Tissue conductivity scaffold material

Abstract

Disclosed herein is a bioactive polymer for forming a tissue scaffold, the polymer comprising: a first monomer for binding water; the second monomer is used for endowing the stent with mechanical properties; optionally a third monomer for binding to a natural or synthetic peptide or protein (NSPP); and a fourth monomer for imparting a phase change behavior, wherein the stent forms a malleable structure after hydration. Preferably, the first monomer is OEGMA; the second monomer is PLA / HEMA; the third monomer is NAS; and the fourth monomer is NIPAAm, and the polymer comprises an amount of OEGMA from about 1 mol% to about 15 mol%; the amount of PLA / HEMA is from 5 mol% to about 50 mol%; the amount of NAS is from 0 to about 15 mol%; and the amount of NIPAAm does not exceed 85 mol%.

Description

[0001] Related applications

[0002] This application claims conventional priority from Australian provisional patent application 2022901056 filed on 21 April 2022. The disclosure of AU'056 is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention relates to biocompatible polymers, particularly polymers that can be used to form tissue scaffolds. In particular, the scaffolds can be used for tissue repair and regeneration.

[0004] The present invention relates to prefabricated porous scaffolds that can guide tissue integration, tissue interface regeneration, and drug delivery applications. One embodiment of the invention relates to the formation of malleable scaffolds that can be administered via arthroscopic or open surgical intervention. When in contact with body tissue, the scaffolds exhibit adhesive properties and suture properties, enabling the fixation of one or more tissues to support tissue regeneration and / or tissue interface integration.

[0005] In another embodiment, the present invention relates to a tissue conductive medical filler. In another embodiment, the polymers of the present invention can form a tissue scaffold. In yet another embodiment, the scaffold forms a malleable structure upon hydration. In yet another embodiment, the scaffold can form in situ upon elevated body temperature following administration.

[0006] It is contemplated that the present invention may be used in tissue engineering applications. This includes cosmetic and therapeutic applications. Although the present invention will be described below with reference to preferred embodiments thereof, those skilled in the art will recognize that the spirit and scope of the present invention may be embodied in many other forms. Background Art

[0007] Any discussion of prior art throughout this specification should not be considered as an admission that such prior art is widely known or forms part of the common general knowledge in the field.

[0008] Tissue engineering is a biomedical engineering discipline that integrates cellular, engineering, material approaches and appropriate biochemical and physicochemical factors to restore, maintain, improve or replace different types of biological tissues. Tissue engineering generally involves the use of cells placed on a tissue scaffold to form new viable tissue for medical purposes, but is not limited to applications involving cells and tissue scaffolds. In practice, the term is closely associated with applications that repair or replace part or all of a tissue (e.g., bone, cartilage, blood vessels, bladder, skin, muscle, etc.). Typically, the tissue involved requires certain mechanical and structural properties for its normal functioning. The term also refers to the use of cells in artificially created support systems (e.g., artificial pancreas or bioartificial liver) to perform specific biochemical functions.

[0009] Scaffolds are materials engineered to induce desired cellular interactions, thereby facilitating the formation of new functional tissues for medical purposes. Cells are typically seeded into these structures, which support three-dimensional tissue formation. Scaffolds mimic the extracellular matrix of native tissue, recreating the in vivo environment and allowing cells to influence their own microenvironment. They typically fulfill at least one of the following objectives: allowing cell attachment and migration; delivering and retaining cellular and biochemical factors; enabling the diffusion of important cellular nutrients and expression products; and / or exerting certain mechanical and biological influences to alter cellular behavior.

[0010] Material selection is an important aspect in the production of scaffolds. The materials used can be natural or synthetic and can be biodegradable or non-biodegradable. In addition, they must be biocompatible, i.e. they do not cause any adverse effects on cells. For example, silicone is a synthetic, non-biodegradable material that is commonly used as a drug delivery material, while gelatin is a biodegradable natural material that is commonly used in cell culture scaffolds. The optimal material for each application will inevitably be different and will depend on the desired mechanical properties of the material. For example, tissue engineering of long bone defects will require a rigid scaffold with a compressive strength similar to that of cortical bone (100-150 MPa), which is much higher than that of a scaffold used for skin regeneration.

[0011] There are several versatile synthetic materials that can be used in a variety of different stent applications. One commonly used material is polylactic acid (PLA), a synthetic polymer. PLA is a polyester that degrades in the human body to form lactic acid, a naturally occurring chemical that is easily cleared from the body. Similar materials are polyglycolic acid (PGA) and polycaprolactone (PCL). PLA is often combined with PGA to create polylactic-co-glycolic acid (PLGA). This is particularly useful because the degradation of PLGA can be tailored by varying the weight percentages of PLA and PGA. This tunability, along with its biocompatibility, makes it a very useful material for stent fabrication.

[0012] Scaffold can also be constructed from natural materials. Protein-based materials (such as collagen or fibrin) and polysaccharide materials (such as chitosan or glycosaminoglycans (GAG)) have been shown to be suitable in terms of cell compatibility. Among GAGs, hyaluronic acid (which can be combined with a cross-linking agent (such as glutaraldehyde, water-soluble carbodiimide, etc.)) is a commonly used scaffold material. In addition, fragments of extracellular matrix proteins, such as RGD peptides, can also be coupled with non-bioactive materials to promote cell attachment. Another form of scaffold is decellularized tissue, which is produced by chemically extracting cells from tissues and leaving only extracellular matrix. Its advantage is that a matrix with specificity for the desired tissue type can be fully formed. However, decellularized scaffolds may cause immune problems to cells introduced in the future.

[0013] All patents and patent publications cited herein are incorporated by reference in their respective entireties.

[0014] WO 2013 / 091001 (PCT / AU2012 / 001566) relates to polymers, particularly polymers for use as hydrogels, and the use of hydrogels for repairing or restoring tissue. Specifically, the polymers and hydrogels of WO '001 can be used to repair or restore cartilage, particularly articular cartilage. The polymers comprise at least monomers for binding water, monomers for imparting mechanical properties, and monomers for binding extracellular proteins. The hydrogel comprises a polymer comprising at least monomers for binding water and monomers for binding extracellular proteins. The polymers are cross-linked by binding to extracellular matrix proteins to form a hydrogel.

[0015] WO 2017 / 035587 (PCT / AU2016 / 050817) discloses biocompatible materials for tissue regeneration and repair, wherein the bioactive polymer may be in the form of a hydrogel, such as a thermoresponsive hydrogel. The bioactive polymers and resulting hydrogels of WO'587 can be used for bone tissue regeneration. Thus, the reference teaches a method for treating bone defects in a mammal, comprising administering to the mammal a therapeutically effective amount of a hydrogel formed from a bioactive polymer to treat the bone defect.

[0016] WO 2017 / 015703 (PCT / AU2016 / 050653) discloses polymers comprising at least one antimicrobial / analgesic / anti-inflammatory monomeric unit and at least three additional monomeric units that impart properties selected from the group consisting of temperature activation, water solubility, mechanical strength, protein / polysaccharide binding capacity, and combinations thereof. Specifically, WO '703 discloses a polymer wherein the water-soluble monomeric unit is a hydrophilic ethylene glycol (OEGMA) moiety; the mechanical strength-imparting monomeric unit is a poly(lactic acid-co-2-hydroxyethyl methacrylate) (PLA / HEMA); the protein-reactive monomeric unit is an N-acryloyloxysuccinimide (NAS) moiety; and the thermosetting monomeric unit is an N-isopropylacrylamide (NIPAAm) moiety. The antimicrobial / analgesic / anti-inflammatory monomeric unit comprises a methacrylate derivative of salicylic acid (5-HMA or 4-HMA, or a combination thereof).

[0017] WO 2021 / 119727 (PCT / AU2020 / 051332) teaches a composition comprising a polymer and a natural or synthetic peptide or protein (NSPP) such as thymosin beta-4. The polymer comprises a first monomer for binding water, a second monomer for imparting mechanical properties, a third monomer for binding the NSPP, and a fourth monomer for imparting phase change behavior. Specifically, the composition forms a viscous and flowable hydrogel when applied to the body or on the surface of the body, thereby facilitating tissue repair and regeneration. Therefore, WO'727 discloses a method for tissue repair and / or regeneration, which comprises administering the composition by injection or aerosol administration, thereby forming a hydrogel at the body temperature of a mammal.

[0018] All four of the above-mentioned applications were assigned to the present applicant, Trimph IPPty Ltd of Sydney, Australia.

[0019] It is an object of the present invention to overcome or ameliorate at least one of the disadvantages of the prior art, or to provide a useful alternative.

[0020] There is a general need in the art for new scaffolds for tissue repair and / or regeneration. There is a general need in the art for new scaffolds that allow regeneration and integration of tissue interfaces. There is a general need in the art for new scaffolds that allow tissue ingrowth rather than superficial tissue on-growth. There is a general need for scaffolds for tissue repair and / or regeneration that eliminate the critical issue of water solubility. There is a general need for tissue scaffolds that can be manufactured using different processing methods. There is a general need for tissue scaffolds that can be tailored to modify the microenvironment to meet requirements. There is a general need for tissue scaffolds that can be bound to peptides and / or proteins. There is a general need for tissue scaffolds that can be bound to drugs of varying hydrophilic and hydrophobic properties. There is a general need for tissue scaffolds that are non-adhesive to gloves for efficient handling. There is a general need for malleable tissue scaffolds that allow for efficient delivery to the site either arthroscopically or via open surgical intervention. There is a need for tissue scaffolds that can adhere to the treatment site. There is a general need for tissue scaffolds that can be sutured (or stapled) for internal fixation.

[0021] The present invention was developed against this background. Various embodiments of the present invention are useful for one or more of the above-mentioned general needs.

[0022] Specifically, the present invention can be used for soft tissue applications, such as skin grafts to fix graft tissue, preparing sites for future skin grafts, tendon and / or ligament healing and / or enhancement and / or reinforcement and / or stabilization, repair of partial or full-thickness rotator cuff tears (including but not limited to superior capsule reconstruction (SCR)), shoulder arthroplasty, knee arthroplasty and hip arthroplasty, cruciate ligaments (including anterior cruciate ligament, posterior cruciate ligament and medial cruciate ligament, ACL, PCL, MCL) and / or other ligament / tendon tears (including but not limited to Achilles tendon, biceps tendon, patellar tendon) in soft tissue healing and hard tissue applications. The present invention can also be used for drug delivery applications.

[0023] Although the invention will be described with reference to specific examples, it will be appreciated by those skilled in the art that the invention may be embodied in many other forms. Summary of the Invention

[0024] Broadly speaking, the present invention is embodied as a filler, wherein the filler forms a malleable scaffold upon hydration. The scaffold is well tolerated in vivo with minimal inflammatory response and can be used as a universal matrix to support tissue healing and integration of two or more tissue interfaces. The scaffold is conductive to the host tissue, but not inductive. The filler can be used during surgery and hydrated with saline, the patient's own blood, platelet-rich plasma (PRP), platelet-rich fibrin, bone marrow aspirate and / or other blood / cell / tissue products and / or extracts (from autologous and allogeneic sources) to form a malleable scaffold. The resulting scaffold can be administered via arthroscopic or open surgical intervention. When in contact with body tissue, the scaffold has adhesive properties and is easy to suture and surgically staple, thereby being able to fix one or more tissues to support tissue regeneration and / or tissue interface integration.

[0025] According to a first aspect of the present invention, there is provided a polymer for forming a tissue scaffold, the polymer comprising:

[0026] a first monomer for binding water;

[0027] a second monomer for imparting mechanical properties to the scaffold;

[0028] an optional third monomer for conjugation to a natural or synthetic peptide or protein (NSPP); and

[0029] a fourth monomer for imparting phase change behavior;

[0030] The scaffold forms a malleable structure after hydration.

[0031] In an embodiment, the first monomer is selected from the group consisting of: polyether, polyvinyl alcohol (PVA), poly(vinyl pyrrolidone) (PVP), poly(amino acid), and dextran.

[0032] In an embodiment, the polyether is selected from polyethylene glycol (PEG), oligo(ethylene glycol) (OEG), polyethylene oxide (PEO), polyethylene oxide-co-propylene oxide (PPO), co-polyethylene oxide blocks, or random copolymers thereof.

[0033] In an embodiment, the first monomer is oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA).

[0034] In an embodiment, the second monomer is a methacrylate or a random copolymer comprising a methacrylate.

[0035] In an embodiment, the second monomer is selected from: hydroxyethyl methacrylate (HEMA), hydroxyethyl methacrylate poly(lactic acid) copolymer (PLA / HEMA), poly(lactic acid), poly(caprolactone), poly(glycolide), poly(glycolide-co-lactide) or poly(glycolide-co-caprolactone).

[0036] In an embodiment, the second monomer is hydroxyethyl methacrylate poly(lactic acid) (PLA / HEMA).

[0037] In embodiments, the third monomer has an electrophilic functional group that binds to the NSPP.

[0038] In an embodiment, the third monomer is selected from the group consisting of: N-hydroxysulfosuccinimide (SNHS), N-hydroxyethoxylated succinimide (ENHS), and N-acryloyloxysuccinimide (NAS).

[0039] In an embodiment, the third monomer is N-acryloyloxysuccinimide (NAS).

[0040] In embodiments, the fourth monomer has a lower critical solution temperature (LCST) of less than about 37°C.

[0041] In an embodiment, the fourth monomer is selected from the group consisting of: poly(ethylene oxide) / poly(propylene oxide) and poly(N-isopropylacrylamide) (PNIPAAm) homopolymers and copolymers.

[0042] In an embodiment, the fourth monomer is (N-isopropylacrylamide) (NIPAAm).

[0043] In embodiments, the polymer comprises the first monomer in an amount from about 1 mol% to about 15 mol%.

[0044] In embodiments, the polymer comprises the second monomer in an amount from about 5 mol% to 50 mol%.

[0045] In embodiments, the polymer comprises the third monomer in an amount from about 0 mol% to about 15 mol%.

[0046] In embodiments, the polymer comprises the fourth monomer in an amount from about 50 mol% to about 85 mol%.

[0047] In an embodiment, the polymer comprises the first monomer in an amount of about 1 mol% to about 15 mol%; the second monomer in an amount of about 5 mol% to about 50 mol%; the third monomer in an amount of 0 mol% to about 15 mol%; and the fourth monomer in an amount making up the remainder to 100% of the polymer.

[0048] In an embodiment, the first monomer is OEGMA; the second monomer is PLA / HEMA; the third monomer is NAS; and the fourth monomer is NIPAAm, wherein the polymer comprises OEGMA in an amount from about 1 mol% to about 15 mol%; PLA / HEMA in an amount from 5 mol% to about 50 mol%; NAS in an amount from 0 mol% to about 15 mol%; and NIPAAm in an amount not exceeding about 85 mol%.

[0049] According to a second aspect of the present invention, there is provided a tissue scaffold comprising a polymer as defined in accordance with the first aspect of the present invention, wherein the scaffold forms a malleable structure upon hydration.

[0050] In an embodiment, the stent is hydrated intraoperatively. In another embodiment, the stent is hydrated with saline, aqueous solution, autologous or allogeneic blood, cell or tissue products, or a combination thereof.

[0051] In embodiments, the scaffold is hydrated with the patient's own blood, platelet-rich plasma (PRP), bone marrow aspirate, platelet-rich fibrin, or other blood or tissue-derived products, or a combination thereof.

[0052] In embodiments, the scaffold is hydrated with allogeneic blood, platelet-rich plasma (PRP), platelet-rich fibrin, or other blood or tissue-derived products, or combinations thereof.

[0053] In an embodiment, the stent is non-adhesive to surgical gloves. In an embodiment, the stent is adhesive to the treatment site.

[0054] In embodiments, the scaffold is not tissue inductive.

[0055] In embodiments, following administration to a mammal, a scaffold is formed.

[0056] In embodiments, wherein the scaffold is formed at body temperature.

[0057] In embodiments, administration is by injection or spray.

[0058] According to a third aspect of the present invention, there is provided a malleable structure formed from a tissue scaffold defined according to the second aspect of the present invention.

[0059] According to a fourth aspect of the present invention, there is provided a method for preparing a tissue scaffold, the method comprising the following steps:

[0060] dissolving an aqueous solution of the polymer according to the first aspect of the present invention in a buffer solution to obtain a resultant solution; and

[0061] The resulting solution was freeze-dried;

[0062] The morphology of the scaffold can be changed by adjusting the freeze-drying parameters and polymer concentration.

[0063] In embodiments, at least one drug or biological moiety is added to the resulting solution between the dissolution and freeze-drying steps.

[0064] In an embodiment, the drug or biological moiety is selected from the group consisting of: an antibiotic, a growth factor, a live virus, or a combination thereof.

[0065] According to a fifth aspect of the present invention, there is provided a method for preparing a tissue scaffold, the method comprising the following steps:

[0066] dissolving the polymer according to the first aspect of the present invention in an organic solvent to obtain a solution;

[0067] precipitating the solution in water as an antisolvent to obtain a gel; and

[0068] The gel was freeze-dried.

[0069] The morphology of the scaffold can be changed by adjusting the freeze-drying parameters.

[0070] In embodiments, the organic solvent is miscible with water.

[0071] According to a sixth aspect of the present invention, there is provided a method for preparing a tissue scaffold, the method comprising the following steps:

[0072] dissolving the polymer according to the first aspect of the present invention in an organic solvent to obtain a solution; and

[0073] The solution was electrospun.

[0074] According to a seventh aspect of the present invention, there is provided a method for preparing a tissue scaffold, the method comprising:

[0075] dissolving a polymer as defined in the first aspect of the invention in an aqueous solution, autologous blood, a cell or tissue product, or a combination thereof;

[0076] The stent is injectable or sprayable.

[0077] According to an eighth aspect of the present invention, there is provided the use of a polymer according to the first aspect of the present invention in the manufacture of a tissue scaffold, which can be used for: skin grafts to fix grafted tissue, preparing sites for future skin grafts, tendon and / or ligament healing and / or enhancement and / or reinforcement and / or stabilization, repair of partial or full-thickness rotator cuff tears, soft tissue healing in shoulder replacements, knee replacements and hip replacements, cruciate ligament and / or other ligament / tendon tears.

[0078] According to a ninth aspect of the present invention, there is provided use of the tissue scaffold according to the second aspect of the present invention in tissue repair and / or regeneration.

[0079] According to a tenth aspect of the invention, a method is provided for: tissue repair and / or regeneration; supporting skin grafts to fix the grafted tissue; preparing a site for a future skin graft; tendon and / or ligament healing and / or enhancement and / or reinforcement and / or stabilization; repairing partial or full-thickness rotator cuff tears; soft tissue healing in shoulder, knee and hip replacements, cruciate ligaments and / or other ligaments / tendons, the method comprising administering a tissue scaffold according to the second aspect of the invention to a mammal.

[0080] In embodiments, administration is by injection or spray. In embodiments, the scaffold is administered via arthroscopic or open surgical intervention. In embodiments, the scaffold is hydrated after intraoperative administration to form a malleable structure.

[0081] According to the eleventh aspect of the present invention, a tissue scaffold according to the second aspect of the present invention is provided for use in tissue repair and / or regeneration; supporting skin grafts to fix transplanted tissue; preparing sites for future skin grafts; tendon and / or ligament healing and / or enhancement and / or reinforcement and / or stabilization; repairing partial or full-thickness rotator cuff tears; soft tissue healing in shoulder replacement, knee replacement and hip replacement, cruciate ligament and / or other ligament / tendon tears.

[0082] In embodiments, the scaffold is administered by injection or spray. In embodiments, the scaffold is administered by arthroscopic or open surgical intervention. In embodiments, the scaffold is hydrated after intraoperative administration to form a malleable structure.

[0083] According to a twelfth aspect of the present invention, a kit for forming a tissue scaffold is provided, the kit comprising a polymer, wherein the polymer comprises: a first monomer for binding water; a second monomer for imparting mechanical properties to the scaffold; an optional third monomer for binding to a natural or synthetic peptide or protein (NSPP); and a fourth monomer for imparting phase change behavior, wherein the scaffold forms a malleable structure upon hydration.

[0084] Definitions and Terminology

[0085] In describing and claiming the present invention, the following terminology will be used in accordance with the definitions listed below. It should also be understood that the terminology used herein is intended only to describe specific embodiments of the present invention and is not intended to be limiting. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.

[0086] Unless the context clearly requires otherwise, throughout the specification and claims, the words "comprise", "comprising" and the like should be understood to have an inclusive meaning rather than an exclusive or exhaustive meaning; in other words, they should be understood to mean "including but not limited to".

[0087] The terms "preferred" and "preferably" refer to embodiments of the invention that may provide certain benefits under certain circumstances. However, other embodiments may also be preferred, under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not mean that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.

[0088] As used herein, words defining ranges or length limits, such as "1-5," refer to any integer from 1 to 5, i.e., 1, 2, 3, 4, and 5. In other words, any range defined by two explicitly mentioned integers is intended to include and disclose any integers defining the limits as well as any integers included in the range.

[0089] Except in the operating examples or otherwise noted, all numbers used herein indicating the amounts of ingredients or reaction conditions should be understood as being modified by the term "about" in all cases. The examples are not intended to limit the scope of the invention. Hereinafter, or where otherwise noted, "%" should mean "weight %", "ratio" should mean "weight ratio", and "parts" should mean "parts by weight".

[0090] Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values ​​set forth in the specific examples are reported as precisely as possible. Any numerical value, however, inherently contains certain errors necessarily resulting from the standard deviation found in their respective testing measurements.

[0091] This manual uses the following abbreviations:

[0092] ECM extracellular matrix

[0093] EHNS N-Hydroxyethoxylated succinimide

[0094] HEMA Hydroxyethyl Methacrylate

[0095] LA lactic acid

[0096] NAS N-acryloyloxysuccinimide

[0097] NIPAAm N-isopropylacrylamide

[0098] NSPP natural or synthetic peptide or protein

[0099] OEG oligo(ethylene glycol)

[0100] OEGMA Oligo(ethylene glycol) monomethyl ether methacrylate

[0101] PBS Phosphate-buffered saline

[0102] PEG polyethylene glycol

[0103] PEO Polyethylene Oxide

[0104] PLA poly(lactic acid)

[0105] PLA / HEMA Hydroxyethyl Methacrylate Poly(lactic acid)

[0106] PPO Polyethylene oxide-co-propylene oxide

[0107] PVA polyvinyl alcohol

[0108] PVP poly(vinyl pyrrolidone)

[0109] PNPHO Poly(N-isopropylacrylamide-co-(N-acryloyloxysuccinimide)-co-(polylactide / 2-hydroxymethacrylate)-co-(oligo(ethylene glycol) / poly(NIPAAm-co-NAS-co-(PLA / HEMA)-co-OEGMA)

[0112] PPHO Poly(N-isopropylacrylamide-co-(polylactide / 2-hydroxymethacrylate)-co-(oligo(ethylene glycol) / poly(NIPAAm-co-(PLA / HEMA)-co-OEGMA)

[0114] SNHS N-hydroxysulfosuccinimide

[0115] TB4 Thymosin beta-4 BRIEF DESCRIPTION OF THE DRAWINGS

[0117] Preferred embodiments of the present invention will now be described, by way of example only, with reference to the accompanying drawings, in which:

[0118] FIG1( a ) shows the surface of a PNPHO scaffold formed from a polymer of the present invention, Figures 1(b) to 1(d) Its morphology is shown at 100, 50, and 20 μm resolutions, respectively.

[0119] Figure 2 Handling of the PNPHO scaffold after hydration is shown, illustrating the structural stability of the product during handling for efficient manipulation and surgical administration. Figure 2 The sequential steps of stent folding / unfolding are shown.

[0120] Figure 3(a) shows the adhesion test of the PNPHO scaffold on soft tissue (epidermis).

[0121] Figure 3(b) shows the test of the PNPHO scaffold peeling off from the soft tissue (epidermis).

[0122] Figure 4(a) shows the adhesion test of the PNPHO scaffold on a hard surface (simulating hard tissue, such as bone).

[0123] Figure 4(b) shows the peel test of the PNPHO scaffold on a hard surface (simulating hard tissue, such as bone).

[0124] Figure 5(a) shows the PPHO (NAS = 0 mol%) in CD3CN. 1 H NMR spectroscopy. The presence of characteristic monomer peaks confirmed the preparation of PPHO.

[0125] Figure 5(b) shows the comparison between PNPHO and PPHO 1 H NMR spectrum; (a) PPHO 1 HNMR spectrum (NAS = 0 mol%), the presence of characteristic peaks confirmed the preparation of PPHO (NIPAAm = ~90 mol%, PLA / HEMA = ~10 mol%, OEGMA = ~1 mol%); (b) PNPHO 1 H NMR spectrum (NAS = 10 mol%, OEGMA = 5 mol%). PPHO 1 HNMR spectra were collected using CD3CN, PNPHO 1 H NMR spectra were collected in CDCl 3 . The reason for changing the solvent to collect NMR data is that the hydrophobic nature of PPHO prevents its solubility in CDCl 3 .

[0126] Figure 5(c) shows the HPLC chromatogram at 230 nm, showing that PNPHO eluted at 6.2 min and PPHO eluted at 7.2 min. The difference in elution times confirms that PPHO eluted with a higher percentage of acetonitrile than PNPHO and further confirms the hydrophobicity of PPHO compared to PNPHO.

[0127] Figure 6(a) shows the adhesion of PPHO on soft tissue and how it exhibits adhesion despite its inherent hydrophobicity.

[0128] Figure 6(b) shows the adhesion of PPHO on a hard surface to demonstrate the adhesion of the product on simulated hard tissue.

[0129] Figure 7 Shown are the body weight changes of mice implanted with PNPHO and / or PPHO scaffolds over 42 days (time point = days after implantation).

[0130] Figure 8 Digital images of macroscopic observations at various time points over 42 days are shown. Note that any hair regrowth on the mouse's back is independent of the presence of the scaffold. This is a normal regeneration pattern that is generally consistent across all mice.

[0131] Figure 9 Digital images of mice sacrificed on days 7, 14, 21, and 42 after surgery are shown.

[0132] Figure 10 H&E images of skin histology of mouse ID "S-4" at week 1 are shown.

[0133] Figure 11 H&E images of the skin implant site at 1, 2, 3, and 6 weeks after surgery are shown. DETAILED DESCRIPTION

[0134] The present invention will now be described more fully with reference to the accompanying examples and drawings. However, it should be understood that the following description is illustrative only and should not be considered in any way as limiting the generality of the invention described above.

[0135] Certain embodiments of the present invention will now be described in detail. Although the present invention will be described in conjunction with these embodiments, it should be understood that its intention is not to limit the present invention to these embodiments. On the contrary, the present invention is intended to encompass all alternatives, modifications, and equivalents that may be included within the scope of the present invention defined by the claims.

[0136] Those skilled in the art will recognize that many methods and materials similar or equivalent to those described herein can be used to practice the present invention. The present invention is in no way limited to the methods and materials described.

[0137] It should be understood that the invention disclosed and defined in this specification extends to all alternative combinations of two or more of the individual features mentioned or evident from the text or drawings. All these different combinations constitute various alternative aspects of the invention.

[0138] Broadly disclosed herein are uses of tissue-customizable polymers. Scaffolds formed from the polymers preferably form malleable structures upon hydration.

[0139] polymer

[0140] As used herein, the term "polymer" refers to a macromolecule (polymer) composed of repeating structural units (monomers). These subunits are typically linked by covalent chemical bonds. A polymer can be a linear polymer or a branched polymer. The polymer of the present invention is preferably a copolymer comprising three or more different monomers. For example, in one embodiment, the polymer of the present invention includes a first water-binding monomer, a second monomer capable of imparting mechanical properties to the tissue scaffold, and a third monomer having a functional group that binds to the NSPP.

[0141] As used herein, the term "monomer" refers to a structural unit that can be combined to form a polymer, but can also itself be a polymer, or a derivative of a monomer or polymer. This type of monomer is also referred to herein as a "macromer." As used herein, a "macromer" refers to a polymer or oligomer whose molecules each have an end group that acts as a monomer molecule, so that each polymer or oligomer molecule contributes only a single monomer unit to the product polymer chain.

[0142] The polymer of the present invention comprises: a first monomer for binding water; a second monomer for imparting mechanical properties to the tissue scaffold; an optional third monomer for binding to a natural or synthetic peptide or protein (NSPP); and a fourth monomer for imparting phase transition behavior.

[0143] First monomer: water-binding monomer

[0144] As discussed above, the advantages of the tissue scaffolds of the present invention can be attributed, at least in part, to the specific components that make up the polymers of the present invention. A particularly advantageous property of the polymers of the present invention is their water binding capacity. The presence of water in the scaffolds of the present invention provides an environment similar to the natural environment of the damaged tissue (which aids in tissue regeneration) while also providing the scaffold with the desired compressive strength.

[0145] Therefore, the preferred polymers used herein should include monomers or units whose ability to bind water allows the polymer to form a malleable structure when hydrated. In addition, the structure formed thereby should have the desired compressive resistance and elasticity.

[0146] Those skilled in the art will appreciate that the water-binding monomer must be present in the polymers of the present invention in a ratio sufficient to produce a polymer that meets these requirements. Typically, the ratio of water-binding monomer in the polymer is about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:10, about 1:20, about 1:30, about 1:40, or about 1:50 molar ratio of water-binding monomer to mechanical strength monomer. Indeed, the water-binding monomer must not only render the polymer hydrophilic but also impart significant water-binding capacity to the polymer. Thus, the polymers of the present invention should have a water-binding capacity of about 70% to about 500%, about 80% to about 400%, about 90% to 300%, or about 100% to 200%. For example, the water binding capacity of the polymers of the present invention should be about 70%, about 80%, about 90%, about 100%, about 110%, about 120%, about 130%, about 140%, about 150%, about 160%, about 170%, about 180%, about 190%, about 200%, about 210%, about 220%, about 230%, about 240%, about 250%, about 260%, about 270%. , about 280%, about 290%, about 300%, about 310%, about 320%, about 330%, about 340%, about 350%, about 360%, about 370%, about 380%, about 390%, about 400%, about 410%, about 420%, about 430%, about 440%, about 450%, about 460%, about 470%, about 480%, about 490% or about 500%.

[0147] Suitable examples of water-binding monomers include those from which polymers can be synthesized, such as polyethers (e.g., alkylene polyoxides, such as polyethylene glycol (PEG), oligo(ethylene glycol) (OEG), polyethylene oxide (PEO), polyethylene oxide-co-propylene oxide (PPO), co-polyethylene oxide block or random copolymers, polyvinyl alcohol (PVA)), poly(vinyl pyrrolidone) (PVP), poly(amino acids), and dextran, etc. Polyethers, particularly oligo(oxyalkylenes) (e.g., OEG), are particularly preferred because they have the desired water-binding capacity, are readily synthesized and / or commercially available, and are inert in the sense that they elicit minimal or no immune response to tissues into which they are placed.

[0148] In addition, any of a variety of hydrophilic functional groups can be used to make the monomer (and the polymer formed from such monomer) water-soluble. For example, water-soluble functional groups such as phosphate, sulfate, quaternary amine, hydroxyl, amine, sulfonate, and carboxylate can be introduced into the monomer to make it water-soluble.

[0149] Monomers may also be reacted with other compounds to form “macromers.” Thus, the first monomer may optionally be a macromer.

[0150] As the macromonomer, a preferred first monomer is oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA), which is a hydrophilic monomer composed of two hydrophilic monomers, ethylene glycol and methacrylate.

[0151] Preferably, the polymer includes the first monomer in an amount of about 1 mol% to about 15 mol%. In various embodiments, the first monomer may be present in an amount of about 1 mol%, about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, or about 15 mol%. In various embodiments, the first monomer may be present in an amount of about 1 mol% to about 15 mol%, about 2 mol% to about 14 mol%, about 3 mol% to about 13 mol%, about 4 mol% to about 12 mol%, about 5 mol% to about 11 mol%, about 6 mol% to about 10 mol%, about 7 mol% to about 9 mol%, or about 8 mol%.

[0152] Second monomer: monomer that imparts mechanical properties

[0153] As discussed above, the advantageous properties of the tissue scaffolds of the present invention can be attributed in part to the specific components comprising the polymers of the present invention. In some embodiments, the polymers of the present invention can provide additional mechanical properties and adhesiveness to the scaffolds of the present invention.

[0154] It will be understood by those skilled in the art that monomers capable of imparting mechanical properties to the tissue scaffold need to be present in the polymer of the present invention in a ratio sufficient to produce a tissue scaffold with the desired mechanical properties. Typically, the ratio of "mechanical" monomers in the polymer is about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:10, about 1:20, about 1:30, about 1:40, about 1:50 water-binding monomer: mechanical strength monomer molar ratio. Suitable examples of monomers capable of imparting mechanical properties (e.g., compressive resistance) to the scaffold include acrylates, such as hydroxyethyl methacrylate (HEMA); polyesters, such as poly(lactic acid), poly(caprolactone), poly(glycolide); and random copolymers thereof, such as poly(glycolide-co-lactide) and poly(glycolide-co-caprolactone).

[0155] The monomers may also be reacted with other compounds to form a "macromer." As a macromer, a preferred second monomer is hydroxyethyl methacrylate poly(lactic acid) (PLA / HEMA).

[0156] Preferably, the polymer includes the second monomer in an amount of from about 1 mol% to about 50 mol%. In various embodiments, the second monomer can be present in an amount of about 1 mol%, about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, about 15 mol%, about 16 mol%, about 17 mol%, about 18 mol%, about 19 mol%, about 20 mol%, about 21 mol%, about 22 mol%, about 23 mol%, about 24 mol%, about 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol%, about 55 mol%, about 56 mol%, about 57 mol%, about 58 mol%, about 59 mol%, about 60 mol%, about 61 mol%, about %, about 47 mol%, about 48 mol%, about 49 mol%, or about 50 mol%. In various embodiments, the second monomer may be present in an amount of about 1 mol% to about 15 mol%, about 2 mol% to about 49 mol%, about 3 mol% to about 48 mol%, about 4 mol% to about 47 mol%, about 5 mol% to about 46 mol%, about 6 mol% to about 45 mol%, about 7 mol% to about 44 mol%, about 8 mol% to about 43 mol%, about 9 mol% to about 42 mol%, about 10 mol% to about 41 mol%, about 11 mol% to about 40 mol%, about 12 mol% to about 39 mol%, about 13 mol% to about 39 mol%, about 16 mol% to about 39 mol%, about 17 mol% to about 39 mol%, about 18 mol% to about 39 mol%, about 20 mol% to about 39 mol%, about 21 mol% to about 39 mol%, about 22 mol% to about 39 mol%, about 23 mol% to about 39 mol%, about 24 mol% to about 39 mol%, about 25 mol% to about 39 mol%, about 26 mol% to about 39 mol%, about 27 mol% to about 39 mol%, about 28 mol% to about 39 mol%, about 29 mol% to about 39 mol%. % to about 38 mol%, about 14 mol% to about 37 mol%, about 15 mol% to about 36 mol%, about 16 mol% to about 35 mol%, about 17 mol% to about 34 mol%, about 18 mol% to about 33 mol%, about 19 mol% to about 34 mol%, about 20 mol% to about 33 mol%, about 21 mol% to about 30 mol%, about 22 mol% to about 29 mol%, about 23 mol% to about 28 mol%, about 24 mol% to about 27 mol%, or about 25 mol% to about 26 mol%.

[0157] It will be understood by those skilled in the art that the amount of the second monomer has a wider range than that of the other monomers because mechanical strength and adhesion are key factors of the present invention.

[0158] The third monomer: NSPP binding monomer

[0159] As discussed above, the tissue scaffolds used in the present invention can optionally be formed by combining a polymer with a NSPP. In order to effectively combine a polymer with a NSPP, it is preferred to include monomers or units with cross-linking capabilities in the polymer.

[0160] This cross-linking ability means that the polymer can bind to the NSPP and, through binding, cross-link the NSPP, thereby forming a scaffold containing the NSPP. Alternatively, the NSPP acts as a cross-linking agent via a similar mechanism, thereby cross-linking the polymer to form a scaffold.

[0161] In order to produce a polymer capable of binding to an NSPP, one skilled in the art will appreciate that the monomer capable of binding to an NSPP needs to be present in the polymer of the present invention in a ratio sufficient to crosslink with the NSPP, thereby forming a tissue scaffold in the presence of water. Typically, the ratio of "crosslinking" monomer in the polymer is about 15:1, about 10:1, about 5:1, about 4:1, about 3:1, about 2:1, about 1:1, about 1:2, about 1:3, about 1:4, about 1:5, about 1:10, or about 1:15 crosslinking monomer:water-binding monomer.

[0162] Monomers capable of binding to NSPPs typically have electrophilic or nucleophilic functional groups, such that, for example, a nucleophilic functional group on the NSPP can react with an electrophilic functional group on the monomer to form a covalent bond.

[0163] Thus, for example, if the NSPP has a nucleophilic functional group such as an amine, the polymer can have an electrophilic functional group such as N-hydroxysuccinimide (NHS). Other electrophilic functional groups suitable for use in the present invention are N-hydroxysulfosuccinimide (SNHS) and N-hydroxyethoxylated succinimide (ENHS). An example of such a monomer is N-acryloyloxysuccinimide (NAS). On the other hand, if the NSPP has an electrophilic functional group, the polymer can have a nucleophilic functional group such as an amine or a thiol.

[0164] Preferably, the polymer comprises a third monomer in an amount not exceeding 15 mol%. In various embodiments, the third monomer may be present in an amount of about 0 mol%, about 1 mol%, about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, or about 15 mol%. In various embodiments, the third monomer may be present in an amount of about 0 to about 1 mol%, about 1 mol% to about 15 mol%, about 2 mol% to about 14 mol%, about 3 mol% to about 13 mol%, about 4 mol% to about 12 mol%, about 5 mol% to about 11 mol%, about 6 mol% to about 10 mol%, about 7 mol% to about 9 mol%, or about 8 mol%.

[0165] The skilled artisan will appreciate that the polymer may be formed from a hydrophobic composition and thus the third monomer is optional in the polymer.

[0166] The fourth monomer: phase change monomer

[0167] In another embodiment of the present invention, polymer can also include can give support phase change characteristics, thereby ensure the stability of support after application of the fourth monomer. In addition, these phase change characteristics allow the polymer of the present invention to form support, by changing factors such as pH and temperature, can change the various properties (such as viscosity) of support. Support is designed to make the lowest critical solution temperature (LCST) lower than body temperature. Various thermal response and injectable polymers, including poly (ethylene oxide) / poly (propylene oxide) and poly (N-isopropyl acrylamide) (PNIPAAm) copolymers, are all suitable for the present invention.

[0168] Typically, the ratio of phase change monomer in the polymer is a phase change monomer:water binding monomer molar ratio of at least about 3: 1. For example, the ratio can be increased to a phase change monomer:water binding monomer molar ratio of about 10: 1, about 11: 1, about 12: 1, about 13: 1, about 14: 1, about 15: 1, about 16: 1, about 17: 1, about 18: 1, about 19: 1, about 20: 1, about 25: 1, about 30: 1, about 35: 1, about 40: 1, about 45: 1, about 50: 1, about 55: 1, about 60: 1, about 65: 1, about 70: 1, about 75: 1, about 80: 1, and about 85: 1.

[0169] Preferably, the polymer comprises an amount of the fourth monomer that makes up the remainder to 100% of the polymer composition. In an embodiment, the mole percentage (mol %) of the fourth monomer may not exceed about 85%, and is preferably about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 or 85 mol %.

[0170] Other polymer properties

[0171] It will be appreciated by those skilled in the art that by combining different types of monomers, polymers with various properties can be produced. In addition, by introducing specific monomers or functional groups into existing polymers, the properties of polymers can be changed. For example, the copolymerization of HEMA monomers with other monomers (such as methyl methacrylate) can be used to change properties such as swelling and mechanical properties. Monomers can also be reacted with other compounds to form macromonomers (as defined above), which are then included in polymers of the present invention. For example, HEMA and propiolactone can be reacted to form HEMA-polylactic acid polymers (PLA / HEMA), which themselves can be used as monomers in polymers of the present invention. In addition, monomers themselves can also be combinations of monomeric units, which are then introduced into polymers. An example of this type of monomer is oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA), which is a hydrophilic monomer composed of two hydrophilic monomers, i.e., ethylene glycol and methacrylate.

[0172] Preferred polymers of the present invention may also be modified with one or more molecules and / or functional groups. Any moiety or functional group may be used in accordance with the present invention. In some embodiments, the polymer may be modified with polyethylene glycol (PEG), carbohydrates, and / or acyclic polyacetals derived from polysaccharides. Additionally, as discussed above, hydrophilic groups may be introduced into the monomers (and therefore the polymer) to increase the water binding capacity of the polymer.

[0173] In terms of sequence, the copolymer can be a block copolymer, a graft copolymer, a random copolymer, a blend, a mixture and / or an adduct of any of the above polymers and other polymers. The polymer according to the present invention is generally an organic polymer. The polymer of the present invention is preferably biocompatible. In some embodiments, the polymer is biodegradable. In other embodiments, the polymer is both biocompatible and biodegradable.

[0174] Preferred polymers of the present invention may also include other monomers in their structure. For example, the monomers may be polymers such as poly(vinyl alcohol) (PVA), polyesters, acrylic polymers, and ionomers, or monomers of these polymers.

[0175] If it is desired that the polymer be biodegradable or absorbable, one or more monomers with a biodegradable bond (linkage) can be used. Alternatively or additionally, monomers can also be selected so that the reaction product therebetween produces a biodegradable bond. For each method, monomers and / or bonds can be selected so that the resulting biodegradable polymer will degrade or be absorbed within a desired time period (e.g., about 6 hours to about 6 months). Preferably, monomers and / or bonds are selected so that when the polymer degrades under physiological conditions, the resulting product is non-toxic.

[0176] Biodegradable bonds can be chemically or enzymatically hydrolyzable or absorbable. Exemplary chemically hydrolyzable biodegradable bonds include polymers, copolymers, and oligomers of glycolide, lactide, caprolactone, dioxanone, and trimethylene carbonate. Exemplary enzymatically hydrolyzable biodegradable bonds include peptide bonds that can be cleaved by metalloproteinases and collagenases. Other exemplary biodegradable bonds include polymers and copolymers of poly(hydroxy acids), poly(orthocarbonates), poly(anhydrides), poly(lactones), poly(amino acids), poly(carbonates), and poly(phosphonates).

[0177] In the present invention, chemical hydrolysis of lactide results in an increase in the lower critical solution temperature (LCST) of the polymer (by reducing the overall hydrophobicity of the polymer), thereby increasing its bioabsorbability.

[0178] Preferred polymers

[0179] The polymer preferably includes the first monomer in an amount of about 1 mol% to about 15 mol%. In various embodiments, the first monomer may be present in an amount of about 1 mol%, about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, or about 15 mol%. Preferably, the first monomer is OEGMA.

[0180] The polymer preferably includes the second monomer in an amount of about 5 mol% to about 50 mol%. In various embodiments, the second monomer can be present in an amount of about 1 mol%, about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, about 15 mol%, about 16 mol%, about 17 mol%, about 18 mol%, about 19 mol%, about 20 mol%, about 21 mol%, about 22 mol%, about 23 mol%, about 24 mol%, about 25 mol%, about 26 mol%, about 27 mol%, about 28 mol%, about 29 mol%, about 30 mol%, about 31 mol%, about 32 mol%, about 33 mol%, about 34 mol%, about 35 mol%, about 36 mol%, about 37 mol%, about 38 mol%, about 39 mol%, about 40 mol%, about 41 mol%, about 42 mol%, about 43 mol%, about 44 mol%, about 45 mol%, about 46 mol%, about 47 mol%, about 48 mol%, about 49 mol%, about 50 mol%, about 51 mol%, about 52 mol%, about 53 mol%, about 54 mol%, about 55 mol%, about 56 mol%, about 57 mol%, about 58 mol%, about 59 mol%, about 60 mol%, about 61 mol%, about %, about 48 mol%, about 49 mol%, or about 50 mol%. Preferably, the second monomer is PLA / HEMA.

[0181] The polymer preferably includes no more than 15 mol% of the third monomer. In various embodiments, the third monomer may be present in an amount of about 0 mol%, about 1 mol%, about 2 mol%, about 3 mol%, about 4 mol%, about 5 mol%, about 6 mol%, about 7 mol%, about 8 mol%, about 9 mol%, about 10 mol%, about 11 mol%, about 12 mol%, about 13 mol%, about 14 mol%, or about 15 mol%. Preferably, the third monomer is NAS.

[0182] The polymer preferably includes the fourth monomer in an amount that makes up the remainder to 100% of the polymer composition, for example, from about 50 mol% to about 85 mol%. In an embodiment, the mole percentage of the fourth monomer may not exceed about 85 mol%, preferably about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84 or 85 mol%. Preferably, the fourth monomer is NIPAAm.

[0183] The percentages stated herein refer to the final polymer composition and not the feedstock amounts used in forming the polymer.

[0184] In one embodiment, the polymer preferably comprises the first monomer in an amount of from about 1 mol% to about 15 mol%; the second monomer in an amount of from about 5 mol% to about 50 mol%; the third monomer in an amount not exceeding 15 mol%; and the fourth monomer in an amount not exceeding about 85 mol%.

[0185] Preferably, the first monomer is OEGMA, the second monomer is PLA / HEMA, the third monomer is NAS, and the fourth monomer is NIPAAm.

[0186] In another embodiment, preferably, the polymer comprises the first monomer in an amount of about 7 mol%; the second monomer in an amount of about 30 mol%; the third monomer in an amount of about 7 mol%; and the fourth monomer in an amount of about 53 mol%.

[0187] Preferably, the first monomer is OEGMA, the second monomer is PLA / HEMA, the third monomer is NAS, and the fourth monomer is NIPAAm.

[0188] In one embodiment, the polymer of the present invention is a polymer of formula (I):

[0189]

[0190] in

[0191] A is the first monomer (water-binding monomer), such as OEGMA;

[0192] B is the second monomer (a monomer that can impart mechanical properties to the tissue scaffold), such as PLA / HEMA;

[0193] C is a third monomer (a monomer having a functional group that binds to NSPP), such as NAS; and

[0194] D is a fourth monomer (a monomer capable of imparting phase change properties to the scaffold), such as NIPAAm.

[0195] In various embodiments, m is an integer from 1-20; n is an integer from 1-20; p is an integer from 0-20; and q is an integer from 1-20.

[0196] An exemplary polymer of the present invention is represented by Formula (Ia) below:

[0197]

[0198] wherein A is the water-binding monomer OEGMA, B is the reinforcing monomer PLA / HEMA, C is the cross-linking agent NAS, D is the phase-change monomer NIPAAm, and m, n and p, q, x and y are as defined above.

[0199] It will be understood by those skilled in the art that monomers A, B, C and D may be present in the polymer in any order as long as the desired water binding, reinforcement and / or cross-linking capabilities are achieved.

[0200] It has also been discovered that some monomers such as PLA / HEMA, polyesters such as poly(lactic acid), poly(caprolactone), poly(glycolide), and random copolymers thereof (e.g., poly(glycolide-co-lactide) and poly(glycolide-co-caprolactone), as well as other biodegradable and biocompatible polymers, can increase the LCST of the preferred polymers used in the present invention during the in vivo degradation of the biodegradable segment (e.g., PLA), thereby leading to bioabsorption of the polymer. This provides the additional advantage that the polymers used in the present invention can be designed to be biodegradable in vivo.

[0201] The overall size of the preferred polymers used in the present invention can vary depending on factors such as the type of monomers incorporated into the polymer, the type of NSPPs sought to form the scaffold, and the conditions under which the protein is coupled to the polymer. However, preferred polymers used in the present invention can generally be molecules of about 1 kDa to about 100 kDa, about 5 kDa to about 60 kDa, or about 30 kDa. In various embodiments, the polymers of the present invention can be about 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, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85 94, 95, 96, 97, 98, 99, or about 100 kDa.

[0202] PNPHO

[0203] The preferred polymer of the present invention is poly(NIPAAm-co-NAS-co-(PLA / HEMA)-co-OEGMA), or "PNPHO." Preferably, the polymer PNPHO comprises OEGMA in an amount of about 1 mol% to about 15 mol%; PLA / HEMA in an amount of about 5 mol% to about 50 mol%; NAS in an amount not exceeding 15 mol%; and NIPAAm in an amount making up the remainder to 100% of the polymer composition, for example, from about 50 mol% to about 85 mol%.

[0204] The percentages stated herein refer to the final polymer composition and not the feedstock amounts used in forming the polymer.

[0205] The preferred form of polymer PNPHO for use in the present application is the polymer of formula (Ia) depicted above. In addition, x is in the range of 1-1000, y is 1-1000, and m, n, p and q are 1-20.

[0206] It will be understood by those skilled in the art that monomers A, B, C and D may be present in the polymer in any order as long as the desired water binding, reinforcement and / or cross-linking capabilities are achieved.

[0207] PPHO

[0208] Another preferred polymer of the present invention is poly(NIPAAm-co-(PLA / HEMA)-co-OEGMA), or "PPHO." Preferably, the polymer PPHO comprises OEGMA in an amount of about 1 mol% to about 15 mol%; PLA / HEMA in an amount of about 5 mol% to about 50 mol%; and NIPAAm in an amount making up the remainder to 100% of the polymer composition, for example, from about 50 mol% to about 85 mol%. In preferred embodiments, the PPHO comprises about 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or about 15 mol % OEGMA, and / or about 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44 , 45, 46, 47, 48, 49, or about 50 mol % PLA / HEMA, and / or about 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, or about 85 mol % NIPAAm.

[0209] The percentages stated herein refer to the final polymer composition and not the feedstock amounts used in forming the polymer.

[0210] A preferred form of polymer PPHO for use in the present application is a polymer of formula (II) as depicted below: Furthermore, x is 1-1000, y is 1-1000, and m, n and q are 1-20.

[0211] It will be understood by those skilled in the art that monomers A, B, and D may be present in the polymer in any order as long as the desired water binding, reinforcement, and / or cross-linking capabilities are achieved.

[0212] Polymer synthesis

[0213] Those skilled in the art will appreciate suitable methods for synthesizing the preferred polymers used in the present invention, including methods such as ring-opening polymerization, addition polymerization (including free radical polymerization), and condensation polymerization.

[0214] The formation of the preferred polymers PNPHO and PPHO is described in the Examples below.

[0215] Compositions for forming scaffolds

[0216] The present invention also relates to a polymer for forming a tissue scaffold, comprising: a first water-binding monomer; a second monomer that imparts mechanical properties; an optional third monomer that is a NSPP-binding monomer; and a fourth monomer that is capable of imparting phase change properties to the scaffold.

[0217] As used herein, the term "composition" refers to a solid or liquid composition containing the above-mentioned ingredients. In some embodiments, the composition of the present invention may also include other ingredients, such as pharmaceutically acceptable excipients and bioactive agents (e.g., drugs, vitamins and minerals) to help target tissue repair and / or regeneration, and / or provide a method for achieving targeted delivery of bioactive compounds.

[0218] Excipients and bioactive agents

[0219] Pharmaceutically acceptable excipients may be included in the compositions and / or stents of the present invention, including any and all solvents, dispersion media, inert diluents or other liquid solvents, dispersing aids or suspension aids, granulating agents, surfactants, disintegrants, isotonic agents, thickeners or emulsifiers, preservatives, binding agents, lubricants, buffers, oils, etc., to suit the specific dosage form required. Remington (Gennaro, AR, Remington: The Science and Practice of Pharmacy, 21st edition (2006) Lippincott Williams & Wilkins) discloses various excipients and known preparation techniques for pharmaceutical compositions. Unless any conventional excipient is incompatible with a substance or its derivatives, such as producing any adverse biological effect or otherwise interacting with any other component of the pharmaceutical composition in a harmful manner, its use is expected to fall within the scope of the present invention.

[0220] Excipients, such as coloring agents, coating agents, sweetening, flavoring, and perfuming agents, can be present in the composition according to the judgment of the formulator.

[0221] Bioactive agents or pharmaceutical compounds that can be added to the compositions and / or scaffolds of the present invention include proteins, glycosaminoglycans, carbohydrates, nucleic acids, and inorganic and organic bioactive compounds, such as enzymes, antibiotics, anti-tumor agents, local anesthetics, hormones, angiogenic agents, anti-angiogenic agents, growth factors (such as insulin-like growth factor-1 (IGF-1), basic fibroblast growth factor (bFGF) and transforming growth factor-b (TGFb)), antibodies, neurotransmitters, psychotropic drugs, anti-cancer drugs, chemotherapeutic drugs, drugs that affect reproductive organs, genes and oligonucleotides.

[0222] By combining the polymer of the present invention with NSPP, combining with one or more other ingredients, and then freeze-drying the resulting composition, a composition containing ingredients such as excipients and / or bioactive agents can be produced, thereby producing a ready-to-use tissue scaffold.

[0223] The amount of polymer, NSPP and bioactive agent present in the composition will necessarily depend on the specific drug and condition to be treated. One skilled in the art will be aware of appropriate agents and amounts for treating the condition.

[0224] Natural or synthetic peptides or proteins

[0225] NSPP can be important because it provides additional mechanical properties (eg, strength and elasticity) to the scaffold and provides an environment at the repair site that mimics the natural environment, thereby aiding tissue repair and regeneration.

[0226] Importantly, the NSPP contains side chains or other functional groups that, when exposed, can react with functional groups of the NSPP binding monomer, thereby conjugating the NSPP to the polymer via the NSPP binding monomer. Examples of suitable side chains include glutamic acid or lysine side chains.

[0227] The present invention also contemplates the use of variants of NSPP, such as species variants or polymorphic variants. The present invention is intended to encompass all functionally active variants of NSPP that exhibit the same activity. This also includes apoforms and haloforms, post-translationally modified forms, and glycosylated or deglycosylated derivatives of NSPP. These functionally active fragments and variants include, for example, fragments and variants having conservative amino acid substitutions.

[0228] The NSPP used in the present invention is preferably obtained from a recombinant source, but may also be extracted from a natural source or synthesized.

[0229] bracket

[0230] The present invention also relates to a tissue scaffold comprising a polymer according to the present invention and optionally a NSPP, wherein the polymer comprises: a first hydration-binding monomer; a second monomer that imparts mechanical properties; an optional third monomer, namely a NSPP-binding monomer, comprising a functional group capable of binding to the NSPP; and a fourth monomer capable of imparting phase-change properties to the scaffold, wherein the scaffold forms a malleable structure upon hydration. Preferably, the first monomer is OEGMA, the second monomer is PLA / HEMA, the third monomer is NAS, and the fourth monomer is NIPAAm.

[0231] In an embodiment, the stent includes a polymer having the above-mentioned monomers to ensure the stability of the stent after administration. Specifically, the LCST of the stent is below body temperature or about 37°C to avoid the formation of unstable complexes or dissolution in the body. An example of a monomer that can be used for this purpose is NIPAAm.

[0232] In another embodiment, the amount of the first monomer in the polymer does not exceed about 15 mol % to ensure that the LCST of the scaffold is below body temperature, or about 37° C. One example of a monomer that can be used for this purpose is OEGMA.

[0233] In yet another embodiment, the amount of the second monomer in the polymer may be up to 50 mol % to serve as the backbone of the polymer.

[0234] cell

[0235] The scaffolds of the present invention may also include cells to assist in the repair and / or regeneration of target tissues.

[0236] Generally, the cells to be used in the present invention are any type of cells. The cells should be viable when embedded in the scaffold of the present invention.

[0237] In some embodiments, cells that can be embedded in the scaffolds of the present invention include, but are not limited to, mammalian cells (e.g., human cells, primate cells, mammalian cells, rodent cells, etc.), avian cells, fish cells, insect cells, plant cells, fungal cells, bacterial cells, and hybrid cells. In some embodiments, exemplary cells that can be embedded in the scaffolds include stem cells, totipotent cells, pluripotent cells, and / or embryonic stem cells.

[0238] In some embodiments, the exemplary cells that can be embedded in the support of the present invention include, but are not limited to, primary cells and / or cell lines from any tissue. For example, cardiomyocytes, myocytes, hepatocytes, keratinocytes, melanocytes, neurons, astrocytes, embryonic stem cells, adult stem cells, hematopoietic stem cells, hematopoietic cells (e.g., monocytes, neutrophils, macrophages, etc.), ameloblasts, fibroblasts, chondrocytes, osteoblasts, osteoclasts, neurons, spermatocytes, egg cells, hepatocytes, epithelial cells from the lungs, epithelial cells from the digestive tract, epithelial cells from the intestinal tract, epithelial cells from the liver, epithelial cells from the skin, etc., and / or their hybrids, can be embedded in the support of the present invention.

[0239] Exemplary mammalian cells that can be embedded in the scaffolds of the present invention include, but are not limited to, Chinese hamster ovary (CHO) cells, HeLa cells, Madin-Darby canine kidney (MDCK) cells, baby hamster kidney (BHK) cells, NSO cells, MCF-7 cells, MDA-MB-438 cells, U87 cells, A172 cells, HL60 cells, A549 cells, SP10 cells, DOX cells, DG44 cells, HEK 293 cells, SHSY5Y, Jurkat cells, BCP-1 cells, COS cells, Vero cells, GH3 cells, 9L cells, 3T3 cells, MC3T3 cells, C3H-10T1 / 2 cells, NIH-3T3 cells, and C6 / 36 cells.

[0240] In some embodiments, it is desirable that cells are evenly distributed throughout the support. Even distribution can help provide a more uniform tissue-like support that provides a more uniform environment for encapsulating cells. In some embodiments, cells are located on the surface of the support. In some embodiments, cells are located inside the support. In some embodiments, cells are layered within the support. In some embodiments, the support contains different types of cells.

[0241] In some embodiments, the conditions under which cells are embedded in the scaffold are changed to maximize cell viability. For example, in some embodiments, the lower the polymer concentration, the higher the cell viability. In some embodiments, the viability of cells located at the periphery of the hydrogel tends to decrease compared to cells completely encapsulated in the hydrogel. In some embodiments, it may be necessary to adjust and / or change the conditions of the surrounding environment (e.g., pH, ionic strength, nutrient availability, temperature, oxygen availability, osmotic pressure, etc.) to maximize cell viability.

[0242] In some embodiments, cell viability can be measured by monitoring one of the multiple indices of cell viability. In some embodiments, cell viability indicators include but are not limited to intracellular esterase activity, plasma membrane integrity, metabolic activity, gene expression and protein expression. To give just one example, when cells are exposed to fluorescent esterase substrates (such as calcein AM), the esterase substrate is hydrolyzed into a green fluorescent product by intracellular esterase activity, and living cells emit green fluorescence. For another example, when cells are exposed to fluorescent nucleic acid stains (such as ethidium bromide dimer-1 (ethidium homodimer-1)), dead cells emit red fluorescence because their plasma membrane is damaged and therefore permeable to high-affinity nucleic acid stains.

[0243] Typically, the number of cells in the composition is an amount that allows for the formation of a hydrogel according to the present invention. In some embodiments, the amount of cells suitable for forming a hydrogel according to the present invention is from about 0.1% w / w to about 80% w / w, from about 1.0% w / w to about 50% w / w, from about 1.0% w / w to about 40% w / w, from about 1.0% w / w to about 30% w / w, from about 1.0% w / w to about 20% w / w, from about 1.0% w / w to about 10% w / w, from about 5.0% w / w to about 20% w / w, or from about 5.0% w / w to about 10% w / w.

[0244] In some embodiments, the amount of cells in a composition suitable for forming a hydrogel according to the present invention is about 5% w / w. In some embodiments, the concentration of cells in a precursor solution suitable for forming a hydrogel according to the present invention is about 10 to about 1×10 8 cells / mL, about 100 to about 1×10 7 cells / mL, about 1×10 3 to about 1×10 6 cells / mL or approximately 1×10 4 to about 1×10 5 In some embodiments, a single hydrogel comprises a population of the same cells and / or cell types. In some embodiments, a single hydrogel comprises a population of different cells and / or cell types.

[0245] In some embodiments, a single hydrogel can contain at least two different types of cells. In some embodiments, a single hydrogel can contain 3, 4, 5, 10, or more types of cells. By way of example only, in some embodiments, a single hydrogel can contain only embryonic stem cells. In some embodiments, a single hydrogel can contain both embryonic stem cells and hematopoietic stem cells.

[0246] culture medium

[0247] Any of the various cell culture media (including complex culture media and / or serum-free culture media) that can support the growth of one or more cell types or cell lines can be used for growth and / or maintenance cells. Typically, cell culture media contains buffer, salt, energy source, amino acid (such as natural amino acid, non-natural amino acid, etc.), vitamins and / or trace elements. Cell culture media can optionally contain various other ingredients, including but not limited to carbon source (such as natural sugar, non-natural sugar, etc.), cofactor, lipid, sugar, nucleoside, animal-derived component, hydrolyzate, hormone, growth factor, surfactant, indicator, mineral matter, activator of specific enzyme, activation inhibitor of specific enzyme, enzyme, organic matter and / or small molecule metabolite.

[0248] Cell culture media suitable for use in the present invention are commercially available from various sources, such as ATCC (Manassas, VA.). In certain embodiments, one or more of the following culture media are used to grow cells: RPMI-1640 medium, Dulbecco's Modified Eagle's Medium, Minimum Essential Medium Eagle, F-12K medium, Iscove's Modified Dulbecco's Medium.

[0249] Those skilled in the art will recognize that the cells listed herein represent an exemplary list of cells that can be encapsulated in a precursor solution according to the present invention, and are not a comprehensive list.

[0250] application

[0251] The present invention aims to provide a scaffold that supports the natural healing of damaged tissue without inducing the formation of any specific tissue. The goal is to use the present invention to fix one, two, or more tissues to support tissue regeneration and / or tissue interface integration while minimizing the foreign body reaction.

[0252] The tissue scaffold of the present invention is preferably hydrated intraoperatively with physiological saline, the patient's own blood, platelet-rich plasma (PRP), platelet-rich fibrin, and / or other autologous blood / cell / tissue products to form a malleable scaffold. The resulting scaffold can be administered via arthroscopic or open surgical intervention. When in contact with body tissue, the scaffold has adhesive properties and suture properties, thereby being able to fix one or more tissues to support tissue regeneration and / or tissue interface integration.

[0253] The stent of the present invention can preferably be manufactured by using different processing methods.

[0254] The scaffolds of the present invention preferably eliminate the criticality of water solubility.

[0255] The scaffold of the present invention can preferably be adjusted to change its microenvironment to meet different requirements.

[0256] The stents of the present invention are preferably combined with different hydrophilic and hydrophobic drugs.

[0257] The stent of the present invention is preferably non-adhesive to gloves to facilitate effective handling.

[0258] The scaffolds of the present invention are preferably malleable, thereby allowing for efficient delivery to the site via arthroscopic or open surgical intervention.

[0259] The stent of the present invention is preferably adhesive to the treatment site, and the stent of the present invention is preferably sutureable for internal fixation.

[0260] The present invention has been developed for use in skin grafting to secure graft tissue, repair partial or full thickness rotator cuff tears, tendon healing in total shoulder replacements, anterior cruciate ligament (ACL) and / or other ligament / tendon tears.

[0261] Reagent test kit

[0262] The present invention provides a variety of kits comprising one or more scaffolds and / or polymers of the present invention. For example, in one aspect, the present invention provides a kit comprising a scaffold and / or polymer and instructions for use. The kit can include a variety of different scaffolds and / or polymers. The kit can optionally include cells, NSPPs, biologically active compounds, etc. The kit can include any number of additional components or reagents in any combination. Not all combinations are explicitly listed, but each combination is intended to be within the scope of the present invention.

[0263] PNPHO / PPHO composition

[0264] PNPHO was synthesized following the synthetic procedure generally set forth in the applicant's previously published WO 2013 / 091001. PPHO was synthesized in a similar manner except for the addition of NAS.

[0265] pass 1 The synthesis of the PNPHO copolymer was confirmed by H NMR spectroscopy, demonstrating proton peaks for each monomer, which is consistent with that shown in WO2013 / 091001. 1The synthesis of PPHO was confirmed by H NMR spectroscopy, as shown in Figure 5(a), demonstrating the proton peaks of each monomer. Comparative NMR spectra of PNPHO and PPHO are shown in Figure 5(b). The HPLC chromatogram at a wavelength of 230 nm, as shown in Figure 5(c), showed that PNPHO eluted at 6.2 min and PPHO eluted at 7.2 min. The difference in elution time indicates that PPHO eluted with a higher percentage of acetonitrile than PNPHO, thus confirming the hydrophobicity of PPHO compared to PNPHO.

[0266] Characteristic proton peaks of NIPAAm (a and b), NAS (e), PLA / HEMA (f, h, k), and OEGMA (m and n) were detected. The final composition of the copolymer was calculated based on the integration of these peaks for each monomer: NIPAAm (a), NAS (e / 2-f), PLA / HEMA (h), and OEGMA (n / 2). In this study, the copolymer is denoted as PNPHO with the addition of a subscript corresponding to the molar ratio of PLA / HEMA (lactate length) to OEGMA. For example, PNPHO 8(6)3 Represents a copolymer synthesized from 8 mol% PLA / HEMA with a lactate length of 6 and 3 mol% OEGMA. Various copolymers were produced.

[0267] Fabrication of tissue scaffolds using PNPHO / PPHO

[0268] The tissue scaffold of the present invention can be manufactured from PNPHO / PPHO solution using different methods. The obtained PNPHO / PPHO scaffold and its morphology are shown in FIG1 . Figure 2 As shown, after hydration, the scaffold can be shaped and, if necessary, delivered arthroscopically. The product can be used for suturing and stapling. The product is non-sticky to gloves, allowing for efficient handling.

[0269] The product has adhesive properties when in contact with different tissues (as shown in Figures 3, 4 and 6), either in the case of suture / internal fixation or sutureless / internal fixation, thus allowing fixation of one or more tissues in the body.

[0270] Generally, the PHPHO / PPHO scaffold of the present invention can be prepared by freeze-drying a water-soluble composition, wherein a PNPHO / PPHO solution (with or without a peptide / protein component) is dissolved in a buffer solution; different drugs / moieties (antibiotics, growth factors, live viruses) can be added to the solution for embedding in the final product; the resulting solution is freeze-dried, and the morphology of the composite material is modified by adjusting the freeze-drying parameters and the concentration of the PNPHO polymer; the final product is in the form of a ready-to-use scaffold, with or without embedding the biological moiety.

[0271] Exemplary Method 1

[0272] The first method is used when the polymer is water-soluble and involves dissolving an aqueous polymer solution in a buffer solution and freeze-drying the resulting solution, wherein the morphology of the scaffold is modified by adjusting the freeze-drying parameters and the polymer concentration.

[0273] Exemplary Method 2

[0274] In the case of hydrophobic polymers (e.g., PPHO), a second method is used, which involves dissolving the polymer in an organic solvent to obtain a solution; precipitating the solution in water as an antisolvent to obtain a gel; and freeze-drying the gel, wherein the morphology of the scaffold is modified by adjusting the freeze-drying parameters.

[0275] Other preparation methods may include porogen leaching and / or gas foaming.

[0276] Exemplary Method 3

[0277] A method for producing an injectable or sprayable stent is employed, wherein the method comprises dissolving a PPHO polymer in an aqueous solution, autologous blood, a cell or tissue product, or a combination thereof.

[0278] Stent manufacturing

[0279] As previously described, the scaffold according to the present invention is manufactured by freeze-drying PNPHO and / or PPHO to form a porous membrane, such as Figure 7 and Figure 10 scaffolds can also be fabricated by compressing PNPHO and / or PPHO powders. SEM images of both structures show similar microstructures and morphologies; however, scaffolds fabricated using compression were used for in vivo biocompatibility analysis.

[0280] This study aimed to investigate the biodegradability of freeze-dried TM scaffolds in a mouse model. Twenty-four male Balb / c mice, approximately 10 weeks old, were subcutaneously implanted with freeze-dried PNPHO and PPHO scaffolds. Ultrasound measurements were performed weekly to determine in situ presence and volume. Animals were sacrificed on days 7, 14, 21, and 42 after implantation. Biopsy specimens were obtained from the implantation site, fixed in 10% formalin, embedded in paraffin, sectioned, and stained with H&E for histological analysis.

[0281] Surgical procedures and product preparation

[0282] PNPHO and PPHO stents were used. Before surgery, mice were anesthetized with 3% isoflurane. General anesthesia was determined by a lack of response to toe pinching. A 1 cm incision was made in the dorsal skin of the mouse, and the stent was inserted subcutaneously. The incision was then closed with sutures and cleaned as necessary.

[0283] Material handling

[0284] Prior to implantation, the scaffold was hydrated in 2 mL of warm water at approximately 37°C for at least 3 minutes. The scaffold was found to be cohesive and malleable. The scaffold could be easily picked up with forceps and delivered easily and conveniently into the pouch created on the mouse's back.

[0285] recover

[0286] All experimental mice recovered within 5 minutes of anesthesia. The mice were recorded as healthy, with no signs of discomfort or stress. The surgery did not affect the mice's mobility, and after the incision was closed with sutures, no stent protrusion from the incision was detected. The stent appeared to remain in the mice for a period of time.

[0287] Animal health and weight

[0288] With the exception of mouse ID "S-21," no abnormalities were found in the health of the mice during the experiment (no significant weight loss). This mouse showed significant weight loss on day 4 after surgery, but did not show any other abnormal signs or distress; the mouse behaved normally, had no wounds or bite marks, and did not exhibit any hair loss. As all other parameters were normal, the mouse was retained for further observation. Its weight steadily increased on day 7 and returned to normal levels on day 11.

[0289] Macroscopic observation and ultrasonic analysis

[0290] Mice were scanned with high-frequency ultrasound to examine the volume of the scaffold after implantation. Ultrasound was performed immediately after implantation and at time points of 0, 4, 7, 11, and 14. The scaffold was clearly detected immediately after surgery and was evident, but was not detected by ultrasound imaging, likely due to the solid structure of the scaffold.

[0291] Biopsy and autopsy

[0292] At 7, 14, 21, and 42 days, mice from each group were sacrificed and macroscopically scored for the presence of the scaffold. Skin biopsies were collected and preserved in 10% formalin for histological analysis. At all time points, the scaffolds were integrated into the host environment. Compared to historical controls and empirical mouse dissections, the panniculus carnosus layer was found to have greater adhesion to the underlying structures. The skin also showed greater adhesion to the subcutaneous fascia and muscle tissue. The scaffolds are believed to be integrated within the tissue layers of the underlying tissue.

[0293] Histological analysis

[0294] Skin biopsies collected from the implantation site were processed and stained with H&E to analyze the presence of TM scaffolds, cellular infiltration, inflammatory response, and angiogenesis.

[0295] Figure 10 Histological evaluation of the site 1 week later shows the structure of the scaffold with cell infiltration (blue arrow). The scaffold implanted in the mouse back should be located below the panniculus carnosus layer (orange arrow). The underlying area shows a large space with cell infiltration of the scaffold (yellow arrow).

[0296] Figure 11 The results in Figure 3 show that at week 1, the scaffold present in the tissue was infiltrated with numerous cells (red arrows). At weeks 2, 3, and 6, residual scaffold was detectable by histology, while the host tissue had a loose connective tissue structure, likely fascia (green arrows). It is important to note that PNPHO and PPHO are highly soluble in ethanol and were therefore removed during the processing steps. Despite this, there was no sign of scaffold encapsulation, fibrosis, or fibrous tissue formation around the scaffold, confirming the biocompatibility of the product.

[0297] The mild cellular infiltration observed 1 week after implantation is consistent with the early stages of wound healing and foreign body response. No significant multinucleated giant cells were detected in the area of ​​scaffold presence. Extensive cellular infiltration was observed around the sutures, which was expected and considered a typical foreign body response during suture biodegradation. No cellular inflammatory response was observed at 2, 3, or 6 weeks.

[0298] Other Notes

[0299] The scaffolds used in the experiments were handled in a manner that allowed for easy implantation and administration after hydration. All experimental mice recovered immediately after surgery without abnormal signs of discomfort or stress, demonstrating biocompatibility for in vivo life.

[0300] Solid scaffolds were detected in the mice immediately after implantation, indicating that the scaffolds remained in place and were not easily displaced or degraded in the body. None of the mice lost significant weight, indicating the animals' overall health and stability during the experiment. The surgical results showed that the scaffolds used were effective for their intended purpose and that the implantation procedure was generally successful. Macroscopically, the researchers were able to easily identify the sheet-like scaffolds beneath the skin immediately after the implantation procedure. The scaffolds did not become liquid, but remained solid.

[0301] Industrial Applicability

[0302] It should be recognized that the present invention has applicability in surgical and biomedical fields. The PNPHO or PPHO scaffolds of the present invention can be used in any anatomical area where internal fixation of one or more tissues is required to support healing. This includes, but is not limited to, skin grafts to fix transplanted tissues, repair of partial or full-thickness rotator cuff tears, total shoulder replacements, tendon healing in anterior cruciate ligament (ACL) and / or other ligament / tendon tears, etc.

Claims

1. A polymer for forming a tissue scaffold, the polymer comprising: a first monomer for binding water; a second monomer for imparting mechanical properties to the stent; an optional third monomer for conjugation to a natural or synthetic peptide or protein (NSPP); and a fourth monomer for imparting phase change behavior; The scaffold forms a forgeable structure after hydration.

2. The polymer of claim 1, wherein the first monomer is selected from the group consisting of: polyether, polyvinyl alcohol (PVA), poly(vinyl pyrrolidone) (PVP), poly(amino acid), and dextran.

3. The polymer of claim 2, wherein the polyether is selected from: polyethylene glycol (PEG), oligo(ethylene glycol) (OEG), polyethylene oxide (PEO), polyethylene oxide-co-propylene oxide (PPO), copolymers of polyethylene oxide blocks or random copolymers thereof.

4. The polymer of claim 3, wherein the first monomer is oligo(ethylene glycol) monomethyl ether methacrylate (OEGMA).

5. The polymer according to any one of the preceding claims, wherein the second monomer is a methacrylate or a random copolymer comprising a methacrylate.

6. The polymer of claim 5, wherein the second monomer is selected from: hydroxyethyl methacrylate (HEMA), hydroxyethyl methacrylate poly(lactic acid) copolymer (PLA / HEMA), poly(lactic acid), poly(caprolactone), poly(glycolide), poly(glycolide-co-lactide) or poly(glycolide-co-caprolactone).

7. The polymer of claim 6, wherein the second monomer is hydroxyethyl methacrylate poly(lactic acid) (PLA / HEMA).

8. The polymer of any one of the preceding claims, wherein the third monomer has an electrophilic functional group that binds to the NSPP.

9. The polymer of claim 8, wherein the third monomer is selected from the group consisting of: N-hydroxysulfosuccinimide (SNHS), N-hydroxyethoxylated succinimide (ENHS), and N-acryloyloxysuccinimide (NAS).

10. The polymer of claim 9, wherein the third monomer is N-acryloyloxysuccinimide (NAS).

11. The polymer of any preceding claim, wherein the fourth monomer has a lower critical solution temperature (LCST) of less than about 37°C.

12. The polymer of claim 11, wherein the fourth monomer is selected from the group consisting of poly(ethylene oxide) / poly(propylene oxide) and poly(N-isopropylacrylamide) (PNIPAAm) homopolymers and copolymers.

13. The polymer of claim 12, wherein the fourth monomer is (N-isopropylacrylamide) (NIPAAm).

14. The polymer of any preceding claim, wherein the polymer comprises the first monomer in an amount of from about 1 mol% to about 15 mol%.

15. The polymer of any preceding claim, wherein the polymer comprises the second monomer in an amount of from about 5 mol% to about 50 mol%.

16. The polymer of any preceding claim, wherein the polymer comprises the third monomer in an amount of from about 0 to about 15 mol%.

17. The polymer of any preceding claim, wherein the polymer comprises the fourth monomer in an amount of from about 50 mol% to about 85 mol%.

18. A polymer according to any of the preceding claims, wherein the polymer comprises the first monomer in an amount of about 1 mol% to about 15 mol%; the second monomer in an amount of about 5 mol% to about 50 mol%; the third monomer in an amount of 0 to about 15 mol%; and the fourth monomer in an amount making up the remainder to 100% of the polymer.

19. A polymer according to any one of the preceding claims, wherein the first monomer is OEGMA; the second monomer is PLA / HEMA; the third monomer is NAS; and the fourth monomer is NIPAAm, wherein the polymer comprises OEGMA in an amount of about 1 mol% to about 15 mol%; PLA / HEMA in an amount of 5 mol% to about 50 mol%; NAS in an amount of 0 to about 15 mol%; and NIPAAm in an amount not exceeding about 85 mol%.

20. A tissue scaffold comprising a polymer as defined in any one of claims 1 to 19, wherein the scaffold forms a malleable structure upon hydration.

21. The tissue scaffold of claim 20, wherein the scaffold is capable of being hydrated intraoperatively.

22. The tissue scaffold of claim 20 or 21, wherein the scaffold is hydrated with saline, aqueous solution, autologous or allogeneic blood, cell or tissue products, or a combination thereof.

23. The tissue scaffold of claim 22, wherein the scaffold is hydrated with the patient's own blood, platelet-rich plasma (PRP), bone marrow aspirate, platelet-rich fibrin, or a combination thereof.

24. The tissue scaffold of any one of claims 20-23, wherein the scaffold is non-adhesive to surgical gloves.

25. The tissue scaffold of any one of claims 20-24, wherein the scaffold is adhesive to a treatment site.

26. The tissue scaffold of any one of claims 20-25, wherein the scaffold is not tissue inductive.

27. The tissue scaffold of any one of claims 20-26, wherein the scaffold is formed after administration to a mammal.

28. The tissue scaffold of claim 27, wherein the scaffold is formed at body temperature.

29. The tissue scaffold according to claim 27 or 28, wherein the administration is performed by injection or spraying.

30. A malleable structure formed from a tissue scaffold as defined in any one of claims 20 to 29.

31. A method for preparing a tissue scaffold, the method comprising the following steps: dissolving an aqueous solution of a polymer as defined in any one of claims 1 to 19 in a buffer solution to obtain a resulting solution; as well as freeze-drying the obtained solution; The scaffold has a morphology that can be changed by adjusting freeze-drying parameters and the concentration of the polymer.

32. The method of claim 31 , wherein at least one drug or biological moiety is added to the resulting solution between the dissolution and freeze-drying steps.

33. The method of claim 32, wherein the drug or biological moiety is selected from the group consisting of: antibiotics, growth factors, live viruses, or combinations thereof.

34. A method for preparing a tissue scaffold, the method comprising the following steps: dissolving a polymer as defined in any one of claims 1 to 19 in an organic solvent to obtain a solution; precipitating the solution in water as an antisolvent to obtain a gel; as well as freeze-drying the gel; The scaffold has a morphology that can be changed by adjusting freeze-drying parameters.

35. The method of claim 34, wherein the organic solvent is miscible with water.

36. A method for preparing a tissue scaffold, the method comprising the following steps: dissolving a polymer as defined in any one of claims 1 to 19 in an organic solvent to obtain a solution; as well as The solution was electrospun.

37. A method for preparing a tissue scaffold, the method comprising: dissolving a polymer as defined in any one of claims 1 to 19 in an aqueous solution, autologous blood, a cell or tissue product or a combination thereof; The stent is injectable or sprayable.

38. Use of a polymer as defined in any one of claims 1-19 in the manufacture of a tissue scaffold for: skin grafting to fix the grafted tissue, preparing a site for a future skin graft, tendon and / or ligament healing and / or enhancement and / or reinforcement and / or stabilization, repair of partial or full thickness rotator cuff tears, soft tissue healing in shoulder, knee and hip replacements, cruciate ligament and / or other ligament / tendon tears.

39. Use of the tissue scaffold according to any one of claims 20 to 29 in tissue repair and / or regeneration.

40. A method for: tissue repair and / or regeneration; supporting skin grafts to fix the grafted tissue; preparing a site for a future skin graft; tendon and / or ligament healing and / or enhancement and / or reinforcement and / or stabilization; repair of partial or full thickness rotator cuff tears; soft tissue healing in shoulder, knee and hip replacements, cruciate ligament and / or other ligament / tendon tears, the method comprising administering to a mammal a tissue scaffold as defined in any one of claims 20-29.

41. The method of claim 40, wherein the administration is by injection or spraying.

42. The method of claim 40 or 41, wherein the stent is administered by arthroscopic or open surgical intervention.

43. The method of any one of claims 40-42, wherein the scaffold is hydrated after intraoperative administration to form a malleable structure.

44. A tissue scaffold according to any one of claims 20-29, used for: tissue repair and / or regeneration; supporting skin grafts to fix grafted tissue; preparing sites for future skin grafts; tendon and / or ligament healing and / or enhancement and / or reinforcement and / or stabilization; repair of partial or full-thickness rotator cuff tears; soft tissue healing in shoulder, knee and hip replacements, cruciate ligaments and / or other ligament / tendon tears.

45. The tissue scaffold of claim 44, wherein the scaffold is administered by injection or spraying.

46. ​​The tissue scaffold of claim 44 or 45, wherein the scaffold is administered by arthroscopic or open surgical intervention.

47. The tissue scaffold of any one of claims 44-46, wherein the scaffold hydrates after intraoperative administration to form a malleable structure.

48. A kit for forming a tissue scaffold, the kit comprising a polymer, wherein the polymer comprises: a first monomer for binding water; a second monomer for imparting mechanical properties to the scaffold; an optional third monomer for binding to a natural or synthetic peptide or protein (NSPP); and a fourth monomer for imparting phase change behavior, wherein the scaffold forms a malleable structure upon hydration.

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