Enhanced Culture Techniques and Model Development for Skin Regeneration

Deep, (burn) wounds in humans generally heal with the formation of a hypertrophic scar. This severe scar affects appearance and causes serious restrictions on joint mobility. The standard treatment for full-thickness burn wounds is transplantation with a meshed split skin autograft. However, this therapy has several disadvantages. Healing still results in scar formation and morbidity at the donor site may occur. In addition, insufficient donor sites may be present in patients with extensive burns, due to limited availability of healthy skin. Transplantation with autologous keratinocytes is a promising alternative for the treatment of deep (burn) wounds. However, keratinocytes are conventionally cultured in the presence of fetal calf serum (FCS) and mouse feeder layer cells. These xenobiotic materials can be a potential risk for the patient (e.g., transfer of prions). Chapter 2 showed that we could successfully culture keratinocytes without FCS and mouse feeder layers cells. If the keratinocytes were grown on collagen type IV, these cells were able to form a fully differentiated epidermis at least up to passage 4. This indicates that the cultured keratinocytes are still suitable for clinical application. Using our culture technique, sufficient numbers of keratinocytes can be obtained from 1 cm2 skin to potentially cover 400 cm2 of wound surface in 2 weeks. For the development of new (tissue-engineered) anti-scarring therapies, it is essential to have detailed knowledge about scar formation and tissue regeneration. Many studies show that fetal skin has the ability to heal without scar formation. The composition of fetal skin itself probably plays an important role in this process. Thus, knowledge about fetal and adult skin constitution may contribute to the understanding of wound healing. In Chapter 3, we studied the differences between human fetal and adult skin architecture. We found that most differences between fetal and adult skin were present at the level of dermal extracellular matrix (ECM) molecular expression. The expression of fibronectin (FN) and chondroitin sulfate (CS) was higher in fetal skin than in adult skin, whereas elastin was not present in fetal skin at least up to 22 weeks of gestation. In contrast, the fetal epidermis was only slightly different from the adult epidermis in skin. These findings suggest that the presence or absence of certain ECM molecules might be beneficial in promoting adult wound healing. Therefore, a possible therapeutic intervention for improving adult healing is the use of a dermal substitute that contains certain ECM molecules (e.g., FN and CS). To investigate the mechanisms of wound healing, wound model systems are indispensable. No ideal wound model is available at this moment. Animal models CH A PT ER 7