Low-intensity pulsed ultrasound promotes tissue regeneration in rat dental follicle cells in a porous ceramic scaffold

Abstract The aim of this study was to investigate the effects of low-intensity pulsed ultrasound (LIPUS) on the osteogenic differentiation of dental follicle cells (DFCs) in vitro and on the regenerative effects of DFC-OsteoBoneTM complexes in vivo. DFCs were isolated and characterized. In the in vitro study, DFCs were cultured in an osteogenic medium in the presence or absence of LIPUS. The expression levels of ALP, Runx2, OSX, and COL-I mRNA were analyzed using real-time polymerase chain reaction (RT-PCR) on day 7. Alizarin red staining was performed on day 21. The state of the growth of the DFCs that were seeded on the scaffold at 3, 5, 7, and 9 days was detected by using a scanning electron microscope. In our in vivo study, 9 healthy nude mice randomly underwent subcutaneous transplantation surgery in one of three groups: group A, empty scaffold; group B, DFCs + scaffold; and group C, DFCs + scaffold + LIPUS. After 8 weeks of implantation, a histological analysis was performed by HE and Mason staining. Our results indicate that LIPUS promotes the osteogenic differentiation of DFCs by increasing the expression of the ALP, Runx2, OSX, and COL-I genes and the formation of mineralized nodules. The cells can adhere and grow on the scaffolds and grow best at 9 days. The HE and Mason staining results showed that more cells, fibrous tissue and blood vessels could be observed in the DFCs + scaffold + LIPUS group than in the other groups. LIPUS could promote the osteogenic differentiation of DFCs in vitro and promote tissue regeneration in a DFCs-scaffold complex in vivo. Further studies should be conducted to explore the underlying mechanisms of LIPUS.

摘要 本研究旨在探讨低强度脉冲超声（low-intensity pulsed ultrasound, LIPUS）对体外牙囊细胞（dental follicle cells, DFCs）的成骨分化作用，以及其对牙囊细胞-OsteoBone™复合物体内再生的影响。本研究分离并鉴定了牙囊细胞。体外实验中，将牙囊细胞置于成骨培养基中培养，分别施加或不施加低强度脉冲超声干预。于培养第7天采用实时荧光定量聚合酶链反应（real-time polymerase chain reaction, RT-PCR）检测碱性磷酸酶（alkaline phosphatase, ALP）、Runt相关转录因子2（Runx2）、Osterix（OSX）及I型胶原（collagen type I, COL-I）的mRNA表达水平；于第21天进行茜素红染色。通过扫描电子显微镜观察接种于支架上的牙囊细胞在第3、5、7、9天的生长状态。体内实验部分，将9只健康裸鼠随机分为3组实施皮下移植手术：A组为空白支架组，B组为牙囊细胞+支架组，C组为牙囊细胞+支架+低强度脉冲超声组。移植8周后，采用HE染色及Mason染色开展组织学分析。研究结果显示：低强度脉冲超声可通过上调ALP、Runx2、OSX及COL-I基因的表达，促进矿化结节形成，从而增强牙囊细胞的成骨分化能力；细胞可在支架表面黏附并生长，且于第9天时生长状态最佳。HE染色及Mason染色结果表明，相较于其余两组，C组可观察到更多的细胞、纤维组织与血管。综上，低强度脉冲超声可在体外促进牙囊细胞的成骨分化，并在体内促进牙囊细胞-支架复合物的组织再生。未来需开展进一步研究以阐明低强度脉冲超声发挥调控作用的潜在分子机制。