In vitro and in vivo studies on biodegradable matrices for autotransplantation
Abstract: Tissue engineering (TE), one of the most rapidly growing fields of life science, is an interdisciplinary area in which technical, biological and medical expertise co-fertilize on another with the ultimate aim of restoring, maintaining or improving tissues and/or organs. This purpose links TE closely to research concerning reconstructive plastic surgery. At present, TE utilizes two major approaches: I. Autologous cells are cultivated outside the body in vitro and returned as autotransplants to the patient. II. Autologous cells are stimulated to regenerate in the patient - in vivo usually together with a suitable carrier structure or substances that regulates cell function. This approach is referred to as guided tissue regeneration. With regards to full thickness skin wounds, such as deep burn wounds, the belief today is that optimal treatment must achieve restoration of both the dermal and the epidermal regions of the skin. At present, the treatment considered to be the gold standards for treatment of full thickness wounds only restore the epidermis of the wound, with either split thickness skin grafts or cultured keratinocytes. This thesis focuses on approaches allowing restoration of both the dermis and the epidermis. In order to achieve this, different dermal matrices have been characterised and evaluated. A suitable dermal matrix should fulfil a number of demands: I. It should be biodegradable II. Epithelial cells should be able to attach to this matrix III. Epithelial cells should be able to migrate and proliferate on the matrix IV. The dermal matrix should stimulate regeneration of an autologous neodermis (i.e. ingrowth of fibroblasts and angiogenesis) V. It should be possible to store the matrix for long period of time without loss of function VI. Production, use and storage of the matrix should be cost-effective In Paper I and II cell-free dermis was used as a carrier for epithelial cells cultured in vitro. Cell-free dermis fulfils most of the demands enumerated above, and reliable techniques for the cultivation of both keratinocytes and urothelial cells for autotransplantation were developed. A disadvantage associated with the use cell-free dermis is its limited ability to be maintained as a ready-to-use product, primarily due to the complicated and, to date, costly process of production. In Paper III the possibility to harvest urothelial cells with a non-invasive technique was investigated. The majority of patients suffering from hypospadia are children. To be able to harvest cells for transplantation through bladder-washing, without loss of proliferative capacity would be a great advantage. In search for a more convenient product, a poly urethane urea scaffold (PUUR) was investigated in paper IV. An in vitro study followed by a pilot study- in vivo-on healthy volunteers, revealed that the PUUR scaffold possesses great potential as template for dermal regeneration. Unfortunately, this scaffold is not rapidly degraded in vivo. In paper V the suitability of a three-dimensional system consisting of biodegradable macroporous gelatine spheres as carriers for transplantation of keratinocytes into full-thickness wounds was examined in an in vivo study, involving nude rats. Gelatine is an autologous material and thus, biodegradable and the small size of the spheres allow them to be injected with a syringe at location where they are needed. In addition, the pores greatly increase the number of keratinocytes that can be cultured on these spheres. In comparison to treatment with split-thickness skin grafts and cultured keratinocytes in single-cell solution, macroporous gelatine spheres coated with keratinocytes promoted an earlier wound closure and better skin quality. Accordingly, such spheres represent a highly promising and interesting matrix for dermal regeneration and for co-cultivation with epithelial cells.
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