Isolation, Culture And Differentiation Of Rabbit Peripheral Blood Mesenchymal Stem Cells In Vitro And Preparation Of ECM

ObjectiveTo explore how to isolate and culture the rabbit peripheral blood mesenchymal stem cells (PBMSCs) under the conditions of the skin injury, and to observe their differentiation potential into the bone and cartilage cells. The effective method for the isolation and culture of rabbit peripheral blood mesenchymal stem cells will provide the experimental basis for using PBMSCs as seed cells in tissue engineering.Methods1. Six Zelanian rabbits were used to establish skin wound model, and femoral venous blood drawn before and after trauma. Mononuclear cells were purified by density gradient centrifugation.2. The cells were respectively cultured in a-MEM medium and LG-DMEM medium. Growth of cells was observed, and the cell growth curve was determined by MTT. Effects of the cultivation of MSCs were compared between different culture medium.3. MSCs were induced to differentiate into osteoblasts. The P2-generation MSCs were divided into the experimental group and the control group. MSCs were induced by osteogenic inducer in experimental group. In the control group, MSCs were cultured in a-MEM medium which containing 10% fetal calf serum. The cell morphology was observed. Alkaline phosphatase and calcified nodule were respectively detected by alkaline phosphatase staining and alizarin red staining.4. MSCs were induced to differentiate into chondrocytes. The P2-generation MSCs were divided into the experimental group and the control group. MSCs were induced by cartilage induction agent in experimental group. In the control group, MSCs were cultured in a-MEM medium which containing 10% fetal calf serum. The cell morphology was observed. And the expression of typeⅡcollagen was detected by immunohistochemical staining.5. Zelanian rabbits were used as the costicartilages donors. costicartilages were cut and taken to the lab immediately after the execution of the donor rabbits. The costicartilages were immersed into the 1% Triton X-100 and solutions containing of DNAse and RNAse in sequence. According to the different length of time in Triton X-100 solution,4 groups were divided:24-hour group,48-hour group,72-hour group and 96-hour group. After all of the treatments, the samples were collected for HE staining and scanning electronic microscoping to observe the effect of removal of cells in the acellular matrixs and the effect of the four groups are compared.6. Adjust the cell density of 7-day induced MSCs to 3×106/ml, then co-culture the autogenic MSCs and the allogenic acellular matrix for 14 days in vitro. The composites were harvested for scanning electronic microscoping at the 3rd,7th and 14th day to observe the adherence and proliferation of MSCs on the surface of matrixs.Results1. After stimulation of the trauma, the number of the rabbit peripheral blood mesenchymal stem cells was significantly increased (P<0.05).2. Primary mesenchymal stem cells were round, spindle, polygonal. The cells were purified by passage and became into long fusiform. Their growth curve was typical "S"-shaped. It was found that primary cells adhered to the wall after 12h, and gathered after 48h, and lots of cell-colony-units formed in 5 days in a-MEM. The colonies of MSCs increased and the consecutive colonies mixed together in 7-8 days. The Adhesion and convergence time of the cells in a-MEM was earlier than that in LG-DMEM, and the colony number of the former was more than the number of the latter. The difference was statistically significant (P<0.05). It was shown that a-MEM was more suitable than LG-DMEM for PBMSCs to grow.3. Differentiation into osteoblasts:In the experimental group, MSCs may be spindle-shaped, triangular or polygonal. There were a number of coadjacent enation with different sizes and varying length, larger particles in the cytoplasm and oval nuclei. 2-3 nucleolus and cell division phase could be seen. The cells proliferate slowly. In the control group, MSCs remained long spindle-shaped and had strong proliferative ability. Brown or brown-black particles were seen by alkaline phosphatase staining in the cytoplasm of PBMSCs, indicating the presence of alkaline phosphatase. PBMSCs were induced to form calcium nodules, and the clear positivity for the red nodules was shown by alizarin red staining.4. Differentiate into chondrocytes:Rate of cell proliferation in experimental group was lower compared with the control group, and the shape was gradually became form long spindle-shaped to polygonal and circular. With the extended induction time, there were trends to growing close together. In the control group, MSCs remained long spindle-shaped and had strong proliferative ability. Immunohistochemistry results showed that the expression of type II collagen was gradually increased in the experimental group. Cultured after two weeks, there were yellow or brown granules in the cytoplasm. While in the control group, positive cells were not obvious.5. It took at least 96 hours to remove all cells from the allogenic costicartilages with 1% Triton X-100 solution. HE staining showed that chondrocytes of costicartilages samples in 96-hour group were removed entirely. The results of SEM also demonstrated that all the cells of costicartilages samples were removed in the 96-hour group. The cartilage lacunas, without chondrocytes, were clear on the surface of cartilage acellular matrixes.Conclusions1. PBMSCs could be harvested from peripheral blood of wounded rabbits.2.α-MEM was more suitable than LG-DMEM for PBMSCs to grow.3. PBMSCs cultured in vitro can differentiate into osteoblasts and Chondrocytes.4. The detergent-nucleinase method can remove all the cells of costicartilage samples thoroughly by using 1% Triton X-100 solution, without damages to the ECM. It keeps the integrity of structure of COSticartilage acellular matrix.