The Experimental Study Of Low Intensity Pulsed Ultrasound Promote The Osteogenic Differentiation Of RDFCs And RBMSCs

Objective:To investigate the intensity pulsed ultrasound promote the osteogenic differentiation effect of DFCs and BMSCs.Methods:Isolation and culturingof rDFCs and rBMSCs was done. Flow cytometry was done to detect the cell surface marker expression.Multi-directional differentiation was done by alizarin red staining and oil red staining. Alizarin red staining and qRT-PCR was done at the 7d and the21 d respectively to detect the osteogenic effect of LIPUS(30mW/cm2,20min/d) on rDFCs and rBMSCs in vitro.ThenrDFCs and rBMSCs were seeded on the prefabricated-mineralized inducing bone tissue engineering scaffold material. Cell growth condition was detected under electron microscope scanning when cells were seeded in 3,5,7,9d respectively.At last, rDFCs and rBMSCs were seeded on the scaffold and were transplanted in nude mice subcutaneous tissue. Experiment groups:G1:blank scaffold;G2:rDFCs+scaffold;G3:r BMSCs+scaffold;G4:r DFCs+scaffold+LIPUS; G5:r BMSCs+scaffold+LIPUS. The LIPUS parameter was 30mW/cm2,20 min per day. Cells were exposed in LIPUSfor 8w. HE and Masson staining was made to analyse the status of tissue regeneration.Results:rDFCs and rBMSCs were successfully obtainedand mesenchymal stem cell surface marker was expressed. The alizarin red staining and oil red staining results showed that rDFCs and rBMSCs have the ability of multi-directional differentiation.The results of alizarin red staining showed the calcified nodules in LIPUS treated groups were more significantly than that in the control groups. The qRT-PCR results showed the relative expression of ALP, Runx2, OSX and COL-1 in LIPUS treated groups was much higher than that in the control groups. Electron microscope scanning results showed that rDFCs and rBMSCs were able to grow around the scaffold and adhere on the surface of the scaffold. Cells and large numbers of extracelluar matrix could be seen in the material porosity.The samples were collected after they were transplanted into nude mice subcutaneously8 weeks later. The HE staining showed that no cells and newly formed tissues could be obviously seen in the blank group. A few cells and newly formed bone-like tissues could be seen in rDFCs+scaffold group and rBMSCs+scaffold group. More cells and newly formed bone-like tissues could be seen in rDFCs+scaffold+LIPUS group and rBMSCs+scaffold+LIPUS group.Masson staining showed that no cells and newly formed fiberous could be seen in the blank group. A few cells and newly formed fiberous 、 blood vessels could be seen in rDFCs+scaffold group and rBMSCs+scaffold group. More cells and newly formed fiberous、blood vessels could be seen in rDFCs+scaffold+LIPUSgroup and rBMSCs+scaffold+LIPUS group.ConclusionThe osteogenesis differentiation ability of rDFCs and rBMSCs could be enhanced by LIPUS in vitro. The electron microscope scanning results showed that rDFCs and rBMSCs could grow well on the prefabricated-mineralized inducing bone tissue engineering scaffold material. After rDFCs and rBMSCs was seeded on the scaffold, the cell-scaffold complex was transplanted into the subcutaneous of nude mice.The transplant site was exposed by LIPUS andthe results showed that LIPUS could promote the osteogenesis differentiation ability of rDFCs and rBMSCs.This study preliminary suggests that LIPUS can promote the osteogenesis differentiationof rDFCs and rBMSCs, and it provided a reference for the application of LIPUS in periodontal tissue regeneration.