自体腘绳肌腱移植重建前交叉韧带后移植物胶原纤维直径与Mohawk表达水平的相关性研究

A correlation study between the Mohawk expression level and the collagen fiber diameter of hamstring tendon graft after anterior cruciate ligament reconstruction

目的探讨在自体腘绳肌腱移植重建前交叉韧带(anterior cruciate ligament,ACL)术后的移植物塑形稳定阶段,移植物Mohawk(MKX)表达水平与胶原纤维直径的相关性。方法以2018年1月—8月关节镜下自体腘绳肌腱单束重建ACL术后48个月以上、因移除胫骨端内固定物行二次关节镜探查患者的移植物为研究对象。术中对移植物塑形评分;取移植物中央表面标本,采用实时荧光定量PCR(real-time fluorescent quantitative PCR,qRT-PCR)检测ACL移植物MKX表达水平;透射电镜下观察移植物胶原纤维超微结构,测量胶原纤维直径(Dc)、大直径(≥90 nm)胶原纤维直径(DL)、小直径(<90 nm)胶原纤维直径(DS)以及大、小直径胶原纤维数量比值(RL/S)。计算MKX表达水平与胶原纤维直径的相关性。结果共26例患者符合选择标准,其ACL移植物标本纳入研究。ACL重建术至二次关节镜探查时间为52~128个月,平均78.6个月。关节镜下移植物质量评分为3~6分,平均4.8分;塑形良好17例、塑形一般9例。透射电镜观察,ACL移植物均表现为大直径与小直径胶原纤维双峰混合排列,与正常ACL类似。与塑形一般的移植物相比,塑形良好的移植物可见更丰富的大直径胶原纤维,且大、小直径胶原纤维间隔交叉分布更均匀、有序。移植物Dc、DL、DS及RL/S分别为(65.2±9.3)nm、(91.6±10.5)nm、(45.7±8.6)nm、0.73±0.12。移植物MKX相对表达量为1.42±0.11。相关分析显示,移植物MKX表达水平与Dc、DL及RL/S成正相关(r=0.809,P=0.000;r=0.861,P=0.000;r=0.942,P=0.000),与DS无相关(r=0.147,P=0.238)。进一步回归分析示,MKX表达水平可以预测移植物Dc、DL、RL/S(P<0.05)。结论采用自体腘绳肌腱重建ACL移植物达塑形稳定后,MKX表达水平可预测电镜下胶原纤维直径测量结果,提示其可作为移植物胶原合成代谢水平的重要评估依据。

Objective To evaluate the correlation between the Mohawk(MKX) expression level and the collagen fiber diameter of autologous hamstring tendon graft during the stable graft remodeling phase after anterior cruciate l igament(ACL) reconstruction. Methods Between January 2018 and August 2018, patients who underwent arth-roscopic single-bundle anatomical ACL reconstruction with autologous hamstring tendons for at least 48 months and also underwent second-look arthroscopy were enrolled in study. During the second-look arthroscopic procedures, ACL graft biopsies were performed from the surface of central part of the ligament. MKX expressions of ACL grafts were analysed by real-time fluorescent quantitative PCR(qRT-PCR). The ultrastructure of collagen fibers of grafts were evaluated by transmission electron microscopy, which included average diameter of collagen fibers(Dc), average diameter of largediameter collagen fibers(DL), average diameter of small-diameter collagen fibers(DS), and large-small collagen fibers ratio(RL/S). The correlation between MKX expression level and graft collagen fiber diameter was calculated. Results Twentysix patients met the selection criteria and their ACL graft specimens were enrolled in the study. The interval between ACL reconstruction and second-look arthroscopy was 52-128 months, with an average of 78.6 months. Arthroscopic graft remodeling score was 3-6(mean, 4.8). There were 17 cases of excellent remodeling and 9 cases of fair remodeling. All ACL grafts showed typical bimodal distributions of both large-diameter collagen fibers and small-diameter collagen fibers, but the ultrastructural characteristics of the graft collagen fibers were different according to different remodeling status under arthroscopy. The DC, DL, DS, and RL/S of the graft specimens were(65.2±9.3) nm,(91.6±10.5) nm,(45.7±8.6) nm, and0.73±0.12, respectively. The relative expression level of MKX was 1.42±0.11, which was positively linearly correlated with DC, DL, and RL/S, and the correlation coefficient was statistically significant(r=0.809, P=0.000;r=0.861, P=0.000;r=0.942,P=0.000), while there was no significant correlation between DS and relative expression level of MKX(r=0.147, P=0.238).Regression analysis showed that the relative expression level of MKX could predict the DC, DL, and RL/S results of the ACL graft specimens(P<0.05). Conclusion After autologous hamstring tendon grafts stepped into stabilized remodeling phase, MKX expression level could predict the diameter measurement results of collagen fibers and be used as an important evaluation basis for graft collagen anabolic metabolism.