The mixture and PLGA (Mw = 200,000, Jinan Daigang Biomaterial Co., Ltd, China) were used as the two kinds of ink for 3D printing. A mixture of gelatin (10% (w/v)), glycerol (10% (v/v)), and glutaraldehyde (1% (v/v)) was applied as dispersant and binder. Α- tricalcium phosphate (TCP) was synthesized by a mixture of CaCO 3 and CaHPO 4 in a certain ratio in a high temperature calcination reaction. Furthermore, we focused on the influence of LIPUS combined with MBs in different irradiation intensity to promote bone repair, and finally got optimal acoustic parameters. Therefore, we hypothesized that LIPUS combined with MBs could promote the proliferation and bone regenerative repair of bone marrow mesenchymal stem cells (BMSCs) in 3D-printed scaffolds. Ultrasonic lipid microbubbles (MBs) can burst to form acoustic cavitation under the action of ultrasound, and the energy released can start or promote the sonochemical reaction. LIPUS not only acts on osteoblasts, osteoclasts and mesenchymal stem cells to exert osteogenic effects, but also has a positive effect on bone healing and regeneration through its effects on blood vessels and nerves. Preclinical trials have demonstrated the potential of LIPUS in the field of bone tissue repair and regeneration. Low-intensity pulsed ultrasound (LIPUS) is a mechanical stimulation composed of constant periodic amplitude waves with intensity or sonic intensity (SI) ranging from 5 to 100 mW/cm 2. 3D-printing technology can be designed for patients by computers, can adjust shapes and internal 3D structures, and can prepare personalized scaffolds with biocompatibility, which, in comparison to previous methods, can better solve the problem of personalized and accurate repair. The development of biomaterials, stem cells and bone tissue engineering technology provide new hope for bone regeneration. However, it is difficult for traditional scaffold materials to have a good biocompatibility, biodegradability and porous three-dimensional structure and to have the characteristics of bone conduction, bone induction and osteogenesis. Tissue engineering has provided hope for bone repair and is an effective way to repair bone defects, which has been a concern of clinical and basic researchers. It is estimated that more than 2 million bone grafts are performed worldwide each year to provide a solution for cases in which the natural repair of bone is hampered. LIPUS combined with lipid microbubbles on PLGA/TCP scaffolds can promote BMSCs growth and bone differentiation, which is expected to provide a new and effective method for the treatment of bone regeneration in tissue engineering.Ī variety of trauma, tumours, infections, and congenital bone diseases caused by large bone defects, which affect the quality of life and physical and mental health of patients, have been a common clinical problem. After 21 days, scanning electron microscopy experiments showed that osteogenesis was obvious in the PLGA/TCP scaffolds. After 14 days, the type I collagen expression and alkaline phosphatase activity in the scaffold increased significantly compared to those in the control group, and alizarin red staining showed more calcium salt production during osteogenic differentiation.
ResultsīMSCs proliferation was the most significant under the condition of 0.5% (v/v) lipid microbubble concentration, 2.0 MHz frequency, 0.3 W/cm 2 sound intensity and 20% duty cycle. Alizarin red staining was used to evaluate the calcium salt production during osteogenic differentiation. The expression of type I collagen and the activity of alkaline phosphatase were detected. MethodsīMSCs were irradiated with different LIPUS parameters and microbubble concentrations, and the best acoustic excitation parameters were selected. To investigate the effect of low-intensity pulsed ultrasound (LIPUS) combined with lipid microbubbles on the proliferation and bone regeneration of bone marrow mesenchymal stem cells (BMSCs) in poly (lactic-glycolic acid copolymer) (PLGA)/α-tricalcium phosphate (TCP) 3D-printed scaffolds.
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