Osteogenesis and expression of bone morphogenetic protein 2 after bone marrow mesenchymal stem cells combined with calf cortical bone with partial cancellous bone implanted into rabbits

doi:10.3969/j.issn.2095-4344.2017.17.010

Abstract

Abstract:

BACKGROUND: Previous studies have confirmed that rabbit bone marrow mesenchymal stem cells (BMSCs) can differentiate into osteoblasts under osteogenic induction in vitro, stably express the specific phenotype of osteoblasts and have osteogenic ability. Calf cortical bone scaffold with partial cancellous bone has good biocompatibility and degradability, which can be used as a carrier material of bone marrow mesenchymal stem cells.
OBJECTIVE: To combine rabbit BMSCs with calf bone composite according to the basic principles of bone tissue engineering and to observe the osteogenesis in the New Zealand white rabbits after implantation of BMSCs/calf bone composite into the ilium, thereby providing a direct evidence for preliminary clinical application of tissue-engineered bone products.
METHODS: BMSCs/calf cortical bone scaffold with partial cancellous bone (tissue-engineered bone group), simple calf heterogeneous bone (heterogeneous bone group) or autologous iliac bone (autologous iliac bone group) was randomly implanted into the rabbit ilium. The changes of implant surface and tissue reactions around the implant were observed. X-ray examination was performed to observe osteogenic changes at 4, 8, 12, 24 weeks after implantation. Immunohistochemistry staining was used to observe the expression of bone morphogenetic protein 2.
RESULTS AND CONCLUSION: After heterogeneous bone implantation, the wound healed well, and there were no systemic or local inflammation and toxicity reactions in all groups. The X-ray results showed that at postoperative 24 weeks, the implant was basically fused with the host bone in the tissue-engineered bone group, but the fusion was unsatisfactory in the heterogeneous bone group. The process of ossifications from cartilages was observed in all groups by hematoxylin-eosin staining, and bone morphogenetic protein 2 was positive for immunohistochemical staining. Findings from in vivo experiments indicate that rabbit BMSCs seeded onto the calf cortical bone scaffold with partial cancellous bone could construct tissue-engineered bone by osteoinductation in vitro in the rabbits.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Stem Cells, Bone Morphogenetic Proteins, Tissue Engineering

CLC Number:

R394.2

Chen Neng, Shao Yun-feng, Liu Tang, Zhang Xiang-sheng, Xiong Guang-zhong. Osteogenesis and expression of bone morphogenetic protein 2 after bone marrow mesenchymal stem cells combined with calf cortical bone with partial cancellous bone implanted into rabbits[J]. Chinese Journal of Tissue Engineering Research, 2017, 21(17): 2684-2689.

Figures/Tables 1

2.1 实验动物数量分析 60只新西兰大白兔无脱落，全部进入结果分析。各组材料复合体植入兔髂骨后，伤口生长良好，没有全身和局部的炎症和毒性反应，伤口一期愈合。植入材料均未见明显炎症和排斥反应。 2.2 术后X射线片观察于术后4，8，12，24周行X射线片检查，观察各组成骨情况。发现未复合骨髓间充质干细胞的异种骨组植入后与宿主骨愈合能力较组织工程骨组差，但组织工程骨与宿主骨的愈合能力差于自体髂骨移植，见图1。 2.3 苏木精-伊红染色结果于术后4，8，12，24周处死动物，取材石蜡块包埋，切片行苏木精-伊红染色，行组织学观察。 (1)组织工程骨组：术后4周，可见组织工程骨周围有大量软组织紧密粘贴，与宿主骨结合处，形成骨痂。组织工程骨载体内部及边缘可见大量的间充质细胞、成纤维细胞和软骨细胞，表面有较多骨吸收陷窝，内有破骨细胞活动。骨痂内仍可见肥大、固缩、空白陷窝明显的发生退行性变的软骨细胞，以及不同发育阶段的软骨细胞，表现为软骨化骨的过程(图2A)；术后8周，组织工程骨周围及与宿主骨结合处骨痂比较成熟，可见编织骨，成熟骨小梁；术后12周，在组织工程骨周围及与宿主骨结合处有大量的骨痂包围，可见类骨组织、编织骨等。异种骨的内外表面有多量的陷窝，内含有骨细胞、破骨细胞等。横截面可见多量的扩大的哈佛氏管，其中参与成骨的细胞排列成“镶边状”，毛细血管增多、变粗；术后24周，在组织工程骨和宿主骨连接处可见大量的骨小梁形成，内外表面吸收陷窝继续增加，皮质骨内可见扩大的哈佛氏管继续增加，成骨及破骨活动更加活跃，有些部位见新形成板状骨和一些未吸收完全的异种骨。 (2)异种骨：术后4周，异种骨表面可见少量的软组织贴附，且不够紧密，载体边缘可见成纤维细胞、少量软骨细胞，载体内偶尔可见新生的毛细血管；术后8周，异种骨的表面见少量的骨吸收馅窝，皮质骨内部哈佛管无明显变化；术后12周，可见较多软骨细胞，未见类骨组织、编织骨，见新生毛细血管(图2B)；术后24周，皮质骨内部结构变化不明显，类编织骨出现，新生骨尚不成熟。 (3)自体髂骨组：术后4，8周与组织工程骨组类似；术后12周，移植自体髂骨与宿主骨基本愈合；术后24周，移植自体髂骨与宿主骨完全融合。 2.4 BMP-2免疫组化结果于术后4，8，12，24周处死动物，取材石蜡块包埋，切片行BMP-2免疫组化。 (1)组织工程骨组：术后4周，组织工程骨-宿主骨结合处骨细胞强阳性表达。皮质骨扩大的哈佛氏管呈弱阳性表达，异种骨表面的骨吸收陷窝内阳性表达清晰，骨基质呈弱阳性表达；术后8周，异种骨周围及与宿主骨结合处骨痂内骨细胞阳性表达(图3A)；术后12周，组织工程骨的外周可见较强的BMP-2阳性表达，皮质骨内扩大的哈佛氏管内细胞成分增多，围绕内壁的细胞呈阳性表达，同时，哈佛氏管周围数个骨陷窝内的骨细胞阳性表达；术后24周，组织工程骨周边成熟的骨单位和正在发育中的骨单位BMP-2染色均呈阳性；周围骨细胞陷窝也呈阳性表达。 (2)异种组：各时间段均可观察到BMP-2阳性表达，但阳性表达明显较组织工程骨组弱(图3B)。 (3)自体髂骨组：术后4，8周与组织工程骨组类似，但阳性表达明显较组织工程骨组强；术后12周，移植自体髂骨与宿主骨基本愈合；术后24周，移植自体髂骨与宿主骨完全融合。结果显示：BMP-2阳性表达存在于成骨细胞胞浆和及其分泌的类骨质中，异种骨本身、成熟软骨细胞、成熟骨小梁基质中未见BMP-2阳性表达。移植的异种骨哈佛氏管内新生的骨组织及其周围的类骨质BMP-2表达呈阳性。"

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