脂肪间充质干细胞混合成骨细胞与羟基磷灰石/壳聚糖/聚乳酸复合后的体内异位成骨效应
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摘要
背景:成骨细胞缺乏是骨组织工程面临的关键问题,而间充质干细胞联合成骨细胞移植能够获得理想效果。目的:观察脂肪间充质干细胞与成骨细胞复合羟基磷灰石/壳聚糖/聚乳酸支架材料在体内的异位成骨效应。方法:酶消化法和贴壁法分离原代脂肪间充质干细胞、成骨细胞,并进行鉴定。取成骨细胞、脂肪间充质干细胞、成骨细胞+脂肪间充质干细胞混合细胞(比例为1︰1),分别复合羟基磷灰石/壳聚糖/聚乳酸骨修复材料,体外复合培养48 h后,植入SD大鼠背部皮下,作为实验组,以单纯材料植入作为对照组。术后8周取出标本,进行大体观察、组织学观察并计算新骨生成率。结果与结论:(1)脂肪间充质干细胞经成脂、成软骨、成骨诱导后,油红O染色、甲苯胺蓝染色和茜素红染色均为阳性;流式细胞仪检测脂肪间充质干细胞中CD147、CD90、CD105、CD44呈阳性表达(>80%),而CD117、CD34、CD31、CD45则呈阴性表达(<5%);(2)第3代成骨细胞茜素红染色、碱性磷酸酶染色均为阳性;(3)植入8周后大体观察可见材料中有软组织长入,难以分离;(4)植入8周后组织学观察可见各组均有新骨形成,与其他材料组比较,脂肪间充质干细胞+成骨细胞+羟基磷灰石/壳聚糖/聚乳酸组成骨分数最高,差异有显著性意义(P<0.05);(5)结果表明,脂肪间充质干细胞可促进成骨细胞复合羟基磷灰石/壳聚糖/聚乳酸支架材料的异位成骨。
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.
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.
引文
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[22]Zhang J,Zhou S,Zhou Y,et al.Adipose-Derived Mesenchymal Stem Cells(ADSCs)With the Potential to Ameliorate Platelet Recovery,Enhance Megakaryopoiesis,and Inhibit Apoptosis of Bone Marrow Cells in a Mouse Model of Radiation-Induced Thrombocytopenia.Cell Transplant.2016;25(2):261-273.
[23]Lai QG,Sun SL,Zhou XH,et al.Adipose-derived stem cells transfected with p EGFP-OSX enhance bone formation during distraction osteogenesis.J Zhejiang Univ Sci B.2014;15(5):482-490.
[24]Zhang J,Zhou S,Zhou Y,et al.Adipose-Derived Mesenchymal Stem Cells(ADSCs)With the Potential to Ameliorate Platelet Recovery,Enhance Megakaryopoiesis,and Inhibit Apoptosis of Bone Marrow Cells in a Mouse Model of Radiation-Induced Thrombocytopenia.Cell Transplant.2016;25(2):261-273.
[25]Wan W,Zhang S,Ge L,et al.Layer-by-layer paper-stacking nanofibrous membranes to deliver adipose-derived stem cells for bone regeneration.Int J Nanomedicine.2015;10:1273-1290.
[26]Zhang W,Zhang X,Wang S,et al.Comparison of the use of adipose tissue-derived and bone marrow-derived stem cells for rapid bone regeneration.J Dent Res.2013;92(12):1136-1141.
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[28]芮钢,胡宝山,郭元利,等.成骨细胞、血管内皮细胞复合组织工程骨修复骨缺损的实验研究[J].中华骨科杂志,2008,28(2):155-158.
[29]Zhou X,Tao Y,Wang J,et al.Three-dimensional scaffold of type II collagen promote the differentiation of adipose-derived stem cells into a nucleus pulposus-like phenotype.J Biomed Mater Res A.2016;104(7):1687-1693.
[2]Yao W,Lay YE,Kot A,et al.Improved Mobilization of Exogenous Mesenchymal Stem Cells to Bone for Fracture Healing and Sex Difference.Stem Cells.2016;34(10):2587-2600.
[3]Zhang H,Kot A,Lay YE,et al.Acceleration of Fracture Healing by Overexpression of Basic Fibroblast Growth Factor in the Mesenchymal Stromal Cells.Stem Cells Transl Med.2017;6(10):1880-1893.
[4]陆钰,王鑫,甘朝兵,等.多孔纳米羟基磷灰石/壳聚糖支架材料复合成骨细胞的异位成骨研究[J].生物骨科材料与临床研究,2012,9(1):21-25.
[5]Yuan H,Qin J,Xie J,et al.Highly aligned core-shell structured nanofibers for promoting phenotypic expression of v SMCs for vascular regeneration.Nanoscale.2016;8(36):16307-16322.
[6]Zhang CY,Zhang CL,Wang JF,et al.Fabrication and in vitro investigation of nanohydroxyapatite,chitosan,poly(L‐lactic acid)ternary biocomposite.Journal of Applied Polymer Science.2014;127(3):2152-2159.
[7]Bodle JC,Hanson AD,Loboa EG.Adipose-derived stem cells in functional bone tissue engineering:lessons from bone mechanobiology.Tissue Eng Part B Rev.2011;17(3):195-211.
[8]Sakaguchi Y,Sekiya I,Yagishita K,et al.Comparison of human stem cells derived from various mesenchymal tissues:superiority of synovium as a cell source.Arthritis Rheum.2005;52(8):2521-2529.
[9]Feng W,Lv S,Cui J,et al.Histochemical examination of adipose derived stem cells combined withβ-TCP for bone defects restoration under systemic administration of1α,25(OH)2D3.Mater Sci Eng C Mater Biol Appl.2015;54:133-141.
[10]Zhang H,Kot A,Lay YE,et al.Acceleration of Fracture Healing by Overexpression of Basic Fibroblast Growth Factor in the Mesenchymal Stromal Cells.Stem Cells Transl Med.2017;6(10):1880-1893.
[11]Vergroesen PP,Kroeze RJ,Helder MN,et al.The use of poly(L-lactide-co-caprolactone)as a scaffold for adipose stem cells in bone tissue engineering:application in a spinal fusion model.Macromol Biosci.2011;11(6):722-730.
[12]Roskies MG,Fang D,Abdallah MN,et al.Three-dimensionally printed polyetherketoneketone scaffolds with mesenchymal stem cells for the reconstruction of critical-sized mandibular defects.Laryngoscope.2017;127(11):E392-E398.
[13]Fang D,Roskies M,Abdallah MN,et al.Three-Dimensional Printed Scaffolds with Multipotent Mesenchymal Stromal Cells for Rabbit Mandibular Reconstruction and Engineering.Methods Mol Biol.2017;1553:273-291.
[14]Kang ML,Kim JE,Im GI.Vascular endothelial growth factor-transfected adipose-derived stromal cells enhance bone regeneration and neovascularization from bone marrow stromal cells.J Tissue Eng Regen Med.2017;11(12):3337-3348.
[15]Godoy Zanicotti D,Coates DE,Duncan WJ.In vivo bone regeneration on titanium devices using serum-free grown adipose-derived stem cells,in a sheep femur model.Clin Oral Implants Res.2017;28(1):64-75.
[16]Lee SJ,Kim BJ,Kim YI,et al.Effect of Recombinant Human Bone Morphogenetic Protein-2 and Adipose Tissue-Derived Stem Cell on New Bone Formation in High-Speed Distraction Osteogenesis.Cleft Palate Craniofac J.2016;53(1):84-92.
[17]Tabatabaei QomiR,Sheykhhasan M.Adipose-derived stromal cell in regenerative medicine:A review.World J Stem Cells.2017;9(8):107-117.
[18]Shakir M,Jolly R,Khan MS,et al.Nano-hydroxyapatite/chitosan-starch nanocomposite as a novel bone construct:Synthesis and in vitro studies.Int J Biol Macromol.2015;80:282-292.
[19]Godoy Zanicotti D,Coates DE,Duncan WJ.In vivo bone regeneration on titanium devices using serum-free grown adipose-derived stem cells,in a sheep femur model.Clin Oral Implants Res.2017;28(1):64-75.
[20]biocompatible evaluation of natural hydroxyapatite/chitosan composite for bone repair.J Appl Biomater Biomech.2011;9(1):11-18.
[21]Park S,Heo HA,Lee KB,et al.Improved Bone Regeneration With Multiporous PLGA Scaffold and BMP-2-Transduced Human Adipose-Derived Stem Cells by Cell-Permeable Peptide.Implant Dent.2017;26(1):4-11.
[22]Zhang J,Zhou S,Zhou Y,et al.Adipose-Derived Mesenchymal Stem Cells(ADSCs)With the Potential to Ameliorate Platelet Recovery,Enhance Megakaryopoiesis,and Inhibit Apoptosis of Bone Marrow Cells in a Mouse Model of Radiation-Induced Thrombocytopenia.Cell Transplant.2016;25(2):261-273.
[23]Lai QG,Sun SL,Zhou XH,et al.Adipose-derived stem cells transfected with p EGFP-OSX enhance bone formation during distraction osteogenesis.J Zhejiang Univ Sci B.2014;15(5):482-490.
[24]Zhang J,Zhou S,Zhou Y,et al.Adipose-Derived Mesenchymal Stem Cells(ADSCs)With the Potential to Ameliorate Platelet Recovery,Enhance Megakaryopoiesis,and Inhibit Apoptosis of Bone Marrow Cells in a Mouse Model of Radiation-Induced Thrombocytopenia.Cell Transplant.2016;25(2):261-273.
[25]Wan W,Zhang S,Ge L,et al.Layer-by-layer paper-stacking nanofibrous membranes to deliver adipose-derived stem cells for bone regeneration.Int J Nanomedicine.2015;10:1273-1290.
[26]Zhang W,Zhang X,Wang S,et al.Comparison of the use of adipose tissue-derived and bone marrow-derived stem cells for rapid bone regeneration.J Dent Res.2013;92(12):1136-1141.
[27]Lough DM,Chambers C,Germann G,et al.Regulation of ADSC Osteoinductive Potential Using Notch Pathway Inhibition and Gene Rescue:A Potential On/Off Switch for Clinical Applications in Bone Formation and Reconstructive Efforts.Plast Reconstr Surg.2016;138(4):642e-652e.
[28]芮钢,胡宝山,郭元利,等.成骨细胞、血管内皮细胞复合组织工程骨修复骨缺损的实验研究[J].中华骨科杂志,2008,28(2):155-158.
[29]Zhou X,Tao Y,Wang J,et al.Three-dimensional scaffold of type II collagen promote the differentiation of adipose-derived stem cells into a nucleus pulposus-like phenotype.J Biomed Mater Res A.2016;104(7):1687-1693.