Influences Of Focus Low Intensity Pulsed Ultrasound On Bmscs’ Multi-directional Differentiation

Objectives Recent years, the rapid development of fractures due to an aging population and increasing building transport sector caused serious harm effect on our health. Promoting healing, preventing the occurrence of delayed union and nonunion remain a challenge that we may face. Low-intensity pulsed ultrasound draws people’s attention by its low cost, no infection, no trauma, simple treatment, no adverse reactions and good effect. However, the mechanism of LIPUS which promote bone healing is still not clear. We analyze the key that LIPUS promotes bone healing was LIPUS accelerates BMSCs differentiating into bone and vascular endothelial cells to repair damaged bone tissue. Therefore, this paper we will study the impact of BMSCs’ multi-directional differentiation by LIPUS, computing LIPUS increase differentiation rate of BMSCs differentiation. And combinating LIPUS and BMSCs treats bone-defect healing in animal studies.Methods 1.BMSCs was divided into control group, Osteoinduction group(O), Vascular endothelial induction group(V), Double induction group(D) and LIPUS group, Osteoinduction+LIPUS(OL), Vascular endothelial induction+LIPUS group(VL), Double induction+LIPUS group(DL). LIPUS, OL, VL and DL group treated with the LIPUS for 20 min once every day, for 13 consecutive days(LIPUS with frequency=0.6MHz, repetition frequency=1k Hz, duty cycle=20%, Isata of 0.03W/cm2). The others take sham LIPUS by the same method. BMSCs’ multi-directional differential ability was evaluated by morphologic observation, MTT, Electron Microscopy test, Double Immunofluorescence test, Flow Cytometry detection and fluorescent PCR test.2. The 24 female SD rats were randomly divided into four groups: control group, LIPUS group, BMSCs group and BMSCs+LIPUS group(BL). Bone needle with a diameter of 1.6mm as a unified rat bone defects "hole" model. Each group started treating from the moment that defect model established, the continuous observation four weeks. At the treatment of two weeks and four weeks, the rats were taken X-ray imaging, femur samples collected drawings and HE staining test.Results 1.Cellular morphology, Electron Microscopy and Double Immunofluorescence test showed that the morphology changed earlier in irradiation group than in sham control group, the number of cells inducing towards specific direction is also more in irradiation group than in sham control group. Flow Cytometry detection showed that cell differentiation rate LIPUS group reached(11± 0.11), differentiation rate of the control group(5.95±0.05), the difference between the two groups was significant(P<0.05). Differentiation rate of O group reached(17.58±0.046), OL group was(10.39 ±0.08), the difference between the two groups was significant(P<0.05). Differentiation rate of VL group reached(99.46±0.05), V group was(96.97±0.03), the difference between the two groups was significant(P<0.05). Differentiation rate of DL group were reached(34.79±1.21), D group was(24.77±0.3), the difference between the two groups was significant(P<0.05). Fluorescent PCR results showed that all irradiation groups’ osteocalcin gene or v WF gene expression were significantly higher than that in the sham control group. O and OL groups’ v WF gene expression at 5d and V and VL groups’ OC gene expression at 5d is no difference(P> 0.05). The rest irradiation groups’ osteocalcin gene or v WF gene expression was higher than that in the sham control group, and there is a significant difference between the irradiation group and corresponding sham control group(P <0.05).2. Combining LIPUS and BMSCs treats bone defects in rats. The result showed that, LIPUS group and BMSCs groups both had healing effect, but there is no significant difference between the two groups about the test of uncured bone area(P> 0.05). But HE staining found the quality of new born bone was worse than the surrounding normal bone in the two groups. The combined treatment group’s bone healing was significantly better than that in LIPUS group and BMSCS group. At 4 weeks, bone defects almost disappeared. Uncured bone area had a very significant difference(P <0.01) compared the LIPUS and BMSCs group. According to HE staining results, in the combined treatment group, the new born bone tissue had a unity range, neovascularization was also found at the defect area.Conclusion 1. LIPUS can accelerate BMSCs differentiating into osteoblasts and endothelial cells, making it faster generate bone tissue and vascular tissue. When BMSCs was induced by two-direction, BMSCs can differentiate into blood vessels and bone tissue at the same time, which provides a basis for generating vascularized bone tissue in the future. When BMSCs was induced by two-direction, LIPUS enhance the BMSCs’ differentiation rate most obvious.2. Combining LIPUS and BMSCs treat bone healing had the best effect, the combination therapy can significantly reduced bone healing time, and the quality of new bone tissue is the same as the normal bone tissue. After 4 weeks’ irradiation, HE staining image showed that new bone tissue at the bone defect area also observed neovascularization. Prove that LIPUS can promote BMSCs two-direction differentiation at the same time.