The use of fibrin glue in skin grafts and tissue-engineered skin replacements: a review.

Fibrin glue has been widely used as an adhesive in plastic and reconstructive surgery. This article reviews the advantages and disadvantages of its use with skin grafts and tissue-engineered skin substitutes. Fibrin glue has been shown to improve the percentage of skin graft take, especially when associated with difficult grafting sites or sites associated with unavoidable movement. Evidence also suggests improved hemostasis and a protective effect resulting in reduced bacterial infection. Fibrin, associated with fibronectin, has been shown to support keratinocyte and fibroblast growth both in vitro and in vivo, and may enhance cellular motility in the wound. When used as a delivery system for cultured keratinocytes and fibroblasts, fibrin glue may provide similar advantages to those proven with conventional skin grafts. Fibrin glue has also been shown to be a suitable delivery vehicle for exogenous growth factors that may in the future be used to accelerate wound healing.     

Applied tissue engineering in the closure of severe burns and chronic wounds using cultured human autologous keratinocytes in a natural fibrin matrix

Whereas in severe burns cultured human epithelial cells may well serve as a life saving method, the true value of tissue-engineered skin products in chronic wound care has yet to be clearly defined. Among other well-known clinical problems, the engraftment rate of commercially available multilayered "sheet grafts" has been shown to vary extremely. Adherence of transplanted cells to the wound bed--especially in the presence of potential wound contamination-- is one of the crucial aspects of this technique. Keratinocyte suspensions in a natural fibrin sealant matrix can potentially treat a variety of skin defects. In acute burn wounds, as well as in chronic wounds the clinical application of this type of tissue-engineered skin substitute demonstrates the capacity of cultured human autologous keratinocytes in a fibrin sealant matrix to adhere to wound beds, attach and spread over the wound resulting in reepithelialization of both acute and chronic wounds. In full thickness burns the combination of this new tool with allogenic dermis is a promising option to achieve complete dermal-epidermal reconstitution by means of tissue engineering and guided tissue repair. When transferring this technique into the treatment of chronic wounds we found an optimal preparation of such recipient wound beds to be crucial to the success. The additional application of continuous negative pressure (vacuum therapy) and preliminary chip skin grafting to optimally prepare the recipient site may be helpful tools to achieve such well-prepared and graftable surfaces. Prospective controlled comparative studies should be designed to further assess the clinical efficacy of this technique.     

Composite autologous-allogeneic skin replacement: development and clinical application

A major unsolved problem in skin restoration in severe burns is replacement of lost dermis. We report the development and clinical application of a composite grafting technique in which allogeneic skin is the source of dermis, and cultured autologous keratinocytes generate epidermis. Excised burn wounds are resurfaced with unmatched allograft. Immunosuppression from the burn and reduced immunoreactivity of the allograft permit successful allograft engraftment. Keratinocyte cultures are initiated from the patient. Allogeneic epidermis is removed, and the dermal bed is resurfaced with keratinocyte cultures. The allogeneic dermis promotes rapid (less than 7 days) stratification, maturation, and integration of the cultures and the synthesis of anchoring fibrils. One case followed 11 months has shown no evidence of rejection. We reason that removal of the epidermis from allograft eliminates the majority of cells constitutively expressing alloclass II antigens, leaving behind a viable allogeneic dermal bed that serves as an ideal substrate for engraftment and integration of keratinocyte cultures but does not initiate rejection.     

Enhanced function of cultured epithelium by genetic modification: Cell-based synthesis and delivery of growth factors

Skin substitutes, containing cultured keratinocytes of the epidermis (autologous or allogeneic cells), have been used in the treatment of severe burns and other defects of the skin such as chronic ulcers. Our goal is to enhance the functions of the cells used in these skin substitutes by genetic modification. We propose to develop a genetically modified skin graft which would function as a cell-based vehicle for the local synthesis and delivery of wound-healing growth factors. Using retroviral-mediated gene transfer, we have introduced stable copies of the genes encoding platelet-derived growth factor (PDGF-A) or insulin-like growth factor-1 (IGF-1) into cultured human diploid keratinocytes. After stable integration of these genes, the cells secreted significant levels of these growth factors, 744 ng and 502 ng/10(7) cells/24 h for PDGF-A and IGF-1, respectively. The modified cells were grown to confluence, detached as a multicell-layered epithelial sheet, and transplanted to athymic mice.Seven days after transplantation, grafts secreting PDGF-A or IGF-1 differentiated into a stratified epithelium comparable to unmodified cells. Most importantly, the newly synthesized connective tissue layer subjacent to the PDGF-A-modified grafts was significantly thicker and showed an increase in cellularity, vascularity, type I collagen, and fibronectin deposition when compared to control grafts of unmodified cells or grafts expressing IGF-1.These results demonstrated that the function of the cells of a skin substitute can be enhanced by genetic modification and show that PDGF-A secretion from these cells can mediate changes to the cellular, vascular, and extracellular matrix composition of the adjacent dermal tissue. Moreover, these results suggest that a cell-based method for growth factor synthesis and delivery may be a useful approach to promoting tissue repair. (c) 1996 John Wiley & Sons, Inc.     

Clinical interest of cutaneous models reproducedin vitro for severe burn treatment: histopathological and ultrastructural study

The healing of minimal skin lesions is usually obtained by epidermal migration and proliferation from peripheral wound margins. However, cutaneous grafts or reconstituted skin are necessary for severe injuries. Various models have recently been reproduced for this purpose. The aim of this work is to report the histopathologic evolution of burn lesions treated two years ago by autologous epidermis (Genzyme Tissue Repair, Boston, USA). Fifteen patients with severe burns (more than 80% of surface) have been treated. These observations have been based exclusively on biopsies of grafted wounds. Cultured epidermis is rapidly fully differentiated after grafting with temporary hyperplasia and normal strata. At 18 months, rete ridges formation is present only in young patients. Melanocytes and Langerhans' cells repopulated grafts rapidly. The use of cultured epidermis nowadays represents an important improvement in burn treatment.Abbreviations CEA cultured epidermal autologous sheets - TBSA total burn surface area     

Fabrication, quality assurance, and assessment of cultured skin substitutes for treatment of skin wounds

Advances in treatment of skin wounds depend on demonstration of reduced morbidity or mortality either during or after hospitalization. Tissue engineering of skin grafts from cultured cells and biopolymers permits greater amounts of grafts from less donor tissue than conventional procedures. Autologous keratinocytes and fibroblasts isolated from epidermis and dermis of skin may be combined with collagen-based substrates to generate cultured skin substitutes (CSS) with epidermal and dermal components. By regulation of culture conditions, CSS form epidermal barrier and basement membrane, and release angiogenic factors that stimulate vascularization. Prototypes of CSS may be tested for safety and efficacy by grafting to athymic mice which do not reject human tissues. Clinical application of CSS requires establishment of quality assurance assessments, such as, epidermal barrier by measurement of surface hydration, and anatomy by standard histology. Medical benefits of tissue engineered skin for treatment of burns are evaluated quantitatively by the ratio of healed skin to donor skin, and qualitatively by the Vancouver Scar Scale. These benefits may also be extended to other medical conditions including chronic wounds and reconstructive surgery.     

MHC class I manipulation on cell surfaces by gene transfer of anti-MHC class I intrabodies--a tool for decreased immunogenicity of allogeneic tissue and cell transplants

Intrabodies (IB) are suitable tools to down-regulate the expression of cell surface molecules in general. In this work, the appearance of major histocompatibility (MHC) class I molecules on the cell surface could be prevented by the expression of intracellularly localized anti-MHC class I antibodies. The expression of MHC antigens presenting intracellularly synthetised peptides on the cell surface is the predominant reason for immunologic detection and rejection of allogeneic cell and tissue transplants. Allogeneic keratinocyte sheets might be a suitable tool for skin grafting. Within this study primary rat keratinocytes have been transfected with anti-MHC I-IB. Strong IB-expressing cells showed a MHC I "knockout" phenotype. The cells did not exhibit any significant alterations compared to non-transfected cells: the cell growth and the expression of other surface molecules were unaltered. Merely an enhanced intracellular accumulation of MHC I molecules could be detected. Notably, IB-expressing keratinocytes displayed a reduced susceptibility to allogeneic cytotoxic T cells in vitro compared to unmodified cells with a normal level of MHC I surface expression. These MHC I-deficient keratinocytes might be utilized in tissue-engineered allogeneic non-immunogeneic skin transplants. The principle of MHC class I manipulation in general can be used for other allogeneic cell and tissue-engineered transplants as well.     

The use of allogeneic cultivated keratinocytes for the early coverage of deep dermal burns – indications, results and problems

Since 1995, keratinocytes are grown into cultures and used as allografts for the coverage of deep dermal defects in our burn unit. Donor skin samples are mostly acquired from other burn patients. In addition, special methods of skin preservation allow us the use of skin, which has been taken in redundancy for split thickness skin grafting from nonburned patients.Thirty five patients with deep partial thickness burns in the face were treated since 1996 according to the following concept: Dermabrasion or tangential excision was performed before the 5(th) day following trauma. If viable dermis was present, the wounds were covered with sheets of allogeneic cultivated keratinocytes. In cases of deeper defects, autologous skin grafts were applied. In 23 cases, epithelialisation was achieved within 10 days, in 8 patients, a prolonged duration until complete healing was observed. In 5 faces, coverage of residual defects with skin grafts was necessary. The mentioned problems of wound healing occurred from infection, incomplete excision of burn eschar and a depth of the wound which was retrospectively seen too deep for the treatment with keratinocytes. At follow up, patients were examined clinically and functionally with Frey's faciometer(R), which is an instrument for quantification of mimic movements. In cases of uncomplicated healing, a nearly complete restitution was found.Other indications include deep dermal burns in children and the coverage of early excised wounds in adults, with a reasonable amount of viable dermis remaining, both resulting in a significant reduction of donor-site morbidity. In severely burned adults with limited donor sites, it offers the possibility of immediate definite coverage of large areas.     

The role of allogenic fibroblasts in an acute wound healing model

Skin is the first tissue-engineered organ to have been successfully developed in the laboratory, and it has been clinically available for use as epidermal sheets for some time. As refinements in this field of tissue engineering continue, several key issues give cause for concern. One issue is the need to form a more complete dermal analogue before grafting. To this end, fibroblasts may be used in vitro to deposit extracellular matrix components within a basic scaffold, laying down those molecules not endogenous to the material and thereby improving the quality of the skin replacement. Many studies have shown the benefits of in vitro seeding with fibroblasts, but there has been some debate regarding the longevity of such cells after allotransplantation into an immunocompetent host. In this study, the authors set out to determine the longevity of transplanted cells in an immunocompetent porcine model. A total of 24 wounds were made on four female animals, 12 of which were covered with acellular hyaluronic acid dermal matrices, and the remainder of which were covered with matrices seeded with allogenic (male) fibroblasts. After a week in vivo, the wounds were grafted with either split-thickness skin grafts or cultured epithelial autograft. Biopsy specimens were obtained from wounds at varying time intervals and assessed using genetic analysis to determine the survival of allotransplanted cells. No cells were detectable by polymerase chain reaction analysis (sensitivity, 1:100,000) after 7 days in vivo. Subsequent histologic examination demonstrated little difference in wound morphology. The authors conclude that allogenic fibroblasts do not survive transplantation in a porcine wound model.     

Full-thickness grafting of acute eyelid burns should not be considered taboo.

Split-thickness skin grafts are commonly used for the treatment of acute eyelid burns; in fact, this is dogma for the upper lid. Ectropion, corneal exposure, and repeated grafting are common sequelae, almost the rule. It was hypothesized that for acute eyelid burns, the use of full-thickness skin grafts, which contract less than split-thickness skin grafts, would result in a lower incidence of ectropion with less corneal exposure and fewer recurrences. The records of all patients (n = 18) who underwent primary skin grafting of acutely burned eyelids (n = 50) between 1985 and 1995 were analyzed retrospectively. There were 10 patients who received full-thickness skin grafts (12 upper lids, 8 lower lids) and 8 patients who received split-thickness skin grafts (15 upper lids, 15 lower lids). Three of 10 patients (30 percent) who received full-thickness skin grafts and 7 of 8 patients (88 percent) who received split-thickness skin grafts developed ectropion and required reconstruction of the lids (p = 0.02). No articles were found substantiating the concept that only split-thickness grafts be used for acute eyelid burns. The treatment of acute eyelid burns with full-thickness rather than split-thickness skin grafts results in less ectropion and fewer reconstructive procedures. It should no longer be considered taboo and should be carried out whenever possible and appropriate.     

Effect of Mixed Transplantation of Autologous and Allogeneic Microskin Grafts on Wound Healing in a Rat Model of Acute Skin Defect

The treatment of extensive thermal injuries with insufficient autologous skin remains a great challenge to burn surgeons. In this study, we investigated the influence of the ratio of autologous and allogeneic tissue in mixed microskin grafts on wound healing in order to develop an effective method for using limited donor skin to cover a large open wound. Four different mixtures were tested: autologous microskin at an area expansion ratio of 10∶1 with allogeneic microskin at an area expansion ratio of 10∶1 or 10∶3 and autologous microskin at an expansion ratio of 20∶1 with allogeneic microskin at an expansion ratio of 20∶3 or 20∶6. Wound healing, wound contraction, and integrin β1 expression were measured. Mixed microskin grafting facilitated wound healing substantially. The mixture of autologous microskin at an expansion ratio of 10∶1 with the same amount of allogeneic microskin achieved the most satisfactory wound healing among the 4 tested mixtures. Histological examination revealed the presence of obviously thickened epidermis and ectopic integrin β1 expression. Keratinocytes expressing integrin β1 were scattered in the suprabasal layer. Higher levels of integrin β1 expression were associated with faster wound healing, implying that ectopic expression of integrin β1 in keratinocytes may play a pivotal role in wound healing. In conclusion, this study proves that this new skin grafting technique may improve wound healing.     

Drug releasing systems in cardiovascular tissue engineering

Heart disease and atherosclerosis are the leading causes of morbidity and mortality worldwide. The lack of suitable autologous grafts has produced a need for artificial grafts; however, current artificial grafts carry significant limitations, including thrombosis, infection, limited durability and the inability to grow. Tissue engineering of blood vessels, cardiovascular structures and whole organs is a promising approach for creating replacement tissues to repair congenital defects and/or diseased tissues. In an attempt to surmount the shortcomings of artificial grafts, tissue-engineered cardiovascular graft (TECVG), constructs obtained using cultured autologous vascular cells seeded onto a synthetic biodegradable polymer scaffold, have been developed. Autologous TECVGs have the potential advantages of growth, durability, resistance to infection, and freedom from problems of rejection, thrombogenicity and donor scarcity. Moreover polymers engrafted with growth factors, cytokines, drugs have been developed allowing drug-releasing systems capable of focused and localized delivery of molecules depending on the environmental requirements and the milieu in which the scaffold is placed. A broad range of applications for compound-releasing, tissue-engineered grafts have been suggested ranging from drug delivery to gene therapy. This review will describe advances in the development of drug-delivery systems for cardiovascular applications focusing on the manufacturing techniques and on the compounds delivered by these systems to date.     

Immunological challenges associated with artificial skin grafts: available solutions and stem cells in future design of synthetic skin

The repair or replacement of damaged skins is still an important, challenging public health problem. Immune acceptance and long-term survival of skin grafts represent the major problem to overcome in grafting given that in most situations autografts cannot be used. The emergence of artificial skin substitutes provides alternative treatment with the capacity to reduce the dependency on the increasing demand of cadaver skin grafts. Over the years, considerable research efforts have focused on strategies for skin repair or permanent skin graft transplantations. Available skin substitutes include pre- or post-transplantation treatments of donor cells, stem cell-based therapies, and skin equivalents composed of bio-engineered acellular or cellular skin substitutes. However, skin substitutes are still prone to immunological rejection, and as such, there is currently no skin substitute available to overcome this phenomenon. This review focuses on the mechanisms of skin rejection and tolerance induction and outlines in detail current available strategies and alternatives that may allow achieving full-thickness skin replacement and repair.     

Survival of cultured allografts in patients with burns assessed with probe specific for Y chromosome.

The aim of the study was to determine the fate of cultured skin allografts in patients with burns. In situ DNA hybridisation with a Y probe (pHY 2.1) was used to detect cells carrying the Y chromosome (the probe being visualised by the alkaline phosphatase-antialkaline phosphatase method) in biopsy specimens taken from cultured allografts derived from donors of the opposite sex to the recipients (20 patients with burns). Specimens were taken within a week, between one and three weeks, between four and six weeks, and more than six weeks after grafting. Only two of the 27 biopsy specimens contained cells that were the same sex as the donor; both were taken within a week after grafting. In the 25 other specimens the epithelial cells were the same sex as the recipient. Cultured skin allografts showed no evidence of survival in patients with burns, which suggests that they are probably not suitable for long term management of burns but may be useful as short term biological dressings.     

Clinical application of autologous cultured epithelia for the treatment of burn wounds and burn scars

This report presents our experience with autologous cultured human epithelia grafting on burn wounds, burn scars, and skin-graft donor sites in seven patients. Dispersed epidermal cells were cultured with 3T3 cells treated with mitomycin C. After 2 to 3 weeks, cultured epithelia (total 350 to 2250 cm2) were grafted to the wound. The results showed that cultured epithelia grafts did not take so completely compared to the meshed skin grafts used for the coverage of burn wounds. However, cultured grafts placed on aseptic wounds adhered well and showed good appearance. In the histologic findings, normal differentiation of epidermal cells was found. Cultured grafts were bordered from subepidermal granulative tissue with basement membrane. A rete ridge and the adnexal structures were absent in the specimens that adhered to the burn wounds. However, in the specimens that took on abraded wounds, a gently sloping rete ridge and elastic fibers were seen. The histologic findings showed structures resembling normal skin.     

Prevention of burn wound conversion by allogeneic keratinocytes cultured on acellular xenodermis

Deep dermal burns frequently tend to convert into full-thickness skin loss. We found that this wound deepening may be prevented by recombined human/pig skin (RHPS), consisting of human allogeneic keratinocytes cultured on acellular pig dermis. RHPS has skin-like consistency and therefore optimal adhesiveness to the wound. It can be easily removed from the dish and transferred to the recipient. The wound bed has to be prepared by tangential excision or deep dermabrasion to the level of capillary bleeding. RHPS has to be applied ‘upside-down’, with the epidermal layer facing the wound, to avoid the dermal matrix forming a barrier to the nutrients for the keratinocytes. In our practice, more than 70% of early excised or deeply dermabraded wounds grafted with RHPS healed in the course of one week after keratinocyte transplantation. This revised version was published online in July 2006 with corrections to the Cover Date.     

Bone grafts,bone substitutes and orthobiologics: The bridge between basic science and clinical advancements in fracture healing

The biology of fracture healing is better understood than ever before, with advancements such as the locking screw leading to more predictable and less eventful osseous healing. However, at times one’s intrinsic biological response, and even concurrent surgical stabilization, is inadequate. In hopes of facilitating osseous union, bone grafts, bone substitutes and orthobiologics are being relied on more than ever before. The osteoinductive, osteoconductive and osteogenic properties of these substrates have been elucidated in the basic science literature and validated in clinical orthopaedic practice. Furthermore, an industry built around these items is more successful and in demand than ever before. This review provides a comprehensive overview of the basic science, clinical utility and economics of bone grafts, bone substitutes and orthobiologics.     

A Silk Fibroin/Collagen Nerve Scaffold Seeded with a Co-Culture of Schwann Cells and Adipose-Derived Stem Cells for Sciatic Nerve Regeneration

As a promising alternative to autologous nerve grafts, tissue-engineered nerve grafts have been extensively studied as a way to bridge peripheral nerve defects and guide nerve regeneration. The main difference between autogenous nerve grafts and tissue-engineered nerve grafts is the regenerative microenvironment formed by the grafts. If an appropriate regenerative microenvironment is provided, the repair of a peripheral nerve is feasible. In this study, to mimic the body’s natural regenerative microenvironment closely, we co-cultured Schwann cells (SCs) and adipose-derived stem cells (ADSCs) as seed cells and introduced them into a silk fibroin (SF)/collagen scaffold to construct a tissue-engineered nerve conduit (TENC). Twelve weeks after the three different grafts (plain SF/collagen scaffold, TENC, and autograft) were transplanted to bridge 1-cm long sciatic nerve defects in rats, a series of electrophysiological examinations and morphological analyses were performed to evaluate the effect of the tissue-engineered nerve grafts on peripheral nerve regeneration. The regenerative outcomes showed that the effect of treatment with TENCs was similar to that with autologous nerve grafts but superior to that with plain SF/collagen scaffolds. Meanwhile, no experimental animals had inflammation around the grafts. Based on this evidence, our findings suggest that the TENC we developed could improve the regenerative microenvironment and accelerate nerve regeneration compared to plain SF/collagen and may serve as a promising strategy for peripheral nerve repair.     

Development of a dermal matrix from glycerol preserved allogeneic skin

Dermal substitutes can be used to improve the wound healing of deep burns when placed underneath expanded, thin autologous skin grafts. Such dermal matrix material can be derived from xenogeneic or human tissue. Antigenic structures, such as cells and hairs must be removed to avoid adverse inflammatory response after implantation. In this study, a cost-effective method using low concentrations of NaOH for the de-cellularization of human donor skin preserved in 85% glycerol is described. The donor skin was incubated into NaOH for different time periods; 2, 4, 6 or 8 weeks. These dermal matrix prototypes were analyzed using standard histology techniques. Functional tests were performed in a rat subcutaneous implant model and in a porcine transplantation model; the prototypes were placed in full thickness excision wounds covered with autologous skin grafts. An incubation period of 6 weeks was most optimal, longer periods caused damage to the collagen fibers. Elastin fibers were well preserved. All prototypes showed intact biocompatibility in the rat model by the presence of ingrowing blood vessels and fibroblasts at 4 weeks after implantation. An inflammatory response was observed in the prototypes that were treated for only 2 or 4 weeks with NaOH. The prototypes treated with 6 or 8 weeks NaOH were capable to reduce wound contraction in the porcine model. In neo-dermis of these wounds, elastin fibers derived from the prototype could be observed at 8 weeks after operation, surrounded by more random orientated collagen fibers. Thus, using this effective low cost method, a dermal matrix can be obtained from human donor skin. Further clinical studies will be performed to test this material for dermal substitution in deep (burn) wounds.     

Early excision and skin grafting of selected burns of the face and neck

Since 1979, 16 patients with facial and neck burns have been treated with excision and skin grafting within the first 4 days of injury. The injuries were tangentially excised and immediately covered with split-thickness skin grafts. Detailed consecutive results are presented. The patients can be divided into three groups. Group 1 consisted of small subdermal or circumscribed deep dermal burns of the face (n = 8). Healing was quick. Some patients developed signs of overgrafting. As a late result, unevenness and discoloration were seen. Group 2 consisted of mixed deep dermal and subdermal burns of the face and neck (n = 5). Usually, minor areas had to be regrafted. Some patients developed hypertrophic scars at border areas. In the completely excised and grafted area, the skin was smooth, pliable, and discolored. Group 3 consisted mostly of subdermal burns of the face and neck (n = 3). The surgical trauma was significant. Small areas had to be regrafted. Ectropion and microstomia developed. It is concluded that in selected cases of deep dermal and subdermal burns, early excision and skin grafting will result in faster healing and less scarring than expectant treatment.