The effect of fibrin glue on skin grafts in infected sites.

Fibrin bonding of skin grafts to wounds is an essential part of the graft-adherence process. Bacteria, in concentrations greater than 10(5)/gm of tissue, are associated with graft failure. Sixty-five rats were randomly divided into three groups, dorsal split-thickness skin grafts were harvested, and the sites were inoculated with Staphylococcus aureus. After incubation, each wound was quantitatively biopsied and treated with saline, fibrin glue with aprotinin, or fibrin glue alone. We found that the addition of commercially available fibrin glue with or without the antifibrinolytic agent aprotinin is capable of restoring graft adherence to normal levels in graft sites infected with greater than 10(5) bacteria/gm of tissue. Fibrin glue may have potential for increasing skin-graft take in the clinical situation where the graft bed is infected.     

Reversing the detrimental effect of adriamycin on skin grafts in rats

We evaluated in a rat model the effects of a homologous fibrin glue in reversing the effects of Adriamycin on adherence and take of skin grafts. A total of 40 male Fisher rats were used in the study. During the first phase of the experiment, the animals were assigned to either group I (N = 10) receiving normal saline or group II (N = 10) receiving 6 mg/kg Adriamycin by tail vein injection 24 hours before surgery. Skin grafts with and without fibrin glue were placed over wounds in the backs of the animals and adherence was measured at 24 and 48 hours. In the second phase (N = 20), the experiment was repeated, this time evaluating the total area of skin graft take at 7 days. Fibrin glue was found to increase adherence and take of skin grafts in all Adriamycin-treated animals.     

Tissue engineering of cultured skin substitutes

Skin replacement has been a challenging task for surgeons ever since the introduction of skin grafts by Reverdin in 1871. Recently, skin grafting has evolved from the initial autograft and allograft preparations to biosynthetic and tissue-engineered living skin replacements. This has been fostered by the dramatically improved survival rates of major burns where the availability of autologous normal skin for grafting has become one of the limiting factors. The ideal properties of a temporary and a permanent skin substitute have been well defined. Tissue-engineered skin replacements: cultured autologous keratinocyte grafts, cultured allogeneic keratinocyte grafts, autologous/allogeneic composites, acellular biological matrices, and cellular matrices including such biological substances as fibrin sealant and various types of collagen, hyaluronic acid etc. have opened new horizons to deal with such massive skin loss. In extensive burns it has been shown that skin substitution with cultured grafts can be a life-saving measure where few alternatives exist. Future research will aim to create skin substitutes with cultured epidermis that under appropriate circumstances may provide a wound cover that could be just as durable and esthetically acceptable as conventional split-thickness skin grafts. Genetic manipulation may in addition enhance the performance of such cultured skin substitutes. If cell science, molecular biology, genetic engineering, material science and clinical expertise join their efforts to develop optimized cell culture techniques and synthetic or biological matrices then further technical advances might well lead to the production of almost skin like new tissue-engineered human skin products resembling natural human skin.     

Why do skin grafts fail?

Fibrin is shown to be the agent responsible for the adherence of biological dressings and of autografts to wounds. Its presence is associated with graft success, and its absence with graft failure. The results suggest that the deposition of fibrin provides the basis for the anti-bacterial actions of biological dressings and for the sterilization of the wound under adherent autografts. The total number of bacteria per gram of tissue in the wound, though important, is not critical to the result of skin grafting. The mechanism by which different organisms cause grafts to fail is by the production of plasmin and proteolytic enzymes which dissolve the important fibrin scaffold--thus ensuring their own survival. Thus, it is the levels of these (and the numbers of organisms efficient in producing them) which cause success or failure of applied skin grafts.     

Multiple growth factor delivery for skin tissue engineering applications

Administration of exogenous growth factors (GFs) to a damaged site has been investigated for skin tissue regeneration. Among the many types of GFs and cytokines, epidermal growth factor, vascular endothelial growth factor, platelet-derived growth factor, fibroblast growth factor, and hepatocyte growth factor could be specifically used for stimulating molecules in wound healing as well as for recovery of damaged skin tissues. It is speculated that delivered GFs could stimulate various cellular functions, including proliferation, migration, deposition of extracellular matrix molecules, and remodeling of collagen synthesis. Although the physiological wound healing process is complex, engineering strategies for proper delivery of multiple therapeutic GFs could enhance the quality and quantity of regenerated skin tissues. As compared to single delivery of a GF, recent studies have proven that any combination of multiple GFs and/or therapeutic chemical factors synergistically facilitates the regeneration of damaged skin tissues. In order to maximize the stability, bioactivity, intrinsic therapeutic functionality, and efficiency of internal delivery of cargo GFs, it is essential to utilize tissueengineered biomaterials and related composites as implantable platforms. Successful fabrication and development of skin tissue engineering applications as well as subsequent surgical implantation of these platforms might provide clinical treatment for superior skin regeneration. Therefore, the present review summarizes the biological functions, related signaling mechanisms, and recent developments of tissue engineering applications for multiple GF delivery.     

Skin regeneration with fibroblast growth factor 2 released from heparin-conjugated fibrin

Fibroblast growth factor 2 (FGF2) stimulates skin wound healing but does long-term delivery of FGF2 enhance skin regeneration compared to short-term delivery? Heparin-conjugated fibrin (HCF) was used as a vehicle for long-term delivery of FGF2. Fibrin, HCF, FGF2-loaded fibrin, and FGF2-loaded HCF were implanted into full-thickness skin defects of mice. The neoepidermis thickness was significantly larger in the FGF2-loaded HCF group than in the other groups, except for the FGF2-loaded fibrin group. Suprabasal cytokeratin differentiation in squamous neoepithelium was greatest in the FGF2-loaded HCF group. The enhanced skin regeneration accompanying the long-term delivery of FGF2 could be mediated, at least partially, by enhanced neovascularization and cell proliferation in the neodermis.     

The use of skin grafts in postburn contracture release: a 10-year review

Postburn scarring and contracture affecting function remain the most frustrating late complications of burn injury. Various techniques are used to release contractures; the choice depends on their location and/or the availability of unaffected skin adjacent to the contracture or elsewhere. A retrospective review was carried out of the case notes of patients who had skin grafting for the release of postburn contracture at the Burns Unit, City Hospital, Nottingham between May of 1984 and August of 1994 to evaluate the experience over this period. Information was obtained about the burn injury, contracture site, interval between burn and release of contracture, indication, age at first release, intervals between releases, operative details (donor and graft sites), complications and nonoperative treatment, and follow-up to the end of the study period. A total of 129 patients underwent skin grafting for release of contractures as opposed to any other method of correction. Full-thickness skin grafts were used in 81 patients (63 percent) and split-thickness skin grafts in 26 (20 percent). Twenty-two patients (17 percent) had both types used on different occasions. Flame burns (41 percent) were the most common causes, followed by scalds (38 percent). Two hundred thirty-nine sites of contracture were released, with the axilla (59) and the hand/wrist (59) being the most common sites involved, followed by the head/neck region (42). It was found that for the same site, release with split-thickness skin grafts was associated with more rereleases of the contracture than with full-thickness skin grafts. Also, the interval between the initial release and first rerelease was shorter than with full-thickness skin grafts (p < 0.048). It was also noted that children required more procedures during growth spurts, reflecting the differential effect of the growth of normal skin and contracture tissue. Patients reported more satisfaction with texture and color match with the full-thickness skin grafts. There was comparable donor-site and graft morbidity with both graft types. The use of skin grafts is simple, reliable, and safe. Whenever possible, the authors recommend the use of full-thickness skin grafts in preference to split-thickness skin grafts in postburn contracture release.     

Immunohistochemical localization of heparin-binding epidermal growth factor-like growth factor in normal skin and skin cancers

Summary Heparin-binding epidermal growth factor (EGF)-like growth factor is a 22-kDa glycoprotein that was originally identified as a secreted product of cultured human macrophages. Although the growth factor mRNA has been identified in various cells and tissues, the tissue distribution of the protein itself has rarely been demonstrated. In this study, the EGF-like growth factor was detected immunohistochemically in a variety of human skin samples by indirect immunofluorescence using a polyclonal rabbit antiserum raised against residues 26–41 of mature heparin-binding EGF. The keratinocytes of a variety of epithelium-derived structures demonstrated reproducible, specific staining for the EGF. In normal tissues, this staining was prominent in the basal cells of the epidermis and in the epithelial cells lining epidermal appendages such as hair follicles, sebaceous sweat glands and eccrine sweat glands. In addition, specific staining was detected in skin cancers derived from the basal epithelial cell layer, including basal and squamous cell carcinomas of the skin, with no staining detected in melanoma specimens. Immunoreactive heparin-binding EGF was characteristically associated with the surface of cells. With minor exceptions, the immunoreactive sites are identical to the known EGF receptor distribution in the skin, and suggest that keratinocyte-derived heparin-binding EGF may act in concert with other EGF family members in processes such as skin morphogenesis and wound repair, as well as in the development of skin cancers This revised version was published online in November 2006 with corrections to the Cover Date.     

The pectoralis major for single-stage reconstruction of the difficult wounds of the orbit and pharyngoesophagus

This paper reports the results of reconstructions of difficult wounds in the head and neck area with musculocutaneous flaps in 20 patients. Twelve patients had reconstructions following cranial and orbital resections, and eight patients had reconstructions of the pharyngoesophagus. There were four wound complications, all of which healed without further surgical procedures. The pectoralis major musculocutaneous flap continues to be the most versatile flap for reconstructions at a variety of sites in the head and neck area. Contrary to other reports, it has not been bulky, it has been used without previous delays of the skin paddle, it has not required skin grafts to close the donor sites, and it has been used without difficulty in five female patients.     

Differential cytokine expression in skin graft healing in inducible nitric oxide synthase knockout mice

Inducible nitric oxide synthase (iNOS) and its product, nitric oxide, have been shown to play important roles in wound biology. The present study was performed to investigate the role of iNOS in modulating the cytokine cascade during the complex process of skin graft wound healing.Fifteen iNOS-knockout mice and 15 wild-type C57BL/6J mice were subjected to autogenous 1-cm2 intrascapular full-thickness skin grafts. Three animals in each group were killed on postoperative days 3, 5, 7, 10, and 14. Specimens were then analyzed using nonisotopic in situ hybridization versus mRNA of tumor growth factor-beta1, vascular endothelial growth factor, iNOS, endothelial nitric oxide synthase (eNOS), tumor necrosis factor-alpha, and basic fibroblast growth factor, as well as positive and negative control probes. Positive cells in both grafts and wound beds were counted using a Leica microgrid. Scar thickness was measured with a Leica micrometer. Data were analyzed using the unpaired Student's t test.Expression of iNOS was 2- to 4-fold higher in knockout mice than in wild-type mice on postoperative days 5, 7, and 14. Expression of eNOS was 2- to 2.5-fold higher in knockout mice than in wild-type mice on postoperative days 5 and 7. Tumor necrosis factor-alpha expression was 2- to 7-fold higher in knockout mice than in wild-type mice on all postoperative days. In contrast, expression levels of angiogenic/fibrogenic cytokines (vascular endothelial growth factor, basis fibroblast growth factor, and tumor growth factor-beta1) were 2.5- to 4-fold higher in wild-type mice than in knockout mice. Scars were 1.5- to 2.5-fold thicker in knockout mice than in wild-type mice at all time points. All of the above results represent statistically significant differences (p < 0.05).Significantly different patterns of cytokine expression were seen in knockout and wild-type mice. Although the scar layer was thicker in knockout mice, it showed much greater infiltration with inflammatory cells. These data further delineate the modulatory effect of iNOS and nitric oxide in healing skin grafts.     

Preparation and evaluation of a nonproprietary bilayer skin substitute.

Cross-linked, allogeneic, telopeptide-depleted dermal grafts were lyophilized and laminated with silicone rubber elastomer. Resultant bilayers were studied for incorporation into the wound site and capacity to inhibit cutaneous wound contraction in experimental animals. Bilateral full-thickness skin wounds were made in 20 male New Zealand white rabbits. One side was grafted with the processed graft, while the contralateral side remained ungrafted as a control wound. Over 63 days, wound sites were analyzed at intervals on the basis of the extent and rate of wound contraction and by histologic examination. Cutaneous wounds successfully incorporated graft matrix and were significantly inhibited in their rate and extent of wound contraction. Notably, by day 63, grafted wounds retained 71 percent of their original area, whereas ungrafted control wounds retained only 16 percent of their original area. There were no graft rejections, and the bilayer graft's dermal analogue appeared to function as a biodegradable template that physically conformed neodermis to a preestablished pattern while counteracting contractile forces. This investigation suggests that, in experimental animals, the success of bilayer dermal grafts is less dependent on highly specialized and complex preparative techniques than typically has been presumed and that relatively simple, previously published, nonproprietary techniques, when adapted to a bilayer format, yield acceptable results as defined in terms of biocompatibility, capacity for graft incorporation, and inhibition of wound contraction.     

Mesenchymal stem cells are recruited into wounded skin and contribute to wound repair by transdifferentiation into multiple skin cell type

Mesenchymal stem cells (MSCs) can differentiate not only into mesenchymal lineage cells but also into various other cell lineages. As MSCs can easily be isolated from bone marrow, they can be used in various tissue engineering strategies. In this study, we assessed whether MSCs can differentiate into multiple skin cell types including keratinocytes and contribute to wound repair. First, we found keratin 14-positive cells, presumed to be keratinocytes that transdifferentiated from MSCs in vitro. Next, we assessed whether MSCs can transdifferentiate into multiple skin cell types in vivo. At sites of mouse wounds that had been i.v. injected with MSCs derived from GFP transgenic mice, we detected GFP-positive cells associated with specific markers for keratinocytes, endothelial cells, and pericytes. Because MSCs are predominantly located in bone marrow, we investigated the main MSC recruitment mechanism. MSCs expressed several chemokine receptors; especially CCR7, which is a receptor of SLC/CCL21, that enhanced MSC migration. Finally, MSC-injected mice underwent rapid wound repaired. Furthermore, intradermal injection of SLC/CCL21 increased the migration of MSCs, which resulted in an even greater acceleration of wound repair. Taken together, we have demonstrated that MSCs contribute to wound repair via processes involving MSCs differentiation various cell components of the skin.     

The use of human deceased donor skin allograft in burn care

Burns are tissue wounds caused by thermal, electrical, chemical cold or radiation injuries. Deep injuries lead to dermal damage that impairs the ability of the skin to heal and regenerate on its own. Skin autografting following burn excision is considered the current gold standard of care, but lack of patient’s own donor skin or unsuitability of the wound for autografting may require the temporary use of dressings or skin substitutes to promote wound healing, reduce pain, and prevent infection and abnormal scarring. These alternatives include deceased donor skin allograft, xenograft, cultured epithelial cells and biosynthetic skin substitutes. Allotransplantation is the transplantation of cells, tissues, or organs, sourced from a genetically non-identical member of the same species as the recipient. Human deceased donor skin allografts represent a suitable and much used temporizing option for skin cover following burn injury. The main advantages for its use include dermoprotection and promotion of reepithelialisation of the wound and their ability to act as skin cover until autografting is possible or re-harvesting of donor sites becomes available. Disadvantages of its use include the limited abundance and availability of donors, possible transmission of disease, the eventual rejection by the host and its handling storing, transporting and associated costs of provision. This paper will explore the role of allograft skin in burn care, defining the indications for its use in burn management and the future potential for allograft tissue banking.     

The role of serum imbibition for skin grafts

Numerous studies of grafted skin suggest that full-thickness skin grafts are nourished by exudate from the recipient bed called a serum imbibition. However, whether serum imbibition by itself is sufficient for nourishment of skin grafts has not been shown definitely and directly. To clarify the role of serum imbibition, we performed a comparative study between 20 skin grafts and 20 musculocutaneous flaps. The nourishment of the cell in the skin graft is by serum imbibition. That in musculocutaneous flaps is mainly derived from blood supply. We evaluated the nourishment by means of the unique characteristics of the cell cycle. Once cells are put into a synthetic phase, they cannot reverse or stop the progress of the cell cycle. To take advantage of this characteristic of the cell cycle, prewounding methods (40 flaps were lifted once and put back to the original sites prior to the evaluation) were intended for the cells in pre-elevated skin to turn into a proliferating phase. Cells were examined by antibody against proliferating cell nuclear antigen immunohistologically, to determine whether they had turned into the proliferating phase or not. After 3 days, all flaps were reelevated; half (20 flaps) had their muscle layer and the neurovascular bundle removed to make a full-thickness skin graft. The rest (20 flaps) were only lifted. They were sutured back to the original sites. Ten skin grafts and musculocutaneous flaps each were harvested at 3 hours (1st day) and at 11 days (11th day) after the second operation. Bromodeoxyuridine, which is a thymidine analog and is taken into the cells in the synthetic phase, was introduced intraperitoneally 2 hours before the harvest. All flaps and grafts were evaluated histologically and immunohistologically. Proliferating cell nuclear antigen analysis showed that the prewounding method induced the cells of skin grafts and musculocutaneous flaps to proliferate before the implantation. Regarding the bromodeoxyuridine uptake, no significant differences could be seen between skin grafts and musculocutaneous flaps irrespective of their different nourishment. No structural changes, such as degenerative or necrotic, could be seen at the hair follicle and other glands even at the 11th day. Almost all of the layers of skin grafts survived as long as they were checked by light microscopy (hematoxylin and eosin stain). No differences could be seen between musculocutaneous flaps and skin grafts or between the 1st and 11th days in this study. We concluded that serum imbibition is sufficient for nourishment of skin grafts, just as blood supply is sufficient for nourishment of musculocutaneous flaps.     

Topical application of plasma fibronectin in full-thickness skin wound healing in rats

Fibronectin (Fn) has been shown to play an important role in wound healing because it appears to be the stimulus for migration of fibroblasts and epidermal cells. The purpose of this study was to investigate whether topical application of plasma Fn (pFn) improves healing of full-thickness skin wounds in rats. A round section of full-thickness skin (diameter of approximately 15 mm) was resected in rats. Animals were then divided into two groups, and wounds were treated topically with a single application of human plasma albumin (control group) or human pFn (FN group). Wound closure rate, hydroxyproline concentration, and histologic features (immunohistochemical staining) were evaluated. The FN group had a significantly higher wound closure rate and hydroxyproline level in the skin than the control group. Histologic analysis of macrophage and fibroblast migration, collagen regeneration, and epithelialization were significantly increased in the FN group compared with the control group. A single topical application of pFn increased the migration of macrophages, myofibroblasts, and fibroblasts. Moreover, further release of transforming growth factor-beta1 from activated fibroblasts, keratinocytes, and epithelial cells may also contribute to the beneficial effect of pFn on wound healing.     

A potential wound healing-promoting peptide from frog skin

Cutaneous wound healing is a dynamic, complex, and well-organized process that requires the orchestration of many different cell types and cellular processes. Transforming growth factor β1 is an important factor that plays a key role during wound healing. Amphibian skin has been proven to possess excellent wound healing ability, whilst no bioactive substrate related to it has ever been identified. Here, a potential wound healing-promoting peptide (AH90, ATAWDFGPHGLLPIRPIRIRPLCG) was identified from the frog skin of Odorrana grahami. It showed potential wound healing-promoting activity in a murine model with full thickness dermal wound. AH90 promoted release of transforming growth factor β1 through activation of nuclear factor-κB and c-Jun NH2-terminal kinase mitogen-activated protein kinases signaling pathways, while inhibitors of nuclear factor-κB and c-Jun NH2-terminal kinase inhibited the process. In addition, the effects of AH90 on Smads family proteins, key regulators in transforming growth factor β1 signaling pathways, could also be inhibited by transforming growth factor β1 antibody. Altogether, this indicated that AH90 promoted wound healing by inducing the release of transforming growth factor β1. This current study may facilitate the understanding of effective factors involved in the wound repair of amphibians and the underlying mechanisms as well. Considering its favorable traits as a small peptide that greatly promoting generation of endogenous wound healing agents (transforming growth factor β1) without mitogenic effects, AH90 might be an excellent template for the future development of novel wound-healing agents.     

Immediate hair transplantation into a newly closed wound to conceal the final scar on the hair-bearing skin

A surgical incision after suturing usually leaves a visible scar on the hair-bearing skin, even after optimal wound conditions. The conspicuousness of such a scar results from its linear continuity and hairlessness. To prevent this effect, a row of micrografts or minigrafts was inserted between the wound edges immediately after wound closure. The hair grafts that were transplanted were dissected from the discharged skin in the same surgical procedure, if feasible. Otherwise, a mini donor strip was harvested from the mastoid scalp to dissect the hair grafts. The final linear scar was interrupted and concealed sufficiently with the growth of the transplanted hairs. Tension-free closure is required to obtain a satisfactory result with this technique.     

Wet wound healing

Wound treatment in a flexible transparent chamber attached to the perimeter of the wound and containing a liquid has been extensively tested in preclinical experiments in pigs and found to offer several advantages. It protects the wound; the liquid medium or saline in the chamber provides in vivo tissue culture-like conditions; and antibiotics, analgesics, and various molecules can be delivered to the wound through the chamber. The wound chamber causes no injury to the wound itself or to the surrounding intact skin. Topical delivery of, for instance, antibiotics can provide very high concentrations at the wound site and with a favorable direction of the concentration gradient. A series of 28 wounds in 20 patients were treated with a wound chamber containing saline and antibiotics. Most patients had significant comorbidity and had not responded to conservative or surgical management with débridement and delayed primary closure or skin grafts. Six wounds had foreign bodies present; four of these were joint prostheses. Seven patients were on corticosteroids for rheumatoid arthritis, lupus, or chronic obstructive pulmonary disease, and four patients had diabetes. Most patients were treated with the wound chamber in preparation for a delayed skin graft or flap procedure, but one was treated with a wound chamber until the wound healed. Twenty-five of the wounds (89 percent) healed, and five wounds (18 percent) required additional conservative management after the initial chamber treatment and grafting procedure. Of the three wounds that did not heal, one healed after additional chamber treatment, one had a skin graft that did not take, and one required reamputation at a higher level. Antibiotic delivery was less than one intravenous dose daily, which avoided the potential for systemic absorption to toxic levels. Antibiotics such as vancomycin and gentamicin could be used in concentrations of up to 10,000 times the minimal inhibitory concentration. Forty-eight hours after application, 20 percent or more of the original antibiotic concentration was present in the wound chamber fluid. In conclusion, the wound chamber provides a safe, powerful tool in the treatment of difficult infected wounds.     

Wound healing enhancement in leg ulcers: A case report

Wound healing in the skin is a complex biological process in which numerous types of cells, cytokines, growth factors, proteases and extracellular matrix components act in concert to restore the integrity of injured tissue. Cultivated allogenic human keratinocytes have been used for the treatment of various skin defects like burnwounds, surgical wounds, in exfoliative skin diseases and chronic wounds. A new method for wound healing enhancement in leg ulcers using cultured allogenic keratinocytes suspended in fibrin glue and used in spray technique is introduced. Allogenic keratinocytes are supposed to enhance granulation tissue production and to stimulate reepithelisation due to their release of growth factors and thus are able to recreate an active wound. This revised version was published online in July 2006 with corrections to the Cover Date.     

Comparison of proliferation and motile activity between human keratinocytes isolated from skin and oral mucosa

OBJECTIVE: Epithelial wound repair assures the recovery of the epithelial barrier after wounding. During wound healing epithelial cells migrate to cover the wound surface. For healing of skin wounds the skin keratinocytes can be replaced by oral mucosa epithelial cells grown in vitro. The presented experiments were carried out in order to compare the proliferation, morphology, and migration between human keratinocytes isolated from human skin and oral mucosa. MATERIALS AND METHODS: Human epidermal and oral mucosa keratinocytes from primary culture were used in all experiments. Cell motility and shape were determined using computer-aided methods. RESULTS AND CONCLUSIONS: It was demonstrated that although both cell types exhibit the same typical epithelial morphology, oral mucosa keratinocytes locomote significantly faster than skin keratinocytes. They also differ in proliferation activity. Oral mucosa keratinocytes exhibited faster growth and different actin cytoskeleton organisation than skin keratinocytes under in vitro conditions. Autologous oral mucosa keratinocytes may be expanded in vitro and used for skin wound healing in vivo.