The Experimental Study On The Graft Of HAM Loaded With Bone Marrow-Derived MSCs And Keratinocytes And Their Employment In Promoting Refractory Wound Healing In Minipig

Refractory wound often occurs in patients with diabetes mellitus, patients receiving radiotherapy after surgical operation and in patients suffering from radiation injury combined with burns and/or explosive injury. Refractory wound can also occur in nuclear detonation and depleted uranium weapon attacks during wartime. The local tissue wound is usually accompanied with whole body injury. The two aspects deteriorate each other and increase great difficulty to the treatment.Approaches to promote wound healing have been reported recently, including the application of some medicines, new biological and traditional dressings, skin graft, cultured cell layer, cytokines, and transgenic treatment. However, the application of medicines, new biological and traditional dressings didn't produce prominent outcomes; cultured cell layer is thin and fragile; cytokines have short half life, and only promote the wound healing by about 10%. Transgenic method can overcome the disadvantages of cytokine application, but it is hard to control the regulation of gene expression. Although skin graft is the most effective treatment in refractory wound, enough skin autograft and allograft are difficult to obtain. The skin allograft and artificial skin equivalent can cause immune rejection.Our institute has extensively investigated the mechanism of refractory wound healing by using animal models of combined radiation-burn injury and combined radiation-wound injury. A theory has been proposed that the refractory wound refers mainly to the cellular impairment as well as the maladjustment of multi regulating factor network. According to this theory, the most effective treatment is to transplant healthy repairing cells onto the wound surface. Thus, we cultured autologous bone marrow-derived mesinchymal stem cells (MSCs) in vitro, grafted them directly onto such wound surface, and achieved nice wound healing effects. Using cellular tracing technology, we demonstrated that these cells still exist in the wound site 28 days after being grafted. However, the cells grew up in cluster instead of evenly spread on the wound surface. The wound was repaired mainly by hyperplastic collagen. Its covering by epidermic cells wasslow. We conceive that proper biomaterial should be selected to load cells being grafted in order to make the cells grow quickly and evenly. The proliferating keratinocytes should also be added to the graft to promote the wound healing in order to achieve complete tissue regeneration and wound repair. Based on our previous investigations, human amniotic membrane (HAM), a natural high polymer biomaterial composed of thick basement membrane without blood vessels, was chosen as loading material in this experiment. MSCs and keratinocytes were selected as the seed cells of tissue engineering. Autologous bone marrow-derived MSCs and keratinocytes were cultured in vitro, and loaded onto treated HAM separately. The growth state of MSCs and keratinocytes on HAM were examined. Subsequently, both HAM loaded with MSCs and keratinocytes were grafted simultaneously on refractory wounds. Their wound heal promoting functions were carefully examined. The main results of this experiment are as follows:I. Effects of HAM on the growing state of bone marrow-derived MSCs and keratinocytes1. HAM still kept its chaff shape and tenacity after being treated with ammonia of dilute concentration. The MSCs and keratinocytes were easily seeded and growing well. HAM can be absorbed easily when grafted to wound surface. Therefore, HAM is a kind of ideal biomaterial for loading cells.2. MSCs and keratinocytes adhered to HAM quickly and possessed strong proliferating ability after being seeded. Their optima density seeded on HAM is 1.54X105 cells/cm3. After being seeded on HAM, MSCs adhered completely in 12 hours and covered HAM completely in 24 hours; keratrinocyte adhered to HAM completely in 24 hours, and covered the HAM completely in 3 days. Their growth state was fine on HAM.3. Tracing technology revealed that cells grow in single...