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Introduction
As a response to organ injury, regeneration is distinct from repair. Although both are outcomes of the wound healing process that follows injury, regeneration is the restoration of the original, uninjured tissues while repair leads to closure of the wound without restoration of original tissues. In regeneration the wound closes scarlessly with little or no contraction; in repair the wound closes by contraction and formation of scar, a nonphysiological tissue. The contrast between the two healing outcomes is sharp and has major clinical consequences. The clinical outcomes of failure to regenerate are acute or chronic dysfunction of the afflicted organs that frequently results in death. Regeneration of many adult mammalian tissues does not occur spontaneously (unaided by investigators) and has to be induced instead. A number of methods for inducing regeneration of tissues and organs have been described; these methods have achieved various levels of completion, from prototypes to widely used clinical treatments. The oldest method for treatment of failed organs is transplantation, a procedure that replaces rather than regenerates organs. Examples of methods that have been proposed for inducing regeneration or have actually succeeded in inducing regeneration of organs, are the transplantation of stem cells, 3D printing of organs prior to implantation , implantation of decellularized matrices and direct in vivo synthesis, biologically catalyzed by the dermis regeneration template (DRT).

This article summarizes the basic principles of induced organ regeneration (nonspontaneous, aided by the investigator) in the adult mammal for three organs: skin, peripheral nerves (PN) and the conjunctiva. The choice of the three organs discussed was based on the availability in the literature of data related to the basic science of organ regeneration. Although these principles have been developed following studies with animals, mostly rodents, the application of these principles, realized in use of DRT in regeneration of human skin, is being widely practiced in diverse clinical applications.

Quantitative Aspects of Wound Closure
The basic anatomy of these three organs, skin, PN and the conjunctiva, shows that each organ includes three quite different tissues, typically grouped together, and referred to in regeneration studies as the tissue triad, consisting of epithelial tissue, basement membrane and stroma. This classification differs from others used in medical text books; it is particularly useful in comparing the response of the tissues of the three organs to injury, which determines the outcome of wound healing either as regeneration or repair. Epithelial tissue comprises functionally specialized cells only; there is no extracellular matrix (ECM) and the tissue lacks blood circulation. The basement membrane comprises only ECM and typically also lacks blood circulation. The stroma (occasionally referred to as connective tissue) comprises cells of various types, circulating blood and ECM. In skin, the epithelial tissue is the epidermis while the stroma is the dermis; in a PN, the corresponding to epithelial tissue is the myelin sheath while the stroma is the endoneurium; in the conjunctiva, the epithelial tissue lies over loose connective tissue (conjunctival stroma). Basement membranes in these organs share many similarities. The particular value of the tissue triad classification derives from the observation that, following injury in the adult mammal, epithelial tissue regenerates spontaneously (provided stroma is already present), the basement membrane likewise regenerates spontaneously (it is synthesized by the regenerating epithelial cells), while the stroma heals by repair (contraction and scar formation). Since the stroma is injured irreversibly, ongoing studies of induced organ regeneration have focused on regeneration of the stroma. Quantitative experimental studies of tissue and organ regeneration in adult mammals have been based on the anatomically well-defined wound (AWDW) which results from a standardized injury. The AWDW is a volume of freshly injured tissue, generated by excising all nonregenerative tissues (typically stroma), marked by unambiguous anatomical boundaries, and is physically isolated from the environment to prevent loss of wound fluid (exudate) and entry of unwanted tissues or bacteria. A critical requirement for an AWDW is the initial absence of stroma in order to avoid false positive results. The AWDW provides the environment to conduct quantitative studies of wound healing under standardized conditions, and thus provides an opportunity to test independently the experimental conclusions of an investigator. So far, in skin studies, the AWDW has been the full-thickness skin wound produced by surgical excision in a rodent model (typically in the guinea pig or rat); in the PN it has been the fully transected sciatic nerve (typically in the rat or mouse); and in the conjunctiva it has been the fully excised conjunctival stroma (rabbit model). Studies conducted with AWDW in the three organs discussed in this article have yielded useful quantitative information, including the identification of three processes for wound closure and the discovery of a collagen scaffold (dermis regeneration template, DRT) which induces regeneration of all three organs. Normal (healthy, sterile, nonchronic) wounds in each of the three organs discussed here, and probably in other organs as well, close by no more than three processes: Contraction, scar formation and regeneration. Expressing the fractional extent of wound closure that was contributed by each process as C (contraction), S (scar formation) and R (regeneration) we express the wound closure rule as:


 * $$ C + S + R = 1 $$

Each term can be quantitatively reported by measuring, e.g., the fraction of initial wound area that has closed by each process. For example, skin wounds on the back of rodents that close spontaneously, typically yield values such as C = 0.95, S = 0.05, R = 0, while in the human forearm, skin wounds close by C = 0.37, S = 0.63, R = 0. Rodents have mobile skin and wounds close almost entirely by contraction while the skin in the human forearm is much more tightly held to tissues below it and requires extensive formation of scar, in addition to contraction, in order to close. We observe a basic fact of regeneration phenomena: The term R is zero when skin wounds heal spontaneously in the adult mammal. R is also zero when the stumps of a transected rat sciatic nerve (PN) are kept separated by more than 1-2mm. There is an advantage in studying wound healing in terms of wound closure, rather than by following the myriad biological and biochemical processes that participate in the healing process: Emphasis on wound closure brings to focus the intimate relation between these three processes. This focus helps to identify whether contraction or scar formation needs to be controlled in order to achieve regeneration, the desirable outcome of severe injuries. Studies with animals, summarized below, have shown that scar formation is caused by contraction, while regeneration is blocked by contraction. Furthermore, it has been shown that the primary effect of DRT on the wound healing process in the three organs (skin, PN, conjunctiva) is to block contraction of the wound, thereby blocking scar and inducing regeneration.

Wound Contraction and Scar Formation
Immunohistochemical studies have shown that physiological contraction of healing wounds in skin, PN and the conjunctiva takes place by the action of myofibroblasts (MFB), a type of contractile cell that appears in wounds following fibroblast differentiation. During wound contraction MFB are present in assemblies of high cell density and are organized with their long cell axes oriented along a major deformation axis (the main axis along which the wound edges contract). In a rodent model they have generated macroscopic contraction forces amounting to about 0.1N. These forces pull the wound edges towards the wound center. In rodents, closure of skin wounds takes place almost entirely by contraction mediated by MFB; scar formation contributes the remainder of the closure outcome. In the transected PN, the wounded stumps also close by MFB action; quantitative values of C and P for the PN closure process have not been definitively determined. Likewise, in the excised conjunctival stroma, MFB contribute a contractile function to the injured tissues. However, in the presence of DRT in each of the three organs (with variations from one organ to the next), MFB show greatly decreased density (e.g., down to 20% MFB in the presence of DRT relative to the DRT-free skin wound control), dispersion of cell assemblies and randomization of MFB axes in the space of the wound. Wounds that have been treated with DRT either do not contract or contract very little and with great delay. These observations classify DRT as an efficient contraction blocking agent.

Contraction has been identified, based on the available evidence, as the driving force for scar formation. Contraction forces, tensile in skin wounds and compressive in PN wounds, close most of the wound area generated by a standardized injury (full-thickness excision in skin, complete transection in PN, excision of stroma in the conjunctiva). These forces orient the long axes of MFB along a major direction of deformation. It is known that fibroblasts synthesize collagen fibrils with long axes that lie parallel to those of the parent cell. Consistent with this finding, the orientation of collagen fibers synthesized by the cells in contracting wounds have been shown to coincide with the orientation of MFB axes, the latter having previously being oriented by the contraction forces. According to this model, scar, well-known as a tight bundle of highly oriented collagen fibers in skin and PN   , is the result of the normal contraction process in skin wounds and PN wounds. This model explains why contraction blocking by DRT is followed by blocking of scar formation: In the presence of DRT, contraction forces that drive scar formation are blocked and wounds accordingly heal without scar. In the presence of DRT, wounds in skin, PN and the conjunctiva grafts, have been shown to lead to synthesis of physiological stroma (regeneration) followed by synthesis of epithelial tissue (especially if the wound is small).

Dermis Regeneration Template
Dermis Regeneration Template (DRT) is a highly porous Type I collagen scaffold that has the macromolecular structure of a crosslinked protein network. Its unique structure and method of synthesis have been described in detail. Evidence showing that DRT induces regeneration of skin, peripheral nerves  and the conjunctiva in adult animals has been reported. To prepare DRT, collagen is block copolymerized with chondroitin 6-sulfate, a glycosaminoglycan (GAG), in studies with skin and the conjunctiva (but often used without GAG in studies with PN). Three structural features of the scaffold are required for its regenerative activity: average pore size in the region 20-125 µm; estimated degradation half-life of 14 ± 7 days; and scaffold surface that includes the ligands GFOGER and GLOGER as part of the collagen triple helical configuration (gelatin is inactive). The first structural feature ensures that binding of contractile cells to the scaffold surface will indeed take place; the second feature ensures that there is sufficient specific surface inside the porous scaffold to bind on it almost all contractile cells present in the wound; and the third provides the required time window for the binding event to take place. In the presence of DRT, the immunohistochemical data have shown a dramatic reduction in MFB density, dispersion of MFB assemblies and randomization of MFB axes, coinciding with cancellation of the macroscopic wound contraction force. The evidence of dramatic loss of the contractile MFB phenotype is consistent with data obtained by two-photon microscopy showing direct binding between MFB integrins and ligands on the scaffold surface.

Future Directions
The significance of the finding that all three injured organs, skin, peripheral nerves and the conjunctiva, heal by regeneration in the presence of DRT cannot be underestimated: it is the expectation that, in future studies, other injured organs as well may be found to heal by regeneration under similar conditions. Since DRT induces regeneration by blocking contraction of healing wounds, several other organs that are known to heal by a similar mechanism would be expected to heal by induced regeneration in the presence either of DRT or a scaffold with similar critical features.