Justin R. Sharpe

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.

Effect of artificial dermal substitute, cultured keratinocytes and split thickness skin graft on wound contraction

In this study, the effect of different wound treatments on contraction was evaluated in an established porcine model. In two separately conducted experiments full thickness wounds treated with artificial dermal substitute, split thickness skin graft (STSG), meshed STSG applied in combination with cultured keratinocytes or meshed STSG alone were compared with untreated wounds. The surface area of all wounds was quantified at regular time intervals. After 20 days wounds from some groups were subjected to histological analysis to establish the degree of epithelialization. Wounds treated with STSG contracted more than with artificial dermal substitute until day 21. From day 21 to day 35 wounds treated with STSG showed the least contraction. Wounds sprayed with cultured keratinocytes demonstrated a slower rate of contraction than those with meshed STSG alone after 20 days. The untreated control wounds showed a greater rate of contraction and had almost closed by day 20. This study demonstrates that there is a significant difference in contraction between wounds treated with artificial dermal substitute and control wounds and between wounds treated with STSG with cultured keratinocytes and meshed STSG alone. STSG with cultured keratinocytes, unmeshed STSG, and artificial dermal substitute all reduced wound contraction significantly.

Use of a novel porcine collagen paste as a dermal substitute in full‐thickness wounds

A commercially available porcine collagen sheet material has been found previously to be useful as an implant for reconstructive surgery. However, its use as a dermal substitute has been hindered by slow cell penetration and vascularization. A novel paste formulation of this material was investigated for its potential role as a dermal substitute in full‐thickness wounds. A porcine punch biopsy model was initially used to assess the integration of a wide range of material formulations. Selected formulations were then assessed further in a larger wound‐chamber model. Paste formulations were compared with those of sheet and another commercially available dermal regeneration template. The porcine collagen paste became integrated into full‐thickness wounds without rejection and without excessive inflammation. It was detected in wounds up to day 27 postimplantation. Porcine collagen paste was readily infiltrated by host cells by day 2 and supported migrating keratinocytes on its surface. Staining for endothelial cells indicated neovasculature formation as early as day 4 and functional newly formed microvessels were noted at day 7. This was comparable with neovascularization of an alternative and clinically proven dermal regeneration template and was significantly superior to the sheet material formulation at the same time points. Our findings suggest that porcine collagen paste may be suitable as an alternative to current dermal substitutes in full‐thickness wounds.

Progression of wound pH during the course of healing in burns.

The aim of this study was to measure the pH on the wound surface of 30 burn patients and test the hypothesis that wound surface pH is correlated to healing time and burn depth. Inclusion criteria were any adult outpatient with burn injury. Patient age was 17 to 75 years (mean, 44), burn depth ranged from superficial to full thickness with a TBSA of 0.4 to 4%. Cause of burn included scalds, flame burn, and contact burns. On admission, and at each dressing change, the pH on the wound surface was measured. The pH in both healing and nonhealing wounds was found to decrease with each dressing change. At the second dressing change, wounds that went on to heal were found to have a significantly lower pH of 7.32 in comparison with pH 7.73 in wounds that failed to heal and therefore required subsequent grafting (P = .004). Wound pH was also correlated to depth at the second dressing change (superficial = pH 6.05, full thickness = pH 8.0). The correlation between pH and wound outcome could be used as an additional diagnostic tool to predict poor healing in wounds. Early identification of a nonhealing wound may allow a more aggressive treatment regimen, including skin grafting, to bring about rapid wound healing.

A fully autologous co-culture system utilising non-irradiated autologous fibroblasts to support the expansion of human keratinocytes for clinical use

Autologous keratinocytes can be used to augment cutaneous repair, such as in the treatment of severe burns and recalcitrant ulcers. Such cells can be delivered to the wound bed either as a confluent sheet of cells or in single-cell suspension. The standard method for expanding primary human keratinocytes in culture uses lethally irradiated mouse 3T3 fibroblasts as feeder cells to support keratinocyte attachment and growth. In an effort to eliminate xenobiotic cells from clinical culture protocols where keratinocytes are applied to patients, we investigated whether human autologous primary fibroblasts could be used to expand keratinocytes in culture. At a defined ratio of a 6:1 excess of keratinocytes to fibroblasts, this co-culture method displayed a population doubling rate comparable to culture with lethally irradiated 3T3 cells. Furthermore, morphological and molecular analysis showed that human keratinocytes expanded in co-culture with autologous human fibroblasts were positive for proliferation markers and negative for differentiation markers. Keratinocytes expanded by this method thus retain their proliferative phenotype, an important feature in enhancing rapid wound closure. We suggest that this novel co-culture method is therefore suitable for clinical use as it dispenses with the need for lethally irradiated 3T3 cells in the rapid expansion of autologous human keratinocytes.

The effect of topical analgesics on ex vivo skin growth and human keratinocyte and fibroblast behavior

The application of topical analgesics to the donor site of split thickness skin grafts has been proven to be an effective method of pain management but little is known about their effects on wound reepithelialization. This study compares the effect of four analgesics on human keratinocytes and fibroblasts and whole skin explants in vitro to determine whether epithelial cell behavior is affected by topical analgesics. The effect of diclofenac, bupivacaine, lidocaine, and ketorolac was studied at concentrations between 10 mM and 1 nM. The effect on epithelial growth was measured using an ex vivo skin explant model. In addition, cell proliferation, and cytotoxicity were measured in cultured primary human keratinocytes and fibroblasts. Epithelial growth from the explant model was most inhibited by diclofenac with a significant reduction at 100 μM (p=>0.001). Diclofenac also exhibited the strongest inhibitory effect on cell proliferation especially in keratinocytes. Ketorolac was the most cytotoxic. Bupivacaine showed cytotoxicity in a dose‐dependent manner with only the very highest concentrations having a significant inhibitory effect. Lidocaine showed no evidence of cytotoxicity at the concentrations tested in either the in vitro cell studies or the ex vivo explant model. Topical analgesics alter keratinocyte and fibroblast behavior and such inhibition may affect wound healing.

A Novel Skin Substitute Biomaterial to Treat Full-Thickness Wounds in a Burns Emergency Care

A novel porcine collagen-based paste dermal substitute to treat full-thickness wounds has been investigated. A thin split-thickness skin graft or autologous cultured keratinocytes have been combined with dermal replacement biomaterial and applied to full-thickness wounds in a porcine wound chamber preclinical experimental model. The data obtained suggest that: (1) dermal substitute biomaterials may improve wound re-epithelialisation when combined with cultured autologous keratinocytes and (2) porcine collagen paste is able to support split-thickness skin graft survival as well as autologous cultured keratinocyte proliferation. These results demonstrate that the novel porcine collagen paste has a potential as a dermal substitute to treat acute full-thickness wounds and an application for the burns emergency care treatment.