In vivo ectopic osteogenesis of adipose-derived mesenchymal stem cells/osteoblasts combined with hydroxyapatite/chitosan/polylactic acid

doi:10.3969/j.issn.2095-4344.0522

Abstract

Abstract:

BACKGROUND: Osteoblast deficiency is a key problem in bone tissue engineering, and transplantation of mesenchymal stem cells combined with osteoblast can achieve ideal results.
OBJECTIVE: To investigate the in vivo ectopic osteogenesis of adipose-derived mesenchymal stem cells (ADSCs) and osteoblasts (OB) combined with hydroxyapatite (HA)/chitosan (CS)/poly(L-latic acid) (PLLA).
METHODS: ADSCs and OB were obtained by adherent method and enzymatic digestion method. ADSCs, OB and the mixture of ADSCs and OB (at a mixture ratio of 1:1) were cultured with HA/CS/PLLA, respectively. After 48 hours of in vitro culture, cell-scaffold complexes were subcutaneously implanted into the back of Sprague-Dawley rats in corresponding groups, and HA/CS/PLLA without cells was implanted as control group. The rats in each group were killed at 8 weeks postoperatively. The macroscopic and histopathological observations were performed to assess the ectopic osteogenesis potential.
RESULTS AND CONCLUSION: (1) After adipogenic, chondrogenic and osteogenic induction, ADSCs were positive for oil red O, toluidine blue and alizarin red staining. Results from flow cytometry showed that ADSCs were positive for CD147, CD90, CD105 and CD44 with the rate of positivity > 80%, but negative for CD117, CD34, CD131, CD45 with the rate of positivity < 5%. (2) Passage 3 OB were positive for both alizarin red staining and alkaline phosphatase staining. (3) At 8 weeks after implantation, soft tissues grew into the complexes under gross observation. (4) At 8 weeks after implantation, ectopic bone formation was visible in each group. The bone formation was more visible in the ADSCs-OB/HA/CS/PLLA group than the other groups with significant differences (P < 0.05). To conclude, ADSCs can promote the ectopic bone formation of OB in vivo in combination with HA/CS/PLLA scaffold.

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: Osteoblasts, Adipose Tissue, Mesenchymal Stem Cells, Calcium Phosphates, Tissue Engineering

CLC Number:

R394.2

Tian Xue-tao, Wang Xian-xun, Wang Xiao-wei. In vivo ectopic osteogenesis of adipose-derived mesenchymal stem cells/osteoblasts combined with hydroxyapatite/chitosan/polylactic acid[J]. Chinese Journal of Tissue Engineering Research, 2018, 22(21): 3304-3309.

Figures/Tables 1

2.1 细胞形态学观察及染色鉴定结果 ADSCs原代培养4-6 h后开始贴壁，细胞大小不均匀，起初为多角形，梭形，3 d后类成纤维细胞明显增多，四五天时细胞进入对数生长期，六七天时细胞融合接近80%，可消化传代(图1A)；传至第3代后细胞呈长梭形，形态均一，呈漩涡样生长(图1B)，成脂诱导后油红O染色可见红染的脂滴(图1C)，成软骨诱导后可见细胞质蓝染(图1D)，成骨诱导后茜素红染色可见红染的钙化灶(图1E)。骨组织块法获得的成骨细胞贴于细胞瓶底部，24 h后成骨细胞从骨块中爬出，葵花样生长，细胞呈三角形、多角形或梭形(图1F)。传至第3代后细胞呈鳞形，体积较大，多伪足，继续培养可见局部多层生长，并有钙化结节生成(图1G)，培养14 d时茜素红染色可见红染的钙化灶(图1H)，碱性磷酸酶染色可见胞质咖啡样深染(图1I，J)。 HA/CS/PLLA材料呈乳白色，被制成3 mm×3 mm×3 mm的圆柱状中空材料以便于植入体内(图1K-N)。 2.2 ADSCs表面抗原表达 CD147、CD90、CD105、CD44呈阳性表达(> 80%)，而CD117、CD34、CD31、CD45则呈阴性表达(< 5%)，见图2。 2.3 ADSCs和成骨细胞的增殖能力随着培养时间的延长，细胞数逐渐增多，吸光度值增高；ADSCs呈“S”形增殖曲线，第1，2天为平台期，第3-5天为对数生长期，第6天以后细胞密度较大，细胞间接触抑制生长，进入平台期。而成骨细胞呈直线生长，第1，2天为平台期，第3-8天细胞基本呈线性生长，见图3。 2.4 大体观察结果 10只SD大鼠全部存活并进入结果分析，术后伤口愈合良好，无炎性反应，伤口一期愈合，期间进食正常。植入4周后可见材料有软组织长入，包裹，难以分离(图1L，M)。 2.5 苏木精-伊红染色观察组织学形态 ADSCs+成骨细胞+HA/CS/PLLA组：术后8周，移植物已完全被新生的编制骨替代，形成明显软骨陷窝，视野下可见大量钙化的骨组织，并形成骨髓腔，HA/CS/PLLA已被降解、吸收(图4A)。成骨细胞+HA/CS/PLLA组：术后8周，移植物有新生编制骨及大量软骨形成，软骨基质较多，在钙化的骨组织中仍有未完全骨化的软骨。HA/CS/PLLA材料内有大量的纤维、血管、脂肪等结缔组织长入，支架开始降解(图4B)。 ADSCs+HA/CS/PLLA组：术后8周，支架材料表面及周边纤维组织内可见带状、条索状骨组织，内部可见软骨陷窝和软骨基质，周边有大量成骨细胞及未成熟的骨组织(图4C)。 HA/CS/PLLA组：术后8周，支架材料表面及周边纤维组织内可见岛状骨组织，支架材料降解较少，可见少量软骨细胞，周边可见少量成骨细胞和纤维组织(图4D)。 2.6 图像分析结果各组术后8周植入物新骨形成差异明显，ADSCs+成骨细胞+HA/CS/PLLA组成骨分数为(33.8± 3.11)%，成骨细胞+HA/CS/PLLA组为(21.4±3.24)%，两组间比较差异有显著性意义(P < 0.05)；HA/CS/PLLA组为(2.89±0.91)%，ADSCs+HA/CS/PLLA组成骨分数为(13.2± 1.59)%，与ADSCs+成骨细胞+HA/CS/PLLA组比较差异也有显著性意义(P < 0.05)。"

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