脱细胞猪心包膜生物相容性及成骨性能的体内外评价

doi:10.19723/j.issn.1671-167X.2021.04.026

摘要/Abstract

摘要：

目的: 体外检测作为引导性骨再生术(guided bone regeneration, GBR)屏障膜的脱细胞猪心包膜(acellular porcine pericardium,APP)的形貌特性及生物相容性。建立动物模型,检测其体内屏障软组织长入骨缺损的作用及对促成骨的影响。方法: 扫描电镜检测APP膜的超微结构。细胞增殖检测(cell counting kit-8,CCK-8)实验检测人骨髓间充质干细胞(human bone marrow mesenchymal stem cell, hBMSCs)接种于APP膜后第1、3、7天细胞增殖情况;接种后第5天,通过鬼笔环肽+DAPI(4,6-diamidino-2-phenylindole)对细胞骨架及细胞核进行染色,观察hBMSCs的增殖及黏附情况。体内实验建立3只比格犬、18个实验位点的牙槽嵴保存动物实验模型,随机分入APP组(拔牙窝内植入Bio-Oss^®骨粉并覆盖APP膜)、BG组(拔牙窝内植入Bio-Oss^®骨粉并覆盖Bio-Gide^®膜)和空白组(自然愈合),术后4周和12周进行Micro-CT扫描检测各组成骨情况,脱钙后进行HE染色,组织学观察各组愈合情况。结果: 扫描电镜下APP膜具有致密及疏松双层非对称及三维多孔超微结构。体外实验证实APP膜可以促进hBMSCs的增殖及黏附,在接种后第7天APP组细胞数量显著高于BG组(P<0.05)。体内实验拔牙窝整体成骨情况:术后4周,3组新生骨比例差异无统计学意义(P>0.05);术后12周,APP组、BG组间新生骨比例差异无统计学意义(P>0.05),但均高于空白组(P<0.05)。冠方成骨情况:术后4周,APP组及BG组膜下方成骨显著高于空白组(P<0.05),组间差异无统计学意义(P>0.05);术后12周,APP组及BG组膜下方成骨显著高于空白组(P<0.05),组间差异有统计学意义(P<0.05)。颊侧骨嵴顶相对吸收量表明:术后4周,APP组显著低于空白组(P<0.05),BG组低于空白组,差异无统计学意义(P>0.05);术后12周,各组颊侧骨嵴顶继续降低,APP组相对吸收量仍显著低于空白组(P<0.05),BG组低于空白组,差异无统计学意义(P>0.05)。结论: APP膜具有良好的三维结构及细胞相容性,其在GBR中起到良好屏障软组织效果的同时,能够显著促进膜下方成骨及减少颊侧牙槽嵴顶吸收,推测其具有潜在的骨诱导能力。

关键词: 心包膜, 可吸收膜, 人骨髓间充质干细胞, 引导性骨再生术

Abstract:

Objective: To examine the morphology and biocompatibility of a native acellular porcine pericardium (APP) in vitro and to evaluate its barrier function and effects on osteogenesis when used in guided bone regeneration (GBR) in vivo. Methods: First, the morphology of APP (BonanGen^®) was detected using a scanning electron microscope (SEM). Next, for biocompatibility test, proliferation of human bone marrow mesenchymal stem cells (hBMSCs) were determined using cell counting kit-8 (CCK-8) after being seeded 1, 3 and 7 days. Meanwhile, the cells stained with phalloidine and 4,6-diamidino-2-phenylindole (DAPI) were observed using a confocal laser scanning microscopy (CLSM) to view the morphology of cell adhesion and pattern of cell proliferation on day 5. A 3-Beagle dog model with 18 teeth extraction sockets was used for the further research in vivo. These sites were randomly treated by 3 patterns below: filled with Bio-Oss^® and coverd by APP membrane (APP group), filled with Bio-Oss^® and covered by Bio-Gide^® membrane (BG group) and natural healing (blank group). Micro-CT and hematoxylin-eosin (HE) were performed after 4 and 12 weeks. Results: A bilayer and three-dimensional porous ultrastructure was identified for APP through SEM. In vitro, APP facilitated proliferation and adhesion of hBMSCs, especially after 7 days (P<0.05). In vivo, for the analysis of the whole socket healing, no distinct difference of new bone ratio was found between all the three groups after 4 weeks (P>0.05), however significantly more new bone regeneration was detected in APP group and BG group in comparison to blank group after 12 weeks (P<0.05). The radio of bone formation below the membrane was significantly higher in APP group and BG group than blank group after 4 and 12 weeks (P<0.05), however, the difference between APP group and BG group was merely significant in 12 weeks (P<0.05). Besides, less resorption of buccal crest after 4 weeks and 12 weeks was observed in APP group of a significant difference compared in blank group (P<0.05). The resorption in BG group was slightly lower than blank group (P>0.05). Conclusion: APP showed considerable biocompatibility and three-dimentional structure. Performing well as a barrier membrane in the dog alveolar ridge preservation model,APP significantly promoted bone regeneration below it and reduced buccal crest resorption. On the basis of this study, APP is a potential osteoconductive and osteoinductive biomaterial.

Key words: Pericardium, Resorbable membrane, Human bone marrow mesenchymal stem cell, Guided bone regeneration

中图分类号:

R783.1

尤鹏越,刘玉华,王新知,王思雯,唐琳. 脱细胞猪心包膜生物相容性及成骨性能的体内外评价[J]. 北京大学学报（医学版）, 2021, 53(4): 776-784.

YOU Peng-yue,LIU Yu-hua,WANG Xin-zhi,WANG Si-wen,TANG Lin. Biocompatibility and effect on bone formation of a native acellular porcine pericardium: Results of in vitro and in vivo[J]. Journal of Peking University (Health Sciences), 2021, 53(4): 776-784.

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Areas	Time	APP group	BG group	Blank group
Bone area	4 weeks	24.05±1.05	26.48±2.42	29.81±5.46
Bone area	12 weeks	44.05±5.74^c	41.50±6.22^c	21.55±1.56^ab
Bone marrow area	4 weeks	48.52±3.16	47.78±0.55	40.46±4.07^ab
Bone marrow area	12 weeks	32.97±2.22^c	33.06±4.63^c	42.56±2.88^ab
Soft tissue area	4 weeks	14.15±2.57	13.53±1.51	31.12±4.75^ab
Soft tissue area	12 weeks	8.93±1.56^c	12.56±3.45	35.88±3.03^ab
Material area	4 weeks	13.24±3.07	13.58±2.42	-
Material area	12 weeks	14.46±3.91	13.49±1.93	-

Areas	Time	APP group	BG group	Blank group
Bone area	4 weeks	31.26±2.76	30.36±1.72	11.26±0.91^ab
Bone area	12 weeks	42.56±2.88^bc	37.62±1.87^ac	15.09±1.81^abc
Bone marrow area	4 weeks	30.05±1.70	30.59±1.20	10.67±0.71^ab
Bone marrow area	12 weeks	25.69±1.96^c	28.17±2.87	10.57±1.88^ab
Soft tissue area	4 weeks	27.36±2.57	27.98±3.63	78.07±8.54^ab
Soft tissue area	12 weeks	20.35±4.29	22.37±2.69	74.33±1.74^ab
Material area	4 weeks	11.30±1.50	10.74±2.84	-
Material area	12 weeks	11.39±2.21	11.83±2.72	-

Time	APP group	BG group	Blank group
4 weeks	0.89±0.09	1.32±0.33	1.53±0.29^a
12 weeks	1.14±0.20	1.51±0.33	1.78±0.39^a