The Experimental Study Of Rats’ Wound Repair By Human ADM Combined With BMSCs

Background：As a natural protective barrier to the human body, skin plays animportant role in prevent the invasion of the outside microbe and maintain the stability ofthe internal environment. Various causes of skin defects are very common in clinic,currently, the most commonly used treatments are autologous skin graft, allograft orxenograft, and composite skin grafting etc. but these methods have some disadvantagessuch as short of donor site, lack of skin resource, immunological rejection and thetransmission of diseases. Therefore, it’s rather necessary to find a better skin substitute totreat the defect of skin. With the development of tissue engineering, tissue engineeringskin has gradually become the research hotspot, various kind of tissue engineering skinsare applied to clinic, and achieved good results. But, there are still many shortagescompared with the natural human skin. There are diverse of seed cells and supportmaterials to construct tissue engineering skin, it has been a study hot spot for expertsfrom home and abroad to research a better construction method. This research use BoneBarrow Mesenchymal Stem cells (BMSCs) and Acellular Dermal Matrix (ADM) toconstruct tissue engineering skin, to repair skin defect, and expecting to provide atheoretical basis for the clinical treatment of skin defect wound.Objective:1. Study the production method of ADM and evaluate itsbiocompatibility.2. Select BMSCs as seed cells, ADM as support material, to construct tissueengineering skin.3. Study the effect of the wound healing of the animal skin defect with the treatment of tissue engineering skin.Method：1. Choose6-8weeks old SD rats, using the whole bone marrow adherentmethod to separate, cultivate and purify BMSCs.2.Adopting the following methods to identify the BMSCs:(1) MTT method to drawthe growth curve of the third generation of BMSCs.(2)Flow Cytometer detect theexpression of the surface antigens of BMSCs.(3)According to the biologicalcharacteristic of multiple differentiation potential of BMSCs, we did osteogenic andadipogenic introduction.3. Adopting enzyme digestion method to make ADM with the material of humanforeskin.4. To observe the histocompatibility and the structure of ADM through the testamentof SD rat subcutaneous implantation and HE stain.5.Using tissue engineering skin to cover SD rat skin defect wound, record thewound healing time and calculate the wound closure index (WCI). Take wound sampleto proceed HE stain, CK19and Brdu immunohistochemical stain after implantation2weeks, and expecting to evaluate the wound healing situation.Result：1. Using the whole bone marrow adherent culture method to isolate BMSCs,by day5, adherent cells formed distinct colony, some cells began to merge with eachother. By day7cells, more cells gathered, present a long spindle shape or polygonalgrowth, after1:2subculture, cell proliferation increased. It can be grown full of bottlewithin3-4days, the cell morphology was more uniform, expressed a swirling and radialgrowth, with obvious directivity.2. The growth curve showed an S type with the method of MTT. The first1-2dayscells proliferate slowly. Bye day3-5cells proliferate faster, and expressed an exponentialincrease. By day6-8cells entered a plateau, and the growth rate slowed down gradually.3. Flow Cytometer detect the expression of the BMSCs surface antigens, CD34negative, CD44, CD73and CD105were all positivee expression.4. After osteogenic and adipogenic introduction, we can see the orange calcium nodules by alizarin red stain and red fat drops by oil red O stain.5. The ADM we made by enzyme digestion method is milky white, soft texture,high resilience, smooth skin surface, rough leather surface, skin texture clearly visible,uniform pore distribution. HE staining, table components were completely removed andno cells residual in dermis under the microscope.6. After rats’ subcutaneous implantation, take wound sample with HE stain in eachperiod of time. By2weeks mild inflammatory cells infiltration around the implan. By4and6weeks, there were capillaries and fibroblasts could be found in it, and there was noinflammatory cell infiltration.7. After SD rat skin defect wound transplanted with tissue engineering skin, thewound healing rate between any two groups, the difference showed statisticalsignificance (P<0.05), HE staining showed that the wound healing speed between threegroups: group A> group B> group C. immunohistochemical staining showed that: thereare Brdu positive cells expressed in group A, and the expression of CK19in group A wassignificantly higher than that in group B and group C.Conclusion:1. The whole bone marrow adherent method is a reliable isolation andcultivation method for BMSCs.2. The ADM produced by the enzyme digestion, cell components removedcompletely, does not cause the host’s rejection, and has good histocompatibility.3. The tissue engineering skin constructed with BMSCs and ADM has a goodpromotion of wound healing, when it was used on full-thickness skin defect wound.