Wan Wenbing, Zhang Shiwen, Ge Liangpeng, Li Qingtao, Fang Xingxing, Yuan Quan, Zhong Wen, Ouyang Jun, Xing Malcolm
Department of Anatomy, Guangdong Provincial Medical Biomechanical Key Laboratory, Southern Medical University, Guangzhou, People's Republic of China ; Department of Mechanical Engineering, University of Manitoba, Winnipeg, MB, Canada ; Manitoba Institute of Child Health, Winnipeg, MB, Canada.
Department of Mechanical Engineering, University of Manitoba, Winnipeg, MB, Canada ; Manitoba Institute of Child Health, Winnipeg, MB, Canada ; Sichuan University, Chengdu, People's Republic of China.
Int J Nanomedicine. 2015 Feb 12;10:1273-90. doi: 10.2147/IJN.S77118. eCollection 2015.
Bone tissue engineering through seeding of stem cells in three-dimensional scaffolds has greatly improved bone regeneration technology, which historically has been a constant challenge. In this study, we researched the use of adipose-derived stem cell (ADSC)-laden layer-by-layer paper-stacking polycaprolactone/gelatin electrospinning nanofibrous membranes for bone regeneration. Using this novel paper-stacking method makes oxygen distribution, nutrition, and waste transportation work more efficiently. ADSCs can also secrete multiple growth factors required for osteogenesis. After the characterization of ADSC surface markers CD29, CD90, and CD49d using flow cytometry, we seeded ADSCs on the membranes and found cells differentiated, with significant expression of the osteogenic-related proteins osteopontin, osteocalcin, and osteoprotegerin. During 4 weeks in vitro, the ADSCs cultured on the paper-stacking membranes in the osteogenic medium exhibited the highest osteogenic-related gene expressions. In vivo, the paper-stacking scaffolds were implanted into the rat calvarial defects (5 mm diameter, one defect per parietal bone) for 12 weeks. Investigating with microcomputer tomography, the ADSC-laden paper-stacking membranes showed the most significant bone reconstruction, and from a morphological perspective, this group occupied 90% of the surface area of the defect, produced the highest bone regeneration volume, and showed the highest bone mineral density of 823.06 mg/cm(3). From hematoxylin and eosin and Masson staining, the new bone tissue was most evident in the ADSC-laden scaffold group. Using quantitative polymerase chain reaction analysis from collected tissues, we found that the ADSC-laden paper-stacking membrane group presented the highest osteogenic-related gene expressions of osteocalcin, osteopontin, osteoprotegerin, bone sialoprotein, runt-related transcription factor 2, and osterix (two to three times higher than the control group, and 1.5 times higher than the paper-stacking membrane group in all the genes). It is proposed that ADSC-laden layer-by-layer paper-stacking scaffolds could be used as a way of promoting bone defect treatment.
通过在三维支架中接种干细胞进行骨组织工程，极大地改进了骨再生技术，而骨再生技术在历史上一直是一项持续的挑战。在本研究中，我们研究了负载脂肪来源干细胞（ADSC）的逐层纸叠式聚己内酯/明胶静电纺丝纳米纤维膜用于骨再生的情况。使用这种新颖的纸叠方法可使氧气分布、营养供应和废物运输更高效地进行。ADSCs还能分泌成骨所需的多种生长因子。在用流式细胞术对ADSC表面标志物CD29、CD90和CD49d进行表征后，我们将ADSCs接种到膜上，发现细胞发生分化，骨桥蛋白、骨钙素和骨保护素等成骨相关蛋白有显著表达。在体外培养4周期间，在成骨培养基中培养于纸叠膜上的ADSCs表现出最高的成骨相关基因表达。在体内，将纸叠支架植入大鼠颅骨缺损处（直径5毫米，每块顶骨一个缺损）12周。通过微型计算机断层扫描进行研究，负载ADSC的纸叠膜显示出最显著的骨重建，从形态学角度来看，该组占据缺损表面积的90%，产生的骨再生体积最高，骨矿物质密度最高，为823.06毫克/立方厘米。从苏木精-伊红染色和Masson染色结果来看，负载ADSC的支架组新骨组织最为明显。通过对收集的组织进行定量聚合酶链反应分析，我们发现负载ADSC的纸叠膜组骨钙素、骨桥蛋白、骨保护素、骨唾液蛋白、 runt相关转录因子2和osterix的成骨相关基因表达最高（比对照组高两到三倍，在所有基因中比纸叠膜组高1.5倍）。有人提出，负载ADSC的逐层纸叠支架可作为促进骨缺损治疗的一种方法。
