Tissue engineering using hydroxyapatite fiber sheet as a scaffold for bone regeneration culturing mesenchymal stem cells, and application for posterolateral spine fusion as a bone graft

根据2002年对MC3-T3细胞和骨髓细胞的体外研究结果，羟基磷灰石纤维(HAF)可能既可以作为重组人骨形态发生蛋白-2的载体，也可以作为骨再生的支架来支持3D细胞的增殖。2002年，我们制作了大鼠后外侧融合模型。利用这些模型，我们在2003年进行了两项研究，1)评价HAF作为重组人骨形态发生蛋白-2载体的体内应用，为将来在脊柱后外侧融合手术中的临床应用提供依据；2)利用大鼠成骨细胞，评价羟基磷灰石纤维作为成骨细胞支架的生物学特性和实用性。实验1)14周龄Wistar大鼠应用含有重组人骨形态发生蛋白-2(5，10，16μg)的HAF进行双侧L5和L6后外侧融合。植入后取材，评价椎体融合情况。实验2)从1日龄Wistar大鼠颅骨分离出成骨细胞。…用HAFs的孔径和孔隙率另外，本研究的控制率分别为5μm和95%(S0)，250μm和99%(52000)。每种支架接种5×106个细胞。传代培养2周。将含有成骨细胞的支架植入4周龄裸鼠背部皮下。植入后取材。根据病理结果评价骨形成情况。结果1)15μg重组人骨形态发生蛋白-2组术后4周所有标本均有骨融合。结果2)体内培养的细胞均可见软骨内成骨。与S0相比，S2000支架多孔，能够支持细胞的三维增殖。他们可以在体内的支架内引入新的骨形成。但由于它们的机械易碎性，它们的尺寸变小了。因此，我们得出结论，HAF不仅是一种可以接受的材料，可以将rhBMP-2转移到融合部位，从而促进坚实的后外侧脊柱融合，而且还可以作为骨再生的支架，以支持3D细胞的增殖。较少

According to the results of in vitro studies using MC3-T3 cells and bone marrow cells in 2002, hydroxyapatite fibers (HAF) might be useful either as a carrier of rhBMP-2 and a scaffold for bone regeneration to support 3D cell proliferation. In 2002, we have made posterolateral fusion model of rats. Using these models, in 2003, we made two studies, 1) to evaluate usefulness of HAF as a carrier of rhBMP-2 in vivo with future implication of clinical use for posterolateral spinal fusion surgery, 2) to evaluate the biological property and usufulness of hydroxyapatite fiber as a scaffold of osteoblast using rat osteoblastic cells in vivo. Experiment 1) Bilateral posterolateral fusion between L5 and 6 was performed to 14week old Wistar rats using HAF containing rhBMP-2 (5, 10, 16μg). Specimens were retrieved and spinal bone fusion was assessed after implantation. Experiment 2) Osteoblastic cells were isolated from skulls of 1day old wistar rats. Diameter of pores and porosity of HAFs used in … More this study were controlled to be 5μm and 95% (S0), 250μm and 99%(52000) respectively. 5×10^6 cells were seeded in each scaffolds. The subcultures were performed for 2weeks. Scaffolds with osteoblastic cells were implanted in the back of 4-week-old athymic mouse at the subcutaneous site. Specimens were retrieved after implantation. Bone formation was evaluated by pathologic findings. Result 1) Bone fusion was observed at 4 weeks in all specimens in the group of 15μg rhBMP-2. Result 2) Enchondral bone formation was found at all the cells cultured in the scaffolds implanted in vivo. S2000 scaffolds were porose and able to support 3D cell proloferation comparing with S0. They could introduce new bone formation inside the scaffolds in vivo. But they decreased in size due to their mechanical fragility. Thus we conclude that HAF is not only an acceptable material for delivering rhBMP-2 to a fusion site, thereby enhancing solid posterolateral spinal fusion, but also a scaffold for bone regeneration to support 3D cell proliferation. Less