The Study Of Construction Of Artificial Composite Skin By Bone Marrow Mesenchymal Stem Cells

Objective:Construct artificial composite skin by bone marrow mesenchymal stem cells(BMSCs) combined with small intestinal submucosa (SIS) in vitro, then apply it tofull-thickness dermal wounds in rabbits and observe its ability to repair woundscompletely. And then verify its feasibility as artificial composite skin.Methods:(1) Bone marrow was aspirated from rabbits’ proximal tibias or distal femurs bymeans of bone marrow aspiration, and BMSCs were then isolated and purified bydensity-gradient centrifugation and differential adhesion. Cultured the cells byDMEM-F12medium supplemented with fetal bovine serum at a concentration of10%by volume, and incubated them at37℃,5％CO2saturated humidity thermostatincubator. The cells were cultured to third generation in vitro. Observed cellularmorphology and growth status of each generation, then identified features ofthird-generation cells. Marked third-generation BMSCs by transfected withlentiviruses containing a gene that encodes for a green fluorescent(GFP) protein.(2) Acellular SIS was produced by enzyme and detergent, and detectd by HEstaning and electron microscopy. Implant GFP-BMSCs to cellular SIS by the density of106/cm2,and co-culture them for10days to build artificial composite skin. Colonization of BMSCs in SISwas observed by fluorescence microscopy and electron microscopy.(3) Maked three full-thickness skin defect models along the spine of eachJapanese rabbit, and then randomly assigned the wounds to three groups: group A,group B and group C. Transplanted artificial composite skin and SIS respectively ingroup A and group B, and group C is blank group. The conditon of wound healingwas observed everyday and complete wound healing time of each group wererecorded for statistical analysis. Measured wound size of each group on7days,14days,21days and28days after surgery. Cut out tissues from wounds and observetexture and thickness of the tissues. Performed histological examinations of thetissues by means of HE staining to examine organizational changes in wound healing process.Results:（1）Isolated and purified BMSCs by means of density gradient centrifugationand differential adhesion. The morphology of BMSCs was fusiform. BMSCs grewwell and can present as “fish-like” arrangement by clonal proliferation. BMSCs canreach to80%confluence for5-6days in serial subcultivation, and the morphology ofBMSCs became identical. Purified BMSCs can differentiate to fat cells andosteoblasts. Transfected BMSCs grew well and had powerful fluorescence.（2）SIS was proved to be cell-free by means of HE staining and electronmicroscopy. Artificial composite skin was build successfully: observed byfluorescence microscope, it can be find that BMSCs can adhere and colonize to SISand stretch in its surface, adn BMSCs can grew well in SIS. Observed by Scanningelectron microscopy, it can be find that Large number of BMSCs adhere to collagenfibers of SIS.(3)In the experiment of wound healing, complete wound healing time of groupA、group B and group C were20.83±1.17d，23.50±1.05d,27.67±1.03d, the completewound healing time of group A was significantly shorter than the other two groups (P<0.01). Compared group A with other two groups in wound contraction rate onpostoperative7d、14d、21d and28d, the wound contraction rate of group A on everytime point were smaller (P <0.01). Compared group A with the other two groups inwound tissue thickness on postoperative7d、14d、21d and28d, the wound tissuethickness of group A on every time point were softer and thinner (compare with groupB, P <0.05; compared with group C, P <0.01). Therefore, in group A, the woundhealing time was short, the rate of wound retraction was small and the wound tissuewas relatively soft and thin. Besides, HE staining showed that epithelialization speedwas fast in the early period of wound healing and, in the late period of wound healing,capillary and small blood vessel grew richly and collagen fibers reconstructed fastly.The result of HE staining in group A was still significant different compared with theother two groups.Conclusion:(1) The means of density gradient centrifugation and differential adhesion can isolate and purify BMSCs well, and their cellular morphology is consistent.(2) The method of enzyme-detergent can stripped cells in SIS effectively andcompletely. Artificial composite skin can be make by co-culture BMSCs with SIS.(2) Take artificial composite skin made by BMSCs to wounds, can acceleratewound healing by accelerating angiogenesis and promoting epithelialization; and canimprove quality of healing wounds by reducing retraction of wounds, making woundtissue thin and soft.