3D打印技术制备聚磷酸钙/淫羊藿苷骨支架诱导骨髓间充质干细胞成骨分化治疗骨缺损
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摘要
背景:随着组织工程学的快速发展,寻找最佳的支架材料、诱导细胞成骨分化的因子以及分化增殖能力强、来源充足的干细胞,成为骨科领域研究的热点。目的:探讨聚磷酸钙/淫羊藿苷骨支架诱导骨髓间充质干细胞成骨分化治疗骨缺损的效果。方法:采用3D打印技术制备聚磷酸钙/淫羊藿苷复合骨支架,分离和培养兔骨髓间充质干细胞。36只新西兰大白兔根据随机数字法分为对照组和实验组,每组18只,均建立股骨髁骨缺损模型并植入聚磷酸钙/淫羊藿苷骨支架复合体,然后实验组大白兔经股静脉注入骨髓间充质干细胞1.5 m L(1×10~9L~(-1)),对照组大白兔经股静脉注入等量生理盐水。术后4,8,12周每组取6只大白兔,分离培养骨髓间充质干细胞,Transwell小室测定骨髓间充质干细胞迁移能力,q RT-PCR法测定骨髓间充质干细胞Ⅰ型胶原蛋白、CD44 mRNA水平;然后取兔股骨髁标本,X射线片观察复合骨支架以及周围骨痂形成情况,Van-Gieson染色观察骨组织学变化,免疫组化染色法测定骨痂神经生长因子水平。结果与结论:①实验组兔骨髓间充质干细胞迁移数均高于对照组(P <0.05);实验组兔骨髓间充质干细胞Ⅰ型胶原蛋白、CD44 mRNA水平均高于对照组(P <0.05);②X射线检查和组织学观察结果显示实验组骨痂形成速度和效果优于对照组;③实验组骨痂神经生长因子水平均高于对照组(P <0.05);④结果表明3D打印技术制备的聚磷酸钙/淫羊藿苷骨支架可诱导骨髓间充质干细胞向成骨细胞分化,在骨缺损治疗中具有较好效果。
BACKGROUND: With the rapid development of tissue engineering, seeking for optimal scaffold materials, osteogenic factors and abundant stem cells with strong differentiation and proliferation potential has been an issue of concern in the field of orthopedics.OBJECTIVE: To explore the effect of calcium polyphosphate/icariin bone scaffold to induce osteogenic differentiation of bone marrow mesenchymal stem cells for the treatment of bone defects.METHODS: The CPP/ICA composite bone scaffold was prepared using 3 D printing technology. The rabbit bone marrow mesenchymal stem cells were isolated and cultured. Bone defect models were made in 36 New Zealand white rabbits, and equally randomized into control group and experimental group, followed by implantation of calcium polyphosphate/icariin composite bone scaffold. Afterwards, in the experimental group, 1.5 mL of bone marrow mesenchymal stem cells(1×10~9/L) was injected through the femoral vein into the rabbits, while the rabbits in the control group were given the same amount of normal saline via the femoral vein. At 4, 8 and 12 weeks postoperatively, six rabbits from each group were taken to isolate and culture bone marrow mesenchymal stem cells. Transwell chamber assay was used to measure the migration ability of bone marrow mesenchymal stem cells. Fluorescence quantification-reverse transcriptase polymerase chain reaction was used to determine the type I collagen and CD44 mRNA levels in the cells. Rabbit femoral condyle specimens were taken, and X-ray films were used to observe the formation of epiphyses around the composite scaffold. Van-Gieson staining was used to observe the histological changes of bone tissues. Immunohistochemical staining was used to determine the level of epiphyseal nerve growth factors.RESULTS AND CONCLUSION: The number of migrated bone marrow mesenchymal stem cells was significantly higher in the experimental group than the control group(P < 0.05). The type I collagen and CD44 mRNA levels in the cells were also higher in the experimental group than the control group(P < 0.05). Findings from X-ray examination and histological observation showed more osteophytes in the experimental group than the control group. The level of epiphyseal nerve growth factors in the experimental group was significantly higher than that in the control group(P < 0.05). To conclude, the calcium polyphosphate/icariin bone scaffold prepared by 3 D printing technology can induce the osteogenic differentiation of bone marrow mesenchymal stem cells, and has a good effect in the treatment of bone defects.
BACKGROUND: With the rapid development of tissue engineering, seeking for optimal scaffold materials, osteogenic factors and abundant stem cells with strong differentiation and proliferation potential has been an issue of concern in the field of orthopedics.OBJECTIVE: To explore the effect of calcium polyphosphate/icariin bone scaffold to induce osteogenic differentiation of bone marrow mesenchymal stem cells for the treatment of bone defects.METHODS: The CPP/ICA composite bone scaffold was prepared using 3 D printing technology. The rabbit bone marrow mesenchymal stem cells were isolated and cultured. Bone defect models were made in 36 New Zealand white rabbits, and equally randomized into control group and experimental group, followed by implantation of calcium polyphosphate/icariin composite bone scaffold. Afterwards, in the experimental group, 1.5 mL of bone marrow mesenchymal stem cells(1×10~9/L) was injected through the femoral vein into the rabbits, while the rabbits in the control group were given the same amount of normal saline via the femoral vein. At 4, 8 and 12 weeks postoperatively, six rabbits from each group were taken to isolate and culture bone marrow mesenchymal stem cells. Transwell chamber assay was used to measure the migration ability of bone marrow mesenchymal stem cells. Fluorescence quantification-reverse transcriptase polymerase chain reaction was used to determine the type I collagen and CD44 mRNA levels in the cells. Rabbit femoral condyle specimens were taken, and X-ray films were used to observe the formation of epiphyses around the composite scaffold. Van-Gieson staining was used to observe the histological changes of bone tissues. Immunohistochemical staining was used to determine the level of epiphyseal nerve growth factors.RESULTS AND CONCLUSION: The number of migrated bone marrow mesenchymal stem cells was significantly higher in the experimental group than the control group(P < 0.05). The type I collagen and CD44 mRNA levels in the cells were also higher in the experimental group than the control group(P < 0.05). Findings from X-ray examination and histological observation showed more osteophytes in the experimental group than the control group. The level of epiphyseal nerve growth factors in the experimental group was significantly higher than that in the control group(P < 0.05). To conclude, the calcium polyphosphate/icariin bone scaffold prepared by 3 D printing technology can induce the osteogenic differentiation of bone marrow mesenchymal stem cells, and has a good effect in the treatment of bone defects.
引文
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[8]Vasyliev RG, Oksymets VM, Rodnichenko AE, et al.Tissue-engineered bone for treatment of combat related limb injuries. Exp Oncol. 2017;39(3):191-196.
[9]Xing J, Jin H, Hou T, et al. Establishment of a bilateral femoral large segmental bone defect mouse model potentially applicable to basic research in bone tissue engineering. J Surg Res. 2014;192(2):454-463.
[10] Wang X, Tang P, Xu Y, et al. In vitro study of strontium doped calcium polyphosphate-modified arteries fixed by dialdehyde carboxymethyl cellulose for vascular scaffolds. Int J Biol Macromol. 2016;93(Pt B):1583-1590.
[11] Chen L, Song W, Markel DC, et al. Flow perfusion culture of MC3T3-E1 osteogenic cells on gradient calcium polyphosphate scaffolds with different pore sizes. J Biomater Appl. 2016;30(7):908-918.
[12] Wang Z, Wang D, Yang D, et al. The effect of icariin on bone metabolism and its potential clinical application. Osteoporos Int.2018;29(3):535-544.
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[14]陈震东,高辉,徐房添,等.淫羊藿苷促进间充质干细胞成骨分化:为治疗骨缺损提供一个良好的方向[J].中国组织工程研究, 2016,20(50):7594-7600.
[15] Li X, Peng B, Pan Y, et al. Icariin stimulates osteogenic differentiation and suppresses adipogenic differentiation of rBMSCs via estrogen receptor signaling. Mol Med Rep. 2018;18(3):3483-3489.
[16] Nakajima K, Kunimatsu R, Ando K, et al. Comparison of the bone regeneration ability between stem cells from human exfoliated deciduous teeth, human dental pulp stem cells and human bone marrow mesenchymal stem cells. Biochem Biophys Res Commun. 2018;497(3):876-882.
[17] Deng Y, Guo T, Li J, et al. Repair of Calvarial Bone Defect Using Jarid1a-Knockdown Bone Mesenchymal Stem Cells in Rats. Tissue Eng Part A. 2018;24(9-10):711-718.
[18] Zhang F, Peng WX, Wang L, et al. Role of FGF-2 Transfected Bone Marrow Mesenchymal Stem Cells in Engineered Bone Tissue for Repair of Avascular Necrosis of Femoral Head in Rabbits. Cell Physiol Biochem. 2018;48(2):773-784.
[19] Camorani S, Hill BS, Fontanella R, et al. Inhibition of Bone Marrow-Derived Mesenchymal Stem Cells Homing Towards Triple-Negative Breast Cancer Microenvironment Using an Anti-PDGFRβAptamer. Theranostics. 2017;7(14):3595-3607.
[20] Li F, Niyibizi C. Engraftability of Murine Bone Marrow-Derived Multipotent Mesenchymal Stem Cell Subpopulations in the Tissues of Developing Mice following Systemic Transplantation.Cells Tissues Organs. 2016;201(1):14-25.
[21]尹燕雪,李传伟,于爱莲,等.盐霉素通过TGFβ1/Smad信号通路抑制MMP-2、9降低CD44+/CD24-/low表型乳腺癌干细胞迁移和侵袭能力[J].临床与实验病理学杂志, 2016,32(3):262-267.
[22] Cao H, Heazlewood SY, Williams B, et al. The role of CD44 in fetal and adult hematopoietic stem cell regulation.Haematologica. 2016;101(1):26-37.
[23] Scioli MG, Bielli A, Gentile P, et al. Combined treatment with platelet-rich plasma and insulin favours chondrogenic and osteogenic differentiation of human adipose-derived stem cells in three-dimensional collagen scaffolds. J Tissue Eng Regen Med. 2017;11(8):2398-2410.
[24] Fujioka-Kobayashi M, Schaller B, Saulacic N, et al. Growth factor delivery of BMP9 using a novel natural bovine bone graft with integrated atelo-collagen type I:Biosynthesis,characterization, and cell behavior. J Biomed Mater Res A.2017;105(2):408-418.
[25] Han X, Liu H, Kuang X, et al. Silk Fibroin Improves the Release of Nerve Growth Factor from Hydroxyapatite Particles Maintaining its Bioactivity. Curr Drug Deliv. 2018;15(6):879-886.
[26]王裕祥,王卫东,杨应忠,等.NGF、转化生长因子β1、成纤维细胞生长因子在骨折愈合中的作用效果分析[J].湖南师范大学学报(医学版),2018,15(3):95-98.
[27] Chen WH, Mao CQ, Zhuo LL, et al. Beta-nerve growth factor promotes neurogenesis and angiogenesis during the repair of bone defects. Neural Regen Res. 2015;10(7):1159-1165.
[2]Holzapfel BM, Rudert M, Hutmacher DW. Scaffold-based Bone Tissue Engineering. Orthopade. 2017;46(8):701-710.
[3]Qin H, Yang Z, Li L, et al. A promising scaffold with excellent cytocompatibility and pro-angiogenesis action for dental tissue engineering:Strontium-doped calcium polyphosphate. Dent Mater J. 2016;35(2):241-249.
[4]吴曦,彭锐.不同浓度淫羊藿苷对人骨髓间充质干细胞成骨分化的影响[J].中国组织工程研究,2018,22(5):669-674.
[5]Lian F, Zhao C, Qu J, et al. Icariin attenuates titanium particle-induced inhibition of osteogenic differentiation and matrix mineralization via miR-21-5p. Cell Biol Int. 2018;42(8):931-939.
[6]杨志烈,王成龙,赵东峰,等.淫羊藿苷对环磷酰胺化疗导致小鼠骨髓间充质干细胞成骨分化障碍的保护作用[J].中国组织工程研究,2016,20(6):777-784.
[7]Sun BY, Zhao BX, Zhu JY, et al. Role of TGF?β1 expressed in bone marrow?derived mesenchymal stem cells in promoting bone formation in a rabbit femoral defect model. Int J Mol Med.2018;42(2):897-904.
[8]Vasyliev RG, Oksymets VM, Rodnichenko AE, et al.Tissue-engineered bone for treatment of combat related limb injuries. Exp Oncol. 2017;39(3):191-196.
[9]Xing J, Jin H, Hou T, et al. Establishment of a bilateral femoral large segmental bone defect mouse model potentially applicable to basic research in bone tissue engineering. J Surg Res. 2014;192(2):454-463.
[10] Wang X, Tang P, Xu Y, et al. In vitro study of strontium doped calcium polyphosphate-modified arteries fixed by dialdehyde carboxymethyl cellulose for vascular scaffolds. Int J Biol Macromol. 2016;93(Pt B):1583-1590.
[11] Chen L, Song W, Markel DC, et al. Flow perfusion culture of MC3T3-E1 osteogenic cells on gradient calcium polyphosphate scaffolds with different pore sizes. J Biomater Appl. 2016;30(7):908-918.
[12] Wang Z, Wang D, Yang D, et al. The effect of icariin on bone metabolism and its potential clinical application. Osteoporos Int.2018;29(3):535-544.
[13]姜文涛,梅伟,王庆德,等.淫羊藿苷对成骨细胞增殖、分化及矿化的影响[J].中华实验外科杂志,2017,34(3):446-448.
[14]陈震东,高辉,徐房添,等.淫羊藿苷促进间充质干细胞成骨分化:为治疗骨缺损提供一个良好的方向[J].中国组织工程研究, 2016,20(50):7594-7600.
[15] Li X, Peng B, Pan Y, et al. Icariin stimulates osteogenic differentiation and suppresses adipogenic differentiation of rBMSCs via estrogen receptor signaling. Mol Med Rep. 2018;18(3):3483-3489.
[16] Nakajima K, Kunimatsu R, Ando K, et al. Comparison of the bone regeneration ability between stem cells from human exfoliated deciduous teeth, human dental pulp stem cells and human bone marrow mesenchymal stem cells. Biochem Biophys Res Commun. 2018;497(3):876-882.
[17] Deng Y, Guo T, Li J, et al. Repair of Calvarial Bone Defect Using Jarid1a-Knockdown Bone Mesenchymal Stem Cells in Rats. Tissue Eng Part A. 2018;24(9-10):711-718.
[18] Zhang F, Peng WX, Wang L, et al. Role of FGF-2 Transfected Bone Marrow Mesenchymal Stem Cells in Engineered Bone Tissue for Repair of Avascular Necrosis of Femoral Head in Rabbits. Cell Physiol Biochem. 2018;48(2):773-784.
[19] Camorani S, Hill BS, Fontanella R, et al. Inhibition of Bone Marrow-Derived Mesenchymal Stem Cells Homing Towards Triple-Negative Breast Cancer Microenvironment Using an Anti-PDGFRβAptamer. Theranostics. 2017;7(14):3595-3607.
[20] Li F, Niyibizi C. Engraftability of Murine Bone Marrow-Derived Multipotent Mesenchymal Stem Cell Subpopulations in the Tissues of Developing Mice following Systemic Transplantation.Cells Tissues Organs. 2016;201(1):14-25.
[21]尹燕雪,李传伟,于爱莲,等.盐霉素通过TGFβ1/Smad信号通路抑制MMP-2、9降低CD44+/CD24-/low表型乳腺癌干细胞迁移和侵袭能力[J].临床与实验病理学杂志, 2016,32(3):262-267.
[22] Cao H, Heazlewood SY, Williams B, et al. The role of CD44 in fetal and adult hematopoietic stem cell regulation.Haematologica. 2016;101(1):26-37.
[23] Scioli MG, Bielli A, Gentile P, et al. Combined treatment with platelet-rich plasma and insulin favours chondrogenic and osteogenic differentiation of human adipose-derived stem cells in three-dimensional collagen scaffolds. J Tissue Eng Regen Med. 2017;11(8):2398-2410.
[24] Fujioka-Kobayashi M, Schaller B, Saulacic N, et al. Growth factor delivery of BMP9 using a novel natural bovine bone graft with integrated atelo-collagen type I:Biosynthesis,characterization, and cell behavior. J Biomed Mater Res A.2017;105(2):408-418.
[25] Han X, Liu H, Kuang X, et al. Silk Fibroin Improves the Release of Nerve Growth Factor from Hydroxyapatite Particles Maintaining its Bioactivity. Curr Drug Deliv. 2018;15(6):879-886.
[26]王裕祥,王卫东,杨应忠,等.NGF、转化生长因子β1、成纤维细胞生长因子在骨折愈合中的作用效果分析[J].湖南师范大学学报(医学版),2018,15(3):95-98.
[27] Chen WH, Mao CQ, Zhuo LL, et al. Beta-nerve growth factor promotes neurogenesis and angiogenesis during the repair of bone defects. Neural Regen Res. 2015;10(7):1159-1165.