Compositions and methods for improved tissue regeneration

Abstract

Methods, compositions, and kits of parts for preventing or reducing wound contraction. Methods that may include reducing or eliminating a contractile force applied at a tissue wound site by a continuous myofibroblast network. Compositions may include a biocompatible liquid, and a myofibroblast receptor binding agent dispersed in the biocompatible liquid. Kits of parts may include a reservoir and an applicator, such as a nozzle, needle, brush, dropper, or roller.

Description

COMPOSITIONS AND METHODS FOR IMPROVED TISSUE REGENERATIONCross-reference to Related Applications

[0001] This application claims priority- to U.S. Provisional Patent Application No. 63 / 587,302, filed October 2, 2023, which is incorporated by reference herein.Background

[0002] Wounds in mammals, including humans, are closed by wound contraction and scar formation. Wound contraction typically reduces the size of an injured area, and scar formation typically seals the area and achieves secure closure.

[0003] The relevant literature discloses various methods by which wound contraction may be blocked in order to achieve tissue / organ regeneration, such as by achieving the synthesis of new, normal functioning tissue. In most, if not all. situations in which wound contraction has been prevented, there is an absence of scar tissue. These results and other studies have led to the conclusion that scar formation is a process secondary to wound contraction. Therefore, the cancellation of wound contraction implies an absence of scar formation {see references I. V. Yannas, et al. Wound Repair Re gen 25, 177-191 (2017); 1. V. Yannas, et al. npj Regen Med 6, 39 (2021); I. V. Yannas, et al. Proc Nat. Acad Sci USA 86, 933-937 (1989); and I. V. Yannas, Tissue and organ regeneration in adults, Extension of the paradigm to several organs (Springer, ed. 2, 2015)).

[0004] Currently, the blocking of skin wound contraction can be achieved in humans by grafting an injured skin area with a highly porous collagen scaffold, such as INTEGRA® matrix wound dressing (Integra LifeSciences, USA). INTEGRA® matrix wound dressing (Integra LifeSciences, USA) is a (if not the) leading treatment for regenerating skin in humans, e.g.. victims of bums, congenital nevi, chronic skin wounds, and other instances of skin loss. The process of manufacturing INTEGRA® matrix wound dressing (Integra LifeSciences, USA), however, is complex and expensive. The use or application of INTEGRA® matrix wound dressing (Integra LifeSciences, USA) also typically requires the sen-ices of a surgeon or other well-trained medical practitioner.

[0005] Wounds of different organs of mammals, such as humans, are believed to close by the action of myofibroblasts (MFB), which are differentiated forms of fibroblasts. Myofibroblasts typically become differentiated soon after an injury, and can be recognized by staining with the widely used antibody to alpha smooth muscle actin, which is a definitivemarker for MFB. Following differentiation from fibroblasts, MFB typically become connected to each other by specific chemical bonds known as adherence junctions (AJs). Although such MFB-MFB binding has been recognized, the consequences of such binding in wound healing processes has not been pursued.

[0006] It has been reported that the blocking of wound contraction can prevent the formation of scar. Scar formation is the final process for closing a w ound and for sealing off the wound edges securely. Normal scar formation can lead to loss of physiological function of the affected tissue at the site of wound closure, and this can cause tissue and organ dysfunction. Excessive scar formation at an anatomical site can lead to a process called “fibrosis”, which is a leading cause of permanent and often terminal organ dysfunction in the heart, kidneys, liver and other organs. Fibrosis has been reported to cause 45% of all deaths (Wynn, Nat. Rev. Immunol. 4, 583-594 (2004)). However, the cause of fibrosis is not w ell understood, and it is not treated medically today. Although INTEGRA® matrix ound dressing (Integra LifeSciences, USA) is known for blocking scar formation in acute trauma w ounds it has not been used for the treatment of fibrosis, a chronic process that is treated by medical specialists, which are usually distant and unrelated to the acute trauma specialists.

[0007] There remains a need for an improved understanding of wound healing processes, including an understanding of how MFB-MFB binding impacts wound healing processes. There also remains a need for improved methods and compositions for controlling, e.g., cancelling or reducing, w ound contraction, including methods and compositions that are easier to use and / or manufacture compared to currently used methods and compositions.Brief Summary

[0008] Provided herein are improved methods and compositions for controlling wound contraction, including methods that facilitate tissue regeneration by eliminating or minimizing scar formation.

[0009] Surprisingly, it has been discovered that MFB-MFB binding transforms the MFB inside a healing wound into a continuous MFB netw ork, which transfers mechanical forces rapidly and efficiently across the space of the entire wound. This finding contradicts prevailing views of wound contraction, which assume that MFB apply contractile forces independently of each other, thereby requiring separate, extensive cell-by-cell blocking in order to achieve an effective treatment. In contrast, it has been surprisingly discovered thatcell contraction activity is a “cooperative” process that likely depends, at least partially, on close synergy among the MFB. This surprising discovery’ of cooperative activity7, which is a rarely noted phenomenon in cell biology, was an impetus for the development of the methods and compositions herein, which are directed to cancelling or reducing a contractile force of a continuous MFB sheet, such as by fracturing at least a part of the continuous MFB sheet at one or more locations. In some embodiments, the methods described herein may cancel or reduce the mechanical force(s) acting across an entire wound space more efficiently and, possibly, more effectively than blocking individual MFB. Due to the fact that embodiments of the methods described herein may block or reduce wound contraction in a faster and / or more complete manner, it is possible that the methods and compositions herein may improve the quality7of regenerated tissues, thereby leading to a superior medical treatment.

[0010] In one aspect, methods of controlling, e.g., preventing or reducing, wound contraction are provided. In some embodiments, the methods include reducing or eliminating a contractile force applied at a tissue wound site by a continuous myofibroblast network.

[0011] In some embodiments, the methods include contacting a tissue wound site and an amount of a myofibroblast receptor binding agent. A continuous myofibroblast network may be present at the tissue wound site, and the amount of the myofibroblast receptor binding agent may be effective to (i) fracture the continuous myofibroblast network at one or more locations of the continuous myofibroblast network, (ii) reduce or eliminate a contractile force applied at the tissue wound site by the continuous myofibroblast network, or (iii) a combination thereof.

[0012] In another aspect, compositions are provided, such as compositions that may be used to block or reduce wound contraction. In some embodiments, the compositions include a biocompatible liquid, and a myofibroblast receptor binding agent dispersed in the biocompatible liquid. The myofibroblast receptor binding agent may be soluble or partially soluble in the biocompatible liquid.

[0013] As used herein, a material, such as a myofibroblast receptor binding agent, is “soluble” in a liquid when the material has a solubility in the liquid of at least 1 g per 100 mL at room temperature and pressure. As used herein, a material is “partially soluble” in a liquid when the material has a solubility in the liquid that is greater than 0. 1 g and less than 1 g per 100 mL at room temperature and pressure.

[0014] In yet another aspect, kits of parts are provided. In some embodiments, the kits of parts include a reservoir and an applicator, such as a nozzle or a needle. Theapplicator, such as the nozzle or the needle, may be attachable to the reservoir. Any composition disclosed herein may be disposed (e.g., contained, stored, etc.) in the reservoir. The composition may be dispensed from the reservoir with the aid of the applicator.

[0015] Additional aspects will be set forth in part in the description which follows, and in part will be obvious from the description, or may be learned by practice of the aspects described herein. The advantages described herein may be realized and attained by means of the elements and combinations particularly pointed out in the appended claims. It is to be understood that both the foregoing general description and the following detailed description are exemplary and explanatory only and are not restrictive.Brief Description of the Drawings

[0016] FIG. 1A depicts an example of myofibroblasts contracting a wound.

[0017] FIG. IB depicts an example of myofibroblasts contracting a wound.

[0018] FIG. 2A depicts an example of myofibroblasts distributed at an embodiment of a wound site.

[0019] FIG. 2B depicts an example of my ofibroblasts distributed at an embodiment of a wound site.

[0020] FIG. 3A depicts an example of myofibroblasts in a wound grafted with an embodiment of a dermis regeneration template.

[0021] FIG. 3B depicts an example of myofibroblasts in a wound grafted with an embodiment of a dermis regeneration template.

[0022] FIG. 4A depicts an embodiment of a collagen scaffold of a first regenerative activity.

[0023] FIG. 4B depicts an embodiment of a collagen scaffold of a second regenerative activity.

[0024] FIG. 5 depicts a possible configuration of integrin binding to an embodiment of a collagen ligand.

[0025] FIG. 6 depicts an embodiment of a porous graft copolymer.Detailed Description

[0026] Provided herein are compositions, methods, and kits of parts that benefit from the surprising discovery that a myofibroblast (MFB), during normal wound closure by contraction, does not apply a contractile force individually (i.e., separately from other cells).Instead, these contractile cells are believed to act cooperatively by forming a continuous network, which can transfer mechanical forces over most, if not all, of a wound space rapidly and effectively, leading to efficient wound closure. Fracturing the spatially continuous MFB network can be a simpler route towards deactivation of contraction and blocking scar formation than previously used processes, which include capturing individual MFB cells and deactivating them separately (which is done routinely with INTEGRA® matrix wound dressing (Integra LifeSciences, USA)). The compositions, kits, and methods described herein can permit a continuous MFB network to be fractured readily by applying an agent, such a collagen hexapeptide, without (i) the need to employ INTEGRA® matrix wound dressing (Integra LifeSciences, USA), and / or (ii) the sendees of a surgeon or other highly-trained medical practitioner.

[0027] Methods

[0028] Provided herein are methods of controlling wound contraction, such as blocking or reducing wound contraction. In some embodiments, the methods include reducing or eliminating a contractile force applied at a tissue wound site by a continuous myofibroblast network. The reducing or eliminating of the contractile force may be achieved by any manner described herein.

[0029] In some embodiments, the methods include contacting a tissue wound site and an amount of a myofibroblast receptor binding agent. A continuous myofibroblast network may be present at the tissue wound site, and the amount of the myofibroblast receptor binding agent may be effective to (i) fracture the continuous myofibroblast network at one or more locations of the continuous myofibroblast network, (ii) reduce or eliminate a contractile force applied at the tissue wound site by the continuous myofibroblast network, or (iii) a combination thereof.

[0030] In some embodiments, the methods provided herein prevent or reduce scar formation at the tissue wound site. The prevention or reduction of scar formation may eliminate or reduce the risk of fibrosis.

[0031] A myofibroblast receptor binding agent may be applied independently of any other material, or the myofibroblast receptor binding agent may be applied in combination with at least one other material. For example, a myofibroblast receptor binding agent may be present in a liquid prior to the contacting of the tissue wound site. Therefore, the contacting of a tissue wound site and a myofibroblast receptor binding agent may include contacting the liquid and a tissue wound site in any manner, such as by injecting the liquid, spraying theliquid, applying the liquid by brush, dropper, or roller, etc. The liquid, for example, may be sprayed with any known apparatus onto a tissue wound site directly or indirectly, such as by spraying a bandage or other material that is applied to the tissue wound site. The liquid, as a further example, may be injected. The injection may occur at any location (e.g., at or near a tissue wound site) that is effective to contact the liquid and the tissue wound site. A myofibroblast receptor binding agent may be soluble or partially soluble in the liquid.

[0032] Compositions

[0033] Also provided herein are compositions. The compositions, in some embodiments, include a biocompatible liquid, and a myofibroblast receptor binding agent dispersed in the biocompatible liquid. The biocompatible liquid may be an aqueous liquid or an organic liquid, and may include any one or more pharmaceutically acceptable additives, such as a preservative, a buffer, a pharmaceutical agent, a propellant, or a combination thereof.

[0034] In some embodiments, a myofibroblast receptor binding agent is soluble or partially soluble in the biocompatible liquid. A myofibroblast receptor binding agent may be present in a liquid at any concentration, such as at least 1 wt%, at least 5 wt%, at least 10 wt%, at least 25 wt%, or at least 50 wt%, based on the total weight of the liquid and the myofibroblast receptor binding agent. A myofibroblast receptor binding agent may be present in the liquid at a concentration of about 1 \\1% to about 60 wt%, about 1 \\1% to about 50 wt%, about 1 wt% to about 40 wt%, about 1 wt% to about 30 wt%, about 1 wt% to about 20 wt%, or about 1 wt% to about 10 wt%, based on the total weight of the liquid and the myofibroblast receptor binding agent.

[0035] Kits of Parts

[0036] Also provided herein are kits of parts. In some embodiments, the kits of parts include a reservoir and an applicator. The applicator may be attachable to the reservoir. The applicator may be configured to dispense a composition from the reservoir. In some embodiments, the applicator includes a nozzle, a needle, a brush, a dropper, a roller, etc.

[0037] Any of the compositions described herein may be disposed (e.g., contained, stored, etc.) in the reservoir. The reservoir may be a reservoir of a spray bottle. The spraybottle may be an aerosol or non-aerosol spray bottle. The reservoir may be a reservoir of a syringe.

[0038] The applicator may include a nozzle. The nozzle may be attachable to the reservoir, and configured for spraying the composition directly or indirectly, as describedherein, on a tissue wound site. The applicator also may include a needle. The needle may be attachable to a reservoir, and configured for injecting the composition.

[0039] Myofibroblast Receptor Binding Agents

[0040] A myofibroblast receptor binding agent used in the methods, compositions, and kits provided herein may include any agent that binds a myofibroblast receptor. A myofibroblast receptor binding agent may bind to integrin a2pi, integrin a2(12, or a combination thereof.

[0041] In some embodiments, the myofibroblast receptor binding agent includes a hexapeptide, such as a collagen hexapeptide. A collagen hexapeptide may include GFOGER (glycine-phenylalanine-hydroxyproline-glycine-glutamic acid-arginine) and / or GLOGER (gly cine-1 eucine-hydroxyproline-glycine-glutamic acid-arginine).

[0042] Tissue Wound Sites

[0043] A tissue wound site may be present in or on a mammal, such as a human or non-human mammal. An organ, such as a human organ, may include a tissue wound site. A tissue wound site may be present in or on a heart, a kidney, or a liver. Other tissues are envisioned, however, such as nerves, skin. etc.

[0044] All referenced publications are incorporated by reference. Furthermore, where a definition or use of a term in a reference, which is incorporated by reference herein, is inconsistent or contrary to the definition of that term provided herein, the definition of that term provided herein applies and the definition of that term in the reference does not apply.

[0045] While certain aspects of conventional technologies have been discussed to facilitate disclosure of various embodiments, applicants in no way disclaim these technical aspects, and it is contemplated that the present disclosure may encompass one or more of the conventional technical aspects discussed herein.

[0046] The present disclosure may address one or more of the problems and deficiencies of known methods and processes. However, it is contemplated that various embodiments may prove useful in addressing other problems and deficiencies in a number of technical areas. Therefore, the present disclosure should not necessarily be construed as limited to addressing any of the particular problems or deficiencies discussed herein.

[0047] In this specification, where a document, act or item of knowledge is referred to or discussed, this reference or discussion is not an admission that the document, act or item of knowledge or any combination thereof was at the priority date, publicly available, known to the public, part of common general knowledge, or otherwise constitutes prior art under theapplicable statutory provisions; or is known to be relevant to an attempt to solve any problem with which this specification is concerned.

[0048] In the descriptions provided herein, the terms “includes,” “is,” “containing,” “having,” and “comprises” are used in an open-ended fashion, and thus should be interpreted to mean “including, but not limited to.” When methods, devices, compositions, materials, etc. are claimed or described in terms of “comprising” various steps or components, the methods, devices, compositions, materials, etc. can also “consist essentially of’ or “consist of the various steps or components, unless stated otherwise.

[0049] The terms “a.” “an.” and “the” are intended to include plural alternatives, e.g., at least one. For instance, the disclosure of “a liquid”, “a peptide”, and the like, is meant to encompass one, or mixtures or combinations of more than one liquid, peptide, and the like, unless otherwise specified.

[0050] Various numerical ranges may be disclosed herein. When Applicant discloses or claims a range of any type, Applicant’s intent is to disclose or claim individually each possible number that such a range could reasonably encompass, including end points of the range as well as any sub-ranges and combinations of sub-ranges encompassed therein, unless otherwise specified. Moreover, all numerical end points of ranges disclosed herein are approximate. For example, the disclosure states that a myofibroblast receptor binding agent may be present in the liquid at a concentration of about 1 wt% to about 10 wt%, based on the total w eight of the liquid and the myofibroblast receptor binding agent. This disclosure should be interpreted as including concentrations of “about 1 wt%” and “about 10 wt%”. as well as “about 2 wt%”, “about 3 wt%”, “about 4 wt%”. “about 5 wt%”. “about 6 wt%”, “about 7 wt%”, “about 8 wt%”, or “about 9 wt%”, including any sub-ranges among these values.

[0051] As used herein, the term “about” means plus or minus 10 % of the numerical value of the number with which it is being used.EMBODIMENTS

[0052] The following is a non-limiting listing of embodiments of the disclosure.

[0053] Embodiment 1. A method of preventing or reducing wound contraction, the method comprising, consisting essentially of, or consisting of -

[0054] contacting a tissue wound site and an amount of a myofibroblast receptor binding agent;

[0055] wherein a continuous myofibroblast network is present at the tissue wound site.

[0056] Embodiment 2. A method of preventing or reducing wound contraction, the method comprising, consisting essentially of, or consisting of -

[0057] reducing or eliminating a contractile force applied at a tissue wound site by a continuous myofibroblast network.

[0058] Embodiment 3. The method of Embodiment 2, wherein the reducing or the eliminating of the contractile force prevents or reduces scar formation at the tissue wound site.

[0059] Embodiment 4. A composition comprising, consisting essentially of, or consisting of -

[0060] a liquid, such as a biocompatible liquid; and

[0061] a myofibroblast receptor binding agent dispersed in the liquid.

[0062] Embodiment 5. A kit of parts comprising, consisting essentially of, or consisting of -

[0063] (i) a reservoir in which the composition of Embodiment 4 is disposed (e.g., contained, stored, etc.); and

[0064] (ii) an applicator configured for dispending the composition from the reservoir;

[0065] wherein, optionally, the applicator includes (a) a nozzle that is attachable to the reserv oir. and configured for spraying the composition, or (b) a needle that is attachable to the reservoir, and configured for injecting the composition.

[0066] Embodiment 6. The method or composition of any of the preceding Embodiments, wherein the myofibroblast receptor binding agent is present in a liquid, such as the biocompatible liquid, at a concentration of at least 1 wt%, at least 5 wt%, at least 10 wt%, at least 25 wt%, or at least 50 wt%, based on the total weight of the liquid and the myofibroblast receptor binding agent.

[0067] Embodiment 7. The method or composition of any of the preceding embodiments, wherein the myofibroblast receptor binding agent is present in a liquid, such as the biocompatible liquid, at a concentration of about 1 wt% to about 60 wt%. about 1 wt% to about 50 wt%, about 1 wt% to about 40 wt%, about 1 wt% to about 30 wt%, about 1 wt% to about 20 wt%, or about 1 wt% to about 10 wt%, based on the total weight of the myofibroblast receptor binding agent and the liquid.

[0068] Embodiment 8. The method or composition of any of the preceding Embodiments, wherein the amount of the myofibroblast receptor binding agent is effective to-

[0069] (i) fracture the continuous myofibroblast network at one or more locations of the continuous myofibroblast network,

[0070] (ii) reduce or eliminate a contractile force applied at the tissue wound site by the continuous myofibroblast network, or

[0071] (iii) a combination thereof.

[0072] Embodiment 9. The method or composition of any of the preceding Embodiments, wherein the contacting of the tissue wound site and the amount of the myofibroblast receptor binding agent prevents or reduces scar formation at the tissue wound site.

[0073] Embodiment 10. The method or composition of any of the preceding Embodiments, wherein the contacting of the tissue wound site and the amount of the myofibroblast receptor binding agent eliminates or reduces fibrosis, such as fibrosis at the tissue wound site.

[0074] Embodiment 11. The method or composition of any of the preceding Embodiments, wherein the myofibroblast receptor binding agent is present in a liquid, such as the biocompatible liquid, prior to the contacting of the tissue wound site.

[0075] Embodiment 12. The method or composition of any of the preceding Embodiments, wherein the contacting of the tissue wound site and the amount of the myofibroblast receptor binding agent comprises, consists essentially of, or consists of-

[0076] (i) spraying the liquid,

[0077] (ii) injecting the liquid, or

[0078] (iii) a combination thereof.

[0079] Embodiment 13. The method or composition of any of the preceding Embodiments, wherein the myofibroblast receptor binding agent is soluble in the liquid or partially soluble in the liquid.

[0080] Myofibroblast Receptor Binding Agent

[0081] Embodiment 14. The method or composition of any of the preceding Embodiments, wherein the myofibroblast receptor binding agent comprises, consists essentially of, or consists of a collagen hexapeptide.

[0082] Embodiment 15. The method or composition of any of the preceding Embodiments, wherein the collagen hexapeptide comprises, consists essentially of, or consists of-

[0083] (i) GFOGER (glycine-phenylalanine-hydroxyproline-glycine-glutamic acid- arginine),

[0084] (ii) GLOGER (glycine-leucine-hydroxyproline-glycine-glutamic acid- arginine), or

[0085] (iii) a combination thereof.

[0086] Embodiment 16. The method or composition of any of the preceding Embodiments, wherein the myofibroblast receptor binding agent binds to integrin a2pi, integrin a2p2, or a combination thereof.

[0087] Tissue Wound Site

[0088] Embodiment 17. The method or composition of any of the preceding Embodiments, wherein the tissue wound site is present in or on a mammal.

[0089] Embodiment 18. The method or composition of any of the preceding Embodiments, wherein the mammal is a human.

[0090] Embodiment 19. The method or composition of any of the preceding Embodiments, wherein the tissue wound site is in or on an organ.

[0091] Embodiment 20. The method or composition of any of the preceding Embodiments, wherein the organ is a heart, a kidney, or a liver.EXAMPLES

[0092] The present disclosure is further illustrated by the following examples, which are not to be constmed in any way as imposing limitations upon the scope thereof. On the contrary, it is to be clearly understood that resort may be had to various other aspects, embodiments, modifications, and equivalents thereof which, after reading the description herein, may suggest themselves to one of ordinary skill in the art without departing from the spirit of the present disclosure or the scope of the appended claims. Thus, other aspects of this disclosure will be apparent to those skilled in the art from consideration of the specification and practice of the disclosure disclosed herein.

[0093] Example 1 - Testing of Cooperative Cell Activity

[0094] The tests of this example demonstrated that the cancellation of cell-cell cooperativity during wound healing can prevent scar and regenerate tissues.

[0095] This example is directed to a study of wound contraction in three model wounds in animals: excised skin (guinea pig), transected peripheral nerve (rat), and the excised conjunctiva (rabbit).

[0096] Wound contraction was driven by myofibroblasts bound together by adherens junctions (AJ) that conferred cooperative activity to myofibroblasts during wound contraction and synthesis of scar. Grafting with the dermis regeneration template (DRT) canceled cell cooperativity by abolishing AJ connections in myofibroblasts, while also cancelling wound contraction, thereby preventing synthesis of scar and inducing regeneration of excised tissues. The observed definitive prevention of scar synthesis resulted in an exploration of DRT scaffolds to regenerate tissues in several other organs and to prevent fibrosis in humans.

[0097] An event that induced tissue and organ regeneration in a severely injured site of several adult mammals appeared to be blockage of wound contraction (I. V. Yannas, et al. Wound Repair Regen 25, 177-191 (2017); I. V. Yannas, et al. npj Regen Med N 39 (2021)). Inflicting a tissue injury followed by blocking of wound contraction was required in successful attempts at induced regeneration of skin, peripheral nerves, and the eye conjunctiva in animal models, and skin, in humans (I. V. Yannas, et al. npj Regen Med 6, 39 (2021)).

[0098] Regeneration was induced by grafting the wound site with a contractionblocking scaffold based on a highly porous scaffold based on Type I collagen copolymerized with a glycosaminoglygan (GAG), named the dermis regeneration template (DRT) (I. V. Yannas, et al. Proc Nat. Acad Sci USA 86. 933-937 (1989); and I. V. Yannas, Tissue and organ regeneration in adults, Extension of the paradigm to several organs (Springer, ed. 2, 2015)).

[0099] The procedure for DRT synthesis has been disclosed in detail (I. V. Yannas, Tissue and organ regeneration in adults, Extension of the paradigm to several organs (Springer, ed. 2, 2015)). Although organ regeneration has often been considered to be limited to amphibians, recent evidence has shown that the process of regeneration lies dormant in mammals until appropriately activated by injury (I. V. Yannas, et al. npj Re gen Med 6, 39 (2021)). These findings increased the importance of finding out whether induced regeneration of several other organs, including the recently investigated spinal cord (A. Kourgiantaki. et al. npj Regen Med 5, 1-14 (2020)), can become clinical treatments for several terminally diseased organs, as well as contribute to resolution of fibrosis.

[0100] The physiological wound contraction process requires the presence of differentiated fibroblasts, known as myofibroblasts (MFB) (G. Gabbiani, et al. Experientia 27, 549-550 (1971); O Skalli, et al. J Cell Biol 103. 2787-2796 (1986); 1. Darby, et al. Lab Invest 63, 21-29. (1990), B. Hinz, et al. Mol Biol Cell 12. 2730-2741 (2001 )). Although wound contraction and its blocking have been disclosed in detail (I. V. Yannas, Tissue and organ regeneration in adults, Extension of the paradigm to several organs (Springer, ed. 2, 2015)), a remarkable characteristic of MFB during wound contraction has not received attention: the cooperative activity exhibited by MFB during wound contraction and its role in scar formation.

[0101] In this example, evidence is provided, which demonstrates that MFB closes wounds cooperatively, rather than acting as individual cells. Also discussed in this example are the consequences of this finding in tissue regeneration, blocking of scar synthesis, and the prevention of fibrosis.

[0102] Evidence of cell-cell cooperativity during wound contraction

[0103] A well-known characteristic feature of MFB during wound contraction is the presence of stress fibers, which are muscle-like filaments containing alpha-smooth muscle actin (alpha-SMA), which is a widely recognized MFB marker (O. Skalli, et al. J Cell Biol 103, 2787-2796 (1986), I. Darby, et al. Lab Invest 63, 21-29. (1990); B. Hinz, et al. Mol Biol Cell 12, 2730-2741 (2001 )).

[0104] As shown in the literature, stress fibers join at sites of cadherin-type intercellular connections, named adherens junctions (AJs) (B. Hinz, et al. Mol Biol Cell 15, 4310-4320 (2004)), which are known as multiprotein complexes that mediate adhesion of cells of like type in various tissues. Researchers have identified a specific biochemical switch that indicates the differentiation of fibroblasts to MFB: AJs change their cadherin expression from N-cadherin in early, non-contracting wounds to OB-cadherin in mature, strongly contracting wounds. An increase in intercellular transmission of MFB-mediated contractile force, which requires mechanically resistant contacts, w as attributed to the AJs. Following wound closure, MFB undergo spontaneous cell death (apoptosis) (A. Desmouliere, et al. Am J Pathol 146, 56-66 (1995)).

[0105] Clusters of MFB appeared prominently during w ound contraction. Examples are illustrated in skin wounds (FIG. 1A) and peripheral nerve wounds (FIG. IB), which included MFB gathered closely together in fibrous-looking assemblies of a few to several cells. There w as evidence that local contractile events w ere mechanically coordinated by AJs,via synchronization of periodic intracellular Ca+2oscillations between MFBs that were in physical contact (L. Follonier, et al. J Cell Sci 121, 3305-3316 (2008)).

[0106] MFB clusters extended across large swaths of the healing wound in skin (FIG. 2A) and nerve (FIG. 2B), an observation that led to the hypothesis that DRT disrupted a “mechanically coherent” organization of MFB (I. V. Yannas, Wound repair regen 6, 518- 523 (1998)). A related hypothesis of intercellular transmission of MFB -mediated contractile force across a distance was independently supported experimentally. (B. Hinz, et al. Mol Biol Cell 15, 4310-4320 (2004)). Indeed, wound closure by cell-cell transfer of mechanical contraction forces across the entire span of the wound perimeter was most simply explained by cell-cell cooperativity. It appeared futile to contemplate wound closure by invoking individual cell activity7.

[0107] FIG. 1A and FIG. IB depict myofibroblasts contracting examples of wounds physiologically in two animal wound models. In physiologically healing skin wounds and peripheral nen e wounds myofibroblasts close wounds by contraction. FIG. 1A depicts a fullthickness excised dorsal skin wound (guinea pig) 10 days post injury. FIG. IB depicts a stump of transected sciatic nerve (rat) 7 days post injury. MFB, myofibroblasts stained with antibody to alpha-SMA. Scale bars: 100 micron. (I. V. Yannas, Tissue and organ regeneration in adults, Extension of the paradigm to several organs (Springer, ed. 2, 2015)).

[0108] FIG. 2A and FIG. 2B depict myofibroblasts distributed around wounds physiologically in skin and peripheral nen e, respectively; specifically, the figures depict longitudinal section views of physiologically healing skin wounds and peripheral nerve wounds. Myofibroblasts were abundantly present while closing wounds by contraction. FIG. 2A depicts a longitudinal section of an excised full-thickness skin wound (guinea pig, 10 days post injury). Skin wounds contracted in a plane stress field (Vertical arrows). Skin wound edges prior to full-thickness excision were indicated. FIG. 2B depicts longitudinal sections of a transected peripheral nerve (rat) inserted in a silicone tube, relatively inactive regeneratively and used as control (initially 10-mm gap between stumps, specimen was harvested midway between stumps 11 days post injury). Peripheral nerve wounds contracted radially in a circumferential stress field; immunohistochemical localization of myofibroblasts with alpha-SMA antibody. Scale bars: 250 micron. (I. V. Yannas, Tissue and organ regeneration in adults. Extension of the paradigm to several organs (Springer, ed. 2. 2015)).

[0109] At least three characteristics of the MFB population were established during wound contraction that eventually lead to wound closure (FIG. 1A and FIG. IB): MFBdensity was very high, which facilitated cell-cell contacts; extensive MFB clustering took place, which permitted the formation of a dense AJ-bonded network throughout the wound; and long axes of MFB were oriented along a wound axis, which suggested the presence of a macroscopic force vector that directed wound closure. The presumptive force vector was planar in skin wounds and circumferential in a nerve stump following transection.

[0110] All three characteristics of MFB clusters disappeared when a highly porous collagen-based scaffold, named dermis regeneration template (DRT), was in contact with the healing wound tissue. Contact with DRT occurred either as a planar graft in skin and conjunctiva wounds, or as a tube connecting the two stumps in transected nerves.Immunohistochemical staining data, are shown in FIG. 3A for a skin wound and in FIG. 3B for a peripheral nerve wound, and additionally in the excised rabbit eye conjunctiva (I. V. Yannas, et al, npj Regen Med 6, 39 (2021)).

[0111] FIG. 3A and FIG. 3B depict myofibroblasts in wounds grafted with an embodiment of a dermis regeneration template (DRT). FIG. 3A depicts a full-thickness excised dorsal skin wound grafted with DRT (guinea pig, 10 days post injury). FIG. 3B depicts a stump of a transected sciatic nen e tubulated with DRT (rat, 7 days post injury ). Myofibroblasts were stained with antibody to aSMA. F, fibroblasts. Arrows, DRT scaffold struts. Scale bars: 100 pm. (I. V. Yannas, Tissue and organ regeneration in adults, Extension of the paradigm to several organs (Springer, ed. 2, 2015)).

[0112] Reduction of MFB density7in the presence of DRT could be most simply explained in terms of downregulation of MFB differentiation, occasioned (A. Desmouliere, et al. Wound Repair Regen, 13. 7-12 (2005)) by the observed decrease in TGFpl concentration in the presence of DRT, down to 72% of control (E. C. Seller, et al. Biomaterials 33, 4783- 4791 (2012)).

[0113] Extensive binding of contractile cells on the DRT surface was the simplest explanation for dispersion of cell assemblies and disorientation of long axes of cells in the presence of DRT (FIG. 4A, FIG. 4B). Finally, the observed randomization of MFB in DRT- grafted wounds (FIG. 3A, skin; FIG. 3B, nerve) was expected to lead to extensive mutual cancellation of vectors, leading to reduction or even cancellation of the resultant macroscopic contraction force.

[0114] In summary, it was concluded that the decrease in MFB density and clustering, as well as randomization of MFB axes, accounted for the observed cancellation ofmacroscopic wound contraction in skin, nerve and conjunctiva wounds, observed in the presence of DRT.

[0115] Molecular biology of cancellation of cell-cell cooperativity by DRT

[0116] Although the combined microscopic evidence presented above indicates how the macroscopic wound contraction force was cancelled in the presence of DRT, the molecular evidence of MFB-DRT binding required a separate mechanistic interpretation.

[0117] Three-dimensional two-photon microscopy has been extensively used to study the molecular mechanism of DRT-induced cancellation of wound contraction in peripheral nerve wounds. A quantitative in situ study of the density of ligands for adhesion receptors (collagen-binding integrins) on the surface of the porous, three-dimensional collagen matrix was conducted (D. S. Tzeranis, et al. J Roy Soc Interface, 7, 649-661 (2010); D. S. Tzeranis, et al. Ann Biomed Eng 44, 803-815 (2016)).

[0118] It was shown that integrins al 01 and cx2 [31 recognize GFOGER and GLOGER motifs in the collagen molecule (FIG. 5). A quantitative comparison was made between the cell-matrix binding features of two analogs of DRT scaffolds that differed sharply in regenerative activity in a peripheral nerve regeneration study, but differed structurally only in crosslink density (which affected the scaffold degradation half-life) (I. V. Yannas et al. Wound Repair Regen 25, 177-191 (2017); D. S. Tzeranis, et al. J Roy Soc Interface, 7, 649-661 (2010); D. S. Tzeranis, et al. Ann Biomed Eng 44, 803-815 (2016)). Use of the regeneratively active DRT scaffold in the transected nerve resulted in peripheral nerve regeneration of significantly higher quality than was observed with a relatively inactive DRT analog. The collagen-binding ligand concentration on the surface of the active scaffold was observed to be much higher than in the inactive scaffold.

[0119] FIG. 5 depicts integrin binding to a collagen ligand on a DRT surface. The image is based on a cry stallographic study of a complex between the I domain of integrin a2[31 (510) and a critical GFOGER motif (530) located in the triple helical collagen molecule (520) (J. Emsleyet al. Cell 101, 47-56). GFOGER is abbreviation for the collagen hexapeptide glycine-phenylalanine-hydroxyproline-glycine-glutamic acid-arginine (I. V. Yannas, Tissue and organ regeneration in adults, Extension of the paradigm to several organs (Springer, ed. 2, 2015)).

[0120] FIG. 6 depicts an embodiment of a DRT scaffold. Scanning electron microscopic view of DRT, synthesized as a highly porous graft copolymer of type I collagen and chondroitin 6-sulfate. Regenerative activity7requires optimization of average porediameter (range 20-125 gm); half-life for degradation (14 ± 7 days); and presence of an estimated minimal ligand density' (200 pM) for integrins aipi or a2(31 on the collagen surface of DRT. Scale bar 100 pm. (A Kourgiantaki, et al. npj Regen Med 5. 1 -14 (2020)).

[0121] The available data supported the hypothesis that a sufficiently high density of adhesive MFB-DRT binding events competitively inhibited MFB-MFB binding (i.e.. AJ binding) thereby releasing MFB from their cooperative activities. This hypothesis explained the observed rupture of MFB-MFB binding that lead to dismantling of MFB clusters (AJs) in the presence of DRT.

[0122] In the context of the three animal wound models of this example, and based on independent diverse lines of evidence with other species, it was concluded that DRT was a powerful antagonist of cooperative cell activity during wound healing.

[0123] Synthesis of scar tissue by cooperative cells

[0124] During the process of wound contraction MFB becomes involved in a dynamic exchange of phenotypes, primarily including contractile and synthetic phenotypes. The synthetic phenoty pes (mostly synthesis of collagen fibers) appears to dominate as the mechanical stiffness of the substrate increases (I. V. Yannas, et al. Wound Repair Regen 25, 177-191 (2017)).

[0125] In studies of collagen synthesis, it has been independently concluded that the axial orientation of newly synthesized collagen fibers is aligned with the axial orientation of the parent fibroblast (D. E. Birk, et al. “Fibroblasts compartmentalize the extracellular space to regulate and facilitate collagen fibril, bundle, and macro-aggregate formation”. In A. H. Reddi, Ed., Extracellular matrix: Structure and function. (Alan R. Liss. 1985)).

[0126] Since the myofibroblasts in the contractile mechanical field of the healing wound are forced to adopt an oriented configuration along the major deformation axis it is expected that newly synthesized collagen fibers that constitute scar would also possess high orientation along a similar wound axis, as indeed observed by laser light scattering of collagen fibers in dermal scar (A. G. Ferdman, et al. J Invest Dermat of 100, 710-716 (1993)). It was observed that oriented collagen fibers were formed on the plane of the skin wound, while forming circumferentially around the transected nerve. It is concluded from collected data that scar deposition in these models adapted to the physiological anatomic topography of the individual organ.

[0127] It has been previously shown that MFB apoptosis accounts for the observed loss in cellularity during the transition between granulation tissue and scar synthesis (A.Desmouliere, et al. Am J Pathol 146, 56-66 (1995)). It is challenging to attempt an account for the synthesis of scar in the presence of the reported loss in myofibroblast presence due to apoptosis.

[0128] Controlled blocking of scar formation by DRT and its analogs

[0129] A number of detailed morphological and morphometric analyses of wounds produced by excision (animals) or deep bums (humans) were grafted with autologous keratinocyte-seeded DRT (G. F. Murphy, et al. Lab Invest 62, 305-313 (1990); C. C.Compton, et al. J Invest Dermatol 110, 908-916 (1998); C. C. Compton, et al. Lab Invest, 60, 600-612. (1989); and C. E. Butler, et al. Br J Plastic Surg 52, 127-132 (1999)).

[0130] In these studies, emphasis was placed on structural observations both of the regenerated epidermis and dermis in guinea pigs, swine and humans by transmission electron microscopy and other morphological approaches and of the regenerated dermal-epidermal junction (DEJ) in the swine by use of antibodies for structural recognition of specialized structures in the DEJ. Studies with animals showed a regenerated dermis and absence of scar. Studies with humans (21 pediatric bum patients) showed rete ridges (evidence of absence of scar) to have been regenerated from 6 weeks to 1 year after grafting. The subjacent connective tissue “initially healed to form normal scar, but it remodeled dramatically, regenerated elastin, and resembled a true dermis within 4 to 5 years” (C. C. Compton, et al. Lab Invest, 60, 600-612. (1989)).

[0131] The transected sciatic nerve was regenerated in the rat using DRT tubes that connected the two stumps across a 10-mm gap (L. J. Chamberlain et al. Exp Neurology’ 154, 315-329 (1998)). The quality of peripheral nerve regeneration was studied over a period of 60 weeks using histomorphometry and electrophysiological measurements of A-fiber, B- fiber, and C-fiber peaks of the evoked action potentials. The series of studies eventually demonstrated that the regenerated rat sciatic nerve was equivalent in functional quality to that of the nerve autograft. Of specific interest was the finding that the quantitative extent of wound contraction at the nerve stumps (and the corresponding mass of neural scar formed in the case of each DRT analog) was deliberately decreased to various levels by adjusting the degree of contraction-blocking activity' of several DRT analogs that w ere purposely synthesized (E. C. Softer, et al. Biomaterials 33, 4783-4791 (2012)). These findings emphasized the high degree of control over scarring in injured nerves that becomes available by structural modification of DRT and its analogs.

[0132] The excised conjunctival stroma was treated with DRT and the structure of the resulting tissue was viewed by polarized optical microscopy (W. C. Hsu, et al. Invest Ophthalm Visual Sci 41, 2404-2411 (2000)). The regenerated conjunctiva showed a randomly arrayed collection of collagen fibers, which was distinct from the highly oriented array of collagen fibers in the conjunctival scar control.

[0133] To illustrate the phenomenon of cell cooperativity during a contraction process most simply, an in vitro study was conducted where cells contracted individually rather than cooperatively. In this study, researchers (T. M. Freyman, et al. Biomaterials 22, 2883-2892 (2001)) made use of undifferentiated dermal fibroblasts in a standard cell culture medium. Matrix sheets, consisting of dermis regeneration template (DRT) and acting as a planar substrate for cell contraction, were added to the cell culture. Microscopic observation of DRT scaffold struts showed occasional buckling by the cells that were attached to the struts.

[0134] The researchers measured the kinetics of force generated by fibroblasts in vitro seeded at different levels of cell density over a period of 22 hours. Two observations defined the absence of cooperativity in this model. First, the asymptotic force developed by the cells was observed to be linearly proportional to the density of fibroblasts. The data indicated that the contractile force per cell, observed to be of order 1 nN / fibroblast, was independent of cell density. In addition, the value of the time constant observed at different fibroblast density' levels remained approximately constant, indicating that the kinetics of force generation were also independent of fibroblast density'. These results suggested that the fibroblasts in this study acted independently of each other, thereby denying a hypothesis of cooperativity for this in vitro experimental system.

[0135] A brief survey of the literature on the general topic of cooperative cell biology, also referred to as collective cell behavior) (e.g., B. Ladoux, et al. Nat Rev Mol Cell Biol 18, 743-757 (2017)), revealed a relatively small number of studies: these studies were directed primarily towards problems of biological development. Little attention was paid to studies of cell contraction processes. Exceptionally, an in vitro study of a one-dimensional chain model of cell contraction should be noted, which was constructed to provide quantitative information involving physical parameters, such as matrix stiffness, intercellular adhesion and cell chain length involved in the contraction process (S. Li, et al. Biophys J 115:554-564 (2018). This study provided the desired information on relations among the physical variables that were studied but made little reference to physiological contraction processes.

[0136] The approach in this example to study cell-cell cooperativity was focused on the physiological process of contraction during wound healing in three mammalian organs. Described are a collection of cells of same ty pe (myofibroblasts), sharing a common anatomic location (the site of mammalian injury) and functional destination (wound closure), and identified are well-connected myofibroblasts that communicate extensively with each other. These cooperative cell-cell interactions turned out to be of critical importance to the entire wound healing process. Cooperative interactions appeared to control not only wound contraction (and eventual wound closure) but also synthesis of scar, the endpoint of wound healing.

[0137] Excessive scar formation was referred to as fibrosis, a condition which can lead to disruption of organ architecture and loss of tissue function (J-I Jun, et al. J Clin Invest 128, 97-107 (2018)). Organ failure due to fibrosis has been confirmed in the liver (Y.Koyama. et al. J Clin Invest 127, 55-64 (2017)). heart (J. G. Travers et al. Circ Res 118, 1021-1040 (2016)), kidney (B. Kaissling, et al. Biochim Biophys Acta 1832, 931-939 (2013)), and in several other organs. Although the major contributor to fibrosis was known to be the myofibroblast, the process which leads to excessive scar formation is not currently understood (S. Li, et al. Biophys J 115:554-564 (2018), T. A. Wynn, et al. Immunity 44, 450- 462 (2016)).

[0138] Severe fibrosis has been estimated to account for up to 45% of all deaths in the developed world. Several empirical treatments have been proposed for its resolution, including degradation of fibrotic extracellular matrix and elimination of fibrogenic myofibroblasts through their adaptation of various phenotypes, including apoptosis, senescence, dedifferentiation and reprogramming (S. Li, et al. Biophys J 115:554-564 (2018)). Based on the evidence presented above, of persistent scar prevention in different injured organs by DRT, it was postulated that DRT scaffolds could play a definitive role in clinical prevention of fibrosis.

[0139] It was concluded that grafting of wounds with DRT breaks off cell-cell AJ connections thereby blocking the entire wound contraction process. Suppression of cooperativity has led to cancellation of scar synthesis and to introduction of regeneration, a strikingly desirable end point for wound healing. A recent study now shows that the liferestoring process of regeneration is widely embedded in animals, either emerging spontaneously in amphibians or else hidden in mammals, requiring intervention to come into sight.

Claims

Claims1. A method of preventing or reducing wound contraction, the method comprising: contacting a tissue wound site and an amount of a myofibroblast receptor binding agent; wherein a continuous myofibroblast network is present at the tissue wound site; and wherein the amount of the myofibroblast receptor binding agent is effective to (i) fracture the continuous myofibroblast network at one or more locations of the continuous myofibroblast network, (ii) reduce or eliminate a contractile force applied at the tissue wound site by the continuous myofibroblast network, or (iii) a combination thereof.

2. The method of claim 1, wherein the myofibroblast receptor binding agent is present in a liquid prior to the contacting of the tissue wound site.

3. The method of claim 2, wherein the contacting of the tissue wound site comprises spraying the liquid.

4. The method of claim 2, wherein the contacting of the tissue wound site comprises injecting the liquid.

5. The method of claim 2, wherein the myofibroblast receptor binding agent is soluble in the liquid.

6. The method of claim 1, wherein the tissue wound site is present in or on a mammal.

7. The method of claim 6, wherein the mammal is a human.

8. The method of claim 1, wherein the tissue wound site is in or on an organ.

9. The method of claim 8, wherein the organ is a heart, a kidney, or a liver.

10. The method of claim 1, wherein the amount of the myofibroblast receptor binding agent is effective to fracture the continuous myofibroblast network at one or more locations of the continuous myofibroblast network.

11. The method of claim 1 , wherein the amount of the myofibroblast receptor binding agent is effective to reduce or eliminate the contractile force applied at the tissue wound site by the continuous myofibroblast network.

12. The method of claim 1, wherein the contacting of the tissue wound site and the myofibroblast receptor binding agent prevents or reduces scar formation at the tissue wound site.

13. The method of any one of claims 1 to 12, wherein the myofibroblast receptor binding agent comprises a collagen hexapeptide.

14. The method of claim 13, wherein the collagen hexapeptide is selected from the group consisting of GFOGER (glycine-phenylalanine-hydroxyproline-glycine-glutamic acid-arginine) and GLOGER (glycine-leucine-hydroxyproline-glycine-glutamic acid- arginine).

15. The method of any one of claims 1 to 12, wherein the myofibroblast receptor binding agent binds to integrin a2pi. integrin 0.2^2. or a combination thereof.

16. A method of preventing or reducing wound contraction, the method comprising: reducing or eliminating a contractile force applied at a tissue wound site by a continuous myofibroblast network.

17. The method of claim 16, wherein the tissue wound site is present in or on a mammal.

18. The method of claim 17, wherein the mammal is a human.

19. The method of claim 16, wherein the tissue wound site is in or on an organ.

20. The method of claim 19, wherein the organ is a heart, a kidney, or a liver.

21. The method of any one of claims 16 to 20, wherein the reducing or the eliminating of the contractile force prevents or reduces scar formation at the tissue wound site.

22. A composition comprising: a biocompatible liquid; and a myofibroblast receptor binding agent dispersed in the biocompatible liquid.

23. The composition of claim 22, wherein the myofibroblast receptor binding agent is soluble in the biocompatible liquid.

24. The composition of claim 22, wherein the myofibroblast receptor binding agent comprises a collagen hexapeptide.

25. The composition of claim 24, wherein the collagen hexapeptide is selected from the group consisting of GFOGER (glycine-phenylalanine-hydroxyproline-glycine- glutamic acid-arginine) and GLOGER (glycine-leucine-hydroxyproline-glycine- glutamic acid-arginine).

26. The composition of claim 22, wherein the myofibroblast receptor binding agent binds to integrin a2pi, integrin a2 2, or a combination thereof.

27. A kit of parts comprising: a reservoir; the composition of any one of claims 22 to 26. wherein the composition is disposed in the reservoir; and an applicator configured for dispensing the composition from the reservoir.

28. The kit of claim 27, wherein the applicator comprises a nozzle that is attachable to the reservoir, and configured for spraying the composition.

29. The kit of claim 27, wherein the applicator comprises a needle that is attachable to the reservoir, and configured for injecting the composition.

30. The kit of claim 27, wherein the applicator comprises a roller or a brush.

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