血管内皮细胞和α-磷酸三钙促进骨髓基质干细胞成骨分化作用的研究
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摘要
由于肿瘤、外伤、炎症、畸形等因素所造成骨破坏的治疗一直是口腔医学领域的难题，利用组织工程技术构建人工骨进行骨缺损的治疗是目前主要的研究方向之一，而骨组织工程存在的主要问题是所构建的人工骨成骨能力有限。通过建立功能性微血管环境可以满足种子细胞新陈代谢需求，进一步促进新骨形成以及参与细胞间的相互作用。由于血管在骨形成中具有重要作用，使得血管内皮细胞（endothelial cells，ECs）在骨组织工程中应用被广泛关注，但其来源有限，且不易扩增。骨髓基质干细胞(bone mesenchymal stemcells，BMSCs)在骨髓中大量存在，容易获取，且有向血管内皮细胞等多向分化的潜能。因此，我们首先分离了兔的BMSCs作为种子细胞，经内皮细胞条件培养液中诱导培养，利用内皮细胞表面标记物CD34免疫组化染色证明其诱导的细胞为血管内皮细胞。体外以单纯BMSCs为对照组，ECs和BMSCs共培养为实验组，通过碱性磷酸酶活性检测、考马斯亮蓝总蛋白测定、碱性磷酸酶和茜素红染色，结果表明，ECs对BMSCs向成骨细胞分化具有促进作用。将共培养的种子细胞接种在PLGA支架材料上，植入直径为1cm的兔下颌骨缺损模型中，通过软X线和组织化学染色分别对4周和8周的实验动物进行观察，结果显示BMSCs可以在PLGA支架材料上生长且材料具有较好的生物相容性且无细胞毒性，诱导的ECs与BMSCs共培养在PLGA支架材料上，同BMSCs对照组比具有明显的促成骨作用及骨缺损修复能力。结果提示血管化组织工程骨有望成为修复大面积骨缺损的有效的途径。
磷酸钙是天然骨的主要组成成分，作为骨替代物、人工骨和组织工程的支架等具有较好的应用前景。不同结构的磷酸钙中，羟基磷灰石（HA）和磷酸三钙（TCP）由于对人体生理环境具有较强的生物学反应而受到广泛的关注。由于HA的化学结构稳定使其具有较强的抗吸收作用，但其密度高、脆性大、弹性差等原因限制了其广泛应用。TCP按结构分为高温相(α-TCP)和低温相(β-TCP)，其中α-TCP相对于β-TCP具有更好的生物降解性和生物相容性，并且植入人体后反应温和，基本不产热，降解后被生物体迅速吸收和取代，因此具有良好的生物医学应用前景。本研究通过共沉淀法合成了α-TCP，采用XRD、SEM、TEM等方法评价了其物理学及化学性能。通过与大鼠BMSCs共培养，检测MTT以评价材料的生物安全性，通过Real Time PCR检测ALPase、SP7、RUNX2、COL-Ⅰ等成骨细胞相关因子mRNA的表达，探讨α-TCP在体外对BMSCs成骨作用的影响。结果表明，α-TCP能够上调大鼠BMSCs向成骨细胞分化相关基因的表达，证明α-TCP可以促进BMSCs向成骨细胞分化的作用，这就为其在骨组织工程中的应用提供了一定的理论依据。
Tissue engineering strategies often fail to regenerate bones because of inadequatevascularization, especially in the reconstruction of large segmental bone defects. Large volumesof vascular endothelial cells (ECs) that functionally interact with osteoblasts during osteogenesisare difficult to obtain. In this study, we simulated bone healing by co-culturing differentiatedECs and mesenchymal stem cells (MSCs) either on a culture plate or on a polylactide glycolicacid (PLGA) scaffold in vitro. We also evaluated the effect of osteogenesis in repairingrabbitmandible defects in vivo. In this study, MSCs were separated from rabbit as the seed cells.After passage, the MSCs were cultured in an EC-conditioned medium to differentiate into ECs.Immunohistochemical staining analysis with CD34showed that the induced cells had thecharacteristics of ECs and MSCs. The induced ECs were co-cultured in vitro, and the inductionof MSCs to osteoblast served as the control. Alkaline phosphatase (ALP) and alizarin red (AZR)staining experiments were performed, and the Coomassie brilliant blue total protein and ALPactivity were measured. The MSCs proliferated and differentiated into osteoblast-like cellsthrough direct contact between the derived ECs and MSCs. The co-cultured cells were seeded onPLGA scaffold to repair1cm mandible defects in the rabbit. The effectiveness of the repairs wasassessed through soft X-ray and histological analyses. The main findings indicated that MSCssurvived well on the scaffold and that the scaffold is biocompatible and noncytotoxic. Theresults demonstrated that the co-cultured MSC-derived ECs improved MSC osteogenesis andpromoted new bone formation. This study may serve as a basis for the use of in vitroco-culturing techniques as an improvisation to bone tissue engineering for the repair of largebone defects.
Calcium phosphate ceramics of tricalcium phosphate(TCP) and hydroxyl apatite(HA)powders are widely applied in biomedical fields because of their biocompatibility andosteoconductivity.In orthopedic surgery,they are used for filling bone defects as a result of theremoval of diseased or damaged bones. In dentistry, calcium phosphate ceramics are used for the augmentation of deficient mandibular of maxillar ridges. Dense or porous calcium phosphateceramic coatings are often applied on strong and load-bearing core materials for biologicalfixation or osteointegration. In our study,we synthesized the ɑ-TCP, cocultured with rat BMSCsto evaluated the biological safety by MTT, analysis with ALPase mRNA，SP7mRNA, RUNX2mRNA and Col-ⅠmRNA throuh real-time quantitative PCR to investigate the osteogenesisinductivity of ɑ-TCP, and the results proved that ɑ-TCP improve the osteogenesis effection ofBMSCs. It maybe provide a theoretical basis for the further elucidate osteoblast differentiationand regulation mechanism and can apply in short and long-term bone regeneration.
磷酸钙是天然骨的主要组成成分，作为骨替代物、人工骨和组织工程的支架等具有较好的应用前景。不同结构的磷酸钙中，羟基磷灰石（HA）和磷酸三钙（TCP）由于对人体生理环境具有较强的生物学反应而受到广泛的关注。由于HA的化学结构稳定使其具有较强的抗吸收作用，但其密度高、脆性大、弹性差等原因限制了其广泛应用。TCP按结构分为高温相(α-TCP)和低温相(β-TCP)，其中α-TCP相对于β-TCP具有更好的生物降解性和生物相容性，并且植入人体后反应温和，基本不产热，降解后被生物体迅速吸收和取代，因此具有良好的生物医学应用前景。本研究通过共沉淀法合成了α-TCP，采用XRD、SEM、TEM等方法评价了其物理学及化学性能。通过与大鼠BMSCs共培养，检测MTT以评价材料的生物安全性，通过Real Time PCR检测ALPase、SP7、RUNX2、COL-Ⅰ等成骨细胞相关因子mRNA的表达，探讨α-TCP在体外对BMSCs成骨作用的影响。结果表明，α-TCP能够上调大鼠BMSCs向成骨细胞分化相关基因的表达，证明α-TCP可以促进BMSCs向成骨细胞分化的作用，这就为其在骨组织工程中的应用提供了一定的理论依据。
Tissue engineering strategies often fail to regenerate bones because of inadequatevascularization, especially in the reconstruction of large segmental bone defects. Large volumesof vascular endothelial cells (ECs) that functionally interact with osteoblasts during osteogenesisare difficult to obtain. In this study, we simulated bone healing by co-culturing differentiatedECs and mesenchymal stem cells (MSCs) either on a culture plate or on a polylactide glycolicacid (PLGA) scaffold in vitro. We also evaluated the effect of osteogenesis in repairingrabbitmandible defects in vivo. In this study, MSCs were separated from rabbit as the seed cells.After passage, the MSCs were cultured in an EC-conditioned medium to differentiate into ECs.Immunohistochemical staining analysis with CD34showed that the induced cells had thecharacteristics of ECs and MSCs. The induced ECs were co-cultured in vitro, and the inductionof MSCs to osteoblast served as the control. Alkaline phosphatase (ALP) and alizarin red (AZR)staining experiments were performed, and the Coomassie brilliant blue total protein and ALPactivity were measured. The MSCs proliferated and differentiated into osteoblast-like cellsthrough direct contact between the derived ECs and MSCs. The co-cultured cells were seeded onPLGA scaffold to repair1cm mandible defects in the rabbit. The effectiveness of the repairs wasassessed through soft X-ray and histological analyses. The main findings indicated that MSCssurvived well on the scaffold and that the scaffold is biocompatible and noncytotoxic. Theresults demonstrated that the co-cultured MSC-derived ECs improved MSC osteogenesis andpromoted new bone formation. This study may serve as a basis for the use of in vitroco-culturing techniques as an improvisation to bone tissue engineering for the repair of largebone defects.
Calcium phosphate ceramics of tricalcium phosphate(TCP) and hydroxyl apatite(HA)powders are widely applied in biomedical fields because of their biocompatibility andosteoconductivity.In orthopedic surgery,they are used for filling bone defects as a result of theremoval of diseased or damaged bones. In dentistry, calcium phosphate ceramics are used for the augmentation of deficient mandibular of maxillar ridges. Dense or porous calcium phosphateceramic coatings are often applied on strong and load-bearing core materials for biologicalfixation or osteointegration. In our study,we synthesized the ɑ-TCP, cocultured with rat BMSCsto evaluated the biological safety by MTT, analysis with ALPase mRNA，SP7mRNA, RUNX2mRNA and Col-ⅠmRNA throuh real-time quantitative PCR to investigate the osteogenesisinductivity of ɑ-TCP, and the results proved that ɑ-TCP improve the osteogenesis effection ofBMSCs. It maybe provide a theoretical basis for the further elucidate osteoblast differentiationand regulation mechanism and can apply in short and long-term bone regeneration.
引文
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