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

McKay, Ian; Woodward, B.; Wood, Kathryn J.; Navsaria, Harshad; Hoekstra, Harm; Green, C.

doi:10.1016/0305-4179(94)90083-3

Cited by 43 publications

(22 citation statements)

References 3 publications

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“…Interestingly, prolonging the incubation time at the air-liquid interface of the E7-transduced cultures to 21 days, revealed a progressive loss of collagen VII staining of the BM, such that collagen VII-specific staining at the BM was then completely absent. Because collagen VII can normally be synthesized by the keratinocytes under these culture conditions (25), the lack of expression in the HPV-transduced cultures may be due to inhibition of protein synthesis or the active degradation of the protein. We further probed whether the BM function was compromised by investigating the distribution patterns of two other important BM components, collagen IV and laminin V. Both these markers were normally expressed in control cells at both 14 and 21 days.…”

Section: Resultsmentioning

confidence: 99%

The E7 Protein of Cutaneous Human Papillomavirus Type 8 Causes Invasion of Human Keratinocytes into the Dermis in Organotypic Cultures of Skin

et al. 2005

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Human papillomaviruses (HPV) have been implicated in the development of nonmelanoma skin cancer (NMSC). The molecular mechanisms by which these viruses contribute towards NMSC are poorly understood. We have used an in vitro skin-equivalent model generated by transducing primary adult human epidermal keratinocytes with retroviruses expressing HPV genes to investigate the mechanisms of viral transformation. In this model, keratinocytes expressing HPV genes are seeded onto a mesenchyme composed of deepidermalized human dermis that had been repopulated with primary dermal fibroblasts. Expression of the HPV8 E7 gene caused both an enhancement of terminal differentiation and hyperproliferation, but most strikingly, the acquisition of the ability to migrate and invade through the underlying dermis. The basement membrane integrity was disrupted in a timedependent manner in areas of invading keratinocytes, as evidenced by immunostaining of its protein components collagen types VII, IV, and laminin 5. This was accompanied by the overexpression of extracellular matrix metalloproteinases MMP-1, MMP-8, and MT-1-MMP. These results suggest that the cutaneous HPV type 8 that is frequently found in NMSC of epidermodysplasia verruciformis patients may actively promote an invasive keratinocyte phenotype. These findings also highlight the importance of epithelial-extracellular matrixmesenchymal interactions that are required to support cell invasion. (Cancer Res 2005; 65(6): 2216-23)

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Section: Resultsmentioning

confidence: 99%

The E7 Protein of Cutaneous Human Papillomavirus Type 8 Causes Invasion of Human Keratinocytes into the Dermis in Organotypic Cultures of Skin

et al. 2005

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“…It seems obvious that applying a functional skin replacement rather than a pure epidermal replacement most closely resembles the tissue needed for complete skin reconstitution. Several variations on this approach have been described [20,22,24]. Briefly, these include dermisderived lattices, collagen-derived matrices, and cultured substrates designed as acellular and cellular artifical skins, cultured composite skin grafts or cultured cells combined with synthetic skin substitutes.…”

Section: Discussionmentioning

confidence: 99%

Cultured human keratinocytes as a single cell suspension in fibrin glue combined with preserved dermal grafts enhance skin reconstitution in athymic mice full-thickness wounds

Horch

Bannasch

Stark

1999

European Journal of Plastic Surgery

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Cultured human keratinocytes as a single cell suspension in fibrin glue combined with preserved dermal grafts enhance skin reconstitution in athymic mice full-thickness wounds. The technique of transplanting cultured human keratinocytes suspended as single cells in a fibrin-glue matrix (KFGS) has been recently developed to overcome common disadvantages of standard cultured epidermal sheet grafts. The combination of this method with glycerolized (nonvital) xenograft overlays in standardized nude mice full-thickness wounds, as compared to KFGS alone or controls with no grafts, showed enhancement of epithelial regeneration in terms of epithelial thickness and diminished wound contraction during the 6-week follow-up. Total scar thickness was increased after the combined KFGS/xenograft technique. The time taken to complete wound reepithelialization was similar in the two groups. Reconstitution of the dermo-epidermal junction zone as shown by electron microscopy and immunohistochemistry was enhanced by the KFGS+xenograft technique, showing structures resembling rete ridges 6 weeks postoperatively. The combined KFGS/xenograft technique is able to transfer proliferative single keratinocytes. The method simplifies the application when compared to conventional epithelial sheet grafting and reduces wound contraction when compared to pure keratinocyte grafting. Materials and methods Preparation of KFGSHuman keratinocytes were cultured serum-free according to standard protocols. After reaching a confluence of 60-70% (second passage), the KFGS was prepared as described in detail elsewhere [13]. Preparation of glycerol-preserved xenograftsAfter the animals were killed from CO 2 inhalation, adult male Wistar rats were subcutaneously infiltrated with Ringer's solution, shaved, and desinfected and split-thickness skin grafts harvested with an electro-dermatome. Glycerol preservation was performed

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“…9). ture plates (19). Further studies are necessary to assess the clinical po-DISCUSSION tential of this method.…”

Section: Histologymentioning

confidence: 99%

Synergistic Effect of Keratinocyte Transplantation and Epidermal Growth Factor Delivery on Epidermal Regeneration

et al. 2005

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Both keratinocyte transplantation and epidermal growth factor (EGF) delivery stimulate epidermal regeneration. In this study, we hypothesized that the combined therapy of keratinocyte transplantation and EGF delivery accelerates epidermal regeneration compared to the single therapy of either keratinocyte transplantation or EGF delivery. To test this hypothesis, we utilized fibrin matrix as a keratinocyte/EGF delivery vehicle for epidermal regeneration. Full-thickness wounds were created on the dorsum of athymic mice, and human keratinocytes and EGF in fibrin matrix were sprayed onto the wounds to regenerate epidermal layers (group 1). As controls, human keratinocytes in fibrin matrix (group 2), EGF in fibrin matrix (group 3), or fibrin matrix alone (group 4) was sprayed onto the wounds. Spraying keratinocytes suspended in fibrin matrix did not affect the keratinocyte viability, as the cell viabilities before and after spraying were not different. EGF was released from fibrin matrix for 3 days. The wounds were analyzed with histology and immunohistochemistry at 1 and 3 weeks after treatments. Compared with the control groups, initial wound closure rate was highest in group 1. Histological analyses indicated that group 1 exhibited faster and better epidermal regeneration than the other groups. Immunohistochemical analyses showed that regenerated epithelium in groups 1 and 2 stained positively for human involucrin at 3 weeks, whereas the tissue sections of the groups 3 and 4 stained negatively. Human laminin was detected at the dermal-epidermal junction of the regenerated tissues in groups 1 and 2 at 3 weeks and was not detected in groups 3 and 4. The epidermal thickness of the regenerated tissues in group 1 was significantly thicker than that of the other groups at all time points. These results suggest that the combined therapy of keratinocyte transplantation and EGF delivery is more efficacious for epidermal regeneration than each separate therapy alone.

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

Cited by 43 publications

References 3 publications

The E7 Protein of Cutaneous Human Papillomavirus Type 8 Causes Invasion of Human Keratinocytes into the Dermis in Organotypic Cultures of Skin

The E7 Protein of Cutaneous Human Papillomavirus Type 8 Causes Invasion of Human Keratinocytes into the Dermis in Organotypic Cultures of Skin

Cultured human keratinocytes as a single cell suspension in fibrin glue combined with preserved dermal grafts enhance skin reconstitution in athymic mice full-thickness wounds

Synergistic Effect of Keratinocyte Transplantation and Epidermal Growth Factor Delivery on Epidermal Regeneration

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