Safety evaluation of human living skin equivalents.
Human living skin equivalents (LSEs) offer an alternative to the use of split-thickness autografts for the treatment of hard-to-heal wounds. LSEs consist of 4 active components: a well-differentiated stratum corneum derived from epidermal keratinocytes, dermal fibroblasts, and an extracellular collagen matrix. Neonatal foreskins are used as the source of keratinocytes and dermal fibroblasts for the manufacture of LSEs. Following isolation and expansion in vitro, the cells are cultured on a 3-dimensional scaffold to give an upper epidermal layer and supporting dermal layer. The resulting product has the appearance and handling characteristics of human skin. Safety evaluation of LSEs begins with insuring that foreskins are obtained only from healthy infants whose mothers are negative for a panel of adventitious agents. Keratinocyte and fibroblast cell banks are characterized using morphologic, biochemical, and histologic criteria; checked for the absence of contaminating cell types such as melanocytes, macrophages, lymphocytes, and Langerhans cells; subjected to rigorous microbiological testing (with any production materials of biological origin); and evaluated for in vivo tumorigenicity. The consistency of certain key morphologic and functional characteristics are regularly assessed. Because an LSE represents an allogeneic graft, preclinical safety studies include in vitro and in vivo determinations of its potential immunogenicity. Immunocompromised (SCID) mice reconstituted with human leukocytes or engrafted with human fetal hematolymphoid organs have been useful animal models for assessing possible immunologic responses to LSEs. Additional preclinical studies are being conducted to show that LSEs are noncytotoxic and lack allergenic, sensitizing, or irritation potential. (+info)
Enhanced vascularization of cultured skin substitutes genetically modified to overexpress vascular endothelial growth factor.
Cultured skin substitutes have been used as adjunctive therapies in the treatment of burns and chronic wounds, but they are limited by lack of a vascular plexus. This deficiency leads to greater time for vascularization compared with native skin autografts and contributes to graft failure. Genetic modification of cultured skin substitutes to enhance vascularization could hypothetically lead to improved wound healing. To address this hypothesis, human keratinocytes were genetically modified by transduction with a replication incompetent retrovirus to overexpress vascular endothelial growth factor, a specific and potent mitogen for endothelial cells. Cultured skin substitutes consisting of collagen-glycosaminoglycan substrates inoculated with human fibroblasts and either vascular endothelial growth factor-modified or control keratinocytes were prepared, and were cultured in vitro for 21 d. Northern blot analysis demonstrated enhanced expression of vascular endothelial growth factor mRNA in genetically modified keratinocytes and in cultured skin substitutes prepared with modified cells. Furthermore, the vascular endothelial growth factor-modified cultured skin substitutes secreted greatly elevated levels of vascular endothelial growth factor protein throughout the entire culture period. The bioactivity of vascular endothelial growth factor protein secreted by the genetically modified cultured skin substitutes was demonstrated using a microvascular endothelial cell growth assay. Vascular endothelial growth factor-modified and control cultured skin substitutes were grafted to full-thickness wounds on athymic mice, and elevated vascular endothelial growth factor mRNA expression was detected in the modified grafts for at least 2 wk after surgery. Vascular endothelial growth factor-modified grafts exhibited increased numbers of dermal blood vessels and decreased time to vascularization compared with controls. These results indicate that genetic modification of keratinocytes in cultured skin substitutes can lead to increased vascular endothelial growth factor expression, which could prospectively improve vascularization of cultured skin substitutes for wound healing applications. (+info)
Reverse transcription - 3' rapid amplification of cDNA ends-nested PCR of ACT1 and SAP2 mRNA as a means of detecting viable Candida albicans in an in vitro cutaneous candidiasis model.
The presence of viable cells of Candida albicans, in broth or in a reconstructed living skin equivalent, was determined by the detection of amplicons of partial mRNA sequences of the genes encoding fungal actin (ACT1) and secreted aspartyl proteinase 2 (SAP2). The mRNA of both genes were amplified by reverse transcription-3' rapid amplification of cDNA ends-nested polymerase chain reaction. Single bands of ACT1 (315 bp) and SAP2 (162 bp) mRNA were amplified from total RNA extracts of C. albicans grown in yeast carbon base-albumin broth or in living skin equivalent tissue; only the former was amplified from Sabouraud broth-grown organisms. Primer pairs targeted for ACT1 and SAP2 were Candida genus-specific and C. albicans-specific, respectively. The sensitivity limits of the assay were 100 fg of total RNA or 10 cells of C. albicans, by ethidium bromide staining. When C. albicans-infected living skin equivalent was exposed to amorolfine, amplicons of ACT1 and SAP2 mRNA were not detected in total RNA extracts. Non-amplification of the mRNA correlated with the absence of C. albicans growth in Sabouraud agar cultures of living skin equivalent samples. Reverse transcription-3' rapid amplification of cDNA ends-nested polymerase chain reaction of the mRNA encoding specific proteins of an organism has potential application in determining the viability of the organism in tissue, thus monitoring the efficacy of an antimicrobial therapy, and in detecting mRNA expressed in very little amounts in tissue. (+info)
Human melanoma progression in skin reconstructs : biological significance of bFGF.
Human skin reconstructs are three-dimensional in vitro models consisting of epidermal keratinocytes plated onto fibroblast-contracted collagen gels. Cells in skin reconstructs more closely recapitulate the in situ phenotype than do cells in monolayer culture. Normal melanocytes in skin reconstructs remained singly distributed at the basement membrane which separated the epidermis from the dermis. Cell lines derived from biologically early primary melanomas of the radial growth phase proliferated in the epidermis and the basement membrane was left intact. Growth and migration of the radial growth phase melanoma cells in the dermal reconstruct and tumorigenicity in vivo were only observed when cells were transduced with the basic fibroblast growth factor gene, a major autocrine growth stimulator for melanomas. Primary melanoma cell lines representing the more advanced stage vertical growth phase invaded the dermis in reconstructs and only an irregular basement membrane was formed. Metastatic melanoma cells rapidly proliferated and aggressively invaded deep into the dermis, with each cell line showing typical invasion and growth characteristics. Our results demonstrate that the growth patterns of melanoma cells in skin reconstructs closely correspond to those in situ and that basic fibroblast growth factor is critical for progression. (+info)
E-cadherin expression in melanoma cells restores keratinocyte-mediated growth control and down-regulates expression of invasion-related adhesion receptors.
In human epidermis, functional symbiosis requires homeostatic balance between keratinocytes and melanocytes. Compelling evidence from co-culture studies demonstrated a sophisticated, multileveled regulation of normal melanocytic phenotype orchestrated by undifferentiated, basal-type keratinocytes. Keratinocytes control cell growth and dendricity, as well as expression of melanoma-associated cell surface molecules of normal melanocytes. In contrast, melanoma cells are refractory to the keratinocyte-mediated regulation. The loss of regulatory dominance by keratinocytes occurs in concert with down-regulation of E-cadherin expression in melanoma cells. To investigate the potential role of E-cadherin in melanoma-keratinocyte interaction, we transduced E-cadherin-negative melanoma cells with full-length E-cadherin cDNA using an adenoviral vector. Our results show that functional E-cadherin expression in melanoma cells leads to cell adhesion to keratinocytes rendering them susceptible for keratinocyte-mediated control. In a skin reconstruction model, ectopic E-cadherin expression inhibits invasion of melanoma cells into dermis by down-regulating invasion-related adhesion receptors, MelCAM/MUC18 and beta3 integrin subunit, and by induction of apoptosis. Thus, disruption of the E-cadherin-mediated, normal regulatory control from keratinocytes may represent one of the mechanisms accounting for melanocyte transformation. (+info)
Cost-effectiveness modeling of Dermagraft for the treatment of diabetic foot ulcers in the french context.
To assess the cost-effectiveness of Dermagraft(R) (human dermal replacement) in the treatment of the diabetic foot ulcer, compared to standard treatment. A Markov model was developed, to simulate, over a 52-week period, the health status of a cohort of 100 patients with a diabetic foot ulcer treated either with conventional therapy or with Dermagraft(R). The considered health states were: healed, same site recurrence, unhealed not infected, cellulitis, osteomyelitis, amputation and death. Each week, the patient may progress among states according to a set of transition probabilities directly derived from the original clinical trial conducted in the USA. The cost of each health state was estimated by a Delphi panel of French diabetologists (direct costs only, valuated from a societal perspective). A sensitivity analysis was performed. The total number of healed ulcers included first ulcers healed (76.38% for Dermagraft(R) vs. 69.35% for standard treatment; median time to heal is 14-15 weeks for Dermagraft(R) compared with 28-29 weeks for standard treatment) plus recurrences which are subsequently healed within the 52-week period (14.29 for Dermagraft(R) vs. 25.09 for standard treatment; median time to heal is 3-4 weeks for Dermagraft(R) compared with 5-6 weeks for standard treatment). The average expected cost per treated patient (C/E) using standard treatment for the considered 52-week period is 47,418 FF vs. 54,384 FF for Dermagraft(R) (including 18,200 FF for Dermagraft(R) acquisition and 36,184 FF for standard treatment). Because Dermagraft(R) heals more ulcers within 52 weeks, the average cost per healed ulcer is lower (53,522 FF vs. 56,687 FF for standard treatment). The incremental cost-effectiveness ratio of Dermagraft(R) (DeltaC/DeltaE) equals 38,784 FF, indicating the extra investment that the decision-maker has to accept for an additional ulcer healed with Dermagraft(R) compared with conventional treatment. (+info)
Cost-efficacy of cultured epidermal autografts in massive pediatric burns.
OBJECTIVE: To assess the efficacy of cultured epidermal autografts (CEA) for closure of burn wounds in pediatric burn patients with full-thickness burns of more than 90% total body surface area. SUMMARY BACKGROUND DATA: Paucity of donor sites in massive burns makes the use of expanded skin of paramount importance. CEA techniques have been used in burned patients with differing and controversial results. The true impact and the efficacy of such techniques in massive burns remain uncertain. METHODS: Patients with full-thickness burns of more than 90% body surface area treated between May 1988 and May 1998 were studied. Patients grafted with CEA were compared with patients grafted with conventional meshed autografts. Rates of death and complications, length of hospital stay (LOS), hospital cost, acute readmissions for reconstruction, and quality of scars were studied as outcome measures. RESULTS: Patients treated with CEA had a better quality of burn scars but incurred a longer LOS and higher hospital costs. Both groups had comparable readmissions for open wounds, but patients treated with CEA required more reconstructive procedures during the first 2 years after the injury. The incidence of sepsis and pneumonia in both groups was comparable. CONCLUSIONS: Conventional meshed autografts are superior to CEA for containing hospital cost, diminishing LOS, and decreasing the number of readmissions for reconstruction of contractures. However, the use of CEA provides better scar quality such that perhaps future research should focus on bioengineered dermal templates to promote take and diminish long-term fragility. (+info)
Biodegradable poly (lactic acid) microspheres for drug delivery systems.
In connection with aim of maximizing the bio-availability of conventional drugs with minimum side-effects, new drug delivery systems (DDS) continue to attracted much attention. The controlled or sustained release of drugs represents one such approach, and in this regard report upon a study of DDS using biodegradable polymers which include poly (lactic acid) (PLA), poly (glycolic acid), and their copolymers (PLGA). Much attention is being paid to the controlled release of bio-active agents from microcapsules and microspheres made of biodegradable polymers, such as lactic acid homopolymers, as well as copolymers of glycolic acid. (11-21) Microcapsules or microspheres are injectable and able to provide pre-programmed durations of action, offering several advantages over the conventional dosage forms. This article reviews the results of a work program conducted in collaboration with a medical doctor upon DDS using biodegradable microspheres, such as PLA and PLGA. (+info)