[1]王拓△,黄小龙,吴奕江,等.三维打印羟基磷灰石/二氧化锆支架复合诱导多能干细胞来源间充质干细胞构建新型组织工程骨的实验研究[J].中国中医骨伤科杂志,2024,32(05):18-24+30.[doi:10.20085/j.cnki.issn1005-0205.240504]
　WANG Tuo,HUANG Xiaolong,WU Yijiang,et al.Experimental Study on Novel Tissue Engineered Bone Constructed by HA/ZrO2 Scaffold Based on 3D Printing Technology Composite iPSCs-MSCs[J].Chinese Journal of Traditional Medical Traumatology & Orthopedics,2024,32(05):18-24+30.[doi:10.20085/j.cnki.issn1005-0205.240504]
点击复制

三维打印羟基磷灰石/二氧化锆支架复合诱导多能干细胞来源间充质干细胞构建新型组织工程骨的实验研究()
分享到：

《中国中医骨伤科杂志》[ISSN:1005-0205/CN:42-1340/R]

卷:
第32卷
期数:
2024年05期
页码:
18-24+30
栏目:
实验研究
出版日期:
2024-05-15

文章信息/Info

Title:
Experimental Study on Novel Tissue Engineered Bone Constructed by HA/ZrO2 Scaffold Based on 3D Printing Technology Composite iPSCs-MSCs
文章编号:
1005-0205(2024)05-0018-07
作者:
王拓1△; 黄小龙1; 吴奕江1; 杜伟斌1; 韩雷1; 汪灿锋1; 全仁夫1
1浙江中医药大学附属江南医院(杭州市萧山区中医院)(杭州,311200)
Author(s):
WANG Tuo1△; HUANG Xiaolong1; WU Yijiang1; DU Weibin1; HAN Lei1; WANG Canfeng1; QUAN Renfu1
1Jiangnan Hospital Affiliated to Zhejiang Chinese Medical University(Hangzhou Xiaoshan Hospital of Traditional Chinese Medicine),Hangzhou 311200,China.
关键词:
三维打印;  羟基磷灰石/二氧化锆;  诱导多能干细胞;  骨缺损;  骨组织工程
Keywords:
3D printing;  HA/ZrO2;  induced pluripotent stem cells(iPSCs);  bone defect;  bone tissue engineering
分类号:
R-33
DOI:
10.20085/j.cnki.issn1005-0205.240504
文献标志码:
A
摘要:
目的:运用3D打印技术制备羟基磷灰石/二氧化锆(HA/ZrO2)支架,复合诱导性多能干细胞(iPSC)来源的间充质干细胞(MSC)构建新型组织工程骨,观察其修复犬股骨干骨缺损的能力。方法:运用CT扫描数据结合3D打印技术构建犬股骨干缺损部位HA/ZrO2支架,电镜及力学试验分析支架生物力学性能。诱导性多能干细胞通过诱导分化为间充质干细胞形态细胞,构建支架材料与诱导性多能干细胞来源的间充质干细胞体外共培养体系,行细胞毒性等级测定,电镜观察细胞在支架上黏附、生长情况。12只比格犬根据植入材料的不同随机分为3组,每组4只。A组截取犬股骨中段25 mm后空置,B组截骨后植入HA/ZrO2支架,C组截骨后植入复合诱导性多能干细胞来源的间充质干细胞的HA/ZrO2支架。术后X线、CT、力学试验分别观察新生骨长入情况、骨结合能力及力学性能。结果:通过CT扫描精准数据转化为3D打印数据,个体化制备HA/ZrO2复合支架,支架抗压强度为(48.94±0.65)MPa; 免疫荧光检测示诱导性多能干细胞来源的间充质干细胞表达Vimentin,不表达OCT4和Nestin; 支架材料与诱导性多能干细胞来源的间充质干细胞体外共培养细胞毒性等级为0级; 扫描电子显微镜可见梭形的诱导性多能干细胞来源的间充质干细胞在HA/ZrO2支架表面黏附、生长。动物实验术后X线影像学观察显示A组骨缺损断端吸收,骨不愈合; B组和C组支架与宿主骨结合牢固,支架内新生骨填充,有连续性骨痂通过。术后12周CT检测示B组单位体积骨量为(219.45±3.15)mm3/cm3,C组为(222.99±5.97)mm3/cm3,两组差异无统计学意义(P>0.05)。术后12周抗压试验显示B组抗压强度为(52.21±2.41)MPa,C组抗压强度为(52.51±1.35)MPa,两组差异无统计学意义(P>0.05)。结论:3D打印制备的HA/ZrO2支架符合临床个体化治疗原则,具备较强生物力学性能及良好生物相容性,复合诱导性多能干细胞来源的间充质干细胞构建的新型组织工程骨成功修复犬股骨干骨缺损,是理想的骨组织替换材料。
Abstract:
Objective:To observe dogs' bone repairing ability in femoral shaft defect by using novel tissue engineered bone which was constructed by HA/ZrO2 scaffold based on 3D printing technology and induced pluripotent stem cell(iPSC)source mesenchymal stem cells(MSCs).Methods:CT scan data and 3D printing technology were used to construct dog femoral shaft defect HA/ZrO2 scaffold.The biomechanical property of scaffold was analyzed by electron microscope and mechanical test.The iPSCs were induced and differentiated into MSCs morphological cells,and in vitro co-culture system of scaffold and iPSCs-MSCs were constructed.Its cytotoxicity grade was determined.The cell adhesion and growth on the scaffold were observed by electron microscope.12 dogs were divided into 3 groups according to the difference of implant material,with 4 dogs in each group.Group A:dogs' 25 mm mid femur were vacant after osteotomy.Group B:HA/ZrO2 scaffolds were implanted after osteotomy.Group C:HA/ZrO2 scaffolds composite iPSCs-MSCs were implanted after osteotomy.New born bone ingrowth,osseointegration ability and mechanical property were observed by X-ray,CT and mechanical test respectively.Results:HA/ZrO2 composite scaffolds were individually prepared by CT scanning precision data into 3D printed data.Scaffold compressive strength was reached to(48.94±0.65)MPa.Immunofluorescences showed that iPSCs-MSCs expressed Vimentin,while did not express OCT4 and Nestin.The cytotoxicity grade in vitro co-culture of scaffold material with iPSCs-MSCs was 0.Scanning electron microscope(SEM)showed that spindle-shape iPSCs-MSCs adhered and grew on the surface of the HA/ZrO2 scaffold.X-ray examination showed:in group A,the broken end of the fracture was absorbed,and bone nonunion was formed; in group B and C,the scaffold was bonded strongly to the host bone,and the new bone was filled in the stent,with a continuous callus passing through.3D reconstruction of CT after surgery was showed new born bone mass per unit volume was reached to(219.45±3.15)mm3/cm3 in group B,while(222.99±5.97)mm3/cm3 in group C,and there was no significant difference between two groups(P>0.05).Ultimate compressive test in week 12 showed that compressive strength reached to(52.21±2.41)MPa in group B,while(52.51±1.35)MPa in group C,and there was no significant difference between two groups(P>0.05).Conclusion:The HA/ZrO2 scaffolds prepared by 3D printing were in accordance with the principle of clinical individualized treatment,which are with strong biomechanical properties and good biocompatibility.Novel tissue engineered bone constructed by HA/ZrO2 scaffold and iPSCs-MSCs could successfully repair femoral bone defect in dogs,and is an ideal replacement material for bone tissue.

参考文献/References:

[1] FU R,LIU C,YAN Y,et al.Bone defect reconstruction via endochondral ossification:a developmental engineering strategy[J].J Tissue Eng,2021,12:204173142110042.
[2] WU D,MAO F,YUAN B,et al.Minimally invasive percutaneous plate osteosynthesis(MIPPO)combined with onionskin-like autologous bone grafting:a new technique for treatment of tibial nonunion[J].Med Sci Monit,2019,25:5997-6006.
[3] KHIRA Y M,BADAWY H A.Pedicled vascularized fibular graft with Ilizarov external fixator for reconstructing a large bone defect of the tibia after tumor resection[J].J Orthop Traumatol,2013,14(2):91-100.
[4] ZHANG T,WEI Q,ZHOU H,et al.Three-dimensional-printed individualized porous implants:a new “implant-bone” interface fusion concept for large bone defect treatment[J].Bioact Mater,2021,6(11):3659-3670.
[5] QI J,YU T,HU B,et al.Current biomaterial-based bone tissue engineering and translational medicine[J].Int J Mol Sci,2021,22(19):10233.
[6] SHAO R,QUAN R,WANG T,et al.Effects of a bone graft substitute consisting of porous gradient HA/ZrO2 and gelatin/chitosan slow-release hydrogel containing BMP-2 and BMSCs on lumbar vertebral defect repair in rhesus monkey[J].J Tissue Eng Regen Med,2018,12(3):e1813-e1825.
[7] 王拓,全仁夫,杜伟斌,等.3D打印技术制备新型HA/ZrO2梯度复合材料在犬股骨干骨缺损修复中的应用[J].中华解剖与临床杂志,2018,23(5):428-437.
[8] ZHOU L,QUAN R,YANG J,et al.Healing of bone defects by induced pluripotent stem cell-derived bone marrow mesenchymal stem cells seeded on hydroxyapatite-zirconia[J].Ann Transl Med,2021,9(23):1723.
[9] SCHMITZ J P,HOLLINGER J O.The critical size defect as an experimental model for craniomandibulofacial nonunions[J].Clin Orthop Relat Res,1986,205:299-308.
[10] MEZA-MAURICIO J,FURQUIM C P,DOS REIS L D,et al.How efficacious is the combination of substitute bone graft with autogenous bone graft in comparison with substitute bone graft alone in the horizontal bone gain:A systematic review and meta-analysis[J].J Clin Exp Dent,2022,14(8):e678-e688.
[11] HARIMTEPATHIP P,CALLAWAY L F,SINKLER M A,et al.Progressive osteolysis after use of synthetic bone graft substitute[J].Cureus,2021,13(11):e20002.
[12] VLAD M D,FERNANDEZ AGUADO E,GOMEZ GONZALEZ S,et al.Novel titaniumapatite hybrid scaffolds with spongy bone-like micro architecture intended for spinal application:in vitro and in vivo study[J].Mater Sci Eng C Mater Biol Appl,2020,110:110658.
[13] KUSHIOKA J,KAITO T,MAKINO T,et al.Difference in the fusion rate and bone formation between artificial bone and iliac autograft inside an inter-body fusion cage:a comparison between porous hydroxyapatite/type 1 collagen composite and autologous iliac bone[J].J Orthop Sci,2018,23(4):622-626.
[14] ZHANG Q,NETTLESHIP I,SCHMELZER E,et al.Tissue engineering and regenerative medicine therapies for cell senescence in bone and cartilage[J].Tissue Eng Part B Rev,2020,26(1):64-78.
[15] SHAO R X,QUAN R F,HUANG X L,et al.Evaluation of porous gradient hydroxyapatite/zirconia composites for repair of lumbar vertebra defect in dogs[J].J Biomater Appl,2016,30(9):1312-1321.
[16] LUO G,ZHANG Y,WANG X,et al.Individualized 3D printing-assisted repair and reconstruction of neoplastic bone defects at irregular bone sites:exploration and practice in the treatment of scapular aneurysmal bone cysts[J].BMC Musculoskelet Disord,2021,22(1):984.
[17] HALEEM A,JAVAID M,KHAN R H,et al.3D printing applications in bone tissue engineering[J].J Clin Orthop Trauma,2020,11(Suppl 1):S118-S124.
[18] ILAS D C,CHURCHMAN S M,BABOOLAL T,et al.The simultaneous analysis of mesenchymal stem cells and early osteocytes accumulation in osteoarthritic femoral head sclerotic bone[J].Rheumatology,2019,58(10):1777-1783.
[19] KARAKA N,BAY S,TÜRKEL N,et al.Neurons from human mesenchymal stem cells display both spontaneous and stimuli responsive activity[J].PLoS One,2020,15(5):e0228510.
[20] KANGARI P,TALAEI-KHOZANI T,RAZEGHIAN-JAHROMI I,et al.Mesenchymal stem cells:amazing remedies for bone and cartilage defects[J].Stem Cell Res Ther,2020,11(1):492.
[21] MOLLENTZE J,DURANDT C,PEPPER M S.An in vitro and in vivo comparison of osteogenic differentiation of human mesenchymal stromal/stem cells[J].Stem Cells Int,2021:9919361.
[22] LIU G,DAVID B T,TRAWCZYNSKI M,et al.Advances in pluripotent stem cells:history,mechanisms,technologies,and applications[J].Stem Cell Rev Rep,2020,16(1):3-32.
[23] RAMARAJU H,KOHN D H.Cell and material-specific phage display peptides increase iPS-MSC mediated bone and vasculature formation in vivo[J].Adv Healthc Mater,2019,8(9):e1801356.
[24] MIYAZAKI T,HANAMATSU H,XU L,et al.Evaluation of residual human-induced pluripotent stem cells in human chondrocytes by cell type-specific glycosphingolipid glycome analysis based on the aminolysis technique[J].Int J Mol Sci,2019,21(1):231.
[25] ZHOU L,QUAN R,YANG J,et al.Healing of bone defects by induced pluripotent stem cell-derived bone marrow mesenchymal stem cells seeded on hydroxyapatite-zirconia[J].Ann Transl Med,2021,9(23):1723.

备注/Memo

备注/Memo:
基金项目:浙江省医药卫生科技项目(2020KY796)
△通信作者 E-mail:wangtuo1119@126.com
更新日期/Last Update: 2024-05-15