Studies on the interaction of human cells with transplantable materials for skin repair and cardiovascular surgery
Sammanfattning: Tissue engineering is becoming an important field for the development of new tissues in vitro for replacement of diseased or worn out parts of the body. This thesis describes some characteristics of cultured human cells seeded on transplantable materials for skin repair and cardiovascular surgery. Transplantation of sheets of cultured epidermal cells have been widely used for the treatment of large bums and has saved many lives. However, there are some negative features in conjunction with such grafting, e.g. extensive scarring, due to the lack of a functioning dermis at the time of transplantation. To improve the understanding of wound healing in humans, we studied the early events occurring during the reepithelialization process in a human, partial thickness in vitro wound model. The wounds were incubated either submerged in cell culture medium or elevated to the air-liquid interface. The time-course for deposition of a basement membrane was similar between both types of wounds. Collagen type VII and laminin was deposited beneath the migrating keratinocytes while collagen type IV was became detectable after reepithelialization. Terminal differentiation, on the other hand, was complete only in elevated wounds, as indicated by immuno-labeling of involucrin and filaggrin. A biodegradable bovine collagen membrane was studied as a dermal support for healing of human in vivo full thickness wounds and results were obtained 7, 21 or 42 days after grafting. Reepithelialization was complete at day 7, and terminal differentiation was normalized from day 21. A neodermis was generated and matured evident by an increasing number of fibroblasts and a diminishing procollagen-content. A neovasculature was apparent on day 21. To study the potential use of this collagen membrane as a skin substitute, epithelial cells (keratinocytes) and fibroblasts, isolated simultaneously from a biopsy of human skin, were cultured and seeded onto opposite sides of the membrane. Growth characteristics and differentiation were comparable to those on tissue culture plastics, and the technique appeared suitable to achieve a transplantable graft with a confluent or subconfluent epithelium and dermal fibroblasts. This biocompatible collagen membrane may be used as a true dermal implant and also as a supporting matrix for the transplantation of autologous keratinocytes and fibroblasts, and possibly to be used as a novel skin substitute. In order to improve the outcome of cardiovascular prostheses, especially synthetic vascular grafts with small inner diameters, the effects of pre-forming an endothelial cell lining have been investigated. Problems such as thrombus formation and late graft failure can thereby be partly avoided, but little functional data exist, especially from endothelial cells on bioprosthetic tissue. Cultured human saphenous vein endothelial cells were used to in vitro endothelialize ePTFE, a synthetic vascular graft material, and a photooxidized biologic pericardial tissue aimed for use as prosthetic heart valve material. Endothelial cells seeded to achieve immediate confluency were shown to deposit basement membrane proteins within 6 hours after seeding on both types of matrices. The reendothelialization of a 4-mm wide denuded area was shown to comprise both migration and proliferation of endothelial cells, and the process was completed within 4 days on uncoated biologic tissue and 15 days on serum-precoated ePTFE. After complete endothelialization of intact heart valve prostheses, they were exposed to a pulsatile flow in a specially designed flow-device that mediated opening and closing of the valve under conditions that partly mimicked physiological flow conditions. The cell monolayer on the cusps was shown to be intact and the adaptation of cells to the pulsatile flow was verified by the change in morphology and rearrangement of actin-stress fibers. To exclude any procoagulant activity of HSVECs after endothelialization, the expression of tissue factor (TF) was determined. TF mRNA and TF-protein levels were shown to be lower in HSVECs on bioprosthetic material compared to normal cell culture plastic, whereas only a minor difference was seen in levels of functional TF. In conclusion, we showed that cultured HSVECs seeded on bioprosthetic materials withstand pulsatile flow, heal denuded areas and are not apparently procoagulant. This technology may thus be used to enhance the performance of bioprosthetic heart valve prostheses.
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