The relation between wound contraction, scar formation and regeneration

Transcription

1 The relation between wound contraction, scar formation and regeneration Ioannis V. Yannas Massachusetts Institute of Technology, Cambridge, MA, USA Introduction Healing of deep skin wounds in the adult mammal takes place by the processes of wound contraction and scar formation. There is evidence that scar formation is secondary to contraction. Efforts to block scar formation can therefore benefit from strategies for blocking contraction. In this review we provide a semiquantitative description of healing outcomes based on the wound closure rule. Processes of wound contraction, scar formation and induced regeneration are then summarized with emphasis on the description of mechanisms. The discussion below follows reasoning developed in detail in a monograph where a comprehensive list of references prior to 2001 can also be found (Yannas, 2001). Phenomenological description of healing processes We ask the reader to bear with us for a few paragraphs as we focus temporarily on the macroscopic outcome, i.e., the end result of wound healing, rather than on details of the mechanism of healing. Spontaneous vs induced regeneration. Limb regeneration in certain amphibians is a spectacular feat by which an amputated limb spontaneously grows back to its original form and recovers its normal function. It is the prototypical paradigm of regeneration. Very few adult amphibians, and none of the adult mammals, can replicate this feat. Instead, adult mammals, including adult humans, respond to severe injury by a spontaneous repair process. In clear contrast to the adult mammal, however, the mammalian fetus heals its wounds spontaneously by regeneration, provided that the injury has been inflicted at a sufficiently early stage of gestation, typically before the third trimester of gestation (Yannas, 2005c). 1

2 Spontaneous healing is observed, by definition, in the absence of significant experimental manipulation. In contrast, healing by induced regeneration follows from experimental manipulation, such as grafting with scaffolds that have high biological activity and are grafted on severely injured organs either unseeded or seeded with epithelial cells. This process leads to at least partial recovery of physiological structure and function. In clinical terms, it amounts to partial or total replacement of an organ that has stopped functioning due to severe trauma. When an organ can be induced to regenerate, even partially, the patient can be spared most of the problems accompanying alternative procedures that are commonly used. For example, in organ transplantation, the immune response of the host must be suppressed, typically at substantial cost of quality of life or even longevity for the host; in autografting, the donor site contributes significant morbidity, including severe scarring. These problems are obviated by induced regeneration. The tissue triad. A useful classification of tissue types in an organ focuses on three types: epithelia, basement membrane and supporting tissue or stroma. Persistent, independent observations of adult healing following injury have shown that, in a very large number of organs, excised epithelial tissues and its associated basement membrane regenerate spontaneously following excision while the excised stroma does not. Stroma regeneration in an adult occurs, therefore, only if induced by the investigator and, for this reason, it has been defined to be the central problem in studies of induced organ regeneration. The wound closure rule. In this review, only data from full-thickness skin wounds are considered. In order to arrive at a quantitative description of the final outcome of the healing process it is necessary to include data from anatomically well-defined wounds and to use data only from the initial state (injury) and final state (wound closure) of the healing process. Quantitative study of the phenomenology of healing reveals the relative importance of outcomes contributed by three processes: contraction, scar formation and regeneration. We define contraction as the centripetal displacement of perilesional skin both by translation and by mechanical deformation (The term contracture has often been used to describe qualitatively the long-term clinical outcome, such as limb and joint deformation, 2

3 of the wound contraction process.). Following wound closure there is an amount of wound remodeling which must be accounted for by applying a quantitative correction to the numerical data that follow. Errors in description of the final outcome that arise from wound remodeling have been discussed (Yannas, 2001). The relative contribution of each process to wound closure is simply represented by the percentage of initial wound area that has been eventually closed by contraction (C), scar (S) and regeneration (R). Even though these three processes occur simultaneously during healing, their relative contribution to the outcome of healing (wound closure) can be measured separately. Methods of histology and laser light scattering provide means for detecting critical morphological differences among these three tissue types that lead to their assay. This reasoning leads to the wound closure rule, which states that wounds close exclusively by the three processes described above, which means that the fractional contribution of all processes add up to 1 (or the percentage contribution adds up to 100): C + S + R = 100 Here C, S and R represent the percentage of initial wound area closed by each process. To date, only the extent of contraction (C) has been measured directly in several investigations. Such data have been used to derive rough estimates of the other two quantities, S and R in a few cases (Yannas, 2001). Examples of such estimates for a few healing models in animals or humans appear in Table 1 where values of C, S and R for the full-thickness skin wound are compactly presented as [C, S, R]. (See footnote in Table 1 for methodology used.) For example, the result of spontaneous healing of a fullthickness skin wound in the dorsal region of the rabbit can be described in the final state by C = 92 ± 5%, S = 8 ± 5% and R = 0. Omitting the percentage symbol and error limits from these data yields the simple representation of the original data as [92, 8, 0]. 3

4 Table 1. Healing outcomes of full-thickness skin wounds. Data on configuration of the final state. C and R values were directly measured; S values are estimates a (additional data and references in Yannas, 2001). Configuration of final state (C, % contraction; S, % scar; Full-thickness excision of dermis R, % regeneration) General case of organ wound healing [C, S, R] Ideal early fetal healing of dermis-free wound in skin [0, 0, 100] (perfect regeneration model) Spontaneous healing of dermis-free skin wound in [93, 7, 0] Wistar rat dorsum Spontaneous healing of dermis-free skin wound in [72, 28, 0] swine dorsum (Pitman-Moore minipig) Spontaneous healing of dermis-free skin wound in [37, 63, 0] the adult human forearm Spontaneous healing of dermis-free skin wound in [92, 8, 0] Hartley guinea pig dorsum Induced skin regeneration in dermis-free skin wound [28, 0, 72] in Hartley guinea pig dorsum following grafting with DRT b seeded with autologous keratinocytes a Data from studies of spontaneous healing of full-thickness skin wounds (no experimental manipulation). C was determined directly from data on contraction kinetics. A very rough estimate of S was then obtained as S = 100-C. In some cases it was assumed that regeneration of stroma is not observed in spontaneous healing in the adult mammal (R = 0). In studies where regeneration was induced, values of S and R were estimated from histological data. b Dermis regeneration template, a scaffold described in detail (Yannas, 2001). A schematic of a rodent full-thickness skin wound that has closed primarily by contraction (and secondarily by scar formation) (Fig. 1A) as well as one that has closed primarily by induced regeneration (and secondarily by contraction) (Fig. 1B) illustrate the use of the wound closure rule. Fig. 1. Schematic representation of two tissue blocks that have been excised following closure of a full-thickness skin wound in the guinea pig. LEFT, tissue block following wound closure by spontaneous healing. A o, initial wound area. S, fraction of A o which has closed by formation of scar tissue. C, fraction of A o that has closed by contraction. RIGHT, tissue block following closure partly by regeneration. R, fractional coverage of A o by regenerated skin. C, fractional coverage of A o by contraction. 4

5 Representative estimates of C, S and R, tabulated in Table 1, provide insights to the relative importance of these processes in wound closure. In rodents, which have a mobile integument, contraction accounts for almost all of wound closure; in the human, where the skin is tethered to subcutaneous tissues, contraction accounts for little more than a third of the closure process. The last entry in Table 1 illustrates the phenomenon of induced regeneration. For example, spontaneous healing in the guinea pig was described by the outcome [92, 8, 0]; however, following grafting with the keratinocyteseeded DRT scaffold (dermis regeneration template), the outcome was [28, 0, 72], showing a marked reduction in contraction and a large increase in closure by regenerated skin. The reduction in scar to zero is a rough estimate based on available histological data. We stress here that, while the entries for C in Table 1 are based on directly observed data from a number of investigators, the entries for S are rough estimates based on the data. The adult mammal can be induced to heal severe wounds by partial regeneration There is accumulating evidence that the healing process of an injured organ in the adult mammal can be modified to yield a partly or wholly regenerated organ. In almost all such processes the critical reactant supplied by the investigators has been a scaffold synthesized as an analog of the extracellular matrix (ECM), usually seeded with autologous epithelial cells. The most extensive data on induced regeneration are available with skin and peripheral nerves (see review in Yannas, 2001). Data with other organs from the work of several investigators have been presented in a recent volume (Yannas, 2005a). We review the evidence very briefly below. Partial regeneration of skin. A detailed example of induced skin regeneration, originally described as artificial skin, has been described elsewhere (Yannas, 2001). The data describe the structural and functional similarities and differences among normal skin, scar and regenerated skin in the adult guinea pig and the swine following grafting of dermis-free defects with the keratinocyte-seeded dermis regeneration template (DRT), a scaffold characterized by unusual regenerative activity. DRT is a macromolecular network synthesized as a highly porous analog of ECM with highly specific structure that degrades in vivo at a controlled rate. Among other morphological and functional characteristics, partially regenerated skin is mechanically competent, fully vascularized 5

6 and sensitive to touch as well as heat or cold. The regenerated dermal-epidermal junction, with its extensive formations of rete ridges and capillary loops (Fig. 2), leaves no doubt that de novo partially regenerated skin organ is clearly not scar. However, partially regenerated skin differs from physiological skin in the absence of skin appendages (hair follicles, sweat glands, etc.). Capillary loops Rete ridges Capillary loops Fig. 2. Comparison of normal and regenerated skin. LEFT: Schematic of normal skin. RIGHT: Regenerated skin. The dermalepidermal junction of regenerated skin is viewed following immunostaining for Factor VIII in order to visualize the capillary loops inside the epidermal folds (rete ridges). Neither rete ridges nor capillary loops form in scar. Presence of rete ridges is one of two major features that distinguish intact and regenerated skin from scar. Another distinguishing feature is presence of quasirandomly oriented collagen fibers in the intact and regenerated dermis, rather than a highly aligned stroma, as in dermal scar (adapted from Yannas, 2001). Seeding of the template with keratinocytes leads to simultaneous regeneration of a dermis and an epidermis, while omission of seeded cells leads to sequential regeneration of dermis and epidermis. The simultaneous process leads to a clinically desirable result within about 2-3 weeks but is complicated by the need to prepare the seeded template in the clinical setting. The period required for regeneration can be shortened by culture of keratinocytes prior to seeding. The sequential process is obviously simpler to implement clinically. Following grafting, the template induces regeneration of the dermis, and the new dermis is spontaneously epithelialized from the wound margin. Rather than wait for re-epithelialization in the clinical setting, a thin autoepidermal graft is preferably applied on the newly synthesized dermis. Although seeding of DRT with autologous keratinocytes was required to accelerate the kinetics of organ regeneration, seeding was not required to affect the nature of the outcome (i.e., regeneration vs. repair). Neither was seeding with fibroblasts required to affect the outcome. Furthermore, studies of skin wounds under the same experimental conditions as above showed that treatment of the wounds with a large variety of growth 6

7 factors or epidermal cell suspensions or epidermal cell sheets, or with a number of scaffolds based on synthetic polymers, failed to induce dermis regeneration. These and related observations (for review see Yannas, 2001) motivate study of the mechanism by which DRT induces stroma regeneration. In spite of the lack of skin appendages, the cell-free DRT scaffold (clinically referred to as Integra ) that induces regeneration of the dermis has been approved by the US Food and Drug Administration (FDA) for use with massively burned patients and with patients undergoing plastic and reconstructive surgery of the skin. Regeneration of adult organs other than skin. In addition to skin, confirmed observations of partial regeneration using scaffolds with high biological activity (templates) have been also reported with the following adult organs studied in this laboratory: peripheral nerves (Zhang and Yannas, 2005) and conjunctiva (Yannas, 2001). Significant progress in the study of regeneration has been recently reported independently in studies of bone (Mistry and Mikos, 2005), heart valves (Rabkin-Aikawa et al., 2005), articular cartilage (Kinner et al., 2005), urological organs (Atala, 2005), the spinal cord (Verma and Fawcett, 2005). The reader is referred to the relevant publications for further details (see also Yannas, 2005a). Scar formation may strongly depend on the presence of contraction during healing A revealing relationship has been shown to exist between the orientation of axis of a fibroblast (along which mechanical force develops) and the orientation of collagen fibers synthesized by that cell type. During collagen synthesis, fibers are extruded outside the cell with long axes oriented parallel to the long axis of the synthesizing cell. Furthermore, in a wound that was undergoing contraction, the axes of contractile fibroblasts (myofibroblasts) were observed ultrastructurally to be oriented in the plane of the wound surface; out-of-plane orientation of myofibroblasts was negligible (detailed references in Yannas, 2001). It follows from these observations that, in a contracting skin wound, myofibroblasts apparently subject tissues on the wound bed to stresses in the plane of the epidermis (plane tensile stress field). Collagen fibers synthesized by these oriented cells during 7

8 wound contraction should therefore also be oriented in the same plane. Quantitative measurement of the orientation of collagen fibers in dermal scars by laser light scattering showed that the fibers were persistently oriented in the plane of the wound rather than being quasi-randomly oriented, as in physiological dermis (Fig. 3). These considerations suggest the hypothesis that scar formation is the product of collagen fiber synthesis in the presence of the tensile stress field generated by a wound contraction process. Prominent fiber orientation in the plane, one of the hallmarks of scar, should accordingly disappear following cancellation of the tensile mechanical field in the plane of the wound. Blocking of contraction by an appropriate scaffold should cancel such a mechanical field and should block synthesis of oriented fibers (scar). In fact, when scaffolds that block contraction, even to a relatively minor extent, have been used, collagen fibers in the closed wound showed very poor orientation. This discussion leads to the conclusion that scar formation is secondary to wound contraction. Although the orientation of collagen fibers is an important characteristic of stroma, it is not the only one. The structure of the epidermal-dermal junction is an even more striking characteristic: In scar, the rete ridges are absent and the dermal-epidermal interface is flat, totally lacking the specialized vasculature present in normal or partially regenerated skin (see Fig. 2). 8

9 Fig. 3. Quantitative study of orientation of collagen fibers in a healed, full-thickness skin wound in the guinea pig dorsum. TOP: Configuration used to measure collagen fiber orientation in a histological section of scar, harvested from the animal as shown. The laser beam is scattered from the tissue section in a scattering pattern that reveals the orientation of the fibers. Here, the collagen fibers are schematically shown to be oriented along the direction of the major contraction vector. BOTTOM: A composite image which allows comparison of histological (above) and light scattering (below) patterns. Above: morphology of the healed wound by conventional histology, with intact dermis present at extreme left and right, and with scar present in the center of the healed wound. Below: The laser scattering patterns of each of four segments of the same histological tissue section. The two segments of dermis (left and right) show a roughly circular scattering pattern, consistent with high, but not ideal, degree of randomness while the two scar segments in the center show a highly linear pattern, consistent with a highly oriented assembly of collagen fibers. Adapted from Yannas,

10 Antagonistic relation between contraction and regeneration There are several lines of qualitative evidence suggesting that contraction blocks the induction of regeneration. Extensive discussions of the data have appeared elsewhere (Yannas, 2001, 2005b,c). a. The early fetal-to-late fetal transition in mammals: During mammalian development, contraction eventually becomes dominant as a mode of wound closure with eventual loss of regenerative activity. A developmental transition, occurring during late mammalian gestation, leads from healing primarily by regeneration to healing by contraction and scar formation (Soo et al., 2002, 2003; Colwell et al., 2005). b. Amphibian development: Gradual replacement of regeneration by contraction takes place during development of certain amphibian species. During tadpole development (North American bullfrog), contraction gradually becomes dominant at the expense of regeneration. A small component of scar formation is first observed only after metamorphosis of the tadpole to the adult frog; at this adult stage, regeneration has been abolished and contraction accounts for almost all of closure of the defect (Yannas, 2001). c. Blocking of contraction in adult mammals by use of templates: Contraction blockade leads to Induction of regeneration of skin, conjunctiva and peripheral nerves, as discussed immediately above. There is evidence that induced regeneration in adult mammals shares characteristics with early fetal healing (Yannas, 2005c). This finding suggests the hypothesis that the early fetal healing response is dormant (rather than irreversibly lost) in the adult mammal and is therefore subject to reactivation by use of appropriate experimental methodology. However, data from studies of impaired wound healing show a different picture. Although impaired healing in adults is accompanied by loss of contraction, regeneration is not observed. Experimental study of several models of impaired healing of skin wounds has been based on use of pharmacological agents (e.g., steroids), controlled infection, mechanical splinting, or on animal models of genetically impaired healing, such as the diabetic, or obese mouse. In all of these models of impaired wound healing contraction was blocked almost completely; yet, regeneration was not induced (see review in Yannas, 2001). Data from these models clearly show that blocking of contraction is probably required but it certainly does not suffice for regeneration. 10

11 Mechanism of contraction blockade by scaffolds Having highlighted the empirical (and preliminary) evidence for an antagonistic relation between contraction and regeneration in adults, we now seek mechanistic pathways that account for the observations. In the discussion below, it will be hypothesized that myofibroblasts (MFB) are in fact the dominant cell type required for wound contraction in the organism (Desmoulière et al., 2005). The specific feature which provides the most useful operational distinction of MFB differentiation is expression of the α-smooth muscle actin phenotype. There is considerable evidence that myofibroblast differentiation is regulated by at least a cytokine (transforming growth factor-β1), the presence of mechanical tension and an extracellular matrix component (the ED-A splice variant of cellular fibronectin) (Desmoulière et al., 2005). a. Scaffolds appear to block contraction by interfering with the number and organization of myofibroblasts. Scaffolds do not block wound contraction by mechanical splinting action. This has become abundantly clear following observations of a series of scaffolds that differed only in pore size but were otherwise identical in structure and in Young s modulus, i.e., mechanical stiffness (homologous series; pore volume fraction 99.5%). Only scaffolds in the pore size range µm blocked contraction; outside these limiting pore sizes contraction was not blocked (Figs. 4 and 5). If mechanical splinting were to be considered a viable mechanism for for contraction blocking by an active scaffold, scaffolds with identical Young s moduli but differing only in pore size should not show such divergent behavior: all should block contraction. This is not observed (Fig. 5). Another reason for ruling out mechanical splinting is the observation that Young s moduli for these scaffolds is a very low 200 Pa (Harley, 2005), due to a very high pore volume fraction of 99.5% (Fig. 4). A simple calculation shows that such stiffness values are orders of magnitude lower than necessary to provide the scaffold with any significant mechanical splinting capability inside the wound. Two major mechanisms appear to account for reduction of the macroscopic contractile force by scaffolds. The first mechanism depends on reduction of the number of myofibroblasts (MFB) while the second depends on reduction of the effectiveness of forces generated by MFB in the wound. Detailed references are listed in Yannas,

12 Fig. 4. A scaffold with high biological activity: Dermis regeneration template (DRT). Based on a graft copolymer of type I collagen and chondroitin 6-sulfate. Pore volume fraction 99.5%. Average pore diameter, 80 µm. Fig. 5. Contraction blocking activity of a homologous series of scaffolds increases with magnitude of vertical coordinate. Sharp differences in contraction blocking activity due to differences in pore size alone are observed (adapted from Yannas, 2001). By the first mechanism dermis regeneration template (DRT) depletes the wound of MFB. In skin wounds that heal primarily by induced regeneration in the presence of DRT, MFB comprise only about 10% of the total number of fibroblasts; in the absence of DRT, MFB comprise 50% of total number of fibroblasts. Reduction of MFB number probably results either from a built-in feature of DRT structure (absence of collagen fiber banding without loss of triple helical structure) that prevents platelet aggregation on the surface of collagen fibers, probably leading to inhibition of platelet degranulation and concomitant relative depletion of wound form TGF-β1 (a known inductor of MFB differentiation). Another possible mechanism for MFB depletion is based on the observation that TGF-β1 binds avidly, though nonspecifically, on the extensive specific surface of DRT. TGF- β1 binding on the scaffold surface may contribute further to relative unavailability of TGF-β1 in the wound fluid and, accordingly, may deplete cells from the cytokine that is required for MFB differentiation. The second mechanism for contraction blocking by DRT works by reduction of the sum of forces generated by MFB. In the absence of DRT, the wound contracts vigorously; myofibroblasts are densely packed in the plane of the wound with their axes in the plane (Fig. 6 LEFT). In the presence of DRT (Fig. 6 RIGHT) contraction is arrested. Once having migrated inside DRT and become bound on the extensive surface of the highly porous scaffold, the long axes of MFB are oriented out of the plane of the wound, becoming largely ineffective for application of mechanical forces in the plane of the wound (Fig. 6 RIGHT). In such a nearly random assembly of force vectors the sum of forces must be near zero, leading to cancellation of wound contraction, as observed. 12

13 The few cells that remain outside the scaffold are oriented in the plane and are free to generate their full contractile force (Fig. 6 RIGHT). Fig. 6. The DRT scaffold that possesses high regenerative activity reduces the number of myofibroblasts (MFB, brown stain) and has disorganizes the MFBt layer inside a full-thickness skin wound in the guinea pig model. LEFT: Contraction is proceeding vigorously in this untreated skin wound on Day 10 (negative control). A thick, continuous MFB layer is present at the surface of the skin wound. RIGHT: Contraction has been blocked in this skin wound that was treated with the DRT scaffold (Day 10). MFB are dispersed inside the scaffold; cell-cell binding is practically absent and the axes of contractile cells are almost randomly arranged in the space of the wound. Stained with monoclonal antibody against α-smooth muscle actin. Scale bar: ~100 μm. (Courtesy of M.I.T.) b. The structural determinants of scaffold activity have been largely identified (Yannas, 2001). The contraction blocking activity of a scaffold clearly depends on its ability to bind most of the contractile cells in the wound. Accordingly, structural features that control cell-scaffold binding play major roles. For example, fibroblast-drt binding requires participation of specific ligands (short amino acid sequences), in particular those ligands that are specific for the β1 integrins that have been shown to control myofibroblast-matrix binding during contraction. Such ligands are richly present on collagen surfaces but not, for example, on synthetic polymers. Ligand density is another critical feature of scaffold activity; a large concentration of ligands should lead to binding of large numbers of cells on the scaffold, resulting in loss of their ability to scale up contraction forces and leading to blocked contraction. At a very small pore size, cells are prevented from entering inside the scaffold and therefore remain unbound on surface ligands; at very large pore size the specific surface becomes very low (a result simply of sufficient decrease in pore size), corresponding to low levels of the ligand density. Ligand density is accordingly expected to be minimal at large values of pore size. In a homologous series of scaffolds where scaffold members possess increasingly larger pore size, one should therefore expect that contraction blocking activity should go through a maximum, as observed in the range µm (Fig. 5). The observation of an optimal degradation rate is explained as a requirement for presence of an insoluble scaffold over the entire period during which contraction remains active during wound 13

14 healing; loss of solubility too early prevents scaffolds from blocking contraction while persistent insolubility beyond termination of the contraction process interferes sterically with the synthesis of new tissue (Yannas, 2001). Summary Adult mammalian skin wounds close by a combination of wound contraction, scar formation and induced regeneration. Wound contraction establishes a plane tensile stress field in a skin wound which appears to be required for scar formation. Blocking of wound contraction by biologically active scaffolds leads to induced regeneration. It has been hypothesized that the early fetal healing response, which leads to regeneration, is dormant in the adult mammal; if so, it is subject to reactivation by use of appropriate experimental methodology. 14

15 References Atala A. (2005). Regeneration of urologic tissues and organs. Adv. Biochem. Engin./Biotechnol. 94: Colwell AS, Longaker MT, Lorenz HP.(2005). Mammalian fetal organ regeneration. Adv Biochem Eng Biotechnol. 93: Desmoulière A, Chaponnier C, Gabbiani G. (2005). Tissue repair, contraction, and the myofibroblast. Wound Repair Regen. 13(1):7-12. Harley BA (2006). Cell-matrix interactions: Collagen-GAG scaffold fabrication, characterization and measurement of cell migratory and contractile behavior via confocal microscopy. ScD thesis, MIT. Kinner B, RM Capito and M Spector (2005). Regeneration of articular cartilage. Adv. Biochem. Engin./Biotechnol. 94: Mistry, AS and AG MIkos (2005). Tissue engineering for bone regeneration. Adv. Biochem. Engin./Biotechnol. 94:1-22. Rabkin-Aikawa E, JE Mayer Jr and FJ Schoen (2005). Heart valve regeneration. Adv. Biochem. Engin./Biotechnol. 94: Soo, C., Sayah, D. N., Zhang, X., Beanes, S.R., Nishimura, I., Dang, C., Freymiller, E & Ting, K. (2002). The identification of novel wound-healing genes through differential display. Plast Reconstr Surg. 110: ; discussion Soo, C., Beanes, S. R., Hu, F. Y., Zhang, X., Dang, C., Chang, G., Wang, Y., Nishimura, I., Freymiller, E., Longaker, M. T., Lorenz, H. P. & Ting, K. (2003). Ontogenetic transition in fetal wound transforming growth factor-beta regulation correlates with collagen organization. Am J Pathol. 163: Verma P and J Fawcett (2005). Spinal cord regeneration. Adv. Biochem. Engin./Biotechnol. 94: Yannas I.V. (2001). Tissue and Organ Regeneration in Adults. New York: Springer. Yannas I. V. (2005a) editor. Regenerative Medicine. Heidelberg: Springer. Yannas I. V. (2005b) Facts and theories of induced organ regeneration. Adv Biochem Eng Biotechnol. 93, Yannas I.V. (2005c). Similarities and differences between induced organ regeneration in adults and early foetal regeneration. J. Roy. Soc. Interface 2: Zhang M and IV Yannas (2005). Peripheral nerve regeneration. Adv. Biochem. Engin./Biotechnol. 94: