Cultured Keratinocyte Sheets Research Articles

Preserved extracellular matrix in non‐enzymatically detached cultured keratinocyte sheets bolsters the healing effects on grafted burn wounds

Cultured autologous keratinocyte sheets have been proposed for treatments of burn wounds. However, this method has not been successfully translated to clinical practice because of certain limitations, such as inconsistency of engraftment, incidence of infection, spontaneous blistering, and scar contracture. Enzymatic detachment of cultured keratinocyte sheets (KS) from culture dishes was attributed to these negative outcomes. To overcome this problem, we are testing a novel technology that allows the detachment of cultured keratinocyte sheets without using any enzymatic treatment. We compared efficacy of keratinocyte sheets cultured on 2 different dishes: temperature responsive (TRD) and conventional. Cells cultured on TRD were detached by temperature reduction (T‐KS), whereas the cells cultured on conventional dish were detached by enzyme, Dispase (D‐KS). We studied efficacy of these sheets (T‐KS and D‐KS) on both in vitro and in vivo burn wound models and characterized downstream mechanisms underlying differences between the two sheets.In that study, we were found that T‐KS were significantly thicker, preserved more ECM and cytoskeleton proteins, when compared to D‐KS. Therefore, the integrity of T‐KS, tested using mechanical cell dissociation assay, was still intact, while D‐KS was fragmented to multiple pieces. This was correlated with significantly higher number of disassociated cells in D‐KS compared to T‐KS. Also, the reduction in cytoskeleton proteins were associated with significant shrinkage of D‐KS compared to T‐KS. We created in vitro burn skin wound healing model and tested the efficacy of both types of cultured KSs and investigated their proliferation and cellular mechanisms. We found that T‐KS had significantly higher rate of proliferation compared to D‐KS. Also, the activation of MAPK, was lower in the sheet detached by dispase treatment compared to temperature reduction.We wanted to compare wound healing efficacy of cultured T‐KSs and DKSs in 3rd degree ovine burn wounds grafted with cadaver skin. We designed the experiments in such way that closely mimics clinical situation. The burned skin was excised 24 hrs postinjury, followed by grafting with sheep cadaver skin. The formation of KSs was completed at the time of grafted cadaver skin rejection, thus enabling timely transplantation of KSs to the wounds. By using planimetric analysis, we found that wounds treated with T‐KSs had a significantly higher epithelization rate, firmer dermal‐epidermal junction when compared to D‐KSs and control wounds at 14 days of grafting. The vascularization rate tended to be higher in the T‐KSs‐treated wound beds compared to the D‐KS wounds. Next, we have measured a potent growth factor KGF, which is highly expressed in early wound healing. The level of KGF, in the wounds treated with T‐KS tended to be lower reflecting more mature wounds treated with T‐KSs.Taken together, above results suggest overall superior quality of T‐KSs and immature sheet integrity and fragility in D‐KSs were linked to use of enzyme for detachment that disrupted ECM.

Adult Burn Patients With More Than 60% TBSA Involved–Meek and Other Techniques to Overcome Restricted Skin Harvest Availability–The Viennese Concept

Despite the fact that early excision and grafting has significantly improved outcome over the last decades, the management of severely burned adult patients with >/=60% total body surface area (% TBSA) burned still represents a challenging task for burn care specialists all over the world. In this article, we present our current treatment concept for this entity of severely burned patients and analyze its effect in a comparative cohort study. Surgical strategy comprised the use of split-thickness skin grafts (Meek, mesh) for permanent coverage, fluidized microsphere bead-beds for wound conditioning, temporary coverage (polyurethane sheets, Epigard; nanocrystalline silver dressings, Acticoat; synthetic copolymer sheets based on lactic acid, Suprathel; acellular bovine derived collagen matrices, Matriderm; allogeneic cultured keratinocyte sheets; and allogeneic split-thickness skin grafts), and negative-pressure wound therapy (vacuum-assisted closure). The autologous split-thickness skin graft expansion using the Meek technique for full-thickness burns and the delayed approach for treating dorsal burn wounds is discussed in detail. To demonstrate differences before and after the introduction of the Meek technique, we have compared patients of 2007 with >/=60% TBSA (n = 10) to those in a matched observation period (n = 7). In the first part of the comparative analysis, all patients of the two samples were analyzed with regard to age, abbreviated burn severity index, Baux, different entities of % TBSA, and survival. In the second step, only the survivors of both years were separated in two groups as follows: patients receiving skin grafts, using the Meek technique (n = 6), were compared with those without Meek grafting (n = 4). When comparing the severely burned patients of 2007 with a cohort of 2006, there were no differences for age (2007: 46.4 +/- 13.4 vs. 2006: 39.1 +/- 14.8 years), abbreviated burn severity index score (2007: 12.2 +/- 1.0 vs. 2006: 12.1 +/- 1.2) or % TBSA (2007: 72.1 +/- 11.7 vs. 2006: 69.3 +/- 8.7% TBSA). In these two rather small groups of severely burned patients with >/=60% TBSA, the overall survival rate of patients was 70.0% (7/10) in 2007 and 42.9% (3/7) in 2006, respectively. Almost all nonsurvivors in both years died within the first 5 days after admission. If assessing the different treatment modalities of the survivors, we found that although the Meek group patients were older (Meek 48.8 +/- 13.3 vs. non-Meek 26.8 +/- 11.5 years, P = .0381) and had consequently higher Baux scores (Meek 124.0 +/- 2.9 vs. non-Meek 93.8 +/- 8.5, P = .0095) than the non-Meek patients, this seemed to have no effect on length-of-stay (80.5 +/- 9.7 vs. non-Meek 79.8 +/- 33.0 days), hospital length-of-stay (85.7 +/- 14.8 vs. non-meek 84.3 +/- 26.1 days) or number of operations (6.5 +/- 1.0 vs. non-Meek 7.0 +/- 4.1 operations). The achieved results represent a combination of various treatment changes and, therefore, cannot be attributed to a single modality. The Meek technique is one of the technical options to choose from, to achieve permanent skin replacement; we think that it has its place if integrated in a whole treatment concept for management of severely burned patients.

In vitro transfer of keratinocytes: Comparison of transfer from fibrin membrane and delivery by aerosol spray

There are a variety of approaches for the delivery of autologous keratinocytes to restore epithelial coverage of burns wounds that include utilizing cultured keratinocyte sheets, transfer of cultured keratinocytes using a membrane and more recently aerosol spraying of a keratinocyte suspension. The purpose of this study was to compare the effectiveness of direct aerosol delivery of a keratinocyte suspension with a fibrin transfer system to an in vitro wound model consisting of organotypical deepidermalized dermis (DED). A comparison was made between the number of keratinocytes delivered to DED with time, either by transfer from a fibrin membrane or using an aerosol. Additionally, the effect of application time of fibrin membranes to DED, on the number of keratinocytes delivered was investigated and compared with keratinocytes delivered by aerosol at the same time points. After 2 days culture little transfer of keratinocytes had occurred from the fibrin membrane to the DED, whereas 20% more cells were present on the DED than were initially delivered by aerosol spraying. After 7 days, aerosol-delivered cells were found to express cytokeratin K6, indicating a proliferative phenotype. The results from this study show that preconfluent keratinocytes can be delivered by aerosol, and thus may well find application clinically.

Culture confluence regulates gene expression of normal human keratinocytes

Keratinocytes are frequently used to examine efficacy of wound healing products and dermatological agents in vitro. Cultured keratinocyte sheets are also used as autologous or allogenic grafts to promote wound closure. Because it is well known that the expression patterns of keratin genes change when cell cultures reach confluence, we investigated the expression pattern of wound healing-related genes, including growth factors and cytokines. Of additional particular interest is a novel wound healing related factor, secretory leukocyte protease inhibitor (SLPI), which appears to enhance tissue repair. We found that the expression pattern varied for specific genes expressed by keratinocytes as confluence was reached. Specifically, SLPI expression peaked in the early postconfluent state and vascular endothelial growth factor and amphiregulin in the late postconfluent state. Some gene products exhibit autocrine activity, whereas others exert paracrine regulation of growth. These findings indicate that it is critical to define the growth and differentiation state of human keratinocyte cultures to better determine responses and efficacy in vitro to various dermatological/wound care agents tested.

Characterization of hair follicles induced in implanted, cultured rat keratinocyte sheets.

Cultured rat keratinocyte sheets form hair follicles in combination with rat vibrissa dermal papillae when they are transplanted subcutaneously in syngeneic rats and athymic mice. In the present study, the histologic details of these induced follicles were analyzed by preparing cultured sheets mixed with normal rat keratinocytes and green fluorescent protein (GFP)-transgenic rat keratinocytes. Histologic examination demonstrated that some induced follicles maintained their size and morphology for at least 18 weeks, whereas others decreased in size and others totally differentiated into cornified structures between 3 and 6 weeks. The percentage of the grafts with GFP-positive cells decreased during the same period. This finding suggests that some GFP-positive cells were transient-amplifying cells that turned into terminally differentiated cells and were lost during this period. Some large follicles and some small follicles maintained their hair-producing ability and the proliferative activity in their hair matrix for 18 weeks. In addition, one 6-week-old follicle contained label-retaining cells in the outer root sheath. Seven of 25 follicles induced from chimera epithelium contained both GFP-positive cells and GFP-negative cells. These results suggest that stem cells are present in the induced follicle and the induced follicle consists of polyclonally derived cells. The presence of early anagen-like large follicles at week 6 and 9 and a telogen-like small follicle at week 18 also suggests that hair-growth cycle phases proceeded in the induced follicles. In conclusion, the follicles induced in the cultured keratinocyte sheets maintained hair-producing ability and proliferative activity for at least 18 weeks. This and the presence of label-retaining cells suggest that there are stem cells in the induced follicles, which seem to have a hair-growth cycle.

In vitro engineering of human skin‐like tissue

Coverage of large, full-thickness burns presents a challenge for the surgeon due to the lack of availability of the patient's own skin. Currently, tissue engineering offers the possibility of performing a suitable therapeutic wound coverage after early burn excision by using cultured keratinocyte sheets supported by a dermal layer. The aim of this study was to develop and characterize a skin substitute composed of both epidermal and dermal elements. For this purpose we grew keratinocytes and fibroblasts separately for 15 days within two different types of biomaterials. Cells then were co-cultured for an additional period of 15 days, after which samples were taken and processed with either classic or immunohistochemical stainings. Results showed that (1) human fibroblasts and keratinocytes can be cultured on hyaluronic acid-derived biomaterials and that (2) the pattern of expression of particular dermal–epidermal molecules is similar to that found in normal skin. The data from this study suggest that our skin equivalent might be useful in the treatment of both burns and chronic wounds. © 1998 John Wiley & Sons, Inc. J Biomed Mater Res, 40, 187–194, 1998.

In vitro engineering of human skin-like tissue.

Coverage of large, full-thickness burns presents a challenge for the surgeon due to the lack of availability of the patient's own skin. Currently, tissue engineering offers the possibility of performing a suitable therapeutic wound coverage after early burn excision by using cultured keratinocyte sheets supported by a dermal layer. The aim of this study was to develop and characterize a skin substitute composed of both epidermal and dermal elements. For this purpose we grew keratinocytes and fibroblasts separately for 15 days within two different types of biomaterials. Cells then were co-cultured for an additional period of 15 days, after which samples were taken and processed with either classic or immunohistochemical stainings. Results showed that (1) human fibroblasts and keratinocytes can be cultured on hyaluronic acid-derived biomaterials and that (2) the pattern of expression of particular dermal-epidermal molecules is similar to that found in normal skin. The data from this study suggest that our skin equivalent might be useful in the treatment of both burns and chronic wounds.

Use of cyanoacrylate glue to prepare cultured keratinocyte sheets for grafting

We describe a method to prepare keratinocyte cultured sheets for grafting based on the use of an organic glue (n-butyl-2-cyanoacrylate). It is a rapid and easy method in which the cultured layers of keratinocytes remain with a surface area similar to the original cultures.

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.

Reconstruction of human skin from glycerol-preserved allodermis and cultured keratinocyte sheets

The aim of this project was to reconstruct human skin from glycerol-preserved dermis and layers of cultured keratinocytes for use in the treatment of deep burns and ulcers. Glycerol-preserved cadaver skin from the Euro Skin Bank was treated with Dispase II or PBS, under various conditions, to find the best method of retaining the physical structure of the dermis while removing the epidermis and remnants of dead dermal cells which might provoke an allogeneic reaction in a graft recipient. Monoclonal antibodies LH39 and LH7.2, with specificity for basement membrane determinants, showed that treatment with Dispase II resulted in separation of the epidermis from the dermis with concomitant loss of all cellular elements from the dermal layer (as judged by Hand E staining). However, when sheets of cultured keratinocytes were applied to the treated dermis and cultured for several days, the keratinocytes attached and regenerated a new basement membrane.

Cultured epithelium for the treatment of skin defects

A review of the clinical applications of cultured keratinocyte sheets in skin and epithelial defects is reported. The improvement in keratinocyte cell culture techniques and formation of epithelial sheets has allowed the use of these cultured sheets for the grafting of skin defects. Application of cultured epidermal grafts in extensive burn lesions seems to be the most obvious use. However, acceleration of wound healing is also important in other types of wounds to decrease morbidity, hospitalization time and cost of treatment. Both autogeneic and allogeneic cultured keratinocytes have been used to cover several types of wounds. They provide a temporary barrier controlling evaporation and preventing wound infection. Clinical studies with allografts indicate that keratinocytes produce wound healing factor(s) resulting in rapid heating of chronic ulcers and in reduction of hypertrophic scar formation. The secretion of such growth factor(s) survives deep freezing. Therefore cryopreservation seems to be worth...

Cultured Keratinocyte Sheets Enhanced Spontaneous Re-Epithelialization in a Dermal Explant Model of Partial-Thickness Wound Healing

An in vitro model for partial-thickness cutaneous wound healing is described in which the influence of variables present in vivo, such as blood-borne factors and inflammatory cells, is eliminated. Dermal sheets of porcine skin are maintained in culture at the air-liquid interface in serum-free medium, and re-epithelialization from the keratinocytes of the hair infundibula can be studied. Dermal sheets of different thicknesses harvested from various depths were first evaluated for viability and regenerative potential in serum-supplemented medium. Mid-dermal explants, 20/1000 inch thick, showed the greatest epithelial outgrowth from the appendigeal keratinocytes and the longest viability in vitro. Explants of this type were used in all subsequent experiments. The effects of growth factors on re-epithelialization of the explants were studied in a serum-free environment. Epidermal growth factor, cholera toxin, bombesin, and insulin-like growth factor alone and in various combinations were applied to the explant surface in aqueous solutions by micropipette. Outgrowth was assessed by computerized morphometric analysis (RS/1 program by BBN) at days four and eight. Among all factors tested, cholera toxin alone and in combination with insulin-like growth factor produced the greatest epithelial outgrowth. Nevertheless, topical applications of growth factors failed to induce complete re-epithelialization within the experimental time frame. In contrast, explants to which cultured human keratinocyte sheets were topically applied regenerated a confluent and regularly stratified epidermis within 6 d.

In vitro culture of disaggregated rabbit keratinocytes

A technique is described for the culture of disaggregated rabbit keratinocytes and the production of confluent sheets of immunologically pure keratinocytes suitable for use in transplantation studies. The benefits of various feeder layers, nutrients and growth hormones were examined by the use of paired controls. The ability of the cultured keratinocyte sheets to be removed from the culture vessel intact and survive as viable autografts was demonstrated, thus establishing a new animal model for the investigation of allograft rejection responses to cultured keratinocytes. The benefits of using the rabbit model for such studies and the advantages of this culture technique over a previously described method of rabbit keratinocyte cell culture are discussed.