Bone morphogenetic protein 2 transfected bone marrow mesenchymal stem cells and fibrin glue to promote tendon-bone healing after anterior cruciate ligament reconstruction

doi:10.3969/j.issn.2095-4344.1522

Abstract

Abstract:

BACKGROUND: The combination of bone marrow mesenchymal stem cells and fibrin glue can improve the early rupture strength and stiffness of the tendon-bone interface after anterior cruciate ligament reconstruction, and promote the early healing of graft and bone tunnel.
OBJECTIVE: To explore the effect of bone morphogenetic protein 2 transfected bone marrow mesenchymal stem cells combined with fibrin glue on the tendon-bone healing after the reconstruction of the anterior cruciate ligament.
METHODS: Adenovirus vector Ad-GFP-bone morphogenetic protein 2 was transfected into bone marrow mesenchymal stem cells, and the expressions of GFP gene and bone morphogenetic protein 2 were detected after 48 hours. Fifty-four New Zealand white rabbits purchased from the Medical Animal Center of Chongqing were randomly divided into blank group, experimental group and control group, with 18 rats in each group. The anterior cruciate ligament reconstruction model was established in each rat. In the experimental group, after 48 hours of transfection, the transfected bone marrow mesenchymal stem cells were mixed with fibrin glue and injected into the tendon-bone interface. In the control group, non-transfected bone marrow mesenchymal stem cells were mixed with fibrin glue and injected into the tendon-bone interface. In the blank group, no treatment was given. After 4, 8, 12 weeks, the femur-tendon-tibia specimens were taken for histological and biomechanical examinations.
RESULTS AND CONCLUSION: (1) After transfection of 48 hours, the cells expressed green fluorescence under the fluorescence microscope, and the transfection efficiency was (86.1±6.3)%. The expression of cytoplasmic bone morphogenetic protein was positive as shown by SP immunohistochemical test, and the positive band of bone morphogenetic protein 2 was detected by western blot. (2) Findings from the hematoxylin-eosin staining showed best better tendon-bone healing in the experimental and control group than the blank group, and the tendon-bone healing was further better in the experimental group than in the control group. (3) The biomechanical results showed that the maximum load and stiffness were ranked as follows: the experimental group > control group > blank group (P < 0.05). Therefore, bone morphogenetic protein 2-transfected bone marrow mesenchymal stem cells combined with fibrin glue can further enhance the tendon-bone healing after anterior cruciate ligament reconstruction.

中国组织工程研究杂志出版内容重点：干细胞；骨髓干细胞；造血干细胞；脂肪干细胞；肿瘤干细胞；胚胎干细胞；脐带脐血干细胞；干细胞诱导；干细胞分化；组织工程

Key words: Anterior Cruciate Ligament, Bone Morphogenetic Proteins, Bone Marrow, Mesenchymal Stem Cells, Fibrin Tissue Adhesive, Tissue Engineering

CLC Number:

Zhu Lequan, Lu Jianhua, Ran Junling, Yang Weibao, Wang Hui. Bone morphogenetic protein 2 transfected bone marrow mesenchymal stem cells and fibrin glue to promote tendon-bone healing after anterior cruciate ligament reconstruction[J]. Chinese Journal of Tissue Engineering Research, 2019, 23(1): 30-34.

Figures/Tables 1

2.1 基因转染效果转染48 h后，荧光显微镜下可见细胞表达绿色荧光，见图1，提示GFP阳性表达，转染效率为(86.1±6.3)%；SP免疫组织化学检测显示，细胞胞浆内可见棕黄色染色，见图2，即骨形态发生蛋白2表达阳性；Western-blot检测显示，转染骨髓间充质干细胞可见较强骨形态发生蛋白2表达条带，见图3。 2.2 实验动物数量分析实验选用54只新西兰大白兔，实验组与对照组造模过程中如有动物死亡，则按照实验方法重新造模，将动物补齐，最终保证每组有18只新西兰大白兔。 2.3 实验动物大体观察结果实验组、对照组所有兔膝关节切口均愈合，重建的前交叉韧带完整，饮食、活动及精神状态正常，未发生异位骨化。 2.4 苏木精-伊红染色结果见图4。空白组术后第4周，腱-骨界面结合不紧密，其内主要为成纤维细胞及一些炎性细胞；术后第8周，腱-骨界面结合仍不紧密，可见少量无序排列的胶原纤维及少量血管生成；术后第12周，腱-骨界面出现大量排列不规则的胶原纤维，并聚集成束。对照组术后第4周大部分腱-骨界面局部结合较紧密，其内主要为新生的肉芽组织，可见较多不规则排列的胶原纤维，少量血管出现于肌腱内；术后第8周，腱-骨界面结合较4周时紧密，大量规则排列的胶原纤维集结成束，并沿隧道长轴排列；术后第12周，腱-骨界面可见少量规则排列的软骨细胞。实验组术后第4周腱-骨界面结合紧密，可见少量排列不规则的软骨细胞，少量骨质长入肌腱内，同时可见大量胶原纤维；术后第8周，腱-骨界面可见大量排列规则的软骨细胞；术后第12周，腱-骨界面有大量软骨生成，出现胶原纤维-软骨细胞移行带变化，形成了韧带-纤维软骨-钙化软骨-骨的结构，接近正常前交叉韧带结构。 2.5 生物力学检测结果随着术后时间的延长，各组最大载荷与刚度均呈现逐渐增高趋势；相同时间点下，实验组、对照组最大载荷与刚度均高于空白组，并且实验组最大载荷与刚度均高于对照组(P < 0.05)，具体结果见图5。"

References

[1] 颜林飞.关节镜下膝关节前交叉韧带重建远期疗效及影响因素研究[J].数理医药学杂志,2018,31(3):366-367.

[2] 宋洋,杜庆钧,欧阳毅,等.关节镜下前内侧束紧缩、后外侧束保残重建术治疗前交叉韧带损伤的临床疗效[J].广西医学, 2018,40(1):44-47,64.

[3] 陆定贵.关节镜下后交叉韧带损伤重建技术研究进展[J].右江医学, 2018, 46(1):103-107.

[4] Murray MM, Fleming BC.Use of a bioactive scaffold to stimulate anterior cruciate ligament healing also minimizes posttraumatic osteoarthritis after surgery.Am J Sports Med. 2013;41(8):1762-1770.

[5] 柴双,吕松岑.前交叉韧带重建术后促进腱-骨愈合因素的研究进展[J].医学综述,2017,23(4):718-723.

[6] Hu J, Yao B, Yang X, et al.The immunosuppressive effect of Siglecs on tendon-bone healing after ACL reconstruction.Med Hypotheses. 2015; 84(1):38-39.

[7] 张明宇,张宪,杨镇,等.PRP明胶海绵复合物在前交叉韧带重建术后腱骨愈合的作用[J].中国矫形外科杂志,2017,25(8):737-742.

[8] 崔巍,吕伟,时剑辉,等.关节镜下自体骨膜包裹6股腘绳肌腱移植前交叉韧带重建的中期疗效评价[J].中国现代药物应用, 2016,10(17):114-116.

[9] 余刚,徐斌,涂俊,等.外周血间充质干细胞复合富血小板血浆对腱骨界面愈合的影响[J].安徽医科大学学报,2017,52(5):770-773.

[10] 逯代锋,杨传东,董锋,等.酸性成纤维细胞生长因子复合胶原蛋白对兔前交叉韧带重建后腱-骨愈合的影响[J].临床骨科杂志, 2017,20(6):752-756.

[11] 石斌,刘玉杰,王志刚,等.体外冲击波对兔ACL重建腱骨愈合影响的组织学观察[J].军医进修学院学报,2010,31(10):951-953.

[12] 陈平.人脂肪源干细胞在促进兔前交叉韧带重建术后腱骨界面愈合的实验研究[D].广州:南方医科大学,2017.

[13] Dang PN, Dwivedi N, Phillips LM, et al. Controlled Dual Growth Factor Delivery From Microparticles Incorporated Within Human Bone Marrow-Derived Mesenchymal Stem Cell Aggregates for Enhanced Bone Tissue Engineering via Endochondral Ossification.Stem Cells Transl Med. 2016;5(2):206-217.

[14] Solorio LD, Phillips LM, McMillan A, et al. Spatially organized differentiation of mesenchymal stem cells within biphasic microparticle-incorporated high cell density osteochondral tissues. Adv Healthc Mater. 2015;4(15):2306-2313.

[15] Tian F, Ji XL, Xiao WA, et al. CXCL13 promotes the effect of bone marrow mesenchymal stem cells (MSCs) on tendon-bone healing in rats and in C3HIOT1/2 cells.Int J Mol Sci. 2015;16(2):3178-3187.

[16] Kanazawa T, Soejima T, Noguchi K, et al. Tendon-to-bone healing using autologous bone marrow-derived mesenchymal stem cells in ACL reconstruction without a tibial bone tunnel-A histological study. Muscles Ligaments Tendons J. 2014;4(2):201-206.

[17] 刘杨,孙大磊,王毅,等.BMSCs和FG作为种子细胞和支架材料修复大鼠牙槽骨缺损的效果[J].口腔医学,2017,37(4):307-310.

[18] 周丽斌,徐冰心,丁瑞英,等.应用纤维蛋白胶复合细胞膜片构建人工软骨[J].中山大学学报(医学科学版),2017,38(1):122-127.

[19] 周维锋,童松林,徐建杰,等.骨髓间充质干细胞促进前交叉韧带重建后腱-骨愈合的生物力学研究[J].中华创伤杂志, 2013,29(7):667-670.

[20] 赵旭,卢燃,陈溯.TiO2纳米管负载BMP-2指节肽对骨髓间充质干细胞增殖与分化的影响[J].北京口腔医学,2017,25(5):241-244.

[21] 冯倩,曾祥伟,杨瑞,等.骨髓间充质干细胞成骨分化相关信号通路及其与BMP-2关系的研究进展[J].山东医药,2017,57(48):97-99.

[22] 张道鑫,韩庆斌,李新志,等.含骨形态发生蛋白2的植骨材料在股骨髓内钉术后骨不连中的应用[J].国际外科学杂志, 2018,45(3):164-168.

[23] 陈启刚,胡永军,胡海,等.锁定接骨板结合重组人骨形态发生蛋白骨修复材料治疗跟骨骨折的临床分析[J].安徽医药, 2018,22(4):703-706.

[24] 石正松.Ad-BMP-2/BMSCs/DBM对兔股骨头坏死修复的实验研究[J].桂林:桂林医学院,2016.

[25] 向盈盈,宋飞,杨向红.骨形态发生蛋白-2复合纤维蛋白缓释胶修复骨缺损的实验研究[J].昆明医科大学学报,2016,37(3):27-30.

[26] 卢秋野,李文远,孙军,等.保留胫骨止点半腱肌重建兔前交叉韧带腱骨愈合实验分析[J].吉林医药学院学报,2017,38(2):97-99.

[27] 毕方刚,严世贵.促进前交叉韧带重建后腱骨愈合方法的研究进展[J].中华关节外科杂志:电子版,2015,9(3):407-411.

[28] Chen B, Li B, Qi YJ, et al. Enhancement of tendon-to-bone healing after anterior cruciate ligament reconstruction using bone marrow-derived mesenchymal stem cells genetically modified with bFGF/BMP2.Sci Rep. 2016;6:25940.

[29] Schwarting T, Schenk D, Frink M, et al. Stimulation with bone morphogenetic protein-2 (BMP-2) enhances bone-tendon integration in vitro.Connect Tissue Res. 2016;57(2):99-112.

[30] 刘国华,何进,倪同伟,等.可调悬吊钛板在前交叉韧带重建手术中的应用16例[J].交通医学,2018,32(1):37-38,42.

[31] 张合,赵洪波,曹成明,等.双束和单束重建前交叉韧带对膝关节稳定性和膝关节退变的前瞻性随机对照研究[J].河北医科大学学报, 2018,39(1): 44-48,53.

[32] 何衍高,梁春雨,张德新,等.应用腓骨长肌腱和腘绳肌腱重建前交叉韧带的疗效比较[J].实用骨科杂志,2018,24(2):185-188.