Research of the cartilago regeneration using ultrasonic stimulus and the hyaluronic acid

目标。我们首次证明，通过低强度脉冲超声（LIPUS）刺激，可以增强培养的软骨细胞的增殖，并促进在三维（3-D）培养系统中生长的原代软骨细胞中ix型胶原的产生。此外，我们还确定了细胞内信号通路MAPK/MAPKK和Integrin/PI3K/Akt参与软骨细胞增殖过程的机械转导的潜在机制之一。关节软骨组织取自刚屠宰的猪跖指关节。将胶原凝胶和培养基复合材料中分离的软骨细胞添加到i型胶原蜂窝海绵中。最终细胞密度调整为2×10^6个细胞/孔/ml。LIPUS刺激软骨细胞培养，每天20分钟。用抗ii型和抗ix型胶原抗体检测培养后第2周软骨细胞和t细胞的表型，检测ii型和ix型胶原的产生。免疫染色和Western blot分析使用以下一抗：ii型和ix型胶原蛋白，Akt, p-Akt， Cyclin B1和D1， MAPK, p-MAPK， FAK, p-FAK, Paxillin, p-Paxillin, β-catenin， β-1整合素，α- 1,2和v整合素。软骨细胞增生，胶原海绵表面形成胶原基质。通过将软骨细胞暴露于LIPUS，含有ii型胶原蛋白和蛋白多糖的更厚的基质层积累起来。在两周的培养期间，LIPUS刺激下培养的软骨细胞增殖率明显高于对照组。Western blot结果显示，与对照组相比，LIPUS刺激组ix型胶原、Cyclin B1和D1、p-FAK和p-Akt的表达更为强烈。而两组间MAPK、p-MAPK及ii型胶原的表达差异无统计学意义。在以胶原海绵为载体的三维培养系统中，LIPUS刺激促进了培养软骨细胞的增殖和ix型胶原蛋白的产生，从而创造了类似软骨组织的环境。少

Objective. We have shown for the first time that it is possible to enhance the proliferation of cultured chondrocytes and promote the production of type-IX collagen in primary chondrocytes grown on a three-dimensional (3-D) culture system through low-intensity pulsed ultrasound (LIPUS) stimulation. Moreover, one of the underlying mechanisms of intracellular signaling pathways such as MAPK/MAPKK and Integrin/PI3K/Akt involved in mechano-transduction during the proliferation of chondrocytes was identified.Methods. Articular cartilage tissue was obtained from the metatarso-phalangeal joints of freshly slaughtered porcines. Isolated chondrocytes in collagen gel and culture medium composites were added to type-I collagen honeycomb sponges. The final cell density was adjusted to 2×10^6 cells/well/ml. LIPUS stimulation was applied to the chondrocyte cultures for 20 minutes daily. Anti-type-II and anti-type-IX collagen antibodies were used to investigate the phenotype of the chondrocytes and t … More o detect type-II and type-IX collagen production at post-culture week 2. For immuno-staining and Western blot analysis, the following primary antibodies were used: type-II and type-IX collagen, Akt, p-Akt, Cyclin B1 and D1, MAPK, p-MAPK, FAK, p-FAK, Paxillin, p-Paxillin, β-catenin, β-1 Integrin, and α-1, 2 and v Integrins.Results. Chondrocytes proliferated and a collagenous matrix formed on the surface of the collagen sponge. By exposing chondrocytes to LIPUS, thicker layers of matrix containing type-II collagen and proteoglycan accumulated. The rate of proliferation of chondrocytes cultured under LIPUS stimulation was significantly higher than in the control group during the two-week culture period. In Western blot analysis, type-IX collagen, Cyclin B1 and D1, p-FAK, and p-Akt were found to be more strongly expressed in the LIPUS stimulation group than in the control. However, there was no difference between the two groups in MAPK, p-MAPK and type-II collagen expression.Conclusion. LIPUS stimulation promoted the proliferation of cultured chondrocytes and the production of type-IX collagen in a 3-D culture system using collagen sponge as a vehicle to create an environment similar to cartilaginous tissue. Less