Robin Martin

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.

Retroviral labeling of Schwann cells: In vitro characterization and in vivo transplantation to improve peripheral nerve regeneration

Transplantation of Schwann cells (SCs) is a promising treatment modality to improve neuronal regeneration. Identification of the transplanted cells is an important step when studying the development of this method. Genetic labeling is the most stable and reliable method of cell identification, but it is still unclear whether it has deleterious effect on SC characteristics. Our aim was to achieve a stable population of SCs transduced with the lacZ gene at a high frequency using a retroviral vector in vitro, and to follow the labeled SC in vitro to assess their viability and phenotypic marker expression. Furthermore, we transplanted lacZ‐labeled SCs in a conduit to repair peripheral nerve to investigate their effect on nerve regeneration in vivo. Rat and human SCs were cultured and transduced with an MFG lacZ nls marker gene, achieving a transduction rate of 80% and 70%, respectively. Rat SCs were kept in culture for 27 weeks and examined every 4 weeks for expression of lacZ, viability, and phenotypic marker expression of GFAP, p75, MHC I and II. Throughout this period, transduced rat SCs remained viable and continued to proliferate. The proportion of cells expressing lacZ dropped only by 10% and the expression of phenotypic markers remained stable. Transduced human SCs were followed up for 4 weeks in culture. They proliferated and continued to express the lacZ gene and phenotypic marker expression of GFAP and p75 was preserved. Primary culture of transduced rat SCs were transplanted, syngeneically, in a conduit to bridge a 10 mm gap in sciatic nerve and the grafts were examined after 3 weeks for the presence and participation of labeled SCs and for axonal regeneration distance. Transplanted transduced rat SCs were clearly identified, taking part in the regeneration process and enhancing the axonal regeneration rate by 100% (at the optimal concentration) compared to conduits without SCs. Thus, retroviral introduction of lacZ gene has no deleterious effect on SCs in vitro and these SCs take part and enhance nerve regeneration in vivo. GLIA 34:8–17, 2001.

The potential for eye bank limbal rings to generate cultured corneal epithelial allografts.

Purpose. Patients with severe limbal deficiencies are unable to maintain a stable corneal surface. If sheets of cultured allogeneic corneal epithelium could be prepared from eye banked corneal limbal rings, which are normally discarded after keratoplasty, the sheets may be beneficial for grafting onto patients with limbal stem cell deficiencies. Method. Biopsies of limbal tissue (2–3 mm2) removed from organ-cultured corneal limbal rings or from fresh whole globes were either trypsinized or set up as explants to assess their potential for corneal epithelial cell production. Results. Several biopsies were taken from each of 21 organ-cultured limbal rings and 10 fresh cadaveric globes. Cultures were generated from every cadaveric eye (10/10), although not all biopsies from the same eye gave rise to cultures. Confluent sheets of cultured cells were also produced successfully from limbal rings that had been in organ culture for up to 25 days, but the success rate from limbal ring material was variable (14/21). An analysis of parameters associated with each limbal ring was carried out in an attempt to identify the reasons for the different efficiencies of epithelial production. No obvious single parameter correlation was detected, although there was a trend to poorer efficiency with increased donor age. Conclusions. Confluent sheets of cultured corneal epithelial cells, suitable for grafting, can be produced from limbal tissue taken from eye bank organ-cultured corneas, although it takes longer, on average, to reach confluence (17–21 days) than an equivalent sample from a fresh eye (9–12 days).

Upward migration of cultured autologous keratinocytes in Integra artificial skin: a preliminary report

The combination of cultured autologous keratinocytes with the dermal regeneration template Integra™ could offer increased possibilities for reconstructive surgery and wound healing. A single‐step application of cells, centrifuged deep into an Integra™‐like matrix at the silicone–matrix junction, has been described but might prove technically complex for clinical use. We have investigated the possibility of simplifying this procedure by applying cultured cells directly to the underside of the Integra™ or directly to the wound bed immediately prior to grafting. The objective was to see whether cells would migrate through the matrix in an upward direction. We tested the principle of this concept using a pig wound healing model. Integra™ was seeded directly with cultured cells and grafted onto fresh full‐thickness wounds, or unseeded Integra™ was applied to freshly excised wound beds that had just been seeded with the same number of cells. Biopsies were taken at 3, 7, 11, and 14 days. Histological sections showed that the cells moved through the Integra™ to give a confluent surface epithelium. Direct seeding onto the Integra™ was the most efficient method. Transduction of cultured autologous keratinocytes in vitro with a MFGlacZnls retrovirus confirmed that the epidermis was derived from the cultured autologous keratinocytes. (WOUND REP REG 2003;11:132–138)

Demonstration of epidermal transfer from a polymer membrane using genetically marked porcine keratinocytes

The culture of keratinocytes on flexible membranes has been proposed as a means to simplify, accelerate and improve the efficiency with which proliferating cells are delivered to full thickness or non-healing skin defects. However, there have been no studies that monitor the transfer of cells from such membranes to the wound bed. We have used a porcine model of lacZ gene marked cultured autologous keratinocyte grafting to demonstrate unambiguously the transfer of cultured cells to cutaneous wounds from the EpiGen polymer membrane developed by Smith & Nephew Group plc. Full thickness wounds enclosed within rigid chambers were first grafted with autologous de-epidermalised dermis (DED). Keratinocytes were cultured on EpiGen membranes and applied to the wound beds 7 days after the DED grafts. Epidermal remnants persist within the DED and the resultant epidermis is therefore, a mixture of wound regeneration and delivered cultured cells. Unequivocal evidence for keratinocyte transfer from the membrane was obtained through the observed macroscopic surface staining for lacZ transduced cells and lacZ positive cells detected in sections through deeper layers of epidermal tissue. This method offers a general approach for evaluating the efficiency of keratinocyte delivery using upside-down flexible membrane transfer.

Long-term effect of vital labelling on mixed Schwann cell cultures.

Schwann cell transplantation following neuronal injury could encourage regeneration of spinal cord as well as improving peripheral nerve gap repair. In order to gain a better understanding of the role of transplanted Schwann cells in vivo, it is essential to be able to follow their behaviour after transplantation. Our aim was to evaluate the suitability of two vital fluorescent labels on the proliferation rate and phenotypic stability of Schwann cells, in either pure culture or mixed co-culture. Primary cultures of Schwann cells were obtained from Dark Agouti and Lewis neonatal rats and labelled with H33342 and PKH26, respectively. In mixed cultures, a 50 : 50 mixture of Dark Agouti and Lewis Schwann cells was present. Labelled cultured cells were examined at 1, 2 and 4 weeks for viability and phenotypic marker expression of S100, GFAP, p75, MHC I, MHC II and compared with corresponding unlabelled cells. The results showed that although there was no deleterious interaction in the mixed cultures, the viability was reduced by the labelling after 2 weeks. Labelled cells could be distinguished up to 4 weeks, but there was leakage of H33342 label after 2 weeks. Labelled Schwann cells showed reduced expression of phenotypic markers, especially p75 when labelled with H33342. In conclusion, H33342 and PKH26 can be used as fluorescent markers of Schwann cells for short-term studies, for a maximum of 2 weeks, but different markers may be needed for longer experiments.

Retroviral gene transfer into porcine keratinocytes following improved methods of cultivation.

We embarked on a program examining the application of cultured epithelial sheets to skin wounds in pigs using retroviral gene transfer as a means to follow the grafted cells. In the past similar studies have been hampered by an inability to grow porcine keratinocytes without seeding at an extremely high density. In this study we found that excellent results could be achieved with Opti-MEM-1 (Gibco BRL Life Technologies) containing 1 per cent foetal calf serum, 0.5 mM Ca2+ and no other growth factors or stimulants. Keratinocytes were plated on gamma-irradiated 3T3 feeders on surfaces which had previously been coated with rat tail collagen I. Keratinocyte cultures were established at a seeding density of 5 x 10(4) cm-2. The yield of cells from 1 cm2 of skin was sufficient to set up a 75 cm2 flask. Cultures reached 80-90 per cent confluence in 7-10 days, after which they were passaged 1:3 multiple times, taking 3-4 days to reach the same confluency. Allowing cultures to remain confluent for 1 week was sufficient to allow Dispase removal of an intact sheet. Using these techniques porcine keratinocytes were transduced at an average frequency of 25.3 per cent (+/- 14.0 SEM) with the retroviral vector MFG lacZ nls by growth on the gamma-irradiated retroviral producer line GP + envAm12.

Nitric Oxide Production in Burns: Plasma Nitrate Levels Are Not Increased in Patients with Minor Thermal Injuries

BACKGROUND Recent studies have suggested that adults who sustain burns of less than 15% total body surface area display elevated plasma nitrate levels, indicating increased production of nitric oxide. The present study was initiated to confirm whether plasma nitrate is elevated in minor burn injury and, if so, whether it heralds the onset of a systemic inflammatory response to that injury. METHODS Plasma samples were taken from 98 control and 10 burns patients. RESULTS The mean plasma nitrate level for nine burns patients with a mean total body surface area burnt of 7.65% (range, 4-15%) was 42.83 micromol/L on day 1. This was not significantly different from that of a control population of 98 preoperative plastic surgery patients: 36.91 micromol/L (p = 0.162). Eight of 10 burns patients showed a decrease in plasma nitrate to 27.47 micromol/L by day 3 (p = 0.046). Elevated nitrate levels were seen in 2 of 10 burns patients. One had concurrent smoke-inhalation injury preceding multiple organ dysfunction, and one was treated with a cream containing cerium nitrate (Flammacerium, Duphar Laboratories, Southhampton, United Kingdom). CONCLUSIONS For patients who sustain minor burns, plasma levels of nitrate decrease from those of mean normal controls with time unless there is multiple organ dysfunction or the patient receives extraneous nitrate.