Tissue and Organ Regeneration in Adults

In the adult mammal, injury to the stroma is typically irreversible and leads to formation of nonphysiological scar tissue (repair). Every organ in the adult can be irreversibly injured, resulting in repair with scar formation. In certain organs injury is irreversible when it leads to damage of specific tissues while in others it becomes irreversible when the injury exceeds a critical size.

The topic of this volume, induced regeneration, is a process in which physiological tissue, rather than scar, is deliberately synthesized at the anatomical site of the adult host that has been irreversibly injured. This approach is embodied in the collagen scaffold regeneration paradigm, based on five empirical rules, which explains at each of the scales of tissue, cell, and molecule, the mechanism of induced regeneration.

Injury to the skin, peripheral nerves, and several other organs in adults is reversible, leading to spontaneous regeneration, whenever it is limited to the epithelial tissues covering or lining the organ and has not damaged the basement membrane. Injury becomes irreversible, leading to repair, whenever it penetrates the basement membrane and enters into the stroma which is closely attached to the basement membrane.

The epidermis in the skin and the myelin sheath in peripheral nerves both spontaneously regenerate. The basement membranes in both organs also regenerate spontaneously. The dermis in the skin and the endoneurial stroma in a peripheral nerve do not regenerate spontaneously (nonregenerative); they both heal by repair.

The experimental space that is most suitable for a study of induced organ regeneration consists of a volume of freshly injured tissue, generated by excising all nonregenerative tissues (typically stroma), which is marked by unambiguous anatomical boundaries, and is physically contained to prevent loss of wound exudate and entry of extraneous tissues or bacteria. These characteristics define the anatomically well-defined wound, referred to also as “defect” in this volume. With skin, the full-thickness excisional skin wound (dermis-free defect) in the rodent or the swine satisfies these criteria. In the peripheral nerve, the fully transected nerve (sciatic, typically) in the rat or mouse, with stumps separated by a critical gap length is a widely used defect that satisfies these criteria. The partial thickness or incisional skin wounds and the hemisectioned nerve trunk do not meet the criteria for an anatomically well-defined defect.

The full-thickness excisional dermis-free defect is suitable for experimental study of induced regeneration (synthesis) of skin and of its tissue components using a variety of reactants that are implanted in the defect.

The synthesis of an epidermis and of a basement membrane (BM) was most simply accomplished by culturing dissociated keratinocytes in a defined media. A dermis that lacked an epidermis was simply synthesized by grafting the defect with dermis regeneration template (DRT). Regenerated skin, complete with a physiological epidermis, BM, rete ridges, and a dermis, was synthesized without adenexa most simply using the keratinocyte-seeded DRT. Adenexa (hair follicles, sebaceous glands) were also regenerated when the keratinocyte seeding protocol included cells from dermal papillae.

Synthesis of a myelin sheath around axons elongating from neurons in culture was observed to occur when Schwann cells were present but not in their absence. Encasement of myelinated axons by basement membrane required the presence of not only the neuron but also laminin, one of the macromolecular components of basement membrane.

Several configurations yielded nerve trunks with useful physiological properties. These experimental configurations yielded regenerated nerve trunks with physiological or nearly physiological properties, some of which have clinical value.

The critical axon elongation, L _c , a measure of the gap length across which nerve fibers can elongate in a tubulated gap, was determined for a large number of configurations. It was used to rank-order a large number of experimental configurations used to induce peripheral nerve regeneration across gaps of variable length. Results obtained using either a rat or mouse were normalized, yielding a large database of literature data based on both animal models.

Minimal requirements for synthesis of tissues have been determined. In studies with skin, no dermal elements were required for the synthesis of the epidermis with its associated basement membrane (BM); nor were any epithelial elements required for synthesis of the dermis. Likewise, in studies with peripheral nerves, synthesis of the myelin sheath with its BM took place in the absence of endoneurial elements.

No interaction between epithelial and mesenchymal tissues appears to be required in order to synthesize the epithelial tissues (epidermis, myelin sheath) by themselves or the stroma (dermis, endoneurium) by itself in skin and peripheral nerves. An interaction between epithelial and mesenchymal tissues appears, however, to be required for the synthesis of “transition tissues” that line the interface between epithelial tissues and stroma.

Scar formation appears to be a derivative to wound contraction, resulting, as it does from synthesis of collagen fibers, in the tensile field generated during wound contraction. It appears, therefore, that spontaneous repair in adults results primarily from the contraction process rather than scar formation.

Several lines of evidence show that wound contraction antagonizes regeneration. The evidence includes studies of healing in the developing frog, rabbit ear, human oral mucosa, and skin wounds in the axolotl, as well as several lines of evidence based on multiyear use of the dermis regeneration template (DRT) in skin, peripheral nerve, and conjunctiva wounds. In a quantitative study of peripheral nerve regeneration, the evidence clearly shows an inverse relation between contraction and regeneration.

Maximum blocking of contraction and highest quality of regeneration coincided with use of a single scaffold, dermis regeneration template (DRT), with narrowly defined structure. Features required for regenerative activity of DRT were the pore size (in the range 20–125 µm), degradation half-life (optimally 14 ± 7 days), and a density of ligands for collagen-binding integrins α1β1 and α2β1 that exceeded approximately 200 µΜ α1β1 or α2β1 ligands. Pore size controls cell entry into the scaffold and provides adequate specific surface for cell adhesion; the half-life provides a time window during which cells and scaffold can make contact; and the ligand density of collagen-binding integrins α1β1 and α2β1 enables the adhesive interaction between integrins and scaffold surface which affects profoundly the assembled morphology and function of contractile cells. The facts confirm the presence of an insoluble surface with high degree of specificity, which modifies the phenotype of contractile cells. These considerations introduce the concept of surface biology, a biology that derives its functions from specific interactions of cells with an insoluble solid surface.

A theory which explains the data on spontaneous and induced regeneration based on use of collagen scaffolds is presented in the form of a regeneration paradigm. The paradigm is grounded in a set of five empirical rules that govern regeneration in animal models and in the clinic. A detailed mechanistic explanation of the regeneration process interprets the data using evidence that extends from the organ scale down to the scale of the molecule. Use of the scaffold regeneration paradigm explains mechanistically the available data on scarless healing in the early mammalian fetus, the successful use by several investigators of decellularized matrices to regenerate several organs, and suggests a proposed solution to the problem of the fibrotic liver. This chapter ends with a general theory of regeneration based on the speculative premise that early fetal healing, which is regenerative, is eventually turned off reversibly, rather than irreversibly, before birth; it lies dormant during the lifetime of the organism until it is turned back on by a suitable stimulus.