Anthony Metcalfe

Tissue engineering of replacement skin: the crossroads of biomaterials, wound healing, embryonic development, stem cells and regeneration

Advanced therapies combating acute and chronic skin wounds are likely to be brought about using our knowledge of regenerative medicine coupled with appropriately tissue-engineered skin substitutes. At the present time, there are no models of an artificial skin that completely replicate normal uninjured skin. Natural biopolymers such as collagen and fibronectin have been investigated as potential sources of biomaterial to which cells can attach. The first generation of degradable polymers used in tissue engineering were adapted from other surgical uses and have drawbacks in terms of mechanical and degradation properties. This has led to the development of synthetic degradable gels primarily as a way to deliver cells and/or molecules in situ, the so-called smart matrix technology. Tissue or organ repair is usually accompanied by fibrotic reactions that result in the production of a scar. Certain mammalian tissues, however, have a capacity for complete regeneration without scarring; good examples include embryonic or foetal skin and the ear of the MRL/MpJ mouse. Investigations of these model systems reveal that in order to achieve such complete regeneration, the inflammatory response is altered such that the extent of fibrosis and scarring is diminished. From studies on the limited examples of mammalian regeneration, it may also be possible to exploit such models to further clarify the regenerative process. The challenge is to identify the factors and cytokines expressed during regeneration and incorporate them to create a smart matrix for use in a skin equivalent. Recent advances in the use of DNA microarray and proteomic technology are likely to aid the identification of such molecules. This, coupled with recent advances in non-viral gene delivery and stem cell technologies, may also contribute to novel approaches that would generate a skin replacement whose materials technology was based not only upon intelligent design, but also upon the molecules involved in the process of regeneration.

Spatial and temporal changes in Bax subcellular localization during anoikis

Bax, a member of the Bcl-2 family, translocates to mitochondria during apoptosis, where it forms oligomers which are thought to release apoptogenic factors such as cytochrome c. Using anoikis as a model system, we have examined spatial and temporal changes in Bax distribution. Bax translocates to mitochondria within 15 min of detaching cells from extracellular matrix, but mitochondrial permeabilization does not occur for a number of hours. The formation of Bax oligomers and perimitochondrial clusters occurs concomitant with caspase activation and loss of mitochondrial membrane potential, before nuclear condensation. Cells can be rescued from apoptosis if they are replated onto extracellular matrix within an hour, whereas cells detached for longer could not. The loss of ability to rescue cells from anoikis occurs after Bax translocation, but before the formation of clusters and cytochrome c release. Our data suggest that Bax regulation occurs at several levels, with formation of clusters a late event, and with critical changes determining cell fate occurring earlier.

Expression of genes involved in early cell fate decisions in human embryos and their regulation by growth factors

Little is understood about the regulation of gene expression in human preimplantation embryos. We set out to examine the expression in human preimplantation embryos of a number of genes known to be critical for early development of the murine embryo. The expression profile of these genes was analysed throughout preimplantation development and in response to growth factor (GF) stimulation. Developmental expression of a number of genes was similar to that seen in murine embryos (OCT3B/4, CDX2, NANOG). However, GATA6 is expressed throughout preimplantation development in the human. Embryos were cultured in IGF-I, leukaemia inhibitory factor (LIF) or heparin-binding EGF-like growth factor (HBEGF), all of which are known to stimulate the development of human embryos. Our data show that culture in HBEGF and LIF appears to facilitate human embryo expression of a number of genes: ERBB4 (LIF) and LIFR and DSC2 (HBEGF) while in the presence of HBEGF no blastocysts expressed EOMES and when cultured with LIF only two out of nine blastocysts expressed TBN. These data improve our knowledge of the similarities between human and murine embryos and the influence of GFs on human embryo gene expression. Results from this study will improve the understanding of cell fate decisions in early human embryos, which has important implications for both IVF treatment and the derivation of human embryonic stem cells.

Regeneration of the ear after wounding in different mouse strains is dependent on the severity of wound trauma.

The replacement and restoration of tissue mass after organ damage or injury in adult higher vertebrates is critical to the architecture and function of the organ. If replacement occurs with scar tissue, this often results in adverse effects on function and growth as well as an undesirable cosmetic appearance. However, certain mammals, such as the MRL/MpJ mouse, have shown a restricted capacity for regeneration, rather than scar tissue formation, after an excisional ear punch wound. To investigate the changes in tissue architecture leading to ear wound closure, initial ear wounding studies with a 2‐mm clinical biopsy punch were performed on MRL/MpJ mice, by using C57BL/6 mice as a nonregenerative control strain. In contrast to previously reported studies on mouse ear regeneration, we observed that C57BL/6 mice in fact showed a limited regenerative capacity. One explanation for this difference could be attributed to the method of wounding used; both previous studies on mouse ear regeneration used a thumb punch, whereas our approach was to use a clinical biopsy punch. This approach led us to further investigate whether the severity of trauma applied influenced the rate of wound healing. We, therefore, compared the effects of the sharp clinical biopsy punch with that of a cruder thumb punch, and introduced a third strain of mouse, Balb/c, known to be a slow‐healing strain. A new method to quantify ear punch hole closure was developed and a histologic investigation conducted up to 4 months after wounding. Image analysis data showed a reduction in original ear wound area of 85% in MRL/MpJ mice at 4 weeks and of 91.7% over 4 months by using a biopsy punch. In contrast, the crude thumb punch methodology resulted in an increase in wound area of up to 58% in Balb/c ears; thought to be due to increased necrosis of the wound site. All biopsy‐punched wound areas plateaued in healing between days 28 and 112. Only 5 of 80 MRL/MpJ mouse ears showed no residual holes macroscopically after 28 days. Histologically, all strains of mice healed their ear wounds in a similar manner involving re‐epithelialization, blastema‐like formation, dermal extension, blood vessel formation, chondrogenesis, folliculogenesis, and skeletal muscle and fat differentiation. However, all regenerative features were more pronounced and accelerated in MRL/MpJ mice when compared with C57BL/6 and Balb/c biopsy‐punched mouse ears. Developmental Dynamics 226:388–397, 2003.

Interleukin-10 reduces scar formation in both animal and human cutaneous wounds: results of two preclinical and phase II randomized control studies.

Cutaneous scarring affects up to 100 million people per annum. There is no effective scar reducing/preventing therapeutic developed to date. Interleukin (IL)‐10 is an anti‐inflammatory and antifibrotic cytokine. In the embryo it is important for scarless wound repair. We investigated the effect on wound healing and scarring of a double deletion of the IL‐10 and IL‐4 genes in a knockout (KO) mouse model, and also the effect of exogenous addition of recombinant human (rh) IL‐10 into rat and human cutaneous incisions. Mouse study: Two incisions were made on the dorsal skin of 20 double IL‐4/IL‐10 KO mice and 20 wild‐type (WT) controls. Rat study: Three concentrations of rhIL‐10 were investigated. Four incisions were made on the dorsal skin of 30 rats. Each rat received two concentrations. Each incision receiving a concentration of rhIL‐10 was matched with a control incision, which received either placebo or standard care. Human study: Eight concentrations of rhIL‐10 were investigated. Four incisions were made on each arm of 175 healthy volunteers. Four incisions received four different concentrations, which were matched with four control incisions that received either standard care or placebo. KO mice healed with poor scar histology and increased inflammation. rhIL‐10–treated rat incisions healed with decreased inflammation, better scar histology, and better macroscopic scar appearance. rhIL‐10–treated human incisions at low concentrations healed with better macroscopic scar appearance and less red scars. IL‐10 is an important cytokine in wound healing and its suppression of inflammation and scarring is demonstrated in mice and rats with a translational effect in humans.

Avotermin for scar improvement following scar revision surgery: a randomized, double-blind, within-patient, placebo-controlled, phase II clinical trial.

Background: Skin scarring is associated with psychosocial distress and has a negative effect on quality of life. The transforming growth factor (TGF)-&bgr; family of cytokines plays a key role in scarring. TGF-&bgr;3 improves scar appearance in a range of mammalian species. This study was performed to assess the efficacy of intradermal avotermin (TGF-&bgr;3) for the improvement of scar appearance following scar revision surgery. Methods: Sixty patients (35 men and 25 women; age, 19 to 78 years; 53 Caucasians; scar length, 5 to 21 cm) received intradermal avotermin (200 ng/100 &mgr;l/linear cm wound margin) and placebo to outer wound segments immediately after, and again 24 hours after, complete (group 1) or staged (group 2) scar revision surgery. A within-patient design was chosen to control for interindividual factors that affect scarring. The primary efficacy variable was a total scar score derived from a visual analogue scale, scored by a lay panel from standardized photographs from months 1 through 7 following treatment. Results: Primary endpoint data from the combined surgical groups showed that avotermin significantly improved scar appearance compared with placebo (total scar score difference, 21.93 mm; p = 0.04). Profilometry showed a greater reduction in scar surface area from baseline with avotermin treatment compared with placebo, significant in group 2 at months 7 and 12 (difference, 41.99 mm and 25.85 mm, respectively; p = 0.03 for both comparisons). Histologic analysis from group 2 showed that, compared with placebo treatment, collagen organization in avotermin-treated scars more closely resembled normal skin in 14 of 19 cases. Avotermin was well tolerated. Conclusion: Avotermin administration following scar revision surgery is well tolerated and significantly improves scar appearance compared with placebo. CLINICAL QUESTION/LEVEL OF EVIDENCE: Therapeutic, I. Figure. No caption available.

Harnessing wound healing and regeneration for tissue engineering

Biomedical science has made major advances in understanding how cells grow into functioning tissue and the signalling mechanisms used to achieve this are slowly being dissected. Tissue engineering is the application of that knowledge to the building or repairing of organs, including skin, the largest organ in the body. Generally, engineered tissue is a combination of living cells and a supporting matrix. Besides serving as burn coverings, engineered skin substitutes can help patients with diabetic foot ulcers. Today, most of these ulcers are treated with an approach that includes antibiotics, glucose control, special shoes and frequent cleaning and bandaging. The results of such treatments are often disappointing and ineffectual, and scarring remains a major problem, mechanically, cosmetically and psychologically. Within our group we are attempting to address this by investigating novel approaches to skin tissue engineering. We are identifying novel therapeutic manipulations to improve the degree of integration between a tissue engineered dermal construct and the host by both molecular manipulation of growth factors but also by understanding and harnessing mechanisms of regenerative biology. For the purpose of this summary, we will concentrate primarily on the latter of these two approaches in that we have identified a novel mouse mutant that completely and perfectly regenerates skin and cartilaginous components following ear injury. This experimental animal will allow us to characterize not only novel genes involved in the regeneration process but also to utilize cells from such animals in artificial skin equivalents to assess their behaviour compared with normal cells. This approach should allow us to create a tissue-engineered substitute, which more closely resembles the normal regional microanatomy and physiology of the skin, allowing better integration to the host with minimal or no scarring.

Characterizing regeneration in the vertebrate ear

We have previously shown that MRL/MpJ mice have a capacity for regeneration instead of scar formation following an ear punch wound. Understanding the differences that occur between scar‐free regeneration or repair with scarring will have great impact upon advances in skin tissue engineering. A key question that remains unanswered in the MRL/MpJ mouse model is whether regeneration was restricted to the ear or whether it extended to the skin. A histological analysis was conducted up to 4 months post‐wounding, not only with 2‐mm punch wounds to the ear but also to the skin on the backs of the same animals. MRL/MpJ mouse ear wounds regenerate faster than control strains, with enhanced blastema formation, a markedly thickened tip epithelium and reduced scarring. Interestingly, in the excisional back wounds, none of these regenerative features was observed and both the C57BL/6 control and MRL/MpJ mice healed with scarring. This review gives an insight into how this regenerative capacity may be due to evolutionary processes as well as ear anatomy. The ear is thin and surrounded on both sides by epithelia, and the dorsal skin is devoid of cartilage and under greater tensile strain. Analysis of apoptosis during ear regeneration is also discussed, assessing the role and expression of various members of the Bcl‐2 family of proteins. Ongoing studies are focusing on de novo cartilage development in the regenerating ear, as well as understanding the role of downstream signalling cascades in the process. Identification of such signals could lead to their manipulation and use in a novel tissue‐engineered skin substitute with scar‐free integration.

Location of injury influences the mechanisms of both regeneration and repair within the MRL/MpJ mouse

The adult MRL/MpJ mouse regenerates all differentiated structures after through‐and‐through ear punch wounding in a scar‐free process. We investigated whether this regenerative capacity was also shown by skin wounds. Dorsal skin wounds were created, harvested and archived from the same animals (MRL/MpJ and C57BL/6 mice) that received through‐and‐through ear punch wounds. Re‐epithelialization was complete in dorsal wounds in both strains by day 5 and extensive granulation tissue was present by day 14 post‐wounding. By day 21, wounds from both strains contained dense amounts of collagen that healed with a scar. The average wound area, as well as α‐smooth muscle actin expression and macrophage influx were investigated during dorsal skin wound healing and did not significantly differ between strains. Thus, MRL/MpJ mice regenerate ear wounds in a scar‐free manner, but heal dorsal skin wounds by simple repair with scar formation. A significant conclusion can be drawn from these data; mechanisms of regeneration and repair can occur within the same animal, potentially utilizing similar molecules and signalling pathways that subtly diverge dependent upon the microenvironment of the injury.

Peripheral nerve regeneration in the MRL/MpJ ear wound model

The MRL/MpJ mouse displays an accelerated ability to heal ear punch wounds without scar formation (whereas wounds on the dorsal surface of the trunk heal with scar formation), offering a rare opportunity for studying tissue regeneration in adult mammals. A blastema‐like structure develops and subsequently the structure of the wounded ear is restored, including cartilage, skin, hair follicles and adipose tissue. We sought to assess if the MRL/MpJ strain also possessed an enhanced capacity for peripheral nerve regeneration. Female MRL/MpJ and C57BL/6 mice were wounded with a 2‐mm excisional biopsy punch to the centre of each ear and two 4‐mm excisional biopsy punches to the dorsal skin. Immunohistochemical dual staining of pan‐neurofilament and CD31 markers was used to investigate reinnervation and vascularisation of both the dorsal surface of the trunk and ear wounds. The MRL/MpJ mouse ear exhibited a significantly (P > 0.01) higher density of regenerated nerves than C57BL/6 between 10 and 21 days post‐wounding when the blastema‐like structure was forming. Unlike dorsal skin wounds, nerve regeneration in the ear wound preceded vascularisation, recapitulating early mammalian development. Immunohistochemical data suggest that factors within the blastemal mesenchyme, such as aggrecan, may direct nerve regrowth in the regenerating ear tissue.