3D打印胶原/羟基磷灰石支架对骨髓间充质干细胞成骨分化的作用研究

doi:10.3877/cma.j.issn.2096-0263.2020.03.001

中华老年骨科与康复电子杂志 ›› 2020, Vol. 06 ›› Issue (03) : 123 -127. doi: 10.3877/cma.j.issn.2096-0263.2020.03.001

所属专题：文献；

基础研究

3D打印胶原/羟基磷灰石支架对骨髓间充质干细胞成骨分化的作用研究

邬波¹^,^†(

), 柳椰², 马旭¹, 智春升³, 杜明昌², 翟良全², 杨政博², 王佳媛⁴, 王译晗⁴

^1. 110044　沈阳市骨科医院关节外科；110044　辽宁省骨关节病重点实验室
^2. 110044　沈阳市骨科医院关节外科
^3. 110044　沈阳市骨科医院骨病肿瘤科
^4. 110044　辽宁省骨关节病重点实验室

收稿日期:2020-03-20 出版日期:2020-06-05
通信作者: 邬波
基金资助:
辽宁省自然科学基金指导计划(20180550795)

Effect of 3D printed collagen / hydroxyapatite scaffold on osteogenic differentiation of bone marrow mesenchymal stem cells

Bo Wu¹(), Ye Liu², Xu Ma¹, Chunsheng Zhi³, Mingchang Du², Liangquan Zhai², Zhengbo Yang², Jiayuan Wang⁴, Yihan Wang⁴

^1. Department of Joint Surgery, Shenyang Orthopaedic Hospital, Shenyang 110044, China; Key Laboratory of Osteoarthropathy of Liaoning Province, Shenyang 110044, China
^2. Department of Joint Surgery, Shenyang Orthopaedic Hospital, Shenyang 110044, China
^3. Department of Orthopaedic and Oncology, Shenyang Orthopaedic Hospital, Shenyang 110044, China
^4. Key Laboratory of Osteoarthropathy of Liaoning Province, Shenyang 110044, China

Received:2020-03-20 Published:2020-06-05
Corresponding author: Bo Wu

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引用本文：

邬波, 柳椰, 马旭, 智春升, 杜明昌, 翟良全, 杨政博, 王佳媛, 王译晗. 3D打印胶原/羟基磷灰石支架对骨髓间充质干细胞成骨分化的作用研究[J]. 中华老年骨科与康复电子杂志, 2020, 06(03): 123-127.

Bo Wu, Ye Liu, Xu Ma, Chunsheng Zhi, Mingchang Du, Liangquan Zhai, Zhengbo Yang, Jiayuan Wang, Yihan Wang. Effect of 3D printed collagen / hydroxyapatite scaffold on osteogenic differentiation of bone marrow mesenchymal stem cells[J]. Chinese Journal of Geriatric Orthopaedics and Rehabilitation(Electronic Edition), 2020, 06(03): 123-127.

目的

探讨3D打印胶原/羟基磷灰石支架对骨髓间充质干细胞成骨分化的作用。

方法

分离SPF级雄性SD大鼠骨髓间充质干细胞，实验分为：对照组、浸提组、诱导组及浸提诱导组，MTT法检测对照组，浸提组的细胞增殖情况，对比分析各组细胞的碱性磷酸酶（ALP）活性，对各组细胞诱导培养17天后进行茜素红染色，观察钙结节染色情况。

结果

MTT结果显示，浸提组与对照组在不同时间点的OD值比较，差异无统计学意义（P>0.05），ALP活性结果显示，不同时间点各组与对照组相比，差异均有统计学意义（P<0.05）；不同时间点浸提诱导组与浸提组相比，差异均有统计学意义（P<0.05）；诱导组在48 h及72 h与浸提组相比，差异均有统计学意义（P<0.05）。茜素红染色结果显示，对照组细胞无钙结节点，浸提组及诱导组镜下肉眼可明显观察到红色区域染色，镜下观察可见钙结节点，浸提诱导组所产生的钙结节点的数量、大小以及染色的颜色深度均明显优于其他各组。

结论

3D打印胶原/羟基磷灰石支架具有生物相容性好，可促进BMSCs向成骨分化，对细胞毒性低等特点，适宜用作骨缺损的治疗。

关键词: 胶原, 羟基磷灰石, 间充质干细胞, 成骨分化

Objective

To investigate the effect of 3D printed collagen/ hydroxyapatite scaffold on osteogenic differentiation of bone marrow mesenchymal stem cells.

Methods

Rat BMSCs were isolated from rat bone marrow. Cell proliferation and differentiation were detected by MTT and ALP activity. After inducing cell culture for 17 days, cells in each group were stained with alizarin red to observe the staining of calcium nodules.

Results

The MTT results showed that there was no significant difference in OD between the extraction group and the control group at different time point (P>0.05). ALP activity results showed that the extraction group had significant differences compared with the control group at different time point (P<0.05); the extraction induction group had significant differences at different time point compared with the extraction group (P<0.05); At 48 h and 72 h, the group had significant differences compared with the extraction group (P<0.05). Alizarin red staining showed that the cells in the control group had no calcium knot nodes. The red area staining was clearly observed under the microscope in the extraction group and the induction group, and the calcium knot nodes were observed under the microscope. The number, size and color depth of the knot nodes were significantly better than those of the other groups.

Conclusion

The scaffold has good biocompatibility, can promote the differentiation of BMSCs to osteogenesis, and has low cytotoxicity. It is suitable for the treatment of bone defect.

Key words: Collagen, Hydroxyapatite, Mesenchymal Stem Cells, Osteogenic differentiation

图2 浸提组的骨髓间充质干细胞

表1 不同时间点两组BMSCs中的MTT结果（OD，±s，每组复孔数=8）

组别	24 h	48 h	72 h	96 h	F值	P值
对照组	0.681±0.017	0.942±0.034	1.436±0.043	1.514±0.039	1060.99	<0.01
浸提组	0.676±0.018	0. 921±0.028	1.428±0.035	1.506±0.041	1244.957	<0.01
F值	0.332	1.670	0.223	0.121
P值	0.574	0.217	0.644	0.733

表1 不同时间点两组BMSCs中的MTT结果（OD，±s，每组复孔数=8）

组别	24 h	48 h	72 h	96 h	F值	P值
对照组	0.681±0.017	0.942±0.034	1.436±0.043	1.514±0.039	1060.99	<0.01
浸提组	0.676±0.018	0. 921±0.028	1.428±0.035	1.506±0.041	1244.957	<0.01
F值	0.332	1.670	0.223	0.121
P值	0.574	0.217	0.644	0.733

表2 不同时间点各组BMSCs中ALP活性结果（U/g，±s，每组复孔数=8）

组别	24 h	48 h	72 h	96 h	F值	P值
对照组	10.42±0.36	12. 54±0.58	12.13±0.61	11.73±0.54	23.685	<0.01
浸提组	14.71±1.79^a	18.38±1.83^a	17.25±1.63^a	15.912±1.58^a	7.035	0.001
诱导组	16.42±1.62^a	23.16±1.93^ab	20.85±1.95^ab	17.96±2.04^a	20.133	<0.01
浸提诱导组	17.37±2.06^ab	25.83±2.12^ab	20.44±1.86^ab	18.46±2.06^a	28.085	<0.01
F值	31.537	92.369	52.753	26.818
P值	<0.01	<0.01	<0.01	<0.01

表2 不同时间点各组BMSCs中ALP活性结果（U/g，±s，每组复孔数=8）

组别	24 h	48 h	72 h	96 h	F值	P值
对照组	10.42±0.36	12. 54±0.58	12.13±0.61	11.73±0.54	23.685	<0.01
浸提组	14.71±1.79^a	18.38±1.83^a	17.25±1.63^a	15.912±1.58^a	7.035	0.001
诱导组	16.42±1.62^a	23.16±1.93^ab	20.85±1.95^ab	17.96±2.04^a	20.133	<0.01
浸提诱导组	17.37±2.06^ab	25.83±2.12^ab	20.44±1.86^ab	18.46±2.06^a	28.085	<0.01
F值	31.537	92.369	52.753	26.818
P值	<0.01	<0.01	<0.01	<0.01

图3~6 各组BMSCs培养19天钙结节染色结果。图3　对照组；图4　浸提组；图5　诱导组；图6　浸提诱导组

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