Bernard Delaey

In vitro and in vivo biocompatibility of dextran dialdehyde cross-linked gelatin hydrogel films.

The biosafety of a new hydrogel wound dressing material consisting of dextran dialdehyde cross-linked gelatin was evaluated (i) in vitro in cultures of dermal fibroblasts, epidermal keratinocytes, and endothelial cells, three cell types which play a major role in the process of cutaneous wound healing, and (ii) in vivo by subcutaneous implantation studies in mice. The cytotoxicities of this hydrogel, two semi-occlusive polyurethane dressings (Tegaderm and OpSite), and a hydrocolloid dressing (DuoDERM) were compared by measuring cell survival with the tetrazolium salt reduction (MTT) assay after incubations of the wound dressing samples for up to 6 d, in the presence of--but not in direct contact with--the cells. In vitro, the degree of cytotoxicity of the new hydrogel was greater in keratinocyte cultures than in fibroblast and endothelial cell cultures, and increased upon longer incubation time. In keratinocyte cultures, the semi-occlusive polyurethane dressings, the hydrocolloid, and the hydrogel dressings induced low, high and acceptable degrees of cytotoxicity, respectively. The toxicity of the isolated hydrogel components was assessed in Balb MK keratinocyte cultures. In these cells, epidermal growth-factor-stimulated thymidine incorporation into DNA was higher in the presence of gelatin. By contrast, concentrations of dextran dialdehyde as low as 0.002% were found to significantly decrease thymidine incorporation (P < 0.01). Subcutaneous implantation studies in mice showed that in vivo the hydrogel was biocompatible since the foreign body reaction seen around the implanted hydrogel samples was moderate and became minimal upon increasing implantation time. These results indicate that dextran dialdehyde cross-linked gelatin hydrogels have an appropriate biocompatibility.

In vitro release characteristics of bioactive molecules from dextran dialdehyde cross-linked gelatin hydrogel films

Hydrogel films, prepared by cross-linking of gelatin with dextran dialdehydes (weight ratio 2:1), and containing either fluorescein isothiocyanate dextran (Mw 70000) or polypeptides were evaluated in terms of their release characteristics and mechanical properties upon increasing storage time at 4 degrees C. Important changes in release kinetics and mechanical properties of the cross-linked gelatin films were observed, especially during the first week after the hydrogel production. Rheological and NMR measurements showed that the mechanical properties of the gelatin hydrogel films were improved with increasing storage time. It appeared that the process of chemical cross-linking and physical structuring of the gelatin hydrogel matrix did not occur instantaneously and substantially influenced the polypeptide release patterns. Cross-linked gelatin hydrogels were found to be appropriate release systems for medium-term sustained delivery of biologically active epidermal growth factor (EGF), but release characteristics were strongly dependent on the nature of the protein which was incorporated.

Lyophilized keratinocyte cell lysates contain multiple mitogenic activities and stimulate closure of meshed skin autograft-covered burn wounds with efficiency similar to that of fresh allogeneic keratinocyte cultures.

For several years, grafting with allogeneic keratinocyte cultures has been used successfully as a wound-healing therapy both by us and by many other groups. Since their postgrafting survival time is limited, the effect of these cultures is generally explained by the production of wound repair-stimulating factors that promote proliferation and migration of resident cells. In this study we show that lysates of cultured keratinocytes contain mitogenic activity for keratinocytes, endothelial cells, and fibroblasts. In addition, the lysates inhibit the contraction of collagen gels by human skin fibroblasts. On the basis of these observations and of in vivo data obtained by ourselves and others, we have evaluated the effect of total keratinocyte lysates on the healing of meshed skin autograft-covered burn wounds. Twenty burn wounds were tangentially excised and autografted with one to three meshed conventional skin transplants. An area treated with a gel containing lysated keratinocyte cultures was compared with an area treated with placebo-gel in terms of epithelialization on day 5. In six patients an additional fresh keratinocyte alloculture was applied as a positive control. Results indicate that the newly formed epithelium (difference between percentage of epithelialization on day 5 and on day 0) was 31.1 percent in the treated area compared with 16.5 percent in the placebo area. This result is comparable with the value obtained by treatment with fresh keratinocyte allocultures, namely, 33.8 percent. These figures show a twofold stimulation of epithelialization.

Efficacy and safety of the freeze-dried cultured human keratinocyte lysate, LyphoDerm 0.9%, in the treatment of hard-to-heal venous leg ulcers.

LyphoDermTM (XCELLentis, Belgium) is an end‐sterilized, freeze‐dried lysate from cultured allogeneic epidermal keratinocytes, formulated into a hydrophilic gel. Its efficacy and safety were evaluated, in combination with standard care (hydrocolloid dressing and compression therapy), in 194 patients suffering from hard‐to‐heal (lasting more than 6 weeks and not responding to conventional therapy) venous leg ulcers. Two control groups received standard care, with or without vehicle, respectively. Patients had a median age of 67.5 years and the majority were females (61%). The median duration of the ulcer was 43 weeks and in 39% of the subjects it had been present for more than 1 year. Thirty‐eight percent of the patients in the standard care + LyphoDermTM group had complete ulcer healing within 24 weeks (primary end point) compared to 27% of patients in the standard care + vehicle pooled groups (P = 0.114) in the “as treated” intent‐to‐treat cohort (37% vs. 27% in the “as randomized intent‐to‐treat cohort; p = 0.137). In the subgroup of patients with enlarging ulcers, the difference between the two groups was significant (30% vs. 11%; p = 0.024 in the “as treated” intent‐to‐treat cohort and 31% vs. 9%; p = 0.005 in the “as randomized” intent‐to‐treat cohort). LyphoDermTM was well tolerated and safe, and no differences in the frequency of adverse events were noted between the treatment groups. Although the primary objective of the study was not achieved, the exploratory analysis carried out in patients with enlarging ulcers suggests that LyphoDermTM could offer a new prospect for the treatment of patients with venous ulcers that may prove to be a significant adjunct to the overall provision of care.

A Prospective Multicenter Study of the Efficacy and Tolerability of Cryopreserved Allogenic Human Keratinocytes to Treat Venous Leg Ulcers

Allogeneic human keratinocyte cultures have been used to treat burn wounds, donor sites, and chronic skin ulcers with some success. Cryopreservation of these cultures allows for the production of large standardized batches that are readily available for use. The aim of the study presented in this report was to study effects of cryopreserved cultured allogenic human keratinocytes (Cryo Ceal) on chronic lower extremity wounds. Parameters were measured to study efficacy, tolerability, pain associated with chronic wounds, and quality of life of patients. Twenty-seven patients with hard-to-heal venous leg ulcers received a maximum of 9 applications of Cryo Ceal in a prospective, uncontrolled multicenter study lasting 48 weeks. Eleven out of 27 patients (41%; 95% CI: 22%-61%) had complete wound closure within 24 weeks (1 week). The time required for complete wound closure in these 11 patients ranged from 4.1 to 24.9weeks. Only 1 patient had recurrence of the ulcer at 48 weeks. Local (wound) pain scores decreased from a mean of 2.5 at baseline to 0.9 at week 24. Fifty percent of the patients attained a pain score of 0 after 12 weeks and remained stable at this score until the end of the study. Overall, the patient quality of life was better at week 24, compared to baseline values. The treatment was well tolerated, and wound infection was the most frequently occurring adverse event.

Healing of full-thickness wounds in pigs: effects of occlusive and non-occlusive dressings associated with a gel vehicle.

This study, based upon a pig model, was conducted to investigate the effects of moist and dry healing conditions on wound closure (epithelialization, granulation tissue, contraction) of full-thickness wounds. Thirty-two full-thickness square wounds (3 cm x 3 cm) covered with either an occlusive polyurethane dressing (Tegaderm) or a non-occlusive dressing (Melolin) were evaluated. The effect of the presence or the absence of a gel (3% Idroramnosan) was also investigated with both dressings. The dressings were renewed twice a week. The time required for wound closure was 19.2 +/- 1.6 days for Tegaderm and 26.6 +/- 3.0 days (means +/- SD) for Melolin, respectively. The healing time of the full-thickness porcine wounds was significantly (P < 0.001) reduced by the occlusive dressing. Equivalent results were found with the 3% gel, indicating that the gel can be used as a neutral vehicle. The healing rate, calculated according to Gilmans method, was also significantly (P < 0.001) enhanced by the occlusive dressing. This progression was 0.073 +/- 0.004 cm/day and 0.050 +/- 0.009 cm/day (means +/- SD) for Tegaderm and Melolin, respectively. The contribution of contraction to wound closure was similar in all wounds, indicating that the occlusive dressing did not have an effect on wound contraction. Histological evaluation was performed on full-thickness skin biopsies of whole wound harvested from the time of wound closure to 3 months after. At any time point, no significant histological variations were observed between the different treated wounds. This study demonstrates in a porcine model that for full-thickness wounds, as for split-thickness wounds, occlusive dressing enhances healing rate and shortens the time for wound repair. The shortened healing time is a function primarily of the effect of occlusive dressing on epithelialization, especially the third phase of wound resurfacing.

Lysates from Cultured Allogeneic Keratinocytes Stimulate Wound Healing after Tympanoplasty

In the past, cultured keratinocyte allografts have been used with benefit in the treatment of burn wounds and leg ulcers. Since in burn wounds autologous and allogeneic fresh keratinocyte cultures were found to give similar favorable results as lysates of allogeneic cultured cells, the authors investigated whether this lysate mixed in an antibiotic suspension would also accelerate the epithelial healing after routine tympanoplasty. In a double blind setting the healing process in 50 consecutive tympanoplasty ears was studied: an acceleration of healing of 8 days was observed in the lysate-treated group (39.25 days) as compared with the control group (47.23 days). The percentage of ears which healed within 6 weeks (after 5 weekly applications of 200 microliters suspension in both groups) was significantly higher in the treated group (61%) than in the control population (36%). Although the therapeutical effect of the keratinocyte lysate in this study is believed to be due primarily to its mitogenic activity through growth factors or cytokines, at present it is still unclear which growth factors are involved and which combinations of these factors have to be present to modulate the different stages of the complex healing processes.

Treatment of chronic postoperative otorrhea with cultured keratinocyte sheets.

Cultured allogeneic ear keratinocyte sheets were used to treat 26 ears presenting with long-standing (average 37 months) chronic otorrhea, resistant to regular treatment, long after surgery for atresia (n = 8), cholesteatoma (n = 10), and chronic otitis media (n = 8). Complete epithelial healing and cessation of otorrhea were obtained in 18 cases (69%), following an average of 2.2 weekly applications. Temporary epithelial healing lasting at least 3 months was observed in 3 patients (12%) subsequently needing repeated applications. Lack of complete epithelialization was documented in 5 cases (19%). In 3 of those 5 cases, a reason could be determined. The authors speculate that the allocultured keratinocytes are able to promote migration and proliferation of resident cells at the wound edges, despite their short survival time, by release of keratinocyte-stimulating factors.

Healing of full‐thickness wounds treated with lyophilized cultured keratinocyte cell lysate in genetically diabetic mice

The effect of a lyophilized cell lysate prepared from cultured human keratinocytes on the healing of full‐thickness wounds was evaluated in an impaired healing model. Full‐thickness wounds (8 mm in diameter) were made on the dorsal areas of female genetically diabetic mice C57 BL/KsJ (db/db) and their normal (db/+) littermates. Wounds were covered with an occlusive polyurethane film dressing and were treated for 5 days either with the lyophilized cell lysate from cultured human keratinocytes prepared in phosphate‐buffered saline solution or with phosphate‐buffered saline solution. In normal (db/+) mice, all wounds were closed 16 days after wounding, and more than 90% of the wound closure was due to wound contraction. Wound contraction accounted for a similar extent of wound closure in both lyophilized cell lysate‐treated and phosphate‐buffered saline solution‐treated wounds. In contrast, in the diabetic (db/db) mice, after histologic examination of the wounds 32 days after wounding, four of ten lyophilized cell lysate‐treated wounds and four of seven phosphate‐buffered saline‐treated wounds were found to be closed. Moreover, applications of lyophilized cell lysate from cultured human keratinocytes to full‐thickness wounds in diabetic db/db mice significantly decreased the contribution of contraction to wound closure. Day 32 after wounding, contraction contribution to wound closure amounted to 57.7%± 4.7% and 80.4%± 3.2% (mean ± standard error of the mean, p < 0.005) of the initial wound areas, respectively, for lyophilized cell lysate‐treated and phosphate‐buffered saline solution‐treated wounds. At this time of wound healing, the thickness of the dermis was increased 1.7‐fold by the keratinocyte cell lysate treatment, but neither epithelial migration from the wound edges nor the thickness of the regenerated epithelium were significantly affected. In conclusion, in diabetic (db/db) mice the application of lyophilized cell lysate from cultured human keratinocytes influenced the healing of the dermis and wound contraction, but had no effect on reepithelialization.