The Role of Fibroblasts in Tissue Engineering and Regeneration

T. Wong; J.A. McGrath; H. Navsaria

Disclosures

The British Journal of Dermatology. 2007;156(6):1149-1155. 

In This Article

Clinical Applications

Cultured fibroblasts may be used to augment tissue repair in a variety of conditions ranging from acute and chronic wounds through to their application in aesthetic and reconstructive surgery.[31] Dermal replacements may be acellular or cellular and the incorporation of an epidermal component composed of differentiated keratinocyte layers onto a cellular dermal substrate leads to the formation of a bilayered skin substitute. There are currently a range of dermal and combined replacement matrices licensed for a variety of clinical applications and these are summarized in Table 1 .

In addition, from a commercial aspect, the manufacture of tissue-engineered products is rigorous and complex, requiring adherence to good manufacturing practices and quality assurance. Some important considerations in tissue engineering include ensuring asepsis and prevention of contamination, cryopreservation of the tissue-engineered product to provide adequate shelf-life and also from a business viewpoint, minimizing cost and maximizing the convenience of use by the customer.[32]

Dermal equivalents and bilayered skin substitutes have been used to treat a range of chronic nonhealing wounds, which have been defined as a result of an abnormal physiological environment and the subsequent failure to heal in an orderly and timely manner.[33,34] The release of different growth factors and cytokines by the keratinocytes and fibroblasts in tissue-engineered skin has been evaluated and revealed that living skin substitutes produce the correct concentration and combination of growth factors important for efficient and effective wound repair as well as providing the necessary ECM components.[35,36] In addition, one of the advantages of using skin substitutes is that they can be cryopreserved and that following thawing of a cultured dermal substitute, the fibroblasts retain the ability to proliferate and produce appreciable amounts of VEGF, hepatocyte growth factor, basic FGF, TGF-β1 and IL-8.[36]

Dermagraft-TC (TransCyte®, Advanced BioHealing, La Jolla, CA, U.S.A.) and Dermagraft® (Advanced BioHealing) are successful dermal substitutes both consisting of a dermis produced by human neonatal fibroblasts incorporated into biodegradable polyglactin (VicrylTM). However, in Dermagraft-TC the substitute is frozen and the fibroblasts become nonviable whereas Dermagraft® remains a living substitute. The presence of rapidly proliferating fibroblasts is therefore thought to stimulate healing, as it remains metabolically active. Apligraf® was the first bilayered living skin equivalent produced consisting of cultured neonatal fibroblasts embedded in bovine collagen type I with cultured neonatal keratinocytes seeded on top.[37] Both Apligraf® and Dermagraft® are licensed for use by the US Food and Drug Administration and have been used to treat chronic nonhealing venous ulcers and diabetic foot ulcers.

Fibroblasts in chronic venous ulcers have reduced collagen synthetic capacity, a lack of response to stimulatory TGF-β and an increased proportion of the fibroblasts are senescent.[38,39] Studies on diabetic ulcer fibroblasts have also shown altered morphology and reduced proliferative capacity.[40] Results of a large, controlled study on venous ulcers revealed that treatment with Apligraf® and compression therapy led to complete wound healing threefold faster than using compression therapy alone and that it was particularly effective for the hard-to-heal ulcers.[14] A recent Cochrane review studied nine randomized controlled trials of skin grafts for venous leg ulcers and concluded that there is evidence that bilayer artificial skin, used in conjunction with compression bandaging, increases the chance of healing a venous ulcer compared with compression and a simple dressing.[41] In addition, a randomized, controlled multicentre trial investigated the effect of Dermagraft® on the healing of chronic diabetic foot ulcers and found that the ulcers healed significantly more rapidly with Dermagraft® than with conventional treatment alone.[42] As yet there has not been a clinical trial for pressure ulcers, although a case series of 13 patients with pressure ulcers treated with Apligraf® over a period of 10 months resulted in just over half the patients responding to treatment, and the proportion of individuals who failed to respond had stage III and IV ulcers.[33] It is thought that Apligraf® is safe and that it should be considered as an early intervention to halt ulcer progression.[33]

Burn injuries may be divided into partial thickness burns, involving loss of epidermis and papillary dermis, and full-thickness burns where damage is deeper with more extensive dermal injury.[43] Superficial partial thickness burns may result in full regeneration where tissue integrity is restored by re-epithelialization without scar formation, in comparison with full-thickness burns where there is lack of dermis and tissue repair inevitably occurs with scarring.[43] Nevertheless, all burn injuries can lead to major loss of fluid and proteins, and increased susceptibility to infection requiring immediate attention. Nonbiological topical treatments as well as biological dressings may be used, and application of tissue-engineered skin substitutes as temporary dressings is also effective, providing protection for the deeper structures (e.g. tendons and bone) as well as pain relief and promoting wound healing.[43] Alloderm® (LifeCell Corporation, Woodlands, TX, U.S.A.) is a commercially available acellular dermal matrix derived from human skin and used as a temporary biological dressing in burns. Alternatively, there are a variety of tissue-engineered products available. The use of a bilayered skin substitute such as Apligraf® or OrCel® (Ortec International Inc., New York, NY, U.S.A.) requires just one procedure for skin replacement; OrCell consists of a bilayer of normal human allogeneic keratinocytes and fibroblasts in a type I bovine collagen sponge. In addition, treatment of burns may involve two stages where a dermal template with artificial epidermis initially allows autogenous neovascularization and autologous fibroblast migration into the artificial dermal scaffold.[44] Following formation of the neodermis, the temporary epidermis is removed and replaced with an epidermal autograft. An example of this type of product is IntegraTM (Integra LifeSciences, Plainsboro, NJ, U.S.A.), which is composed of a dermal template consisting of collagen and chondroitin-6-sulphate with a temporary silicone epidermal layer.[44]

Traditionally, for extensive burns injuries, autografts are harvested and meshed in order to treat a larger body surface area.[45] However, pretreated cadaver skin or tissue-engineered products are often required as a temporary dressing for large wounds where there is a lack of healthy donor sites, for instance in burns with more than 60% body surface area involvement. Currently, it is thought that the advantage of using Apligraf® as opposed to cadaver skin as a biological dressing is that Apligraf® is readily available, of reproducible quality and does not predispose to infectious disease transmission.[46] Furthermore, Apligraf® incorporates neonatal fibroblasts which have higher proliferative rates and offer the possibility of producing near normal dermis.[46] Overall, incorporation of fibroblasts in tissue substitutes is important as it acts as the key orchestrator in wound healing and enhances re-epithelialization.

Epidermolysis bullosa (EB) is a heterogeneous group of mechanobullous disorders characterized by skin fragility. The application of tissue-engineered skin substitutes has been used with variable success for treating acute and chronic wounds in EB as well as for covering wounds following the surgical release of fused digits to improve hand function.[47,48,49] In a recent prospective study of nine patients with different types of EB (EB simplex-Dowling-Meara and Weber-Cockayne, Herlitz junctional EB and recessive dystrophic EB), Apligraf® was used to treat chronic wounds and cover wounds following hand surgery.[49] The results were encouraging and demonstrated more rapid healing of wounds with reduced symptoms of pain, pruritus, bleeding and improved function following treatment with Apligraf®.

The use of fibroblasts for cell-based therapy and gene therapy for the treatment of recessive dystrophic EB has also been studied. Recessive dystrophic EB is characterized by reduced or absent anchoring fibrils in the skin due to abnormal collagen VII production and mutations in the COL7A1 gene. Although keratinocytes produce more collagen VII in vivo, gene-corrected dystrophic EB fibroblasts may offer greater potential in gene therapy than gene-corrected dystrophic EB keratinocytes as the gene-corrected dystrophic EB fibroblasts produced greater collagen VII staining at the mouse dermal-epidermal junction.[50] Moreover, gene-corrected recessive dystrophic EB fibroblasts and allogeneic fibroblasts have been injected intradermally into athymic nude mice, resulting in human collagen type VII deposition at the basement membrane zone.[51] Gene-corrected recessive dystrophic EB fibroblasts expressing human collagen type VII injected intravenously into the mouse tail vein homed to the wound on the mouse back and deposited human collagen type VII at the dermal-epidermal junction.[52] This finding may have significant implications for future human trials of gene therapy using gene-corrected autologous fibroblasts in individuals with dystrophic EB.

In a case report of a rapidly progressive ulcerative pyoderma gangrenosum on the leg of a young woman, conventional treatment using systemic corticosteroids and ciclosporin were used in conjunction with Apligraf®.[53] Use of Apligraf® led to accelerated ulcer healing with minimal scar contracture and rapid pain relief, and allowed prompt reduction of ciclosporin (4 mg kg-1 daily) without any disease recurrence. Although this is only one case report of the use of Apligraf® for pyoderma gangrenosum, the advantage of using a readily available skin substitute in this particular instance is that it avoids the necessity of harvesting autologous skin for expansion and the subsequent possibility of inducing a pathergic response.[53] Other dermatological conditions in which Apligraf® has been applied and led to successful wound healing include hydroxyurea-induced leg ulcers, bullous morphoea ulcers and ulcerative sarcoidosis.[54,55,56]

Tissue-engineered skin substitutes have also been used for the treatment of wounds following cancer excision. They have the advantage of promoting normal tissue repair without the need to induce donor-site defects as well as allowing monitoring for local tumour recurrence.[57] The use of Dermagraft® for covering intraoral defects following oral squamous cell carcinoma was investigated in a group of seven patients, and it was demonstrated that there was successful incorporation and complete closure of the wounds by day 11 with no evidence of fibrosis.[57] Furthermore, the efficacy and safety of Apligraf® for treating wounds following Moh's or excisional surgery has also been evaluated in a prospective randomized and blinded trial of 12 patients.[58] One group of six patients was treated with Apligraf® while the wounds of the other group of six patients were allowed to heal by secondary intention. The results showed that although there was no significant difference in healing rates and comorbid factors between the two groups, the Apligraf® group produced better cosmetic results in terms of pigmentation, vascularity, pliability and height of the scar.[58]

In terms of cosmetic surgery, the use of injectable fillers for facial rejuvenation is currently widespread and popular, and autologous fibroblasts (Isolagen®; Isolagen Technologies Inc., Paramus, NJ, U.S.A.) are being used as an alternative to other traditional fillers such as bovine collagen (e.g. Xyderm, Xyplast), human collagen (e.g. Dermalogen) and synthetic materials (e.g. silicone), and there is evidence that autologous fibroblast injections can improve the appearances of facial wrinkles and depressed scars.[59] The advantage of injecting a live and dynamic autologous filler is obvious as it not only leads to longer-term and sustained correction but also eliminates the problem of hypersensitivity and foreign body granulomatous reactions. Furthermore, there have been no reports of hypertrophic scarring or keloid scarring following the intradermal injection of autologous fibroblasts, suggesting that fibroblast proliferation and collagen synthesis is naturally regulated by cell-cell and cell-ECM contact and negative feedback.[59]

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