Preparation Of Deep-Degree Burned Dermal Matrix And Evaluation Of Its Biological Properties

Objective:As the largest organ of the human body, skin plays a very important role in regulating body temperature, excreting metabolic waste, maintaining the internal environmental stability, and etc. However, skin is the most vulnerable to be damaged in our daily life, war, trauma or burn. A slightly injured skin can be self-repaired by its own regeneration capabilities according to the templation of the remanent undamaged cells and tissue. Nevertheless, large and severe damaged skin has lost its self-repairing capabilities due to the severe damaged stem cells and tissue. Nowadays, there are still many difficulties for the treatment of large area deep burn, such as scar contracture after healing, insufficiency of donor skin sources, or dissimilar skin allograft immune rejection, and etc. With the birth of the tissue engineering, people began to build various skin substitutes by the application of molecular biology, cell biology, tissue engineering and medical science theories and technologies. Therefore, how to use various techniques and measures to repair and rebuild the damaged skin in the early stages of the skin damage has becoming a new direction.Acellular dermal matrix (ADM), which completely retained in basement membrane between the epidermis and the dermis, is a scaffold of keeping the extracellular matrix structure by removing all the cells in the epidermis and the dermis through the physical and chemical methods. It maintains integrated three-dimensional collagen structure and high biocompatibility, is a good skin substitute. ADM has a similar tissue element with autologous skin, so it can be used as an ideal skin grafts. Antigenicity was eliminated by removing the cell ingredient of epidermis and dermis, so that immunological rejection would not occur on the receptor wound. The scaffold presents a good condition for the receptor fibroblasts to colonize and vascular endothelial cells to induce angiogenesis. Then the resettled fibroblasts produce mature matrices in this cell-free allograft dermal scaffold. Furthermore, the existence of the basement membrane is very beneficial to the proliferation and differentiation of epithelial cells. But due to the immunogenicity of basement membrane and the limited source of xenogenous skin from well-preserved body with ethical restricts and diseases infectious danger, the application of ADM is limited greatly.The other way, heterogeneous acellular dermis is another source of the dermal matrix. Swine has many similarities with human being skin in aspects of organization structure and immunity, is the best source for heterogeneous dermis. The preparation of xeno-ADM is almost in the same way with the allo-ADM, so after that, cell ingredient was removed and the intact ECM was retained. But due to the differences of species, xeno-ADM has stronger immunogenicity than allo-ADM. Xeno-ADM has such defects as:stronger immunogenicity, low-speed vascularization, and potential risk of heterologous virus infection.In our previous studies, we found there is a special layer of dermal tissue which was not burned to necrosis in the deep Ⅱ and mixed degree burned skin, in which the disorders and dysfunction of cells, and also the morphological changes are visible. But with the improvement of local environment, the above phenomena disappear and the skin could recover to normal morphology and function. The thermal injury to dermis could induce the production of plenties of substances, which were named active peptides of ECM, could induce inflammatory reactions, cell migrations and proliferations and etc. The peptides of ECM could induce lethal complications such as shock, arrhythmia. Eschar is the other name of the denatured skin, and temporary dressings were usually covered the wound after the excision of eschar. Eschar grinding was proposed to preserve the denatured dermis in recent years, and the latter could accelerate the wound healing. However, the residual active peptides could still produce adverse impacts on the victims. So that we put forward a new theory to regenerate the burned dermis to be a novel scaffold, which retains collagen structure and contains few active cytokines to accelerate the healing of burned wound.In this study, we processed the deep burned skin with tissue engineering technologies, and the scaffold was named Deep-degree burned dermal matrix (DDBDM). The physical and biological properties would be evaluated. Further analysis, by using the biotech cytokine microarray, was then conducted to test the cytokines in DDBDM, and the outcome would disclose the effects of decellularization on burned dermis.Materials and methods:1. The buildup and the assessment of deep burn modelWistar rats, Kunming mice, and Balb/c mice were chosen as the experimental animals, and heated metal and water were chosen to build the deep burn model. The better method was optimized according to the homogeneity of burned depth and model repeatability.2. The preparation of DDBDM and the assessment of its physical and biological properties.2.1 The preparation of DDBDM and method optimization:The burned skin was cut down and decellularized by vibrating in different concentrations of trypsin-Triton X-100 solutions for different times. The groups were devided as:A for 0.1%trypsin 0.1%Triton X-100, B for 0.25% trypsin 0.5% Triton X-100 and C for 0.5%trypsin 1% Triton X-100. After that, continuous viberation is carrying on in sterile PBS solution until it becomes clear. The method was optimized by the integrity and the extent of decellularization of DDBDM.2.2 The properties of DDBDM were assessed by the general and histological observation, the physical tests, and biological compatibility tests.2.2.1 The assessment of general performance of DDBDM2.2.1.1 To observe and record the appearance, colour and elasticity of DDBDM.2.2.1.2 Physical property test:To measure the mechanical strength and degradation ratio to assess the general properties of DDBDM.2.2.1.3 Histological examination and assessment:To observe the changes of collagen arrangement by using HE staining.2.2.2 The assessment of biological compatabilityAccording to part 6 of the national standards of medical equipment biological evaluation method of the People’s Republic of China:Local reactions after implantation test (GB/T 16886.6-1997), the DDBDM was implanted under the dorsum skin of mice, and ADM was control. Then after 1,2,3 and 4 weeks, the extent of fibrosis and inflammation, the types, numbers and distribution of inflammatory cells on the surface between DDBDM and tissue, the necrosis or not of DDBDM, the extent of dissolution, and the formation of granuloma were all observed to assess the histological compatability of DDBDM.3. The differences of cytokines between DDBDM and ADM.The Balb/c mice was chosen as the research object, and the Raybiotech micro-array cytokine chips were used to test the changes of 308 cytokines among the normal skin, burned skin, ADM and DDBDM. The impact of burn and decellularization on the changes of cytokines were compared and the mechanisms were analysed.Results:1. Both the heated rod and hot bath are reliable methods for building stable and replicable deep burned animal model. However, for this research, hot bath could make more stable and uniformed burned depth model.2. DDBDM could be successfully prepared by viberating the burned skin in the solution of 0.25% trypsin 0.5% Triton X-100 at 37 ℃, and could satisfy the basic requirements of dermal scaffold.3. DDBDM possesses favourable biological compatability, which was testified by the implantation examination.4. There is no more harmful cytokines in DDBDM than in ADM were examined after the tests of micro array chips, and meanwhile, some kind of cytokine in DDBDM could decrease the cicatrization and accelerate the wound healing.Conclusions:In the present study, the preparation method of the DBBDM was determined. From the differences of the cytokines between the ADM and DDM, it could be seen that the DBDDM had not excessive harmful cytokines injury to the body. So the DBDDM could be an ideal material of the dermal matrix. If the effective method is taken to decrease the inflammatory factors which could be harmful to the wound healing and the beneficial factors is utilized, the adverse responses will be alleviated and the good responses, including seed cells infiltrating, angiogenesis and matrix rebuilding, will be promoted. Because the DBBDM come from autologous dermal matrix, the histoincompatibility existed in the xenogeneic or allogeneic dermal matrix could be avoided. However, the present study has some limitations. The experiments about regulating the key cytokines were not conducted, so that the effect of these cytokines on the body is unknown. It is not clear whether the burn wound healing would be promoted using the DBDDM as dermal matrix instead of the ADM. The future study will be conducted to solve these problems.