Incorporation of niacinamide, L-carnosine, hesperidin, and a Heat Shock Protein 70 homologue into collagen matrices : implications on dermal fibroblast activity and wound healing

Abstract

Background: The treatment of severe burns and other dermal defects is extremely challenging. This is due to the fact dermal regeneration does not occur spontaneously. Current treatment approaches are governed by our understanding of adult and embryonic tissue replacement and renewal, as well as our concepts on tissue engineering and stem cell biology. The use of collagen-based scaffolds will prevail in the years to come. Revisiting and changing the biochemical composition of engineered matrices might hold the key to improved and accelerated wound healing, tissue regeneration and ultimately total skin replacement. Aims: Here the author explores the combined effects of niacinamide, L-carnosine, hesperidin, and Biofactor HSP® on fibroblast differentiation and activity. The manipulation of both in vitro and in vivo wound healing is pursued through the use of these actives. The aims of this thesis are thus to determine what strategies are used to improve the clinical outcome of deep dermal injuries and burn wounds. This study wishes to attain the in vitro and in vivo manipulation of dermal fibroblast activity through the combined use of niacinamide, L-carnosine, hesperidin and Biofactor HSP®. In so doing achieve enhanced fibroblast collagen production, promote fibroblast proliferation without terminal differentiation and promote cellular longevity in a dose dependant manner. This study will also develop an enhanced dermal regeneration scaffold intended for the treatment of full-thickness dermal injuries. L-carnosine, hesperidin, and Biofactor HSP® will be incorporated after the assessment of the optimal concentrations of these key components. The effect of such an enhanced scaffold on the process of epithelialisation, wound closure, wound contraction and vascularisation will be determined. Furthermore, this study will also shed light on the mode of action of collagen-based scaffold mediated wound healing. Materials and Methods: This study utilised two experimental models from which data was obtained. The first model of the study employed in vitro cell cultures. Normal human dermal fibroblasts were used to access the effects of Niacinamide, L-carnosine, hesperidin, and Biofactor HSP® (an HSP70 homologue) on fibroblasts proliferation, differentiation and collagen production. The optimal concentrations of actives were determined in vitro. Testing parameters included; microscopic morphological cell analysis, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT) bromide cell viability and proliferation determination, cell senescence assessment, calorimetric collagen detection, sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) analysis on cell lysates, and in vitro wound healing dynamics. The second model used 16 female Sprague-Dawley rats and served as an in vivo wound healing model. Each rat received a conventional collagen-based scaffold (control) and an enhanced scaffold (test). Sample material was collected after termination at day 7 (8 rats) and 28 (8 rats). The data obtained was used to compare the quality and efficacy of dermal repair and regeneration mediated by each implant in vivo. Comparisons were made through the employment of macroscopic and microscopic analyses of the wounds. Results: The data obtained in study demonstrated that Niacinamide (0.31 mg/ml), L-carnosine (0.10 mg/ml), hesperidin (0.48 mg/ml) and Biofactor HSP® (5.18 ìg/ml) promoted in vitro fibroblast collagen production and proliferation in a dose dependant manner. Cellular differentiation was not altered in the process and the exposed fibroblasts retained the replicative capacity without morphotype transition. Moreover, senescent fibroblasts were shown to continue with proliferation during the exposure but rapidly reached senescence once the stimulus was removed. Results also demonstrated a significant difference in the in vitro wound healing of the cells exposed to the supplemented culture media. Formulation exposed cells presented with enhanced proliferation and migration which resulted in accelerated wound closure compared to the control groups. Collagen-based scaffolds were used as delivery systems for niacinamide, L-carnosine, hesperidin, and Biofactor HSP®. Results demonstrated that the incorporation of the mentioned active components into collagen-based scaffolds resulted in a reduction in the activities of both lymphocytes and monocytes on day 7. The results revealed no distinction between test and control treatments with regards to epithelialisation, wound closure and wound contraction. Picrosirius red, Haematoxylin and Eosin, and Masson’s trichrome stained sections confirmed the variable morphology of the healed wounds. Both treatment groups presented with densely organised superficial type I-like collagen fibres with looser deeper type III-like collagen fibres under polarised light. The collagen deposition in the deeper regions characteristically demonstrated a basket weave appearance. In addition, no significant difference was found in the vascularisation of the healed wounds of both the control and test treatments.