Experimental Study Of LIPUS Enhances Cell Proliferation And Chondrocyte Differentiation Of Bone Marrow Mesenchymal Stem Cells

ObjectiveTo observe the effects of low intensity pulsed ultrasound(LIPUS) on cultured bone marrow mesenchymal stem cells(BMSCs) proliferation from whole bone marrow adherence to plastic method in vitro. And to examine the synergistic effects of LIPUS and TGF-?1 on cartilage matrix formation and chondrocyte differentiation of BMSCs cultured in vitro. MethodFirst, the femurs and tibiae cleared of the connective tissue and epiphyses were collected from the sacrificed four-week-old Japanese white rabbit. The standard isolation of whole bone marrow adherence to plastic method was used and after the cells were cultured and purified, we obtained enough BMSCs of high purity, strong vitality and homogeneous biological characteristics. Morphological analyses was carried out with optical microscope, the synthesis and secretion of collagen type II was examined through immunohistochemistry and cell surface markers such as CD34?CD45?CD90 and CD105 were assessed by flow cytometry. Second, Cell Counting Kit-8 assay was performed to detect cell survival and proliferation of bone marrow mesenchymal stem cells after low intensity pulsed ultrasound treatment in different parameters. Also, morphological analyses was carried out with optical microscope again. Third, bone marrow mesenchymal stem cells were divided into four groups as follows: blank control group underwent the same experimental treatment with no TGF-?1 addition and the ultrasound power off, TGF-?1 group received TGF-?1 in the culture medium, LIPUS group received the LIPUS stimulation, and T-L group received both the TGF-?1 in the culture medium and LIPUS stimulation. After the intervention in each group, observation and analysis were carried out in 1, 3, 7 days. The synthesis and secretion of collagen type II was examined through immunohistochemistry. The synthesis and secretion of cartilage extracellular matrix was examined through green and red solid dyeing assay. And the gene m RNA expression of SOX9, COL2A1 and MMP13 were examined through RT-PCR assay. ResultsUsing whole bone marrow adherence to plastic method in vitro could obtain enough BMSCs of high purity, strong vitality and homogeneous biological characteristics, with a form of fusiform or spindle shape. The result of immunohistochemistry of collagen type II is negative. For flow cytometry analysis, BMSCs were positive for CD90 and CD105, but negative for CD34 and CD45, the positive rate was 99.78%, 99.52%, 0.35% and 0.56%, respectively. This verified the cultured cells were undifferentiated homogeneous biological BMSCs. Cell proliferation and colony formation of different parameter groups were different but significantly higher than that of the control group. There were more intensive cell colonies, which formed a typical cell clusters of focal hyperplasia in spiral shaped dense array after 24 h post-stimulation. In the CCK-8 assay, the result revealed that LIPUS at 60 m W/cm2 for 15 minutes significantly improved the cell proliferation of BMSCs after treatment and the optical density was the maximum compared with other groups(P<0.05). For immunohistochemistry assay, collagen type II were positive in group T, L and T-L, especially in group T-L. For green and red solid dyeing assay, LIPUS and TGF-?1 could both improve the synthesis and secretion of cartilage extracellular matrix in 1, 3 and 7 days after treatment, especially in group T-L, more red dyeing areas were observed. The expression levels of chondrocyte differentiation related genes SOX9, COL2A1 and MMP13 were analyzed by quantitative real-time RT-PCR. LIPUS and TGF-?1 could up-regulated m RNA expressions of SOX9 and COL2A1 and down-regulated m RNA expressions of MMP13 in 1, 3 and 7 days after treatment, respectively. When treated BMSCs with LIPUS and TGF-?1 together, it can be seen that m RNA expressions of SOX9 and COL2A1 were up-regulated and MMP13 was down-regulated apparently(P<0.05). ConclusionThe method of whole bone marrow adherence to plastic in vitro can obtain pure and enough isolated rabbits BMSCs. These cells have high purity, strong vitality and homogeneous biological characteristics with no differentiation. The simple, easy to handle, and relatively inexpensive method is of great significance and valuable to implement. The LIPUS stimulation cell device built by ourselves can be used normally. LIPUS can improve cell proliferation of BMSCs after post-treatment for some appropriate parameters. There was some difference between different intensity and time of LIPUS treatment. Parameters with intensity of 60 m W/cm2 for 15 minutes is the optimum to improve the proliferative efficiency in this study. LIPUS and TGF-?1 can both promote cartilage matrix formation and chondrocyte differentiation in BMSCs in vitro. There is a synergistic effect when applied the two treatments together.