lamellae. AF can be subdivided into three regions: inner AF, middle AF and outer AF. The inner AF arises along with endochondral formation of the vertebrae. The outer AF arises as a separate cell condensation with slower matrix formation. Lamellae of inner AF comprises predominantly of type II collagen and fibrochondrocytes, while those of outer AF are comprised of type I collagen and fibroblasts. A population of pancake shaped interlamellar cells as well as elastin fibers are also found within the lamellae, in vertebral attachments, and at the NP-AF interface. Large proteoglycans (PGs; for example aggrecan and versican) and type I and VI collagen permeate interlamellar and translamellar ECM. (Hsieh A. H. and Tworney J. D., J. Biomech., 2010, 43(1): 137-156).

[0203] A large number of coordinated signals originating from the cells of the notochord and floor plate of the embryonal neural tube are instrumental in disc embryogenesis. Key signals include, but are not limited to, sonic hedgehog (Shh), Wnt, noggin, Pax family of transcription factors (e.g., Pax 1 and Pax 9), Sox family of transcription factors (Sox5, Sox6 and Sox) andTGF- . (Smith et al., 2011, Dis Model Mech. 4(1): 31-41). Herniation and IVD degenerationare associated with changes in inflammatory and immune cytokine profiles, including, but notlimited to, the activation of Th1-related cytokines (e.g. IFN ) as well as Th17-related cytokines(e.g., IL-4, IL-6, IL-12 and IL-17). (Shamji et al., 2010, Arthritis & Rheumatism, 62(7): 1974- 1982).

[0204] A potential stem cell niche comprised of progenitor cells that are positive for Notch1, Delta4, Jagged1, CD117, Stro-1 and Ki67 has been identified in intervertebral discs of a number of animals, including humans. It has been reported that the IVD tissue compartments comprise a slow growing zone in the AF as well as the NP regions, (Henriksson et al., 2009, SPINE, 34(21): 2278-2287). c. Dental Tissue Matrix

[0205] A tooth has three anatomical divisions (crown, root and neck), and four structural components (enamel, dentin, cementum and pulp).

[0206] Enamel is the hardest, most mineralized biological tissue in the human body. It is composed of elongated hydroxyapatite crystallites bundled into rods or prisms, interspersed with crystalline interrods filling the interstitial space. Enamel cells, known as ameloblasts, are responsible for enamel development. Ameloblastin, TRAP and enamelin are key proteins found in enamel tissue whereas the enamel matrix is devoid of collagen, composed primarily of amelogenin. An intricate orchestration of signaling factors, such as BMPs (e.g., BMP-2, BMP- 4, BMP-7), FGFs (e.g., FGF-3, -4, -9, -20), Wnt-3, 10a, 10b and transcription factors, such as, p21, Msx2 and Lef1 is responsible for morphogenesis of enamel. Self-assembly of amelogensATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 to form amelogenin nanospheres play a role in nucleation of hydroxyapatite crystallization and enamel mineralization. Matrix processing enzymes, such as MMP-20, kallikrein-4 (KLK4), also known as enamel matrix serine protease-1 (EMSP-1), are involved in the complete elimination of the protein matrix and replacement with a mineralized matrix. (Fong et al., 2005, J. Dent. Educ., 69(5): 555-570). Ameloblasts arise from epithelial stem cells of ectodermal origin. They are lost after tooth eruption leaving no adult human ectodermal stem cells in the mature enamel. In contrast, rodent enamel retain a niche of epithelial stem cells, known as apical bud cells, for continuous enamel production. (Ulmer et al., 2010, Schweiz Monatsschr Zahnmed, 120:860-872).

[0207] Dentin is a hard, yellowish and elastic living connective tissue compartment with biomechanical properties similar to bone. The formation of dentin is driven by mesenchymally derived mature odontoblasts that are fully differentiated and nondividing and that form a single layer underneath the dentin in a mature tooth. A series of epithelial-mesenchymal interactions regulates odontoblast differentiation from neural crest cells in the first branchial arch and frontonasal processes. Mature dentin is comprised of a mantle, composed of intertubular and peritubular dentin made of a collagen fibril matrix, with odontoblast cell processes extending into dentin tubules. The dentin matrix is primarily composed of collagens (e.g., types I, III and V) as well as other matrix proteins, including, but not limited to, phosphorylated and nonphosphorylated matrix proteins, proteoglycans, growth factors, metalloproteinases, alkaline phosphatase serum derived proteins, and phospholipids. (Fong et al., 2005, J. Dent. Educ., 69(5): 555-570). No stem cells have been identified in mature dentin.

[0208] The periodontium consists of tissues supporting the tooth crown, including a nonmineralized periodontal ligament (PDL) sandwiched between layers of mineralized tissues, including the cementum, alveolar bone and dentin. Cementum is a thin mineralized layer covering the dentin. Cementoblasts are cells responsible for cementum matrix secretion and subsequent mineralization. When cementoblasts become entrapped within cementum matrix, they are termed cementocytes. Cementoblasts are ectomesenchymal, being derived from neural crest cells, similar to PDL and alveolar bone. Like bone and dentin, cementum is a collagenous mineralized tissue that hardens upon formation of carbonated hydroxyapatite. (Fong et al., 2005, J. Dent. Educ., 69(5): 555-570).

[0209] PDL is a space between cementum and alveolar bone. It represents a replacement of the dental follicle region in immature developing teeth. Mature PDL contains mostly periodontal fibroblasts as well as stem cells, known as the periodontal ligament stem cells (PDLSCs). TheATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 immature dental follicle is also a source of mesenchymal stem cells, known as dental follicle stem cells (DFSCs). (Fong et al., 2005, J. Dent. Educ., 69(5): 555-570).

[0210] Several dental stem cell markers have been identified. Stro-1 and Stro-4 are commonly used dental stem cell markers for all dental mesenchymal stem cells. Dental stem cells originating from the neural crest have the neural marker, nestin. An osteoblast marker, osteocalcin, is also used as a stem cell marker for DPSCs. Similarly, SCAPs express Oct-4, Nanog, SSEA-3, SSEA-4, TRA-1-60 and TRA-1-81. (Ulmer et al., 2010, Schweiz Monatsschr Zahnmed, 120:860-872). d. Fascial Tissue Matrix

[0211] Fascial tissue compartments form a layer of fibrous tissue found throughout the body surrounding softer and more delicate organs, including but not limited to muscles, groups of muscles, blood vessels, nerves, etc. Fascial tissue originates from the embryonic mesenchyme. Fasciae form during the development of bones, muscles and vessels from the mesodermal layer of the embryo. Fascial tissue can be categorized into three types depending on location: (1) superficial fascial tissue, which is found beneath the integument throughout the body, usually blending with the reticular layer of the dermis; (2) deep fascial tissue comprising dense fibroareolar connective tissue surrounding muscles, bones, nerves and blood vessels; and (3) visceral or subserous fascia, which suspends organs within their cavities and wraps them in layers of connective tissue membranes. (Chapter IV. Myology, Section 3. Tendons, Aponeuroses, and Fasciae, Gray's Anatomy of the Human Body, 20thEdition, Re-edited by Lewis, W. H., Lea & Febiger, Philadelphia, 1918, Bartleby.com, New York, 2000).

[0212] The fibroareolar connective tissue of fascia comprises four kinds of cells: (1) flattened lamellar cells, which may be branched or unbranched (branched lamellar cells contain clear cytoplasm and oval nuclei and project multidirectional processes that may unite to form an open network, such as in the cornea; unbranched lamellar cells are joined end to end. (2) Clasmatocytes, which are large irregular vacuolated or granulated cells with oval nuclei. (3) Granule cells, which are ovoid or spherical in shape. (4) Plasma cells of Waldeyer, usually spheroidal, characterized by vacuolated protoplasm. e. Ligament Tissue Matrix

[0213] The term “ligaments” as used herein refers to dense regular connective tissue comprising attenuated collagenous fibers that connect bones at joints. Ligament ECM is composed of type I and type III collagens together with other proteoglycans and glycoproteins. Mesenchymal stem cells have been found in the human anterior cruciate ligament that exhibitATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 multilineage differentiation potential, like bone-derived mesenchymal stem cells. (Cheng et al., 2010, Tissue Engg. A, 16(7):2237-2253). f. Synovial Tissue Compartment

[0214] The synovial membrane is composed of fibrous connective tissue and lines the joint cavity of synovial joints. It is made up of a layer of macrophage (type A) and fibroblast-like (type B) synoviocytes and a loose sublining tissue. Synovial fluid is secreted by synovial cells lining the synovial membrane in the joint capsule. It is a viscid, mucoalbuminous fluid, rich in hyaluronic acid. It acts as a lubricating fluid, facilitating the smooth gliding of the articular surface. Functional mesenchymal stem cell niches have been identified as resident to synovial lining and subsynovial tissue. These cells are positive for the artificial nucleoside,iododeoxyuridine (IdU) as well as MSC markers such as PDGFR , p75 and CD44 and havechondrogenic potential. (Kurth et al., Arthritis Rheum., 2011, 63(5): 1289-1300). Synovial fluid-derived MSCs have also been identified; and these have higher chondrogenic potential as compared to bone marrow-derived MSCs, (Koga et al., 2008, Cell Tissue Res., 333: 207-215). Synovial MSCs and MPCs have been shown to prevent degeneration due to intervertebral disc disease (IVU) and to be useful for cartilage tissue engineering, (Miyamoto et al., 2010, Arthritis Res. Ther., 12: R206-218; Lee et al., 2010, Tissue Engg. A, 16(1): 317-325). g. Tendon Tissue Compartment

[0215] Tendons are specialized connective tissue compartments that connect bone to muscle. Tendon cells are embedded amongst a parallel group of collagenous fibers that secrete a unique ECM containing collagens, large proteoglycans, and small leucine rich proteoglycans that function as lubricators and organizers of collagen fibril assembly. A unique tendon stem / progenitor cell (TSPC) niche has been identified amongst the parallel collagen fibrils surrounded by ECM. The TSPCs exhibit osteogenic potential. Biglycan and fibromodulin are key tendon ECM components that direct TSPC fate through BMP signaling. These TSPCs are positive for bone marrow derived stem cell markers such as Stro-1, CD146, CD90 and CD44 but not for CD18. TSPCs do not express hematopoietic markers; such as CD34, CD45 and CD117, or the endothelial marker CD146. (Bi et al., 2007, Nat. Med., 13(10): 1219-1227). h. Vasculature Tissue Compartment

[0216] The vascular wall is made of three concentric zones with distinct cellular composition, all mesodermal in origin: the tunica intima, containing predominantly mature differentiated endothelial cells (EC), the tunica media, containing mature and differentiated smooth muscle cells, and the tunica adventitia, containing mature fibroblasts. (Tilki et al., 2009, Trends Mol.ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 Med. 15(11): 501-509). Endothelial progenitor cells (EPCs), meaning cells that exhibit clonal expression, stemness characteristics, adherence to matrix molecules and an ability to differentiate into endothelial cells (ECs) have been implicated in the formation of new blood vessels through angiogenesis and postnatal vasculogenesis. EPCs have many characteristic cell surface markers, including, but not limited to, CD34, AC133, KDR (VEGFR-2), Tie-2 and ligand for UEA-1 lectin, (Tilki et al., 2009, Trends Mol. Med.15(11): 501-509; Melero-Martin and Dudley, 2011, Stem Cells, 29: 163-168; Pascilli et al., 2008, Exp. Cell Res., 315: 901-914).

[0217] EPC niches have been identified in the bone-marrow, peripheral cord blood and vascular wall matrix. Bone-marrow derived and cord blood EPCs essentially may be proangiogenic hematopoietic progenitor cells (HPCs), circulating in the blood and committed to myeloid lineage. (Tilki et al., 2009, Trends Mol. Med.15(11): 501-509). The vascular wall stem and progenitor cells (VW-EPCs) reside in distinct zones of the vessel wall within subendothelial space, known as avasculogenic zone, within the vascular adventitia, forming vascular wall-specific niches. Fetal and adult arterial and venous blood vessel walls have also been found to harbor resident niches for a variety of stem and progenitor cells, such as EPCs, smooth muscle progenitors, HSCs, MSCs, mesangial cells coexpressing myogenic and endothelial markers, neural stem cells (NSCs), etc. (Tilki et al., 2009, Trends Mol. Med.15(11):501-509). The VW-EPCs are CD34(+)VEGFR-2(+)Tie-2(+)CD31( )CD144( ). Proliferatingand differentiating VW-EPCs become CD144(+).

[0218] During embryogenesis, there is evidence of the existence of a hemangioblast (giving rise to endothelial and hematopoietic cells) and hemogenic endothelium, originating from precursors resident in the vascular wall. However, whether adult VW also contains ancestral progenitor hemangioblasts giving rise to both VW-EPCs as well as VW-HSCs is not known. Vascular wall also contains resident pericyte-like cells in the subendothelial spaces. These pericyte-like cells serve as a cellular reservoir for VW-MSCs, which can differentiate into colonies with osteogenic and chondrogenic markers. (Tilki et al., 2009, Trends Mol. Med. 15(11): 501-509). i. Epithelial Tissue Matrix i. Placental Tissue Matrix

[0219] The fetal adnexa is composed of the placenta, fetal membranes, and umbilical cord. The term placenta is discoid in shape with a diameter of 15-20 cm and a thickness of 2-3 cm. The fetal membranes, amnion and chorion, which enclose the fetus in the amniotic cavity, and the endometrial decidua extend from the margins of the chorionic disc. The chorionic plate is a multilayered structure that faces the amniotic cavity. It consists of two different structures: theATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 amniotic membrane (composed of epithelium, compact layer, amniotic mesoderm, and spongy layer) and the chorion (composed of mesenchyme and a region of extravillous proliferating trophoblast cells interposed in varying amounts of Langhans fibrinoid, either covered or not by syncytiotrophoblast).

[0220] Villi originate from the chorionic plate and anchor the placenta through the trophoblast of the basal plate and maternal endometrium. From the maternal side, protrusions of the basal plate within the chorionic villi produce the placental septa, which divide the parenchyma into irregular cotyledons (Parolini, O. et al., 2008, Stem Cell, 2008, 26:300-311).

[0221] Four regions of fetal placenta can be distinguished: an amniotic epithelial region, an amniotic mesenchymal region, a chorionic mesenchymal region, and a chorionic trophoblastic region. ii. Amniotic Membrane

[0222] Fetal membranes continue from the edge of the placenta and enclose the amniotic fluid and the fetus. The amnion is a thin, avascular membrane composed of an inner epithelial layer and an outer layer of connective tissue that, and is contiguous, over the umbilical cord, with the fetal skin. The amniotic epithelium (AE) is an uninterrupted, single layer of flat, cuboidal and columnar epithelial cells in contact with amniotic fluid. It is attached to a distinct basal lamina that is, in turn, connected to the amniotic mesoderm (AM). In the amniotic mesoderm closest to the epithelium, an acellular compact layer is distinguishable, composed of collagens I and III and fibronectin. Deeper in the AM, a network of dispersed fibroblast-like mesenchymal cells and rare macrophages are observed. It has been reported that the mesenchymal layer of amnion indeed contains two subfractions, one having a mesenchymal phenotype, also known as amniotic mesenchymal stromal cells, and the second containing monocyte-like cells. iii. Chorionic Membrane

[0223] A spongy layer of loosely arranged collagen fibers separates the amniotic and chorionic mesoderm. The chorionic membrane (chorion laeve) consists of mesodermal and trophoblastic regions. Chorionic and amniotic mesoderm are similar in composition. A large and incomplete basal lamina separates the chorionic mesoderm from the extravillous trophoblast cells. The latter, similar to trophoblast cells present in the basal plate, are dispersed within the fibrinoid layer and express immunohistochemical markers of proliferation. The Langhans fibrinoid layer usually increases during pregnancy and is composed of two different types of fibrinoid: a matrix type on the inner side (more compact) and a fibrin type on the outer side (more reticulate). At the edge of the placenta and in the basal plate, the trophoblast interdigitatesATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 extensively with the decidua (Cunningham, F. et al., The placenta and fetal membranes, Williams Obstetrics, 20th ed. Appleton and Lange, 1997, 95-125; Benirschke, K. and Kaufmann, P. Pathology of the human placenta. New York, Springer-Verlag, 2000, 42-46, 116, 281-297). iv. Amnion-Derived Stem Cells

[0224] The amniotic membrane itself contains multipotent cells that are able to differentiate in the various layers. Studies have reported their potential in neural and glial cells, cardiac repair and also hepatocyte cells. Studies have shown that human amniotic epithelial cells express stem cell markers and have the ability to differentiate toward all three germ layers. These properties, the ease of isolation of the cells, and the availability of placenta, make amnionic membrane a useful and noncontroversial source of cells for transplantation and regenerative medicine.

[0225] Amniotic epithelial cells can be isolated from the amniotic membrane by several methods that are known in the art. According to one such method, the amniotic membrane is stripped from the underlying chorion and digested with trypsin or other digestive enzymes. The isolated cells readily attach to plastic or basement membrane-coated culture dishes. Culture is established commonly in a simple medium such as Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 5%-10% serum and epidermal growth factor (EGF), in which the cells proliferate robustly and display typical cuboidal epithelial morphology. Normally, 2-6 passages are possible before proliferation ceases. Amniotic epithelial cells do not proliferate well at low densities.

[0226] Amniotic membrane contains epithelial cells with different surface markers, suggesting some heterogeneity of phenotype. Immediately after isolation, human amniotic epithelial cells express very low levels of human leukocyte antigen (HLA)-A, B, C; however, by passage 2, significant levels are observed. Additional cell surface antigens on human amniotic epithelial cells include, but are not limited to, ATP-binding cassette transporter G2 (ABCG2 / BCRP), CD9, CD24, E-cadherin, integrins 6 and 1, c-met (hepatocyte growth factor receptor), stage- specific embryonic antigens (SSEAs) 3 and 4, and tumor rejection antigens 1-60 and 1-81. Surface markers thought to be absent on human amniotic epithelial cells include SSEA-1, CD34, and CD133, whereas other markers, such as CD117 (c-kit) and CCR4 (CC chemokine receptor), are either negative or may be expressed on some cells at very low levels. Although initial cell isolates express very low levels of CD90 (Thy-1), the expression of this antigen increases rapidly in culture (Miki, T. et al., Stem Cells, 2005, 23: 1549-1559; Miki, T. et al., Stem Cells, 2006, 2: 133-142).ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035

[0227] In addition to surface markers, human amniotic epithelial cells express molecular markers of pluripotent stem cells, including octamer-binding protein 4 (OCT-4) SRY-related HMG-box gene 2 (SOX-2), and Nanog (Miki, T. et al., Stem Cells, 2005, 23: 1549-1559). Two types of umbilical stem cells can be found, namely hematopoietic stem cells (UC-HS) and mesenchymal stem cells, which in turn can be found in umbilical cord blood (UC-MS) or in Wharton's jelly (UC-MM). MSCs from the umbilical cord matrix (UC-MM) are obtained by different culture methods depending on the source of cells, e.g.; MSCs from the connective matrix, from subendothelial cells from the umbilical vein or even from whole umbilical cord explant. They are generally well cultured in DMEM medium, supplemented with various nutritional and growth factors; in certain cases prior treatment of vessels with hyaluronic acid has proved beneficial (Saban, B. et al., J Reprod Immunol, 2004, 61: 67-77). j. Lung Tissue matrix

[0228] The lungs, which are paired organs that fill up the thoracic cavity, constitute an efficient air-blood gaseous exchange mechanism, accomplished by the passage of air from the mouth or nose, sequentially through an oropharynx, nasopharynx, a larynx, a trachea and finally through a progressively subdividing system of bronchi and bronchioles until it finally reaches alveoli where the air-blood gaseous exchange takes place. A resident niche with characteristic multipotent stem cells with c-kit positive surface profiles recently has been identified localized in small bronchioles alveoli. These stem cells express the transcription factors, Nanog, Oct3 / 4, Sox2 and Klf4, that govern pluripotency in embryonic stem cells. (Kajstura, J. et al., 2011, New Engl. J. Med., 364(19):1795-1806)). k. Mammary

[0229] The mammary gland is a hormone sensitive bilayered epithelial organ comprising an inner luminal epithelial layer and an outer myoepithelial layer surrounded by a basement membrane in a stromal fat pad. Mammary stem cells with myoepithelial potential have been identified in their niches in the terminal ducts of mammary gland. (LaBarge, 2007, Stem Cell Rev., 3(2): 137-146). l. Skin Tissue Matrix

[0230] The skin functions as the primary barrier imparting protection from environmental insults. Skin is composed of an outer epidermis and inner dermis separated by a basement membrane (BM), rich in ECM and growth factors. The BM of the epidermal-dermal junction is composed of collagens (e.g., type IV and XVII), laminins, nidogen, fibronectin and proteoglycans that provide storage sites for growth factors and nutrients supporting the proliferation and adhesion of epidermal keratinocytes.ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035

[0231] The epidermis is a solid epithelial tissue comprising keratinocytes that are linked to each other via cellular junctions, such as desmosomes. Keratinocytes are organized into distinct layers, comprising the stratum corneum, stratum granulosum, stratum spinosum and stratum basale. The epidermal matrix is made up of hyaluronan and other proteoglycans, including but not limited to, desmosealin, glycipans, versican, perlecan, and syndecans. (Sandjeu and Haftek, 2009, J. Physiol. Pharmacol. 60 (S4): 23-30). Epidermal desmosomes are multimeric complexes of transmembrane glycoprotein and cytosolic proteins with the keratin cytoskeleton. Desmosal proteins of the epidermis predominantly belong to the cadherin, Armadillo and plakin superfamilies.

[0232] The underlying dermis is connective tissue comprised primarily of fibroblasts with occasional inflammatory cells. Embedded within the dermis are also epidermal appendages, such as hair follicles and sebaceous glands, as well as nerves and cutaneous vasculature. The dermal ECM is essentially made of type I, III and V collagens and elastin together with noncollagenous components such as glycoproteins, proteoglycans, GAGs, cytokines and growth factors. Dermal collagens help mediate fibroblast-matrix interactions through a number of cell surface receptors and proteoglycans, such as 1-integrins. (Hodde and Johnson, 2007, Am. J. Clin. Dermatol.8(2): 61-66).

[0233] During embryonic development, the epidermis originates from the ectoderm, while the dermis differentiates from the mesoderm. Following gastrulation, as mesenchymal stem cells of mesodermal origin populate the skin, they send signals to the single epidermal layer for initiation of epidermal stratification and direct the positioning of outgrowths of epidermal appendages, such as the hair follicles and sebaceous glands. Along with the mesenchyme, the basal layer of the epidermis organizes into a basement membrane that is rich in ECM proteins and growth factors. A number of different signaling pathways have been implicated in skin morphogenesis, including but not limited to Notch, Wnt, mitogen activated protein kinase(MAPK), nuclear factor- B (NF- B), transcriptional regulator, p63, the AP2 family oftranscription factors, CCAAT / enhancer binding protein (C / EBP) transcriptional regulators, interferon regulatory 6 (URF6), grainyhead-like 3 (GRHL3) and Kruppel-like factor (KLF4). (Blanpain and Fuchs, 2009, Nat. Rev. Mol. Cell. Biol., 10(3): 207-217). m. Muscle Tissue Matrix

[0234] The muscular tissue compartments are comprised of contractile muscle tissue. These can be of three kinds: skeletal muscle associated with the skeletal system; cardiac muscle associated with the heart; and smooth muscle associated with the vasculature and gastrointestinal tract. Skeletal muscle tissue fibers are striated and are voluntary in function.ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 Cardiac muscle fibers have characteristic intercalated discs and are involuntary in function. Smooth muscle tissue is comprised of spindle shaped cells and is involuntary in function.

[0235] Skeletal muscles are composed of a population of quiescent myogenic precursor cells known as satellite cells with muscle regenerating and self-renewal properties, as well as a population of multipotent muscle-derived stem cells (MDSC) with multilineage differentiation potential, such as mesodermal lineages including, but not limited to, myogenic lineages, osteogenic lineages, chondrogenic lineages, endothelial and hematopoetic lineages, and ectodermal lineages, including not limited to neuron-like cells. (Xu et al., 2010, Cell Tissue Res., 340: 549-567).

[0236] Endogenous cardiac stem cells have also been identified in cardiac stem cell niches. (Mazhari and Hare, 2007, Nat. Clio. Pract. Cardiovasc, Med., 4(S1): S21-S26).

[0237] Vascular smooth muscle cells are derived from embryonic cardiac neural crest stem cells, as well as proepicardial cells and endothelial progenitor cells. Smooth muscle differentiation is dependent on a combination of factors, including but not limited to Pax3, Tbx1, FoxC1 and serum response factor, interacting with microenvironment components of the ECM, such as BMPs, Wnts, endothelin (ET)-1, and FGF8. In the adult, vascular smooth muscle cells undergo constant degeneration, repair and regeneration by the concerted efforts of both multipotent bone-derived mesenchymal cells as well as smooth muscle stem cells resident within vascular smooth muscle tissue. (Hirschi and Majesky, 2004, The Anatomical Record, Part A, 276A: 22-33). n. Nerve Tissue Matrix

[0238] The neural tissue compartments are comprised of neurons and the neuroglia, embedded with the neural matrix. Neural tissue is ectodermal in origin, derived from the embryonic neural plate. Neural tissue is primarily located within the brain, spinal cord and nerves.

[0239] Resident neural stem cell niches have been identified in the adult mammalian brain, restricted to the subventricular zone as well as to the lateral ventricle and dentate gyrus subgranular zone of the hippocampus. Astrocytes, which are star-shaped nerve cells, serve as both neural stem cells as well as supporting niche cells secreting essential growth factors that provide support for neurogenesis and vasculogenesis. The basal lamina and associated vasculogenesis are essential components of the niche. Embryonic molecular factors and signals persist within the neural stem cell niches and play critical role in neurogenesis. Neural stem cells have VEGFR2, doublecortin and Lex (CD15) markers. Major signaling pathways implicated in neurogenesis include but are not limited to Notch, Eph / ephrins, Shh, and BMPs. (Alvarez-Buylla and Lim, 2004, Neuron, 41: 683-686).ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035

[0240] Returning to the Figures, embodiments of the present disclosure will now be described.

[0241] In an exemplary embodiment, with reference to Fig.1, method 100 describes a method of composing an allograft tissue matrix to be administered to a subject to repair, reconstruct, and / or augment native tissue matrices. At sub process 102 of method 100, a composition comprising at least a POP solution and an allograft tissue matrix solution is prepared.

[0242] Sub process 102 is described with reference to Fig.2. As noted above, sub process 102 comprises preparing and mixing a solution of POP and a solution of allograft tissue matrix to form a composition. The resulting composition, formed in vitro, may be referred to as a matrix composition.

[0243] In some embodiments, the matrix compositions described herein comprise at least one buffer. The buffer solution may comprise, but are not limited to, phosphate buffered saline (PBS), Dulbecco’s Phosphate Buffered Saline, HEPES, Trizma® base, sodium bicarbonate, Tris-EDTA buffer solution, water, saline (e.g., NaCl in water), distilled water, phosphate (e.g., sodium phosphate), acetate, and / or double distilled water. In some embodiments, the buffer solution comprises saline having a concentration of NaCl of 0.1% NaCl, 0.2% NaCl, 0.3% NaCl, 0.4% NaCl, 0.5% NaCl, 0.6% NaCl, 0.7% NaCl, 0.8% NaCl, 0.9% NaCl, 1.0% NaCl, 2.0% NaCl, 3.0% NaCl, 3.0% NaCl, 4.0% NaCl, and 5.0% NaCl in water. In some embodiments, the dried composition has a buffer solution content of about 1% to about 5% w / v and / or about 0.5% to about 70% w / v, wherein the buffer solution is a buffered saline, such as buffered saline. In some embodiments, the matrix compositions comprise at least one buffer at a concentration of about 1mM to about 1M (e.g., 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 and 1,000 mM). In some embodiments, the at least one buffer is present at 20 mM. In some embodiments, the buffer is present at about 10 mM to about 30 mM, about 15 mM to about 30 mM, about 20 mM to about 30 mM, about 25 mM to about 30 mM, about 10 mM to about 25 mM, about 15 to about 25 mM, about 20 mM to about 25 mM, about 10 mM to about 20 mM, or about 15 mM to about 20 mM.

[0244] At step 103 of sub process 102, a solution of POP is prepared (referred to herein interchangeably as the first solution). In some embodiments, POP is added as a lyophilized powder to a solution. In some embodiments, the lyophilizing comprises performing lyophilization for a predetermined time period, wherein the predetermined time period is about 24 hours to about 72 hours. In embodiments, the method further comprises rehydrating the dried composition, wherein the rehydrating comprises submerging the dried composition in an aqueous buffer for a predetermined time period and / or wherein the predetermined time periodATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 is about 1 minute to about 5 days, about 2 minutes to about 4 days, about 3 minutes to about 3 days, about 4 minutes to about 2 days, about 5 minutes to about 1 day, about 6 minutes to about 12 hours, about 7 minutes to about 6 hours, about 8 minutes to about 1 hour, about 9 minutes to about 30 minutes, and / or about 10 minutes to about 15 minutes. In embodiments, the drying and the rehydrating are performed iteratively. In embodiments, the composition is a resuspension of lyophilized partially ordered polypeptide in an aqueous buffer.

[0245] The POP solution prepared may comprise POP in buffer solution at about 100 M to about 3 mM, about 150 M to about 2 mM, about 200 M to about 1.5 mM, about 250 M to about 1.25 mM, about 500 m to about 1 mM, about 600 M to about 900 M, and / or about 700 m to about 800 M. In other words, the solution prepared at step 107 of method 106 may comprise about 0.1% by weight POP to about 100% by weight POP, about 0.1% by weight POP to about 85% by weight POP, and / or about 50% by weight POP to about 90% by weight POP. The solution can comprise a variety of concentrations of POPs, where higher concentrations result in greater rigidity and stability.

[0246] In some embodiments, the POP is present in solution at a concentration between about 200 μM and about 3 mM. In some embodiments, the POP solution is present at a concentration between about 300 μM and about 2.9 mM, about 400 μM and about 2.8 mM, about 500 μM and about 2.7 mM, about 600 μM and about 2.6 mM, about 700 μM and about 2.5 mM, about 800 μM and about 2.4 mM, about 900 μM and about 2.3 mM, about 1.0 mM and about 2.2 mM, about 1.1 mM and about 2.1 mM, about 1.2 mM and about 2.0 mM, about 1.3 mM and about 1.9 mM, about 1.4 mM and about 1.8 mM, or about 1.5 mM and about 1.7 mM. In some embodiments, the POP is present at a concentration of about 1 mg / mL to about 500 mg / mL. (e.g., 1, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 and 500 mg / mL). In embodiments, the matrix composition comprises between about 200 M and about 2.5 mM of the POP. In some embodiments, the POP is present at a concentration of about 1 mg / mL to about 150 mg / mL. (e.g., 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 or 150 mg / mL). In some embodiments, the POP is present at a concentration of about 50 mg / mL to about 500 mg / mL, about 100 mg / mL to about 500 mg / mL, about 150 mg / mL to about 500 mg / mL, about 200 mg / mL to about 500 mg / mL, about 250 mg / mL to about 500 mg / mL, about 300 mg / mL to about 500 mg / mL, about 350 mg / mL to about 500 mg / mL, about 400 mg / mL to about 500 mg / mL, about 450 mg / mL to about 500 mg / mL, about 1 mg / mL to about 400 mg / mL, about 50 mg / mL to about 400 mg / mL, about 100 mg / mL to about 400 mg / mL, about 150 mg / mL to about 400 mg / mL, about 200 mg / mL to about 400 mg / mL, about 250 mg / mL to about 400ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 mg / mL, about 300 mg / mL to about 400 mg / mL, about 350 mg / mL to about 400 mg / mL, about 1 mg / mL to about 300 mg / mL, about 50 mg / mL to about 300 mg / mL, about 100 mg / mL to about 300 mg / mL, about 150 mg / mL to about 300 mg / mL, about 200 mg / mL to about 300 mg / mL, about 250 mg / mL to about 300 mg / mL, about 1 mg / mL to about 200 mg / mL, about 50 mg / mL to about 200 mg / mL, about 100 mg / mL to about 200 mg / mL, about 150 mg / mL to about 200 mg / mL, about 1 mg / mL to about 100 mg / mL, or about 50 mg / mL to about 100 mg / mL. In some embodiments, the POP is present at a concentration of about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL , about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, about 20 mg / mL, about 21 mg / mL, about 22 mg / mL, about 23 mg / mL, about 24 mg / mL, about 25 mg / mL, about 26 mg / mL, about 27 mg / mL, about 28 mg / mL, about 29 mg / mL, about 30 mg / mL, about 31 mg / mL, about 32 mg / mL, about 33 mg / mL, about 34 mg / mL, about 35 mg / mL, about 36 mg / mL, about 37 mg / mL, about 38 mg / mL, about 39 mg / mL, about 40 mg / mL, about 41 mg / mL, about 42 mg / mL, about 43 mg / mL, about 44 mg / mL, about 45 mg / mL, about 46 mg / mL, about 47 mg / mL, about 48 mg / mL, about 49 mg / mL, about 50 mg / mL, about 51 mg / mL, about 52 mg / mL, about 53 mg / mL, about 54 mg / mL, about 55 mg / mL, about 56 mg / mL, about 57 mg / mL, about 58 mg / mL, about 59 mg / mL, about 60 mg / mL, about 61 mg / mL, about 62 mg / mL, about 63 mg / mL, about 64 mg / mL, about 65 mg / mL, about 66 mg / mL, about 67 mg / mL, about 68 mg / mL, about 69 mg / mL, about 70 mg / mL, about 71 mg / mL, about 72 mg / mL, about 73 mg / mL, about 74 mg / mL, about 75 mg / mL, about 76 mg / mL, about 77 mg / mL, about 78 mg / mL, about 79 mg / mL, about 80 mg / mL, about 81 mg / mL, about 82 mg / mL, about 83 mg / mL, about 84 mg / mL, about 85 mg / mL, about 86 mg / mL, about 87 mg / mL, about 88 mg / mL, about 89 mg / mL, about 90 mg / mL, about 91 mg / mL, about 92 mg / mL, about 93 mg / mL, about 94 mg / mL, about 95 mg / mL, about 96 mg / mL, about 97 mg / mL, about 98 mg / mL, about 99 mg / mL, about 100 mg / mL, about 101 mg / mL, about 102 mg / mL, about 103 mg / mL, about 104 mg / mL, about 105 mg / mL, about 106 mg / mL , about 107 mg / mL, about 108 mg / mL, about 109 mg / mL, about 110 mg / mL, about 111 mg / mL, about 112 mg / mL, about 113 mg / mL, about 114 mg / mL, about 115 mg / mL, about 116 mg / mL, about 117 mg / mL, about 118 mg / mL, about 119 mg / mL, about 120 mg / mL, about 121 mg / mL, about 122 mg / mL, about 123 mg / mL, about 124 mg / mL, about 125 mg / mL, about 126 mg / mL, about 127 mg / mL, about 128 mg / mL, about 129 mg / mL, about 130 mg / mL, about 131 mg / mL, about 132 mg / mL, about 133 mg / mL, about 134 mg / mL, about 135 mg / mL, about 136 mg / mL, about 137 mg / mL, aboutATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 138 mg / mL, about 139 mg / mL, about 140 mg / mL, about 141 mg / mL, about 142 mg / mL, about 143 mg / mL, about 144 mg / mL, about 145 mg / mL, about 146 mg / mL, about 147 mg / mL, about 148 mg / mL, about 149 mg / mL, or about 150 mg / mL including all ranges, subranges and values therebetween.

[0247] In some embodiments, the POP solution described herein comprises at least one buffer solution. The buffer solution may comprise, but are not limited to, phosphate buffered saline (PBS), Dulbecco’s Phosphate Buffered Saline, HEPES, Trizma® base, sodium bicarbonate, Tris-EDTA buffer solution, water, saline (e.g., NaCl in water), distilled water, phosphate (e.g. sodium phosphate), acetate, and / or double distilled water. In some embodiments, the buffer solution comprises saline having a concentration of NaCl of 0.1% NaCl, 0.2% NaCl, 0.3% NaCl, 0.4% NaCl, 0.5% NaCl, 0.6% NaCl, 0.7% NaCl, 0.8% NaCl, 0.9% NaCl, 1.0% NaCl, 2.0% NaCl, 3.0% NaCl, 3.0% NaCl, 4.0% NaCl, and 5.0% NaCl in water. In some embodiments, the dried POP has a buffer solution content of about 1% to about 5% w / v and / or about 0.5% to about 70% w / v, wherein the buffer solution is a buffered saline, such as buffered saline. In some embodiments, the POP solutions comprise at least one buffer at a concentration of about 1mM to about 1M (e.g., 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 and 1,000 mM). In some embodiments, the at least one buffer is present at 20 mM. In some embodiments, the buffer is present at about 10 mM to about 30 mM, about 15 mM to about 30 mM, about 20 mM to about 30 mM, about 25 mM to about 30 mM, about 10 mM to about 25 mM, about 15 to about 25 mM, about 20 mM to about 25 mM, about 10 mM to about 20 mM, or about 15 mM to about 20 mM.

[0248] The POPs used to prepare the POP solution in sub process 102 can be produced using the exemplary protocol described below.

[0249] Prior to sub process step 103, POPs are first cloned into a modified pet24 vector using a process known as recursive directional ligation by plasmid reconstruction (Pre-RDL). Single- stranded oligomers encoding the desired sequences are annealed into cassettes with CC and GG overhangs, allowing concatemerization and ligation into the pet24 vector. This is used to create a library of ELP and polyalanine cassettes which can be strung together through multiple cycles of Pre-RDL to form the final compositions. Plasmids are transfected into chemically competent EB5a cells for cloning and BL21(DE3) cells for protein expression.

[0250] Protein expression is performed in E. coli using fermentation to achieve high density cell culture, followed by protein isolation and purification. Starter cultures of a semi-complex medium are inoculated from cryostocks of recombinant E. coli (BL21 derived) containing POPs producing genes and grown overnight. The starter culture is used to inoculate a largerATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 volume primary culture of the same medium, which is grown for 6 to 8 hours and then used to inoculate the fermenter containing the same medium. The E. coli culture is grown as a fed batch fermentation using a glucose feed as the provided carbon source, at a high agitation speed with supplied oxygen and pH control, in order to yield high density cell culture. The E. coli is induced to turn on protein expression during exponential phase of growth. The culture is grown under induction conditions to allow for high levels of protein production. The E. coli culture is harvested and remaining medium is removed by centrifugation. Resulting cell paste is resuspended with 1x phosphate buffered saline (PBS) at a 5:1 vol / wt ratio. The resuspension is run through two passes in a microfluidizer at 10,000 psi in order to lyse cells. Cell lysate is treated with 0.5% polyethyleneimine (PEI) to remove contaminating DNA and insoluble cell waste and centrifuged at 12,000 x g for 10min at 4C to remove cell waste. The resulting supernatant containing the soluble protein target is collected. Protein is purified from this solution using four rounds of hot / cold centrifugation. In brief, the solution is heated at 45°C for 1 hour, causing the desired protein to aggregate and fall out of solution as an insoluble fraction. Once aggregated, the mixture is centrifuged at 5,000 x g for 10 minutes at 37°C in order to form a protein pellet of the target protein. The supernatant is discarded. The protein pellet is resuspended in 1x PBS (5:1 vol / wt) and incubated at 4°C, while shaking, for 4 hours in order to solubilize the protein back into solution. Once fully resuspended, the soluble protein solution is centrifuged at 12,000x g for 10 minutes at 4°C to remove insoluble waste. After centrifugation the cold supernatant containing the target protein is collected. The cycle of hot / cold centrifugation is repeated at least 3 times, achieving a 95% purity of our target protein, as determined by SDS-PAGE. The purified protein solution is then treated for endotoxin removal using a chemical treatment and filtration. Protein solution is brought into a salt free water background using dialysis with multiple buffer exchanges. The final, salt free, protein solution is filtered sterilized and then lyophilized for final storage.

[0251] A variety of POP formulations can be produced with the above exemplary protocol. In embodiments, the POPs produced can include a plurality of disordered domains and a plurality of ordered domains, where each disordered domain independently may comprise a PG or GP motif and each ordered domain independently may comprise an alpha helix. In embodiments, each disordered domain may comprise an amino acid sequence of (VPGXG)n, wherein X may be any amino acid except proline and n may be an integer greater than or equal to 1, and each ordered domain comprises a polyalanine motif. In embodiments, each disordered domain may comprise an amino acid sequence of (VPGXG)n, wherein each X may be independently Val or Ala, and wherein n may be an integer from 2 to 50, from 10 to 40, from 15 to 35, and / or fromATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 20 to 30. X may be an alternating iteration of Ala to Val in a ratio from 10:1 to 1:10. The polyalanine motif may comprises (A)m, wherein m may be an integer from 5 to 50. In embodiments, each disordered domain may comprise an amino acid sequence of (GXGVP)n (SEQ ID NO: 1), wherein X is any amino acid except proline and n is an integer greater than or equal to 1, and each ordered domain may comprise a polyalanine motif. Each disordered domain may comprise an amino acid sequence of (GXGVP)n(SEQ ID NO: 2), X may be independently Val or Ala, and n may be an integer from 2 to 50, from 10 to 40, from 15 to 35, and / or from 20 to 30. In embodiments, an alternating iteration of Ala to Val in the amino acid sequence of (GXGVP)n(SEQ ID NO: 27 or SEQ ID NO: 28) may be a ratio ranging from 10:1 to 1:10. The polyalanine motif may comprise one or more of (A)n(SEQ ID NO: 3), K(A)nK (SEQ ID NO: 4), D(A)nK (SEQ ID NO: 5), GD(An)K (SEQ ID NO: 6), or GK(An)K (SEQ ID NO: 7), wherein n may be an integer from 2 to 100. In an example, the polyalanine motif may comprise one or more of (A)25 (SEQ ID NO: 8), K(A)25K (SEQ ID NO: 9), D(A)25K (SEQ ID NO: 10), GD(A25)K (SEQ ID NO: 11), or GK(A25)K (SEQ ID NO: 12). In embodiments, the POP may have a transition temperature of heating (Tt-heating). The Tt-cooling may be dependent on the concentration of the partially ordered polypeptide in the solution. The Tt- heating and the Tt-cooling may range from about 10 °C to about 45 °C. The POP may form a solid aggregate above the Tt-heating.

[0252] While the POP may form an aggregate above the Tt-heating due to physical crosslinks from helical domain swapping, this does not preclude the use of additional chemical crosslinking techniques to further modulate mechanical properties.

[0253] In embodiments, a crosslinker may be added to the POP solution. The crosslinker may be a covalent crosslinker. The covalent crosslinker may by a chemical crosslinker, a pH-based crosslinker, may utilize click chemistry, ultraviolet light, or a combination thereof. When ultraviolet light is used, a UV functionalized POP may be used. In embodiments, the chemical crosslinker may be an amine reactive crosslinker. In embodiments, the chemical crosslinker may be configured to crosslink residues, motifs, and the like of the POPs. To this end, the chemical crosslinker may comprise at least one crosslinker or a derivative thereof selected from the group consisting of tetrakis (hydroxymethyl) phosphonium chloride (THCP), glutaraldehyde, pentanedial crosslinkers, NHS ester crosslinkers, NHS-maleimide crosslinkers, NHS-pyridyldithiol crosslinkers, (1R,8S,9s)-Bicyclo[6.1.0]non-4-yn-9-ylmethyl- N-succinimidyl carbonate, dibenzyocyclooctyne-N-hydroxysuccinimidyl ester, propargyl-N- hydroxysuccinimidyl ester, maleimide-PEG2-succinimidyl ester, Azido-dPEG®4-NHS ester, 3- (2-Pyridyldithio)propionic acid N-hydroxysuccinimide ester, 3-Maleimidobenzoic acid N-ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 hydroxysuccinimide ester, methyltetrazine-NHS ester, bromoacetic acid N- hydroxysuccinimide ester, O,O’-Bis[2-(N-Succinimidyl-succinylamino)ethyl]polyethylene glycol, maleimide-PEG8-succinimidyl ester, iodoacetic acid N-hydroxysuccinimide ester, alkyne-PEG5-N-hydroxysuccinimidyl ester, maleimide-PEG6-succinimidyl ester, 6- maleimidohexanoic acid N-hydroxysuccinimide ester, LC-SMCC (succinimidyl-4-(N- maleimidomethyl)cyclohexane-1-carboxy-(6-amidocaproate)), azido-dPEG®8-NHS ester, PEG4-SPDP (PEGylated, long-chain SPDP crosslinker), SMPH (succinimydl-6-((b- maleimidoproprionamido)hexanoate), LC-SPDP (succinimidyl 6-[3(2- pyridyldithio)propionamido]hexanoate), O-[N-(3-maleimidopropionyl)aminoethyl]-O’-[3-(N- succinimidyloxy)-3-oxopropyl]triethylene glycol, O-[N](3-Maleimidopropionyl)aminoethyl]- O’-[3-(N-succinimidyloxy)-3-oxopropyl]heptacosaethylene glycol, SBAP (succinimidyl 3- bromoacetamido)propionate), SPDP-dPEG®4-NHS ester, acid-dPEG®5-NHS ester, O,O’- Bis[2-(N-succinimidyl-succinylamino)ethyl]polyethylene glycol, SMPT (4- succinimidyloxycarbonyl-alpha-methyl-alpha(2-pyridyldithio)toluene), phthalimidooxy- dPEG®4-NHS ester, acid-dPEG®9-NHS ester, Fmoc-N-amido-dPEG®8-NHS ester, SPDP- dPEG®8-NHS ester, and Fmoc-N-amido-dPEG®4-NHS ester. In embodiments, the crosslinking may be performed after FractomerTMaggregation and / or on ice. In embodiments, the crosslinking may be performed above Tt-heating of the POPs. In embodiments, the chemical crosslinker may be added to the solution at a ratio of the POP to chemical crosslinker of about 1 to about 10, about 2 to about 10, about 3 to about 10, about 4 to about 10, about 5 to about 10, about 1 to about 1, about 2 to about 1, about 3 to about 1, about 4 to about 1, about 5 to about 1, about 10 to about 1, about 20 to about 1, about 30 to about 1, about 40 to about 1, about 50 to about 1, about 100 to about 1, about 200 to about 1, about 300 to about 1, about 400 to about 1, about 500 to about 1, about 1,000 to about 1, about 2,000 to about 1, about 3,000 to about 1, about 4,000 to about 1, about 5,000 to about 1, and / or about 10,000 to about 1. In embodiments, the chemical crosslinker is added to the composition at a ratio of chemical crosslinker to the composition based on a number of available crosslinking sites on each partially ordered polypeptide, wherein each crosslinking site is a lysine residue, an aspartic acid residue, an amine reactive site, or a carboxylic group reactive site. In embodiments, the ratio of chemical crosslinker to the composition is about 6 to about 1. In embodiments, the crosslinking comprises adding a crosslinker to the composition and periodically inverting the composition.

[0254] In some embodiments, no crosslinker is added to the POP solution.ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035

[0255] At step 104 of sub process 102, a solution of allograft tissue matrix is prepared (referred to herein interchangeably as the second solution). In embodiments, the allograft tissue matrix can be derived from a tissue comprising any one of the tissue sources above, as will be described in more detail later. In some embodiments, the allograft tissue matrix is added as a lyophilized power to a solution. In some embodiments, the solution comprises between about 200 μM and about 3 mM of the allograft tissue matrix. In some embodiments, the solution comprises an allograft tissue matrix at a concentration of about 300 μM and about 2.9 mM, about 400 μM and about 2.8 mM, about 500 μM and about 2.7 mM, about 600 μM and about 2.6 mM, about 700 μM and about 2.5 mM, about 800 μM and about 2.4 mM, about 900 μM and about 2.3 mM, about 1.0 mM and about 2.2 mM, about 1.1 mM and about 2.1 mM, about 1.2 mM and about 2.0 mM, about 1.3 mM and about 1.9 mM, about 1.4 mM and about 1.8 mM, or about 1.5 mM and about 1.7 mM. In some embodiments, the allograft tissue matrix is present in an amount of about 1 mg / mL to about 500 mg / mL. (e.g., 1, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 and 500 mg / mL). In some embodiments, the allograft tissue matrix is present in an amount of about 1 mg / mL to about 150 mg / mL. (e.g., 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 and 150 mg / mL). In some embodiments, the allograft tissue matrix is present in an amount of about 50 mg / mL to about 500 mg / mL, about 100 mg / mL to about 500 mg / mL, about 150 mg / mL to about 500 mg / mL, about 200 mg / mL to about 500 mg / mL, about 250 mg / mL to about 500 mg / mL, about 300 mg / mL to about 500 mg / mL, about 350 mg / mL to about 500 mg / mL, about 400 mg / mL to about 500 mg / mL, about 450 mg / mL to about 500 mg / mL, about 1 mg / mL to about 400 mg / mL, about 50 mg / mL to about 400 mg / mL, about 100 mg / mL to about 400 mg / mL, about 150 mg / mL to about 400 mg / mL, about 200 mg / mL to about 400 mg / mL, about 250 mg / mL to about 400 mg / mL, about 300 mg / mL to about 400 mg / mL, about 350 mg / mL to about 400 mg / mL, about 1 mg / mL to about 300 mg / mL, about 50 mg / mL to about 300 mg / mL, about 100 mg / mL to about 300 mg / mL, about 150 mg / mL to about 300 mg / mL, about 200 mg / mL to about 300 mg / mL, about 250 mg / mL to about 300 mg / mL, about 1 mg / mL to about 200 mg / mL, about 50 mg / mL to about 200 mg / mL, about 100 mg / mL to about 200 mg / mL, about 150 mg / mL to about 200 mg / mL, about 1 mg / mL to about 100 mg / mL, or about 50 mg / mL to about 100 mg / mL. In some embodiments, the allograft tissue matrix is present at an amount of about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL , about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 mg / mL, about 19 mg / mL, about 20 mg / mL, about 21 mg / mL, about 22 mg / mL, about 23 mg / mL, about 24 mg / mL, about 25 mg / mL, about 26 mg / mL, about 27 mg / mL, about 28 mg / mL, about 29 mg / mL, about 30 mg / mL, about 31 mg / mL, about 32 mg / mL, about 33 mg / mL, about 34 mg / mL, about 35 mg / mL, about 36 mg / mL, about 37 mg / mL, about 38 mg / mL, about 39 mg / mL, about 40 mg / mL, about 41 mg / mL, about 42 mg / mL, about 43 mg / mL, about 44 mg / mL, about 45 mg / mL, about 46 mg / mL, about 47 mg / mL, about 48 mg / mL, about 49 mg / mL, about 50 mg / mL, about 51 mg / mL, about 52 mg / mL, about 53 mg / mL, about 54 mg / mL, about 55 mg / mL, about 56 mg / mL, about 57 mg / mL, about 58 mg / mL, about 59 mg / mL, about 60 mg / mL, about 61 mg / mL, about 62 mg / mL, about 63 mg / mL, about 64 mg / mL, about 65 mg / mL, about 66 mg / mL, about 67 mg / mL, about 68 mg / mL, about 69 mg / mL, about 70 mg / mL, about 71 mg / mL, about 72 mg / mL, about 73 mg / mL, about 74 mg / mL, about 75 mg / mL, about 76 mg / mL, about 77 mg / mL, about 78 mg / mL, about 79 mg / mL, about 80 mg / mL, about 81 mg / mL, about 82 mg / mL, about 83 mg / mL, about 84 mg / mL, about 85 mg / mL, about 86 mg / mL, about 87 mg / mL, about 88 mg / mL, about 89 mg / mL, about 90 mg / mL, about 91 mg / mL, about 92 mg / mL, about 93 mg / mL, about 94 mg / mL, about 95 mg / mL, about 96 mg / mL, about 97 mg / mL, about 98 mg / mL, about 99 mg / mL, about 100 mg / mL, about 100 mg / mL, 101 mg / mL, about 102 mg / mL, about 103 mg / mL, about 104 mg / mL, about 105 mg / mL, about 106 mg / mL , about 107 mg / mL, about 108 mg / mL, about 109 mg / mL, about 110 mg / mL, about 111 mg / mL, about 112 mg / mL, about 113 mg / mL, about 114 mg / mL, about 115 mg / mL, about 116 mg / mL, about 117 mg / mL, about 118 mg / mL, about 119 mg / mL, about 120 mg / mL, about 121 mg / mL, about 122 mg / mL, about 123 mg / mL, about 124 mg / mL, about 125 mg / mL, about 126 mg / mL, about 127 mg / mL, about 128 mg / mL, about 129 mg / mL, about 130 mg / mL, about 131 mg / mL, about 132 mg / mL, about 133 mg / mL, about 134 mg / mL, about 135 mg / mL, about 136 mg / mL, about 137 mg / mL, about 138 mg / mL, about 139 mg / mL, about 140 mg / mL, about 141 mg / mL, about 142 mg / mL, about 143 mg / mL, about 144 mg / mL, about 145 mg / mL, about 146 mg / mL, about 147 mg / mL, about 148 mg / mL, about 149 mg / mL, or about 150 mg / mL, including all ranges, subranges and values therebetween.

[0256] In some embodiments, the allograft tissue matrix solutions described herein comprise at least one buffer. The buffer solution may comprise, but are not limited to, phosphate buffered saline (PBS), Dulbecco’s Phosphate Buffered Saline, HEPES, Trizma® base, sodium bicarbonate, Tris-EDTA buffer solution, water, saline (e.g., NaCl in water), distilled water, phosphate (e.g., sodium phosphate), acetate, and / or double distilled water. In some embodiments, the buffer solution comprises saline having a concentration of NaCl of 0.1%ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 NaCl, 0.2% NaCl, 0.3% NaCl, 0.4% NaCl, 0.5% NaCl, 0.6% NaCl, 0.7% NaCl, 0.8% NaCl, 0.9% NaCl, 1.0% NaCl, 2.0% NaCl, 3.0% NaCl, 3.0% NaCl, 4.0% NaCl, and 5.0% NaCl in water. In some embodiments, the dried allograft tissue matrix has a buffer solution content of about 1% to about 5% w / v and / or about 0.5% to about 70% w / v, wherein the buffer solution is a buffered saline, such as buffered saline. In some embodiments, the allograft tissue matrix solutions comprise at least one buffer at a concentration of about 1mM to about 1M (e.g., 25, 50, 100, 150, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950 and 1,000 mM). In some embodiments, the at least one buffer is present at 20 mM. In some embodiments, the buffer is present at about 10 mM to about 30 mM, about 15 mM to about 30 mM, about 20 mM to about 30 mM, about 25 mM to about 30 mM, about 10 mM to about 25 mM, about 15 to about 25 mM, about 20 mM to about 25 mM, about 10 mM to about 20 mM, or about 15 mM to about 20 mM.

[0257] Prior to sub process 102, the allograft tissue matrix used to prepare the allograft tissue matrix solution could be produced by the following exemplary protocol.

[0258] The allograft tissue matrix is derived from a source and processed. In embodiments, the allograft tissue matrix is derived from a source. In embodiments, the source comprises a tissue. In embodiments, the source tissue comprises a skin tissue, an amnion tissue, an artery tissue, a cartilage tissue, a connective tissue, a chorion tissue, a colon tissue, a non-calcified dental tissue, a dermal tissue, a duodenal tissue, an endothelial tissue, an epithelial tissue, a fascial tissue, a gastrointestinal tissue, a gingival tissue, a growth plate tissue, an intervertebral disc tissue, an intestinal mucosal tissue, an intestinal serosal tissue, a ligament tissue, a liver tissue, a lung tissue, a mammary tissue, a membranous tissue, a meniscal tissue, a muscle tissue, a nerve tissue, an ovarian tissue, a parenchymal organ tissue, a pericardial tissue, a periosteal tissue, a peritoneal tissue, a placental tissue, a skin tissue, a spleen tissue, a stomach tissue, a synovial tissue, a tendon tissue, a testes tissue, an umbilical cord tissue, a urological tissue, a vascular tissue, a vein tissue, other non-calcified tissues, and a combination thereof.

[0259] In embodiments, the source of the tissue is a mammalian donor. In embodiments, the source of the tissue is a human donor. In embodiments, the human donor is a living donor. In embodiments, the human donor is a cadaveric donor. In embodiments, the tissue donor is the intended recipient of a decellularized allograft tissue matrix. In embodiments, the tissue is a tissue derived from an autologous tissue. In embodiments, the tissue is a tissue comprising a tissue derived from an allogeneic tissue. In embodiments, the tissue is a tissue comprising a tissue derived from a xenogeneic tissue. In embodiments, the tissue comprises skin, bone, cartilage, nerve, muscle, ligament, or vascular tissue derived from an autologous skin, bone,ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 cartilage, nerve, muscle, ligament, or vascular tissue. In embodiments, the tissue comprises skin, bone, cartilage, nerve, muscle, ligament, or vascular tissue derived from an allogenic skin, bone, cartilage, nerve, muscle, ligament, or vascular tissue. In embodiments, the tissue comprises skin, bone, cartilage, nerve, muscle, ligament, or vascular tissue derived from an xenogenic skin, bone, cartilage, nerve, muscle, ligament, or vascular tissue.

[0260] In embodiments, the tissue comprises a cartilage tissue selected from the group comprising a hyaline cartilage tissue, a fibrocartilage tissue, an elastic cartilage tissue, and a combination thereof. In embodiments, the tissue comprises a non-calcified dental tissue. In embodiments, the tissue comprises an epithelial tissue selected from the group comprising a cutaneous epithelial tissue, a mucous epithelial tissue, a serous epithelial tissue, and a combination thereof. In embodiments, the tissue comprises a fascial tissue selected from the group comprising a superficial fascia, a deep fascia, a visceral fascia, and a combination thereof. In embodiments, the tissue comprises a ligament tissue selected from the group comprising a capsular ligament, an extra-capsular ligament, an intracapsular ligament, a cruciate ligament, and a combination thereof. In embodiments, the tissue comprises a muscle tissue selected from the group comprising a cardiac muscle tissue, a skeletal muscle tissue, a smooth muscle tissue, and a combination thereof. In embodiments, the tissue comprises a nerve tissue derived from a nerve tissue-rich organ or at least one fragment thereof. In embodiments, the tissue comprises a skin tissue selected from the group comprising an epidermal tissue, a dermal tissue, a basement membrane tissue; and a combination thereof. In embodiments, the tissue comprises a vascular tissue selection from the group comprising arteries, arterioles, capillaries, venules, and veins.

[0261] Once the allograft tissue matrix is obtained from the source tissue it can then be decellularized by the following exemplary process. In embodiments, the allograft tissue matrix is a decellularized allograft tissue matrix. The decellularized allograft tissue matrix comprises ECM from which unwanted cells and cell fragments have been removed. In embodiments, the tissue is autologous to the intended recipient of the decellularized allograft tissue matrix. In embodiments, the tissue is allogeneic to intended recipient of the decellularized allograft tissue matrix. In embodiments, the tissue is xenogeneic to the intended recipient of a decellularized allograft tissue matrix.

[0262] To begin, tissue is isolated from its source. In embodiments, the source is tissue. In embodiments, the source is skin, bone, cartilage, nerve, muscle, ligament, or vascular tissue. The tissue can then be excised or separated from its source. The excised tissue can then undergo a processing procedure before decellularization. In embodiments, the processing procedureATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 comprises cutting, mincing, grinding milling and heating the tissue. In embodiments, decellularization is a procedure comprising mechanical, enzymatic and chemical means to remove cellular material while leaving the ECM biopolymer components structurally and / or chemically intact. In embodiments, the enzymatic means comprises proteases. In embodiments, the enzymatic means comprise lipases, colipases, and nucleases. In embodiments, the mechanical means comprise agitation, sonication, pressure, or freeze-thaw cycles. In embodiments, the chemical means comprise a detergent. In embodiments, the detergent comprises sodium deoxysulfate, Triton X-100, Sodium lauryl Ether Sulphate, alkaline solutions, acidic solutions, and polar extraction solvents.

[0263] In embodiments, the allograft tissue matrix derived from the source tissue is referred to as an allograft tissue matrix composition. In embodiments, the allograft tissue matrix composition comprises the decellularized allograft tissue matrix in a volume of about 10% v / v to about 100% v / v (e.g., 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, or 100% v / v, including any values or ranges therein). In embodiments, the decellularized allograft tissue matrix has a volume comprising about 10% v / v to about 100% v / v, about 20% v / v to about 100% v / v, about 30% v / v to about 100% v / v, about 40% v / v to about 100% v / v, about 50% v / v to about 100% v / v, about 60% v / v to about 100% v / v, about 70% v / v to about 100% v / v, about 80% v / v to about 100% v / v, about 90% v / v to about 100% v / v, about 10% v / v to about 80% v / v, about 20% v / v to about 80% v / v, about 30% v / v to about 80% v / v, about 40% v / v to about 80% v / v, about 50% v / v to about 80% v / v, about 60% v / v to about 80% v / v, about 70% v / v to about 80% v / v, about 10% v / v to about 60% v / v, about 20% v / v to about 60% v / v, about 30% v / v to about 60% v / v, about 40% v / v to about 60% v / v, about 50% v / v to about 60% v / v, about 10% v / v to about 40% v / v, about 20% v / v to about 40% v / v, about 30% v / v to about 40% v / v, or about 10% v / v to about 20% v / v, including all ranges and values therein. In embodiments, the allograft tissue matrix composition comprises the decellularized allograft tissue matrix in a volume of about 10% v / v to about 90% v / v. In embodiments, the allograft tissue matrix composition comprises the decellularized allograft tissue matrix in a volume of about 10% v / v to about 50% v / v.

[0264] At step 105 of sub process 102, solutions of POP and allograft tissue matrix are mixed below the Tt-heating of the POP to form a composition. In some embodiments, solutions of POP and allograft tissue matrix are mixed. In some embodiments, the POP is added to the mixture concurrently with the allograft tissue matrix. In some embodiments, the POP is added to the mixture before the allograft tissue matrix. In other embodiments, the POP is added to the mixture after the allograft tissue matrix. In some embodiments, the POP is mixed at higherATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 levels with the allograft tissue matrix. In some embodiments, the POP is mixed at the same level with the allograft tissue matrix. In some embodiments, the POP is mixed at a lower level with the allograft tissue matrix. In some embodiments, only the POP is in the mixture. In some embodiments, the POP and allograft tissue matrix are present in a ratio of 95:5, 90:10, 70:30, 50:50, 30:70, 10:90, or 5:95 (POP: allograft tissue matrix), including all ranges, subranges, and values therebetween.

[0265] Returning to Fig. 1, at step 106 of method 100, the prepared matrix composition is administered to a subject.

[0266] In some embodiments, the matrix composition is administered to a subject at an amount of about 1 mg / mL to about 500 mg / mL. (e.g., 1, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475 and 500 mg / mL, including all ranges and values therein).

[0267] In some embodiments, the matrix composition is administered to a subject at an amount of about 1 mg / mL to about 300 mg / mL. (e.g., 1, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, and 300 mg / mL, included all ranges and values therein). In some embodiments, the matrix composition is administered to a subject at an amount of about 1 mg / mL to about 150 mg / mL (e.g., 1, 5, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, 120, 125, 130, 135, 140, 145 and 150 mg / mL, including any ranges and values therein). In some embodiments, the matrix composition is administered to a subject at an amount of about 50 mg / mL to about 500 mg / mL, about 100 mg / mL to about 500 mg / mL, about 150 mg / mL to about 500 mg / mL, about 200 mg / mL to about 500 mg / mL, about 250 mg / mL to about 500 mg / mL, about 300 mg / mL to about 500 mg / mL, about 350 mg / mL to about 500 mg / mL, about 400 mg / mL to about 500 mg / mL, about 450 mg / mL to about 500 mg / mL, about 1 mg / mL to about 400 mg / mL, about 50 mg / mL to about 400 mg / mL, about 100 mg / mL to about 400 mg / mL, about 150 mg / mL to about 400 mg / mL, about 200 mg / mL to about 400 mg / mL, about 250 mg / mL to about 400 mg / mL, about 300 mg / mL to about 400 mg / mL, about 350 mg / mL to about 400 mg / mL, about 1 mg / mL to about 300 mg / mL, about 50 mg / mL to about 300 mg / mL, about 100 mg / mL to about 300 mg / mL, about 150 mg / mL to about 300 mg / mL, about 200 mg / mL to about 300 mg / mL, about 250 mg / mL to about 300 mg / mL, about 1 mg / mL to about 200 mg / mL, about 50 mg / mL to about 200 mg / mL, about 100 mg / mL to about 200 mg / mL, about 150 mg / mL to about 200 mg / mL, about 1 mg / mL to about 100 mg / mL, or about 50 mg / mL to about 100 mg / mL. In some embodiments, the composition is administered at an amount of about 1 mg / mL, about 2 mg / mL, about 3 mg / mL, about 4 mg / mL, about 5 mg / mL, about 6 mg / mL , about 7 mg / mL, about 8 mg / mL, about 9 mg / mL, about 10ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 mg / mL, about 11 mg / mL, about 12 mg / mL, about 13 mg / mL, about 14 mg / mL, about 15 mg / mL, about 16 mg / mL, about 17 mg / mL, about 18 mg / mL, about 19 mg / mL, about 20 mg / mL, about 21 mg / mL, about 22 mg / mL, about 23 mg / mL, about 24 mg / mL, about 25 mg / mL, about 26 mg / mL, about 27 mg / mL, about 28 mg / mL, about 29 mg / mL, about 30 mg / mL, about 31 mg / mL, about 32 mg / mL, about 33 mg / mL, about 34 mg / mL, about 35 mg / mL, about 36 mg / mL, about 37 mg / mL, about 38 mg / mL, about 39 mg / mL, about 40 mg / mL, about 41 mg / mL, about 42 mg / mL, about 43 mg / mL, about 44 mg / mL, about 45 mg / mL, about 46 mg / mL, about 47 mg / mL, about 48 mg / mL, about 49 mg / mL, about 50 mg / mL, about 51 mg / mL, about 52 mg / mL, about 53 mg / mL, about 54 mg / mL, about 55 mg / mL, about 56 mg / mL, about 57 mg / mL, about 58 mg / mL, about 59 mg / mL, about 60 mg / mL, about 61 mg / mL, about 62 mg / mL, about 63 mg / mL, about 64 mg / mL, about 65 mg / mL, about 66 mg / mL, about 67 mg / mL, about 68 mg / mL, about 69 mg / mL, about 70 mg / mL, about 71 mg / mL, about 72 mg / mL, about 73 mg / mL, about 74 mg / mL, about 75 mg / mL, about 76 mg / mL, about 77 mg / mL, about 78 mg / mL, about 79 mg / mL, about 80 mg / mL, about 81 mg / mL, about 82 mg / mL, about 83 mg / mL, about 84 mg / mL, about 85 mg / mL, about 86 mg / mL, about 87 mg / mL, about 88 mg / mL, about 89 mg / mL, about 90 mg / mL, about 91 mg / mL, about 92 mg / mL, about 93 mg / mL, about 94 mg / mL, about 95 mg / mL, about 96 mg / mL, about 97 mg / mL, about 98 mg / mL, about 99 mg / mL, about 100 mg / mL, 101 mg / mL, about 102 mg / mL, about 103 mg / mL, about 104 mg / mL, about 105 mg / mL, about 106 mg / mL , about 107 mg / mL, about 108 mg / mL, about 109 mg / mL, about 110 mg / mL, about 111 mg / mL, about 112 mg / mL, about 113 mg / mL, about 114 mg / mL, about 115 mg / mL, about 116 mg / mL, about 117 mg / mL, about 118 mg / mL, about 119 mg / mL, about 120 mg / mL, about 121 mg / mL, about 122 mg / mL, about 123 mg / mL, about 124 mg / mL, about 125 mg / mL, about 126 mg / mL, about 127 mg / mL, about 128 mg / mL, about 129 mg / mL, about 130 mg / mL, about 131 mg / mL, about 132 mg / mL, about 133 mg / mL, about 134 mg / mL, about 135 mg / mL, about 136 mg / mL, about 137 mg / mL, about 138 mg / mL, about 139 mg / mL, about 140 mg / mL, about 141 mg / mL, about 142 mg / mL, about 143 mg / mL, about 144 mg / mL, about 145 mg / mL, about 146 mg / mL, about 147 mg / mL, about 148 mg / mL, about 149 mg / mL, or about 150 mg / mL including all ranges, subranges and values therebetween. In some embodiments, the matrix composition is administered at about 15 mg / mL to about 35 mg / mL, about 20 mg / mL to about 35 mg / mL, about 25 mg / mL to about 35 mg / mL, about 15 mg / mL to 30 mg / mL, about 20 mg / mL to about 30 mg / mL, about 25 mg / mL to about 30 mg / mL, about 15 mg / mL to about 25 mg / mL, or about 20 mg / mL to about 25 mg / mL.ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035

[0268] In some embodiments, the matrix composition can be used as an allogenic tissue graft. In some embodiments, the composition is administered to the subject in a region of interest. In some embodiments, the POP and allograft tissue matrix are mixed in vitro, shaped and then implanted in situ in the subject. In some embodiments the POP and allograft tissue matrix composition are a shapeable liquid, semisolid or molded semisolid prior to administration.

[0269] At step 107 of method 100, and when the matrix composition is administered to the subject as a flowable material, the administered composition is aggregated upon exposure to temperatures above Tt-heating (e.g., body temperature). In some embodiments, the composition is soluble below LCST. In some embodiments, the composition has a transition temperature of heating (Tt-heating) and a transition temperature of cooling (Tt-coo1ing). In some embodiments, the transition temperature of heating (Tt-heating) and transition temperature of cooling (Tt-cooling) may be identical. In some embodiments, the transition temperature of heating (Tt-heating) is greater than the transition temperature of cooling (Tt- cooling). In some embodiments, the Tt-heating may be dependent on the concentration of the POP in the composition. The Tt-heating and the Tt-cooling may range from about 10 °C to about 45 °C. In some embodiments the Tt-heating and the Tt-cooling may range from about 10 °C to about 40 °C, about 15 °C to about 35 °C, and / or about 20 °C to about 30 °C. In some embodiments, the composition forms a solid aggregate above the Tt-heating.

[0270] In some embodiments, the POP solution is mixed with an allograft tissue matrix solution below the Tt-heating of the POP to form a composition and is then administered to a subject. In some embodiments, solutions of POP and allograft tissue matrix are mixed. In some embodiments no allograft tissue matrix is added to the mixture.

[0271] As stated above with reference to step 106 of method 100, the composition is administered to the subject in a region of interest. In some embodiments, the POP is administered concurrently with the allograft tissue matrix. In some embodiments, the POP is administered prior to the allograft tissue matrix. In some embodiments, the POP is administered after the allograft tissue matrix. In some embodiments, the POP is not administered with an allograft tissue matrix.

[0272] In embodiments, upon administration of a composition to a subject the composition flattens and spreads. In embodiments, if the composition comprises POP then the composition spreads and flattens less than compositions without the POP.ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 Uses of the matrix compositions

[0273] In embodiments, the matrix compositions described herein can be used as bulking agents in reconstructive or cosmetic surgery procedures (e.g., for filling voids in tissue or smoothing wrinkles), and as scaffolds for hard and / or soft tissue matrix repair, reconstruction, and / or augmentation.

[0274] In embodiments, in strip or sheet form, decellularized allograft tissue matrixes according to the present invention may be used to provide structural support to other tissues, and also to provide scaffolds for tissue matrix repair, reconstruction, and / or augmentation. Examples of procedures in which decellularized allograft tissue matrixes may be used include, but are not limited to, wound treatment, tissue grafts, volume replacement, facial defect filling and rejuvenation in the aging face. In embodiments, injectable decellularized allograft tissue matrices may also be used for both superficial and deep aesthetic applications, including lip augmentation or rejuvenation of the aging lip to restore shape and contour, fine line filling to reduce the appearance of wrinkles around the eyes or mouth, improvement of nasolabial folds, correction of eyelid deformities, and for deep wrinkle or scar filling.

[0275] In embodiments, the matrix composition has the shape of an anatomical feature. In embodiments, the matrix composition has a shape comprising the shape of a tissue.

[0276] In embodiments, there matrix compositions provided herein can be used for a tissue graft. A graft is a tissue or organ used for transplantation to a patient. A common strategy employed in tissue engineering involves the seeding of decellularized natural ECM or synthetic scaffolds with a variety of different stem or progenitor cells that are capable of regeneration (see, for example, Flynn and Woodhouse, 2008, Organogenesis, 4(4): 228-235; Uriel et al., 2008, Biomaterials, 29: 3712-3719; Flynn, 2010, Biomaterials, 31: 4715-4724; Choi et al., Tissue Engg. C., 16(3): 387-396; Brown et al., 2011, Tissue Engg. C., 17(4): 411-421; Cheng et al., 2009, Tissue Engg. A, 15(2): 231-241; Li et al., 2011, Biomaterials, doi:10:1016 / j.biomaterials.2011.03.008; Butler et al., 2003, Connective Tissue Research, 44(S1): 171-178); Mercuri et al., J. Biomed. Mater. Res. A., 96(2): 422-435); Olson et al., 2011, Chonnam. Med. J. 47:1-13). In embodiments, the matrix compositions can be used for an organ graft. Patients suffering from affected or injured organs may be treated with organ transplantation. In embodiments, the matrix compositions can be used in a bone graft to treat bone defects created during tumor surgery or caused by trauma, congenital skeletal abnormalities, fracture, scoliosis, spinal arthrodesis, or joint and tooth replacement. In embodiments, the matric compositions used in a bone graft further comprise autologous or allogeneic stem cells.ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035

[0277] Described herein is a method of tissue matrix repair, reconstruction, and / or augmentation in a subject, the method comprising: administering a volume of the matrix composition to the subject. In embodiments, the matrix composition is administered in a subject at an administration site. In embodiments, the administration site comprises a region of a naturally-created tissue defect or a surgically-created tissue defect. In embodiments, the administration site comprises places that a tissue naturally exists in the subject. In embodiments, the places that the tissue naturally exists in the subject comprise skin, bone, cartilage, nerve, muscle, ligament, or vasculature.

[0278] In embodiments, the allograft tissue matrix repair, reconstruction, or augmentation aims to restore or replace soft tissues comprising skin, muscles, tendons, ligaments, nerves, and organs. In embodiments, the soft tissue exhibits one or more of an acute injury, surgery incision, laceration, puncture, wound, abrasion, avulsion, pressure ulcers, burns, scarring, acne, eczema, and psoriasis. In embodiments, the soft allograft tissue matrix repair, reconstruction, or augmentation comprises skin grafting, flap surgery, tissue expansion, local tissue rearrangement, composite tissue allotransplantation, and nerve reconstruction. In embodiments, the allograft tissue matrix repair, reconstruction, or augmentation aims to restore or replace hard tissues comprising bones or teeth. In embodiments, the hard tissue exhibits one or more of a wound, fracture, crush injury, facial wound, disease, infection, cleft lip and palate, skeletal dysplasia, degenerative condition, facial contouring, joint reconstruction, spinal reconstruction, and injury from surgery. In embodiments, hard tissue matrix reconstruction comprises bone grafting, bone transport, bone augmentation, bone regeneration, maxillofacial reconstruction, dental implant, cranial reconstruction and orthopedic reconstruction.

[0279] In embodiments, the method further comprises prior to administering the volume of the matrix composition to the subject, preparing a mixture of the partially ordered polypeptide and the allograft tissue matrix at a temperature below a transition temperature of the partially ordered polypeptide, wherein, after administering the volume of the matrix composition to the subject, the matrix composition aggregates after exposure to the subject’s body temperature, which is above the transition temperature of the partially ordered polypeptide.

[0280] In embodiments, the volume of the matrix composition administered to the subject is between about 1 mL and about 1,500 mL (e.g., 1, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, 300, 325, 350, 375, 400, 425, 450, 475, 500, 525, 550, 575, 600, 625, 650, 675, 700, 725, 750, 775, 800, 825, 850, 875, 900, 925, 950, 975, 1,000, 1,025, 1,050, 1,075, 1,100, 1,125, 1,150, 1,175, 1,200, 1,225, 1,250, 1,275, 1,300, 1,325, 1,350, 1,375, 1,400, 1,425, 1,450, 1,475, or 1,500 mL, including any values or ranged therein). In embodiments, a reference matrixATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 composition comprises the same volume and material as the matrix compositions described herein, but without POP.

[0281] Described herein is use of a volume of a partially ordered polypeptide and a volume of allograft tissue matrix to augment tissue in a subject. Described herein is use of a volume of a partially ordered polypeptide and a volume of allograft tissue matrix to replace tissue in a subject. In embodiments, the replaced tissues comprises a skin tissue, a bone tissue, a cartilage tissue, a nerve tissue, a muscle tissue, a ligament tissue, and a vascular tissue.

[0282] Described herein is a method of tissue matrix repair, reconstruction, and / or augmentation in a subject, the method comprising: administering a volume of a partially ordered polypeptide to the subject; administering a volume of allograft tissue matrix to the subject, wherein the administered volume of the allograft tissue matrix is co-located with the administered volume of the partially ordered polypeptide, thereby forming a matrix composition in situ.

[0283] In embodiments, when the matrix composition comprises a pre-administration volume and a post- administration volume measured post-administration, the post- administration volume of the matrix composition being greater than a post- administration volume of a reference matrix composition measured post-administration, the reference matrix composition comprising only allograft tissue matrix. In embodiments, the matrix composition comprises more epithelial cells post-administration than immediately prior to administration. In embodiments, the matrix composition has at least 5%, at least 10%, at least 15%, at least 20%, at least 25% more epithelial cells than immediately prior to administration. In embodiments, the epithelial cells are measured 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks or more post- administration, including any values or ranges therein. In embodiments, the matrix composition comprises more osteocytes post-administration than immediately prior to administration. In embodiments, the matrix composition has at least 5%, at least 10%, at least 15%, at least 20%, at least 25% more osteocytes than immediately prior to administration. In embodiments, the osteocytes are measured 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks or more post-administration, including any values or ranges therein. In embodiments, the matrix composition comprises more chondrocytes post-administration than immediately prior to administration. In embodiments, the matrix composition has at least 5%, at least 10%, at least 15%, at least 20%, at least 25% more chondrocytes than immediately prior to administration. In embodiments, the chondrocytes are measured 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks or more post-administration, including any values or ranges therein. In embodiments, the matrix composition comprises more glial cells post-administration than immediately prior to administration. In embodiments,ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 the matrix composition has at least 5%, at least 10%, at least 15%, at least 20%, at least 25% more glial cells than immediately prior to administration. In embodiments, the glial cells are measured 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks or more post-administration, including any values or ranges therein. In embodiments, the matrix composition comprises more myocytes post-administration than immediately prior to administration. In embodiments, the matrix composition has at least 5%, at least 10%, at least 15%, at least 20%, at least 25% more myocytes than immediately prior to administration. In embodiments, the myocytes are measured 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks or more post-administration, including any values or ranges therein. In embodiments, the matrix composition comprises more fibroblasts post-administration than immediately prior to administration. In embodiments, the matrix composition has at least 5%, at least 10%, at least 15%, at least 20%, at least 25% more fibroblasts than immediately prior to administration. In embodiments, the fibroblasts are measured 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks or more post-administration, including any values or ranges therein. In embodiments, the matrix composition comprises more endothelial cells post-administration than immediately prior to administration. In embodiments, the matrix composition has at least 5%, at least 10%, at least 15%, at least 20%, at least 25% more endothelial cells than immediately prior to administration. In embodiments, the endothelial cells are measured 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks or more post-administration, including any values or ranges therein.

[0284] In embodiments, the matrix composition comprises a pre-administration volume and a post-administration volume measured after administration, the post- administration volume being from about 50% to about 120% of the pre- administration volume (e.g., 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100, 105, 110, 115, or 120%, including any values or ranges therein). In embodiments, an initial projection of the matrix composition measured immediately post- administration is substantially similar to a future projection of the matrix composition measured after administration. In embodiments, each projection of the matrix composition is measured relative to an adjacent tissue not within an administration site. In embodiments, the post-administration volume is measured 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks or more post- administration, including any values or ranges therein.

[0285] The allograft tissue matrices described herein can be developed to resemble the tensile strength or compression modulus of a desired tissue. In embodiments, the matrix composition has a stiffness about the same as or less than a reference matrix composition without partially ordered polypeptide, as measured 1 hour post-aggregation of the matrix composition. In embodiments, matrix composition is derived from a tissue comprising a skin tissue, an amnionATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 tissue, an artery tissue, a cartilage tissue, a connective tissue, a chorion tissue, a colon tissue, a non-calcified dental tissue, a dermal tissue, a duodenal tissue, an endothelial tissue, an epithelial tissue, a fascial tissue, a gastrointestinal tissue, a gingival tissue, a growth plate tissue, an intervertebral disc tissue, an intestinal mucosal tissue, an intestinal serosal tissue, a ligament tissue, a liver tissue, a lung tissue, a mammary tissue, a membranous tissue, a meniscal tissue, a muscle tissue, a nerve tissue, an ovarian tissue, a parenchymal organ tissue, a pericardial tissue, a periosteal tissue, a peritoneal tissue, a placental tissue, a skin tissue, a spleen tissue, a stomach tissue, a synovial tissue, a tendon tissue, a testes tissue, an umbilical cord tissue, a urological tissue, a vascular tissue, a vein tissue, other non-calcified tissues, and a combination thereof. In embodiments, the stiffness is measured about 1 hour to 12 weeks (e.g., 1 hour, 2 hours, 4 hours, 8 hours, 12, hours, 1 day, 1 week, 4 weeks, 8 weeks or 12 weeks, including any values or ranges therein) post-post aggregation of the matric composition, including any values or ranges therein.

[0286] In embodiments, the tissue matrix composition is administered in a subject at a location comprising an administration site. In embodiments, the administration site comprises a region of excised tissue. In embodiments, the matrix composition has more cells present at an administration site after administered than a reference matrix composition without partially ordered polypeptide. In embodiments, the cells are measured 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks or more post-administration, including any values or ranges therein. In embodiments, the cells comprise epithelial cells, osteocytes, chondrocytes, glial cells, myocytes, fibroblasts, and endothelial cells.

[0287] In embodiments, the matrix composition has about the same, or at least 1% more, at least about 2% more, at least about 3% more, at least about 4% more, at least about 5% more, at least about 6% more, at least about 7% more, at least about 8% more, at least about 9% more, and / or at least about 10% more cells present at an administration site post-administration than a reference matrix composition without partially ordered polypeptide. In embodiments, the cells are measured 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks or more post-administration, including any values or ranges therein. In embodiments, the cells comprise epithelial cells, osteocytes, chondrocytes, glial cells, myocytes, fibroblasts, and endothelial cells.

[0288] In embodiments, the matrix composition has about the same or more cells present at an administration site post-administration than a reference matrix composition without partially ordered polypeptide. In embodiments, the cells are measured 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 weeks or more post-administration, including any values or ranges therein. In embodiments,ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 the cells comprise epithelial cells, osteocytes, chondrocytes, glial cells, myocytes, fibroblasts, and endothelial cells.

[0289] In embodiments, the matrix composition can be administered to a subject via a number of routes. In embodiments, administration routes comprise injection, implantation, and topical application. In embodiments, administration comprises implanting via open surgery or minimally invasive surgery. In embodiments, administration comprises injections in a liquid or gel form. In embodiments, syringe pumps can be used to precisely control flow rates during injections.

[0290] In embodiments, the matrix compositions described herein can be administered to augment shapes or volumes in a region of interest of a subject. Specific exemplary shapes or volumes that could be augmented anatomical shapes, or other irregular or indefinite shapes or volumes present in a subject’s body.

[0291] In embodiments, the matrix compositions described herein retain shape at least 2-times longer than otherwise similar compositions comprising an allograft tissue matrix alone. In some embodiments, compositions comprising the POP retains shape at least 3-times, 4-times, 5- times, 6-times, 7-times, 8-time, 9-times, or 10-times longer, or any value or range therein. In some embodiments, the matrix compositions described herein comprising the POP retain shape within a 10% deviation over a period of time. In some embodiments, the period of time comprises about 1 weeks about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 moths, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, about 19 months, about 20 months, about 21 months, about 22 months, about 23 months, or about 24 months, or any value or range therein.

[0292] In some embodiments, the matrix compositions retain volume at least 2-times longer than the matrix compositions comprising an allograft tissue matrix alone. In some embodiments, the matrix compositions comprising the POP retain volume at least 3-times, 4- times, 5-times, 6-times, 7-times, 8-time, 9-times, or 10-times longer, or any value or range therein. In some embodiments, the matrix compositions described herein comprising the POP retain volume within a 10% deviation over a period of time. In some embodiments, the period of time comprises about 1 week, about 2 weeks, about 3 weeks, about 1 month, about 2 months, about 3 months, about 4 months, about 5 moths, about 6 months, about 7 months, about 8 months, about 9 months, about 10 months, about 11 months, about 12 months, about 13 months, about 14 months, about 15 months, about 16 months, about 17 months, about 18 months, aboutATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 19 months, about 20 months, about 21 months, about 22 months, about 23 months, or about 24 months, or any value or range therein. In some embodiments, the matrix composition comprises a pre-administration volume and a post-administration volume.

[0293] In some embodiments, tissue grafts are augmented by the combination of the allograft tissue matrix with a POP which leads to the enhanced long-term maintenance and survival of the tissue graft including conservation of the volume and shape of the tissue graft, improved vascularization, enhanced survival, and reduction in necrosis.

[0294] In embodiments, cells can be added exogenously to the matrix composition. In embodiments, cells can be added to the tissue matrix composition prior to administration in a subject. In embodiments, cells can be added to the matrix composition prior to implantation or injection. In embodiments, cells can be added to the matrix composition during implantation or injection. In embodiments, the exogenous cells added can be autologous cells. In embodiments, the exogenous cells can be any cell selected from the cells listed and described elsewhere herein. In embodiments, the exogenous cells can be any cell selected from the cells listed under “Stem Cells”. Implantable or Injectable Matrix Compositions

[0295] As introduced above, embodiments of the present invention include implantable and injectable matrix compositions, and, optionally, exogenous stem cells, growth factors, and a carrier. According to some embodiments, the matrix composition is in the form of a sheet. According to some embodiments, the matrix composition is in particulate form. According to some embodiments, the matrix composition is in the form of a paste, gel, or slurry. According to some embodiments, the matrix composition is injectable. According to some embodiments, the matrix composition is in a dried, pre-formed shape. According to some embodiments, the matrix composition is in a porous form. According to some embodiments, the matrix composition is in a fibrous form. According to some embodiments, the matrix composition fills a void in a tissue. In embodiments, the tissue comprises skin, bone, cartilage, nerve, muscle, ligament, or vascular tissue. According to some embodiments, the matrix composition is in bulking agent. According to some embodiments, the matrix composition is decellularized allograft tissue matrix with a POP.

[0296] According to some embodiments, the matrix composition is a scaffold for the delivery of growth-inductive factors. According to some embodiments, the matrix composition is growth-inductive. According to some embodiments, the matrix composition is a scaffold for the delivery of cells. According to some embodiments, the matrix composition is a scaffold forATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 the migration cells. According to some embodiments, the matrix composition is a growth- conductive medium for the ingrowth of tissue.

[0297] According to some embodiments of the present invention, implantable matrix compositions include shaped allograft tissue matrices comprising three-dimensional shaped structures formed by a process in which decellularized tissues are broken into smaller components (e.g., by milling or homogenization), then reformed into a three-dimensional structure that is different from the source tissue.

[0298] According to some embodiments of the present invention, the matrix compositions are mechanically or chemically manipulated into a particulate form, which can be resuspended in a liquid (e.g., water or a buffer solution) to form a flowable mass, such as a slurry. The flowable mass may be poured into a mold of a desired shape, in which it may form a porous or sponge- like shaped matrix composition. According to some embodiments, the liquid or particle matrix compositions are manipulated to form a putty, which can then be molded into a desired shape. According to some embodiments, the liquid or particle matrix compositions are manipulated to form a paste. According to some embodiments, the liquid or particle matrix compositions are manipulated to form a gel. According to some embodiments, the gel is formed during the process used to delipidate an allograft tissue matrix. According to some embodiments, the decellularized allograft tissue matrix particles are mixed with a polymer to form a paste or a putty. According to some embodiments, the porous or sponge-like shaped matrix composition is formed by drying the slurry, paste or gel. According to some embodiments, the porous or sponge-like shaped matrix composition is formed by lyophilizing the slurry, paste or gel. According to some embodiments, the porosity of the shaped matrix composition is controlled selecting the amount of liquid relative to the amount of particulate matrix composition particles. According to some embodiments, the porous or sponge-like shaped matrix composition is a solid piece that conforms to the shape of the mold after being dried.

[0299] According to some embodiments, the shaped matrix composition is formed by one or more of the processes of molding a slurry, paste, or gel, machining a sponge-like shaped matrix composition into a different shape, using three-dimensional (“3-D”) printing to deposit a flowable slurry, paste, or gel into a three-dimensional shape, laminating pieces of shaped matrix composition, and other technologies known for use in shaping three-dimensional objects from soft or flowable materials. According to some embodiments, shaped matrix composition may be provided in a lyophilized, cryopreserved, or frozen form.

[0300] According to some embodiments of the present invention, shaped matrix composition may be used to surgically repair defects in a patient. According to some embodiments, shapedATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 matrix compositions are used alone or after being seeded or cultured with appropriate exogenous stem cells. According to some such embodiments, the cells may be either autologous or allogeneic, or a mixture of autologous and allogeneic cells. According to some embodiments, the shaped matrix compositions are provided with substances such as growth factors, proteins, angiogenic factors and / or controlled-release nanotubes / nanoparticles that preferentially secrete factors for specific processes. According to some embodiments, the substances are added to the slurry, paste, or gel before the shaped matrix composition is formed. According to some embodiments, the substances are added to the shaped matrix composition after it is formed.

[0301] According to some embodiments of the present invention, the degradation profile of the shaped matrix composition and a substance therein cause the substance to be released at an appropriate time for growth or healing of tissues to occur. An example of this would be to promote the formation of vasculature necessary to supply cells within or adjacent to a s matrix composition with nutrients. In such an example, specific factors, cells, and other substances, if needed, may be provided at selected locations on the shaped matrix composition to promote angiogenesis at the desired locations. Other factors may be included that do not promote angiogenesis, such that vasculature is formed only where is it desired. As a more specific example, the creation of a kidney using a shaped matrix composition would include such factors and cells needed to preferentially create architecture for renal arteries, renal veins, a ureter, and other features of a functional kidney.

[0302] According to some embodiments of the present invention, the matrix compositions have simple shapes. According to some embodiments, the shaped matrix compositions have complex shapes. According to some embodiments, the matrix compositions have symmetrical shapes. According to some embodiments, the shaped matrix compositions have asymmetrical shapes. According to some embodiments, the matrix compositions have shapes similar to the shapes of anatomical structures. According to some embodiments, the shaped matrix compositions have the shapes of anatomical organs.

[0303] According to some embodiments, the shaped matrix compositions are provided for ex vivo use. According to some embodiments, the shaped matrix compositions are provided for in vivo use (i.e., for implantation). According to some embodiments, the shaped matrix compositions have porosities customized for their intended use. According to some embodiments of the present invention, the shaped matrix compositions have pH customized for their intended use. According to some embodiments, the shaped matrix compositions include cross-linked collagen. According to some embodiments of the present invention, theATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 matrix compositions include cross-linked non-collagen components. According to some embodiments, the shaped matrix compositions have biological polymers that are cross-linked with non-biological (i.e., synthetic) polymers.

[0304] According to some embodiments of the present invention, a method of forming a shaped matrix compositions includes a step of scanning or imaging a portion of a patient's body (e.g., a portion of patient's face or other anatomical structure), then making shaped matrix compositions to replace those anatomical structures. In other embodiments, a shaped matrix composition is made to restore the shape of an anatomical structure. In other embodiments, a shaped matrix composition is made to provide a substitute for a missing anatomical structure.

[0305] According to some embodiments of the present invention, the shaped matrix composition is formed, then cultured in vitro with exogenous cells. When the cells reach a sufficient number, the shaped matrix compositions is implanted for plastic and / or reconstructive surgery.

[0306] According to some embodiments of the present invention, the shaped matrix composition is formed, then cultured in vitro with cells. When the cells reach a sufficient number, the composition is cryopreserved, and then reconstituted when needed for use.

[0307] According to an embodiment of the present invention, mesenchymal stem cells are harvested from a patient in need of a nasal graft, cultured onto a shaped decellularized tissue matrix resembling the patient's own nasal structure. After the cells have differentiated into a sufficient number of chondrocytes, the shaped matrix composition can be provided to the patient as a viable graft.

[0308] According to an embodiment of the present invention, a shaped matrix composition is provided in a lyophilized form. The lyophilized shaped decellularized tissue matrix is rehydrated in the operating room, where it may be combined with such substances as the patient's platelet-rich plasma (PRP), autologous cells such as those obtained from the patient's bone marrow or stromal vascular fraction (SVF) (e.g. SVF from adipose tissue obtained by liposuction), allogeneic cells such as those obtained from a cell bank (e.g., stem cells, progenitor cells or other cell types available from cell banks), or bone marrow and bone marrow components including bone marrow cells (both autologous and allogeneic).

[0309] Embodiments of the present invention include methods of making the various embodiments of shaped matrix compositions described above. Embodiments of such methods will be obvious to those having ordinary skill in the art and possession of the present disclosure.ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035

[0310] In embodiments, the allograft tissue matrix is decellularized. In embodiments, the decellularized allograft tissue matrix is delipidated. In embodiments, the allograft tissue matrix is derived from allograft tissue. In embodiments, decellularized allograft tissue matrix comprises exogenous cells. In embodiments, exogenous cells comprise exogenous stem cells. In embodiments, the exogenous stem cells comprise one or more of hematopoietic stem cells, mesenchymal stem cells, epithelial stem cells, gastrointestinal epithelial stem cells, neural stem cells, and skin stem cells. In embodiments, the decellularized allograft tissue matrix comprises at least one exogenous growth-inductive substance. In embodiments, the at least one exogenous growth-inductive substance is selected from the group comprising bone morphogenic proteins, vascular endothelial growth factor, basic fibroblast growth factor, transforming growth factor beta, platelet-derived growth factor, neural epidermal growth-factor-like 1, and insulin-like growth factor.

[0311] In embodiments, wherein the decellularized allograft tissue matrix is substantially free of native cellular components. In embodiments, substantially free of native cellular components comprises less than 10%, less than 5% or less than 1% of native cellular components. In embodiments, substantially free of native cellular components comprises less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2% or 1% native cellular components, including any values or ranges therein.

[0312] In embodiments, the allograft tissue matrix comprises one or more fragments of: collagen, glycoproteins, proteoglycans, glycosaminoglycans, laminin, extracellular matrix- related proteins, soluble growth factors, inflammatory cytokines and chemokines, and immune mediators.

[0313] In embodiments, the collagens comprise type I collagen 1 chain, a type I collagen 2 chain, a type II collagen 1 chain, a type III collagen 1 chain, a type V collagen 2 chain, a type VI collagen 3 chain, a type VIII collagen 1 chain, a type IX collagen 2 chain, a type XI collagen 1 chain, a type XI collagen 2 chain, a type XII collagen 2 chain, and a type XIV collagen 1 chain.

[0314] In embodiments, the glycoproteins comprise fibrillin 1, alpha-2-Heremans-Schmid glycoprotein, biglycan, extracellular matrix protein 2, fibrinogen beta chain, fibrinogen gamma chain, fibronectin 1, osteonectin, periostin, tenascin C, tenascin N, thrombospondin 1, induced transforming growth factor beta, and vitronectin.

[0315] In embodiments, the proteoglycans comprise heparan sulfate proteoglycan 2, aggrecan core protein, agaropectins, decorin, fibromodulin, lemma glycans, glypicans, ossycans, bone modulatory proteins and leucine-rich repeat proteins at the ends rich in proline / arginine.ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035

[0316] In embodiments, the partially ordered polypeptide and the allograft tissue matrix are present in a ratio of 95:5, 90:10, 85;15, 80:20, 75:25, 70:30, 65:35, 60:40, 55:45, 50:50, 45:55, 40: 60, 35:65, 30:70, 25:75, 20:80, 15:85, 10:90, or 5:95 (partially ordered polypeptide: allograft tissue matrix), including any values of ranges herein. In embodiments, the partially ordered polypeptide and the allograft tissue matrix are present in a ratio of 90:10, 70:30, 50:50, 30:70, or 10:90 (partially ordered polypeptide: allograft tissue matrix). In embodiments, the partially ordered polypeptide and the allograft tissue matrix are present in a ratio of 90:10, 70:30, or 50:50 (partially ordered polypeptide: allograft tissue matrix). In embodiments, the partially ordered polypeptide and the allograft tissue matrix are present in a ratio of 90:10 (partially ordered polypeptide: allograft tissue matrix).

[0317] In embodiments, the allograft tissue matrix is present in an amount of about 1 mg / mL to about 300 mg / mL (e.g.1, 25, 50, 75, 100, 125, 150, 175, 200, 225, 250, 275, or 300 mg / mL, including any values or ranges therein. In embodiments, the allograft tissue matrix is present in an amount of about 25 mg / mL to about 300 mg / mL, about 50 mg / mL to about 300 mg / mL, about 75 mg / mL to about 300 mg / mL, about 100 mg / mL to about 300 mg / mL, about 125 mg / mL to about 300 mg / mL, about 150 mg / mL to about 300 mg / mL, about 175 mg / mL to about 300 mg / mL, about 200 mg / mL to about 300 mg / mL, about 225 mg / mL to about 300 mg / mL, about 250 mg / mL to about 300 mg / mL, about 275 mg / mL to about 300 mg / mL, about 1 mg / mL to about 250 mg / mL, about 25 mg / mL to about 250 mg / mL, about 50 mg / mL to about 250 mg / mL, about 75 mg / mL to about 250 mg / mL, about 100 mg / mL to about 250 mg / mL, about 125 mg / mL to about 250 mg / mL, about 150 mg / mL to about 250 mg / mL, about 175 mg / mL to about 250 mg / mL, about 200 mg / mL to about 250 mg / mL, about 225 mg / mL to about 250 mg / mL, about 1 mg / mL to about 200 mg / mL, about 25 mg / mL to about 200 mg / mL, about 50 mg / mL to about 200 mg / mL, about 75 mg / mL to about 200 mg / mL, about 100 mg / mL to about 200 mg / mL, about 125 mg / mL to about 200 mg / mL, about 150 mg / mL to about 200 mg / mL, about 175 mg / mL to about 200 mg / mL, about 1 mg / mL to about 150 mg / mL, about 25 mg / mL to about 150 mg / mL, about 50 mg / mL to about 150 mg / mL, about 75 mg / mL to about 150 mg / mL, about 100 mg / mL to about 150 mg / mL, about 125 mg / mL to about 150 mg / mL, about 1 mg / mL to about 100 mg / mL, about 25 mg / mL to about 100 mg / mL, about 50 mg / mL to about 100 mg / mL, about 1 mg / mL to about 50 mg / mL, or about 25 mg / mL to about 50 mg / mL, including any values or ranges therein.

[0318] It will be apparent to one of ordinary skill in the relevant art that suitable modifications and adaptations to the compositions, formulations, methods, processes, and applicationsATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 described herein can be made without departing from the scope of any embodiments or aspects thereof. The compositions and methods provided are exemplary and are not intended to limit the scope of any of the specified embodiments. All of the various embodiments, aspects, and options disclosed herein can be combined in any variations or iterations. The scope of the compositions, formulations, methods, and processes described herein include all actual or potential combinations of embodiments, aspects, options, examples, and preferences herein described. The exemplary compositions and formulations described herein may omit any component, substitute any component disclosed herein, or include any component disclosed elsewhere herein. The ratios of the mass of any component of any of the compositions or formulations disclosed herein to the mass of any other component in the formulation or to the total mass of the other components in the formulation are hereby disclosed as if they were expressly disclosed. Should the meaning of any terms in any of the patents or publications incorporated by reference conflict with the meaning of the terms used in this disclosure, the meanings of the terms or phrases in this disclosure are controlling. Furthermore, the foregoing discussion discloses and describes merely exemplary embodiments. All patents and publications cited herein are incorporated by reference herein for the specific teachings thereof. EXAMPLES

[0319] A series of experiments were performed to evaluate the integration and mechanical performance of a matrix composition of partially ordered polypeptide (FractomerTM) and allograft tissue matrices, including bone derived matrix (BDM) and intestinal submucosa matrix (ISM), representative of a variety of soft and hard tissues. The objective was to demonstrate that FractomerTMcan be easily incorporated with allograft tissue matrices using a syringe-based delivery method, and that the resulting matrix compositions exhibit both shape retention and mechanical stiffness under compressive loading. These experiments were designed to validate FractomerTMas a tunable, load-bearing biomaterial platform suitable for clinical applications requiring shape stability and mechanical support. Example 1: A matrix composition combining FractomerTMwith either BDM-based allograft tissue matrix or ISM-based allograft tissue matrix maintains shape fidelity.

[0320] To assess shape retention, the ability of injected matrix compositions to acquire and maintain the shape of a mold after aggregation in the mold was assessed. Molds were fabricated using 5 wt% agar in 0.9% saline, with cylindrical cavities (12 mm diameter, 1 cm height) produced using a biopsy punch. A BDM-based allograft tissue matrix was obtained (AlloFuse®ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 DBM Putty and Gel; AlloSource®). The BDM-based allograft tissue matrix was preloaded in a syringe. Equal parts of the BDM-based allograft tissue matrix and FractomerTMwere combined. ISM was obtained as a dried sheet (OASIS®Matrix Products; Smith and Nephew). An ISM-based allograft tissue matrix was obtained by dicing the dried sheet into strips having ~0.25-1 μm in width and ~1.5-3.5 mm in length and rehydrating the strips in saline. A single 3 mm x 3 mm dried sheet of BDM was rehydrated in 1 ml of saline. The rehydrated strips were then loaded into a syringe. Equal parts of the ISM-based allograft tissue matrix and FractomerTMwere combined. A FractomerTMsolution was prepared as outlined above based on SEQ ID NO: 19. A 1 ml volume of each combined 50:50 FractomerTM / allograft tissue matrix (FIG.3C) was introduced into a respective mold and incubated at body temperature (37°C) for 10 minutes to allow for aggregation. The molds were then removed, leaving the aggregated plugs, as shown in FIG. 4. Visual inspection showed that the aggregated matrix compositions retained the shape of the mold, confirming successful in situ setting. Histological evaluation of the 12 mm plugs (FIG.5) was performed to assess material homogeneity. The plugs were fixed in a solution containing 10% neutral buffered saline (NBS), 1% glutaraldehyde, and 1% tetrakis(hydroxymethyl)phosphonium chloride prior to mounting and hematoxylin and eosin staining. Histological sections revealed a homogenous dispersion of allograft tissue matrix particles within the FractomerTMmatrix, indicating consistent integration and material distribution. Example 2: A matrix composition combining FractomerTMwith either BDM-based allograft tissue matrix or ISM-based allograft tissue matrix exhibits modifiable mechanical properties.

[0321] For mechanical testing, smaller 6 mm diameter plugs were prepared using 0.2 ml of the FractomerTM / allograft tissue matrix compositions of Example 1, following the same molding and incubation process (i.e., aggregation process) as outlined in Example 1. The plugs were subjected to uniaxial compression at a constant linear rate of 0.05 mm / sec until failure. The resulting stress-strain data demonstrated that the Young’s modulus of the composite could be finely controlled by varying the ratio of FractomerTMto allograft tissue matrix. This tunability highlights the potential of FractomerTMas a modular platform capable of providing structural support while adapting to a range of clinical needs.ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035

[0322] Overall, the results confirm that FractomerTMcan be combined with diverse allograft tissue matrices to create matrix compositions that retain shape, exhibit robust mechanical properties, and support uniform allograft dispersion. INCORPORATION BY REFERENCE

[0323] All references, articles, publications, patents, patent publications, and patent applications cited herein are incorporated by reference in their entireties for all purposes. However, mention of any reference, article, publication, patent, patent publication, and patent application cited herein is not, and should not be taken as an acknowledgment or any form of suggestion that they constitute valid prior art or form part of the common general knowledge in any country in the world.

[0324] Exemplary Sequences of Motifs and POP Constructs:

[0325] SEQ ID NO: 1

[0326] [GXGVP]n

[0327] where X is any amino acid except proline and where n is an integer equal to or greater than 1.

[0328] SEQ ID NO: 2

[0329] [GXGVP]n

[0330] where X is Ala or Val and where n is an integer equal to or greater than 1.

[0331] SEQ ID NO: 3

[0332] (A)n

[0333] where n is an integer from 2 to 100.

[0334] SEQ ID NO: 4

[0335] K(A)nK

[0336] where n is an integer from 2 to 100.

[0337] SEQ ID NO: 5

[0338] D(A)nK

[0339] where n is an integer from 2 to 100.

[0340] SEQ ID NO: 6

[0341] GD(An)K

[0342] where n is an integer from 2 to 100.

[0343] SEQ ID NO: 7

[0344] GK(An)KATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035

[0345] where n is an integer from 2 to 100.

[0346] SEQ ID NO: 8

[0347] (A)25

[0348] SEQ ID NO: 9

[0349] K(A)25K

[0350] SEQ ID NO: 10

[0351] D(A)25K

[0352] SEQ ID NO: 11

[0353] GD(A25)K

[0354] SEQ ID NO: 12

[0355] GK(A25)K

[0356] SEQ ID NO: 13

[0357] M[(GVGVP)15-GD(A25)K]6-GWP

[0358] SEQ ID NO: 14

[0359] M[(GVGVP)15-GD(A25)K]4-GWP

[0360] SEQ ID NO: 15

[0361] M[(GVGVP)15-GK(A25)K]6-GWP

[0362] SEQ ID NO: 16

[0363] M[(GVGVP)15-GK(A25)K]4-GWP

[0364] SEQ ID NO: 17

[0365] M[(G[A1:V1]GVP)16-GD(A25)K]6-GWP

[0366] MGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVG VPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGDAAAAAAAAAAAAAAAAAAA AAAAAAKGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGV GVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGDAAAAAAAAAAAAAAAAAA AAAAAAAKGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPG VGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGDAAAAAAAAAAAAAAAAA AAAAAAAAKGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVP GVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGDAAAAAAAAAAAAAAAA AAAAAAAAAKGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAG VPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGDAAAAAAAAAAAAAA AAAAAAAAAAAKGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPG AGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGDAAAAAAAAAAAA AAAAAAAAAAAAAKGWPATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035

[0367] SEQ ID NO: 18

[0368] M[(G[A1:V1]GVP)16-GD(A25)K]4-GWP

[0369] MGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVG VPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGDAAAAAAAAAAAAAAAAAAA AAAAAAKGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGV GVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGDAAAAAAAAAAAAAAAAAA AAAAAAAKGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPG VGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGDAAAAAAAAAAAAAAAAA AAAAAAAAKGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGAGVP GVGVPGAGVPGVGVPGAGVPGVGVPGAGVPGVGVPGDAAAAAAAAAAAAAAAA AAAAAAAAAKGWP

[0370] SEQ ID NO: 19

[0371] M[(G[V4:A1]GVP)15-GD(A25)K]6-GWP

[0372] MGVGVPGVGVPGVGVPGVGVPGAGVPGVGVPGVGVPGVGVPGVGVPGAG VPGVGVPGVGVPGVGVPGVGVPGAGVPGDAAAAAAAAAAAAAAAAAAAAAAAA AKGVGVPGVGVPGVGVPGVGVPGAGVPGVGVPGVGVPGVGVPGVGVPGAGVPGV GVPGVGVPGVGVPGVGVPGAGVPGDAAAAAAAAAAAAAAAAAAAAAAAAAKGV GVPGVGVPGVGVPGVGVPGAGVPGVGVPGVGVPGVGVPGVGVPGAGVPGVGVPG VGVPGVGVPGVGVPGAGVPGDAAAAAAAAAAAAAAAAAAAAAAAAAKGVGVPG VGVPGVGVPGVGVPGAGVPGVGVPGVGVPGVGVPGVGVPGAGVPGVGVPGVGVP GVGVPGVGVPGAGVPGDAAAAAAAAAAAAAAAAAAAAAAAAAKGVGVPGVGVP GVGVPGVGVPGAGVPGVGVPGVGVPGVGVPGVGVPGAGVPGVGVPGVGVPGVGV PGVGVPGAGVPGDAAAAAAAAAAAAAAAAAAAAAAAAAKGVGVPGVGVPGVGV PGVGVPGAGVPGVGVPGVGVPGVGVPGVGVPGAGVPGVGVPGVGVPGVGVPGVG VPGAGVPGDAAAAAAAAAAAAAAAAAAAAAAAAAKGWP

[0373] SEQ ID NO: 20

[0374] M[(G[V4:A1]GVP)15-GD(A25)K]4-GWP

[0375] MGVGVPGVGVPGVGVPGVGVPGAGVPGVGVPGVGVPGVGVPGVGVPGAG VPGVGVPGVGVPGVGVPGVGVPGAGVPGDAAAAAAAAAAAAAAAAAAAAAAAA AKGVGVPGVGVPGVGVPGVGVPGAGVPGVGVPGVGVPGVGVPGVGVPGAGVPGV GVPGVGVPGVGVPGVGVPGAGVPGDAAAAAAAAAAAAAAAAAAAAAAAAAKGV GVPGVGVPGVGVPGVGVPGAGVPGVGVPGVGVPGVGVPGVGVPGAGVPGVGVPG VGVPGVGVPGVGVPGAGVPGDAAAAAAAAAAAAAAAAAAAAAAAAAKGVGVPGATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 VGVPGVGVPGVGVPGAGVPGVGVPGVGVPGVGVPGVGVPGAGVPGVGVPGVGVP GVGVPGVGVP GAGVPGDAAAAAAAAAAAAAAAAAAAAAAAAAKGWP

[0376] SEQ ID NO: 21

[0377] P(X)nG

[0378] where n is an integer from 1 to 15 and X is any amino acid.

[0379] SEQ ID NO: 22

[0380] (B)mP(X)nG(Z)p

[0381] where m, n, and p are independently an integer from 1 to 15 and B, X, and Z are independently any amino acid.

[0382] SEQ ID NO: 23

[0383] [Bp(A)qZr]n

[0384] where B is Lys, Arg, Asp, or Glu, Z is Lys, Arg, Asp, or Glu, n is an integer from 1 to 50, p is an integer from 0 to 2, q is an integer from 1 to 50, and r is an integer from 0 to 2.

[0385] SEQ ID NO: 24

[0386] [(BAs)tZr]n

[0387] where B is Lys, Arg, Asp, or Glu, Z is Lys, Arg, Asp, or Glu, n is an integer from 1 to 50, r is an integer from 0 to 2, s is an integer from 1 to 5, and t is an integer from 1 to 50.

[0388] SEQ ID NO: 25

[0389] [GVGVP]n

[0390] where n is an integer from 1 to 50.

[0391] SEQ ID NO: 26

[0392] [GAGVP]n

[0393] where n is an integer from 1 to 50.

[0394] SEQ ID NO: 27

[0395] [G[A1:V1]GVP]n

[0396] where n is an integer from 1 to 50. When n is 1, SEQ ID NO: 27 is GAGVPGVGVP.

[0397] SEQ ID NO: 28

[0398] [G[V4:A1]GVP]n

[0399] where n is an integer from 1 to 50. When n is 1, SEQ ID NO: 28 is

[0400] GVGVPGVGVPGVGVPGVGVPGAGVP.

[0401] SEQ ID NO: 29

[0402] (A)m

[0403] where m is an integer from 5 to 50.

[0404] SEQ ID NO: 30ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035

[0405] [(SEQ ID NO: 1)n-a-helix]m

[0406] where n is an integer equal to or greater than 1, m is an integer equal to or greater than 1, and a-helix is any polyalanine based a-helix having about 5 to 50 alanine residues.

[0407] SEQ ID NO: 31

[0408] [(SEQ ID NO: 2)n-GX1(A)25X1]m

[0409] where X1is K or D, n is an integer from 10 to 20, and m is an integer from 4 to 8.

Claims

ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 CLAIMS 1. A matrix composition for replacement of tissue, comprising: a partially ordered polypeptide; and an allograft tissue matrix that is not derived from an adipose tissue; and wherein the composition comprises from about 10% v / v to about 90% v / v partially ordered polypeptide. The matrix composition of claim 1, comprising from about 50% v / v to about 90% v / v partially ordered polypeptide.

3. The matrix composition of claim 1, wherein the tissue matrix comprises a soft tissue, a hard tissue, or a combination thereof.

4. The matrix composition of claim 1, wherein the matrix composition has a stiffness about the same as or less than a reference matrix composition without partially ordered polypeptide, as measured 1 hour post-aggregation of the matrix composition.

5. The matrix composition of claim 3, wherein the tissue matrix comprises soft tissue, the soft tissue comprising skin tissue.

6. The matrix composition of claim 3, wherein the tissue matrix comprises hard tissue, the hard tissue comprising bone tissue.

7. The matrix composition of claim 3, wherein the tissue matrix comprises soft tissue, the soft tissue comprising cartilage tissue.

8. The matrix composition of claim 3, wherein the tissue matrix comprises soft tissue, the soft tissue comprising nerve tissue.

9. The matrix composition of claim 3, wherein the tissue matrix comprises soft tissue, the soft tissue comprising muscle tissue.

10. The matrix composition of claim 3, wherein the tissue matrix comprises soft tissue, the soft tissue comprising ligament tissue.ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 11. The matrix composition of claim 3, wherein the tissue matrix comprises soft tissue, the soft tissue comprising vascular tissue.

12. The matrix composition of claim 1, wherein the matrix composition is administered in a subject at an administration site.

13. The matrix composition of claim 12, wherein the administration site comprises a naturally-created tissue defect or a surgically-created tissue defect.

14. The matrix composition of claim 1, wherein the allograft tissue matrix is decellularized.

15. The matrix composition of claim 14, wherein the decellularized allograft tissue matrix is delipidated.

16. The matrix composition of claim 1, wherein the allograft tissue matrix is derived from allograft tissue.

17. The matrix composition of claim 14, wherein the decellularized allograft tissue matrix comprises exogenous cells.

18. The matrix composition of claim 17, wherein the exogenous cells comprise exogenous stem cells.

19. The matrix composition of claim 18, wherein the exogenous stem cells comprise one or more of hematopoietic stem cells, mesenchymal stem cells, epithelial stem cells, gastrointestinal epithelial stem cells, neural stem cells, and skin stem cells.

20. The matrix composition of claim 14, wherein the decellularized allograft tissue matrix comprises at least one exogenous growth-inductive substance.

21. The matrix composition of claim 20, wherein the at least one exogenous growth- inductive substance is selected from the group comprising bone morphogenic proteins,ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 vascular endothelial growth factor, basic fibroblast growth factor, transforming growth factor beta, platelet-derived growth factor, neural epidermal growth-factor-like 1, and insulin-like growth factor.

22. The matrix composition of claim 14, wherein the decellularized allograft tissue matrix is substantially free of native cellular components.

23. The matrix composition of claim 1, wherein the partially ordered polypeptide comprises a plurality of disordered domains and a plurality of ordered domains.

24. The matrix composition of claim 23, wherein each disordered domain independently comprises a PG or GP motif.

25. The matrix composition of either of claims 23 or 24, wherein each ordered domain comprises at least one alpha helix.

26. The matrix composition of either of claims 23 or 24, wherein at least one ordered domain comprises an alpha helix.

27. The matrix composition of either of claims 23 or 24, wherein at least one ordered domain comprises a beta sheet.

28. The matrix composition of either of claims 23 or 24, wherein at least one ordered domain comprises a polyalanine motif or a polyproline motif.

29. The matrix composition of claim 24, wherein each disordered domain comprising a PG motif comprises an amino acid sequence of (GXGVP)n, wherein X is any amino acid except proline and n is an integer greater than or equal to 1, and each ordered domain comprises a polyalanine motif.

30. The matrix composition of claim 24, wherein each disordered domain comprises an amino acid sequence of (GXGVP)n, wherein each X is independently Val or Ala, and wherein n is an integer from 1 to 50.ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 31. The matrix composition of claim 30, wherein a ratio of Ala to Val in the amino acid sequence of (GXGVP)n ranges from 10:1 to 1:

10.

32. The matrix composition of claim 23, wherein each ordered domain comprises a polyalanine motif comprising at least 4 alanine residues.

33. The matrix composition of claim 32, wherein the at least 4 alanine residues are consecutive.

34. The matrix composition of claim 33, wherein at least about 50% of the amino acids in each polyalanine motif are in an alpha-helical conformation.

35. The matrix composition of claim 34, wherein at least about 90% of the amino acids in each polyalanine motif are in an alpha-helical conformation.

36. The matrix composition of claim 32, wherein at least about 50% of the amino acids in the polyalanine motif are alanine residues.

37. The composition of claim 29, wherein the polyalanine motif comprises (A)m, wherein m is an integer from 5 to 50.

38. The matrix composition of claim 1, wherein the partially ordered polypeptide and the allograft tissue matrix are present in a ratio of 90:10, 70:30, or 50:50 (partially ordered polypeptide: allograft tissue matrix).

39. The matrix composition of claim 1, wherein the partially ordered polypeptide has a transition temperature of heating (Tt-heating).

40. The matrix composition of claim 39, wherein the Tt-heating is dependent on the concentration of the partially ordered polypeptide in the matrix composition.

41. The matrix composition of claim 39, wherein the Tt-heating ranges from about 10°C to about 45°C.ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 42. The matrix composition of claim 39, wherein the partially ordered polypeptide forms an aggregate above the Tt-heating.

43. The matrix composition of claim 42, wherein the aggregate is a stable three- dimensional matrix comprising the allograft tissue matrix.

44. The matrix composition of claim 42, wherein the aggregate comprises a plurality of micropores.

45. The matrix composition of claim 1, wherein the matrix composition comprises between about 200 M and about 2.5 mM of the partially ordered polypeptide.

46. The matrix composition of claim 1, wherein the matrix composition is a liquid or semisolid.

47. The matrix composition of claim 1, wherein the matrix composition forms an aggregate upon administration in vivo.

48. The matrix composition of claim 47, wherein the aggregated matrix composition has the shape of an anatomical feature.

49. The matrix composition of claim 48, wherein the aggregated matrix composition has a shape comprising the shape of a tissue.

50. A method of hard or soft tissue matrix repair, reconstruction, or augmentation in a subject, the method comprising: administering a volume of the matrix composition of claim 1 to the subject.

51. The matrix composition of claim 50, wherein the matrix composition is administered in a subject at an administration site.

52. The matrix composition of claim 51, wherein the administration site comprises a region of a naturally-created tissue defect or a surgically-created tissue defect.ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 53. The method of claim 51, wherein the administration site comprises places soft and hard tissue naturally exist in the subject.

54. The method of claim 53, wherein the allograft tissue matrix repair, reconstruction, or augmentation aims to restore or replace a soft tissue comprising skin, muscles, tendons, ligaments, nerves, and organs.

55. The method of claim 54, wherein the soft tissue exhibits one or more of an acute injury, surgery incision, laceration, puncture, wound, abrasion, avulsion, pressure ulcers, burns, scarring, acne, eczema, and psoriasis.

56. The method of claim 54, wherein the soft tissue allograft tissue matrix repair, reconstruction, or augmentation comprises skin grafting, flap surgery, tissue expansion, local tissue rearrangement, composite tissue allotransplantation, and nerve reconstruction.

57. The method of claim 53, wherein the allograft tissue matrix repair, reconstruction, or augmentation aims to restore or replace hard tissues comprising bones or teeth.

58. The method of claim 57, wherein the hard tissue exhibits one or more of a wound, fracture, crush injury, facial wound, disease, infection, cleft lip and palate, skeletal dysplasia, degenerative condition, facial contouring, joint reconstruction, spinal reconstruction, and injury from surgery.

59. The method of claim 58, wherein hard tissue matrix reconstruction comprises bone grafting, bone transport, bone augmentation, bone regeneration, maxillofacial reconstruction, dental implant, cranial reconstruction and orthopedic reconstruction.

60. The method of claim 50, further comprising, prior to administering the volume of the matrix composition to the subject, preparing a mixture of the partially ordered polypeptide and the allograft tissue matrix at a temperature below a transition temperature of the partially ordered polypeptide, wherein, after administering the volume of the matrix composition to the subject, the matrix composition aggregates after exposure to the subject’s body temperature, which is above the transition temperature of the partially ordered polypeptide.ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 61. The method of claim 50, wherein the volume of the matrix composition administered to the subject is between about 1 mL and about 500 mL.

62. A method of hard or soft tissue matrix repair, reconstruction, or augmentation in a subject, the method comprising: mixing a volume of a partially ordered polypeptide and a volume of allograft tissue matrix that is not derived from an adipose tissue to form a matrix composition; and administering a volume of the matrix composition to the subject.

63. The method of claim 62, further comprising mixing the partially ordered polypeptide and the allograft tissue matrix in vitro to form the matrix composition and administering the matrix composition in vivo in the subject.

64. The method of claim 62, wherein the partially ordered polypeptide and allograft tissue matrix are a liquid, semisolid, or molded semisolid prior to administration and following administration, form an aggregate.

65. The method of claim 62, wherein a mixture of the partially ordered polypeptide and the allograft tissue matrix are administered to a subject when the mixture is below the Tt- heating of the partially ordered polypeptide and the mixture forms a solid after exposure to the subject’s body temperature, which is above the transition temperate of the partially ordered polypeptide.

66. The method of claim 65, wherein the administering comprises administering a volume of the matrix composition into the subject.

67. The method of claim 62, wherein the volume of the matrix composition administered to the subject if between about 1 mL and about 500 mL.

68. The method of claim 62, wherein the allograft tissue matrix is delipidated, decellularized, or both.ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 69. Use of a volume of a partially ordered polypeptide and a volume of allograft tissue matrix to replace hard or soft tissue in a subject.

70. A matrix composition, comprising: a polypeptide comprising a plurality of disordered domains and a plurality of ordered domains, wherein each disordered domain independently comprises a PG or GP motif and each ordered domain independently comprises a polyalanine motif; and a allograft tissue matrix that is not derived from an adipose tissue.

71. The matrix composition of claim 70, wherein the allograft tissue matrix is delipidated, decellularized, or both.

72. A method of hard or soft tissue matrix repair, reconstruction, or augmentation in a subject, the method comprising: administering a volume of a partially ordered polypeptide to the subject; administering a volume of allograft tissue matrix that is not derived from an adipose tissue to the subject, wherein the administered volume of the allograft tissue matrix is co- located with the administered volume of the partially ordered polypeptide, thereby forming a matrix composition.

73. The matrix composition of claim 1, wherein when the matrix composition comprises a pre-administration volume and a post-administration volume, the post-administration volume of the matrix composition being greater than a post-administration volume of a reference matrix composition measured at the same time point after administration, the reference matrix composition comprising only allograft tissue matrix.

74. The matrix composition of claim 1, wherein when the matrix composition comprises a pre-administration volume and a post-administration volume measured after administration, the post-administration volume being from about 50% to about 120% of the pre- administration volume.ATTORNEY DOCKET NO.: INSO-008 / 01WO 344681-2035 75. The matrix composition of claim 1, wherein an initial projection of the matrix composition measured immediately post-administration is substantially similar to a future projection of the matrix composition measured at least 1 week after administration.

76. The matrix composition of claim 75, wherein each projection of the matrix composition is measured relative to an adjacent tissue surface not within an administration site.

77. The matrix composition of claim 1, wherein the tissue matrix comprises one or more fragments of: collagen, glycoproteins, proteoglycans, glycosaminoglycans, laminin, extracellular matrix-related proteins, soluble growth factors, inflammatory cytokines and chemokines, and immune mediators.

78. The matrix composition of claim 77, wherein the collagens comprise type I collagen 1 chain, a type I collagen 2 chain, a type II collagen 1 chain, a type III collagen 1 chain, a type V collagen 2 chain, a type VI collagen 3 chain, a type VIII collagen 1 chain, a type IX collagen 2 chain, a type XI collagen 1 chain, a type XI collagen 2 chain, a type XII collagen 2 chain, and a type XIV collagen 1 chain.

79. The matrix composition of claim 77, wherein the glycoproteins comprise fibrillin 1, alpha-2-Heremans-Schmid glycoprotein, biglycan, extracellular matrix protein 2, fibrinogen beta chain, fibrinogen gamma chain, fibronectin 1, osteonectin, periostin, tenascin C, tenascin N, thrombospondin 1, induced transforming growth factor beta, and vitronectin.

80. The matrix composition of claim 77, wherein the proteoglycans comprise heparan sulfate proteoglycan 2, aggrecan core protein, agaropectins, decorin, fibromodulin, lemma glycans, glypicans, ossycans, bone modulatory proteins and leucine-rich repeat proteins at the ends rich in proline / arginine.

Images