PET人工韧带重建前交叉韧带临床、力学及相关基础研究
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摘要
前交叉韧带(anterior cruciate ligament, ACL)损伤导致膝关节不稳定,严重影响膝关节功能,主要采用ACL重建手术治疗,是运动医学领域研究的热点。ACL重建移植物包括自体移植物、异体移植物和人工韧带。早期的人工韧带失败率太高而被临床淘汰。由于自体和异体移植物重建ACL都存在各自的弊端,近年来人工韧带重新受到关注。临床上应用经过改良设计的聚对苯二甲酸乙二醇酯(polyethylene terephthalate, PET)人工韧带重建ACL逐渐增多,但术后超过两年的疗效和安全性研究尚不多。ACL重建术后动态膝关节旋转稳定性评价是衡量ACL重建术疗效的重要内容,通常采用三维步态分析技术或X光立体摄影技术来评价。前者比较成熟,后者设备昂贵,国内极少单位使用。目前临床应用PET人工韧带还存在一定缺陷：人工韧带两端骨隧道部分依靠挤压螺钉维持韧带固定,韧带骨道部分受螺钉挤压导致腱骨愈合受到影响；材料的生物相容性欠佳,成纤维细胞在人工韧带关节内游离纤维部分的表面粘附很少,分布不均匀,细胞增殖能力差,细胞外胶原纤维在人工韧带内分布不均匀,是导致人工韧带失败的主要原因之一。当前的基础研究开始注重改进PET人工韧带材料的生物相容性,但还没有能满足临床实践需要的有效方法。本研究是我们863和973纳米化人工韧带研究课题的一部分内容,拟观察PET人工韧带材料表面经纳米技术修饰后,PET材料的生物相容性能否提高,从而有助于提高人工韧带的远期疗效呢?
为了解目前PET人工韧带移植后的真实情况,本研究对国内多家骨科运动医学临床中心实施的LARS人工韧带重建ACL术后患者进行3-5年临床随访,评价其临床疗效,分析其失败原因,有针对性地开展相关研究；同时,采用三维步态分析技术,定量评价LARS人工韧带重建ACL术后患者的动态膝关节旋转稳定性,并与自体胭绳肌腱移植重建ACL术后患者比较,探讨人工韧带重建的可靠性；同时对PET人工韧带材料进行纳米化处理,观察人骨髓基质干细胞在经过透明质酸、壳聚糖纳米自组装涂层、以及纳米羟基磷灰石涂层表面处理的PET人工韧带材料上的粘附、增殖和分化能力。为进一步深入研究打下基础,提供参考。本研究包括以下三个部分：
第一部分LARS人工韧带重建前交叉韧带多中心临床研究
目的多中心临床研究评价LARS (Ligament Advanced Reinforcement System, LARS)人工韧带重建前交叉韧带(anterior cruciate ligament, ACL)术后3-5年的临床疗效。
方法对国内四家大学附属医院的骨科运动医学临床中心,从2004年8月到2006年7月间,使用LARS人工韧带重建ACL的159个病例进行回顾性临床研究,所选手术患者均保留ACL残端,施行人工韧带ACL重建术。从ACL损伤到接受重建手术的时间平均为5个月。术后平均随访50个月(36-62个月)。疗效评价包括：采用Lachman试验、轴移试验、KT-1000关节动度计评价膝关节稳定性；采用Lysholm评分、Tegner评分、IKDC评分,进行膝关节功能主客观评分；采用MRI和X线片影像学评价膝关节滑膜炎和骨隧道扩大发生；通过评价患者术后恢复运动的时间,了解患者对疗效的主观满意度；对其中68例患者进行Biodex等速肌力测定,以评价股四头肌力和胭绳肌力恢复情况。
结果最终随访时,KT-1000关节动度计测试的结果是侧-侧比较,胫骨前移差值为1.5±1.6mm,膝关节稳定性较术前明显改善(p<.0001)。Lysholm评分由术前65.1±12.3分提高到最后随访时的94.5±7.0分(p<.0001); Tegner运动评分,由术前的3.1±1.6提高到随访时的6.1±1.5(p<.0001),恢复到不受限制运动的时间为术后5.2±1.1月；IKDC评分,正常或几乎正常(A或B)的比例为94%。有80例患者发生不同程度骨隧道扩大,骨道扩大发生率为51%。主观满意度：81%患者非常满意(127/156),12%患者满意(18/156),不满意者为4%(7/156),非常不满意为3%(4/156)。68例患者股四头肌峰力矩患侧占健侧下肢的百分比为93.6±10.7,等速胭绳肌峰力矩患侧占健侧下肢的百分比为95.8±12.0。与LARS人工韧带重建术直接相关的失败病例11例,失败率为7%,其中3例为固定螺钉松动,4例因为定位错误导致关节松动,3例为LARS人工韧带关节内断裂,伴1例膝关节滑膜炎,1例韧带残端处理不当。
结论对于保留有ACL残端的急性和慢性ACL损伤病例应用LARS人工韧带重建ACL,术后随访50个月的主观和客观临床疗效尚好,骨道扩大发生率为51%。术后4年的失败率为7%,包括了螺钉松动、韧带断裂等。LARS人工韧带两端与骨隧道之间愈合不够牢固。临床相关提示,必须探索促进骨组织PET人工韧带两端愈合的有效方法。
第二部分LARS人工韧带重建前交叉韧带术后膝关节稳定性的三维步态分析
目的评价并比较LARS人工韧带和自体腘绳肌腱移植重建ACL术后,患者在步行状态下的动态膝关节旋转稳定性和步态结果。
方法LARS人工韧带和自体腘绳肌腱(HT)移植重建ACL术后1年以上已经恢复运动的患者各8例,正常对照8名。在步态分析实验室,应用装置8台高速红外摄像机的Vicon三维步态分析系统和测力台,采集实验对象步行时的运动学和动力学数据。分别采集每位实验对象在正常速度步行时3次符合要求的测试数据,取平均值,作为每位实验对象的结果。应用Visual 3D软件对采集的运动学和动力学数据进行分析。步态分析指标：运动学,包括时间-距离参数：步速、步长,患侧下肢的站立期时间占步态周期时间的百分比；患侧膝关节屈伸和旋转角度；步态动力学指标：患侧下肢矢状面垂直地面反作用力,并与正常对照组比较。
结果步速、步长、步频,站立期时间占步态周期的百分比,LARS组、HT组与对照组比较,差异无统计学意义(p>0.05)。站立期的矢状面膝关节角度：足跟着地时的屈膝角度,LARS组、HT组和对照组分别为3.0±1.4度、2.8±1.6度和2.74±2.1度；站立期最大屈膝角度和最小屈膝角度,LARS组、HT组、对照组分别为：33.2±9.6度和7.0±3.1度,33.5±10.8度和7.7±3.8度,32.8±5.7度和7.2±3.4度；三组间比较,差异无统计学意义(p>0.05)。膝关节最大内旋角度,LARS组、HT组和对照组分别为6.2±1.6度、5.8±1.3度和5.2±1.8,三组间差异无统计学意义(p>0.05)。膝关节最大外旋角度,LARS组、HT组和对照组分别为7.8+1.1度、8.2±1.4度和5.6±1.9度,HT组和LARS组均显著大于对照组(p<0.001)；HT组和LARS组比较,组间差异无统计学意义(p>0.05)。动力学,三组的矢状面垂直地面反作用力均呈正常的M形曲线；地面反作用力第1峰值,LARS组、HT组和对照组分别为120.8±15.3、119.7±12.8和121.1±19.0；地面反作用力谷值,LARS组、HT组和对照组分别为73.0±12.9、72.6±13.7和68.9±13.0；地面反作用力第2峰值,站立中期力的变化,地面反作用力达到第1峰值的时间,LARS组、HT组和对照组间的差异均无统计学意义(p>0.05)。
结论LARS人工韧带重建和自体HT移植单束重建ACL术后1年步行时的时间-距离参数和矢状面垂直地面反作用力恢复至正常水平。步行时的矢状面膝关节活动度恢复正常,但2组患者的水平面膝关节旋转稳定性未恢复到正常水平。
第三部分PET韧带材料纳米化对骨髓基质干细胞黏附、增殖以及分化影响的实验研究
目的观察人骨髓基质干细胞(human Bone Marrow Mesenchymal Stem Cells, hBMSCs)在经过表面纳米化处理的PET材料表面的黏附、增殖及成骨分化的能力。
方法观察hBMSCs在以下3组PET膜片上的粘附、增殖和分化：PET膜片表面经壳聚糖、透明质酸纳米自组装涂层处理(LBL组)；PET膜片表面经壳聚糖、透明质酸纳米自组装涂层处理后,表面再加纳米羟基磷灰石涂层(HA组),PET膜片未经表面处理(PET组)。扫描电镜(SEM)观察hBMSCs在材料上培养24小时和7天时的细胞形态。检测hBMSCs培养7天后的增殖和碱性磷酸酶(ALP)活性。Real Time PCR检测培养3天时hBMSCs的整合素β1 mRNA表达。
结果电镜下hBMSCs在纳米羟基磷灰石和透明质酸、壳聚糖涂层的PET表面能更好地伸展和黏附,形态明显优于普通PET组。培养7天的细胞增殖、ALP活性定量,HA组和LBL组均显著高于PET组(p<0.001)。HA组细胞增殖,显著高于LBL组(p=0.01)。HA组的ALP活性,显著高于LBL组(p<.001)。hBMSC培养3天的整合素β1 mRNA表达,HA组和LBL组显著高于PET组(p<.001)；HA组显著高于LBL组(p=.003)。
结论PET表面经壳聚糖、透明质酸纳米自组装涂层和纳米羟基磷灰石涂层处理后,能明显促进人hBMSCs在PET膜表面的粘附、增殖和分化。纳米羟基磷灰石涂层的作用最明显。
Anterior cruciate ligament (ACL) injury results in instability of the knee and damage of its function. ACL reconstruction is the main treatment option for ACL injury, and it is always the research focus in sports medicine area. ACL reconstruction grafts include auto graft, allograft and artificial ligament. The previous artificial ligaments have been abandoned because of the high failure rate. As both of autograft and allograft have some drawbacks that could not be overcome, artificial ligament has been recently reconsidered for ACL reconstruction choice. The modified polyethylene terephthalate (PET) artificial ligament has been used for ACL reconstruction clinically, but its clinical outcome and safety over two years after surgery are still unknown and less reported. Currently, basic research of artificial ligament focus on how to improve the biocompatibility of PET material, but the advancement is too minimal to meet the need of clinical practice. Some drawbacks of the modified PET artificial ligament used in clinic have been found with the poor biocompatibility of PET material. The healing between the both ends of the PET artificial ligament with the bone tunnel is not firm, and the fixation of the artificial ligament is dependent on the interference screw. Also, the tissue growth into the free bibers of intra-articular part of the artificial ligament is poor, so the long-term clinical outcome could not be guaranted. If the nano technology surface modification improve the biocompatibility of the PET artificial ligament material, and make for the raise of the long-term outcome of artificial ligament?
In order to understand the current situation and true fact after ACL reconstruction using PET artificial ligament and to solve the questions above-mentioned, a multicenter study was underwent to evaluate the clinical outcome of ACL reconstruction using the Ligament Advanced Reinforcement System (LARS) artificial ligament with 3-to 5-year follow up. The rotational stability of the knee and function of the lower limb during walking were quantitatively evaluated by use of three-dimensional gait analysis technology on patients who underwent ACL reconstruction using LARS artificial ligament and hamstring autograft. Human bone mesenchymal stem cells (hBMSCs) were cultured on PET artificial ligament materials which have been coated with chitosan and hyaluronic acid or nanohydroxyapatite by use of layer-by-layer nano self-assemble, and the adhesion, proliferation and differentiation of the hBMSCs were observed. This study included three parts as follows.
PartⅠAnterior Cruciate Ligament Reconstruction with LARS Artificial Ligment:A Multi-center Study
Purpose Multicenter study to evaluate the clinical outcome of anterior cruciate ligament (ACL) reconstruction by use of the Ligament Advanced Reinforcement System (LARS) artificial ligament with 3-to 5-year follow up.
Methods From August 2004 to July 2006,159 patients with ACL rupture underwent arthroscopic ACL reconstruction with LARS artificial ligament at 4 orthopaedic sports medicine centers. They were retrospectively followed up for 50 months (36 to 62 months). Outcome assessment included physical examination, KT-1000 arthrometer testing, magnetic resonance imaging, radiography, Lysholm score, Tegner score, International Knee Documentation Committee score, and subjective satisfaction rate. Quadriceps and hamstring isokinetic strength was evaluated in 68 patients.
Results The side-to-side difference in anterior translation (injured side-uninjured side) measured by KT-1000 arthrometer was 1.5±1.6 mm postoperatively, and knee stability was significantly improved compared with preoperative data (P<.0001). The Lysholm score improved from 65.1±12.3 points preoperatively to 94.5±7.0 points postoperatively (P<.0001). The Tegner score improved from 3.1±1.6 preoperatively to 6.1±1.5 postoperatively (P<.0001). According to the International Knee Documentation Committee score,94%patients were graded A or B at the last follow up. The rate of bone tunnel enlargement was 51%(80/156). Ninety-three percent of patients were very satisfied or satisfied with their outcome. Quadriceps and hamstring isokinetic peak torque of the injured limb expressed as a percentage of the contralateral limb was 93.6±10.7 and 95.8±12.0, respectively. Total 11 cases underwent the failure with the rate of 7%, including 3 cases with interference screw loose,4 cases with knee laxity due to surgical technical mal-position of bone tunnel,3 cases with artificial ligament rupture and one case accompanied synovitis.
Conclusions ACL reconstruction with LARS artificial ligament used in patients with the ACL stump preserved in the acute and chronic phases has a very good outcome at mean of 50 months'follow up. The overall failure rate for ACL reconstruction with LARS artificial ligament was 7%, and one case with knee synovitis.
PartⅡThree-Dimensional Gait Analysis for Knee Stability After ACL Reconstruction with LARS Artificial Ligament
Purpose To evaluate the dynamic rotation stability of the knee and lower limb function in patients who had undergone anterior cruciate ligament reconstruction by use of LARS artificial ligament and hamstring autograft using single bundle technique.
Methods Three groups were compared:eight LARS artificial ligament reconstruction patients, eight hamstring autograft anterior cruciate ligament reconstruction patients, and 8 matched control.All patients received the operation before 12 months when the gait analysis was performed and they all returned to the sports activity before injury. A 3-dimension motion analysis and force plate system was used to determine sagittal plane kinematics and kinetics of the lower limb during walking. Each subject underwent 3 trials and the mean were as their results. Visual 3D software was used to resolve the data. Variable analysis included step length, step speed, and stance of gait cycle percent, the angle of knee flexion, extension, and rotation. The sagittal vertical ground reaction force was also analyzed.
Results There was no statistically significant difference between the three groups with respect to the step length, step speed, and stance of gait cycle percent (p>0.05).The knee flexion and extension at heel contact in LARS group, HT group and control were 3.0±1.4,2.8±1.6 and 2.7±2.1, respectively. The maximal and minimal knee flexion and extension during stance in LARS group, HT group and control were 33.2±9.6 and 7.0±3.1,33.5±10.8 and 7.7±3.8,32.8±5.7 and 7.2±3.4, respectively; and there was no significant difference between the three groups (p>0.05).The maximal knee internal rotation in LARS group, HT group and control were 6.2±1.6,5.8±1.3, and 5.2±1.8, respectively, and there was no significant difference between groups (p>0.05). The maximal knee external rotation in LARS group, HT group and control were 7.8±1.1,8.2±1.4, and 5.6±1.9, respectively. The values of LARS group and the HT group were more larger than the control, and indicating a significant difference (p<0.001); there was no significant difference between the HT group and the LARS group (p>0.05).The sagittal vertical ground reaction force:the first peak force of LARS group, HT group and control were 120.8±15.3、119.7±12.8 and 121.1±19.0, respectively; the valley force of LARS group, HT group and control were 73.0±12.9、72.6±13.7 and 68.9±13.0, respectively; there was no significant difference between the three groups (p>0.05). As for the second peak force, the force change in the midfoot, and the first peak time, there was no significant difference between the three groups (p>0.05).
Conclusions The time-spatial data, sagittal knee angle, and sagittal vertical ground reaction force return to normal level during walking at 1 year after anterior cruciate ligament reconstruction by use of LARS artificial ligament or hamstring autograft using single bundle technique, but the knee rotation does not resume to normal level.
Part III Effect of Nanoscale Coating for PET Materials on Adhesion, Proliferation and Differentiation of Mesenchymal Stem Cells
Purpose To observe the effect of chitosan, hyaluronic acid and hydroxyapatite nanoscale coating for polyethylene terephthalate (PET) material on adhesion, proliferation and differentiation of human Bone Marrow mesenchymal stem cells (hBMSCs).
Methods The hBMSCs were cultured on the PET coating of nanoscale chitosan, hyaluronic acid and hydroxyapatite, and the adhesion, proliferation and differentiation of hBMSCs were observed. The PET film were divided into three groups based on the surface modification using layer-by-layer of chitosan and hyaluronic acid (LBL group), LBL plus nano hydroxyapatite coating (HA group), or without treatment (PET group). The morphology of hBMSC were observed by scanning electron microscope (SEM) at 24 hours and 7 days after seeding. The proliferation and the alkali phosphatase (ALP) activity of hBMSCs were detected when cultured for 7 days. Real Time PCR was used to measure the mRNA expression of integrinβ1 at 3 days culture.
Results The hBMSCs showed better spreading in the HA group and LBL group than the PET group. The proliferation and ALP activity of hBMSCs of the HA group and LBL group were significantly higher than the PET group (<.001). The proliferation of the HA group was significantly higher than the LBL group (p=0.01), and the ALP activity of the HA group was also higeher than the LBL group (p<.001).As for the mRNA expression of integrinβ1 at 3 days after seeding, the HA group and LBL group were significantly higher than the PET group (<.001), and the HA group showed significantly higher than the LBL group (p=.003).
Conclusions The chitosan, hyaluronic acid and hydroxyapatite nanoscale coating for PET material significantly improve the adhesion, proliferation and differentiation of hBMSCs. The effect of nano hydroxyapatite is more notable.
为了解目前PET人工韧带移植后的真实情况,本研究对国内多家骨科运动医学临床中心实施的LARS人工韧带重建ACL术后患者进行3-5年临床随访,评价其临床疗效,分析其失败原因,有针对性地开展相关研究；同时,采用三维步态分析技术,定量评价LARS人工韧带重建ACL术后患者的动态膝关节旋转稳定性,并与自体胭绳肌腱移植重建ACL术后患者比较,探讨人工韧带重建的可靠性；同时对PET人工韧带材料进行纳米化处理,观察人骨髓基质干细胞在经过透明质酸、壳聚糖纳米自组装涂层、以及纳米羟基磷灰石涂层表面处理的PET人工韧带材料上的粘附、增殖和分化能力。为进一步深入研究打下基础,提供参考。本研究包括以下三个部分：
第一部分LARS人工韧带重建前交叉韧带多中心临床研究
目的多中心临床研究评价LARS (Ligament Advanced Reinforcement System, LARS)人工韧带重建前交叉韧带(anterior cruciate ligament, ACL)术后3-5年的临床疗效。
方法对国内四家大学附属医院的骨科运动医学临床中心,从2004年8月到2006年7月间,使用LARS人工韧带重建ACL的159个病例进行回顾性临床研究,所选手术患者均保留ACL残端,施行人工韧带ACL重建术。从ACL损伤到接受重建手术的时间平均为5个月。术后平均随访50个月(36-62个月)。疗效评价包括：采用Lachman试验、轴移试验、KT-1000关节动度计评价膝关节稳定性；采用Lysholm评分、Tegner评分、IKDC评分,进行膝关节功能主客观评分；采用MRI和X线片影像学评价膝关节滑膜炎和骨隧道扩大发生；通过评价患者术后恢复运动的时间,了解患者对疗效的主观满意度；对其中68例患者进行Biodex等速肌力测定,以评价股四头肌力和胭绳肌力恢复情况。
结果最终随访时,KT-1000关节动度计测试的结果是侧-侧比较,胫骨前移差值为1.5±1.6mm,膝关节稳定性较术前明显改善(p<.0001)。Lysholm评分由术前65.1±12.3分提高到最后随访时的94.5±7.0分(p<.0001); Tegner运动评分,由术前的3.1±1.6提高到随访时的6.1±1.5(p<.0001),恢复到不受限制运动的时间为术后5.2±1.1月；IKDC评分,正常或几乎正常(A或B)的比例为94%。有80例患者发生不同程度骨隧道扩大,骨道扩大发生率为51%。主观满意度：81%患者非常满意(127/156),12%患者满意(18/156),不满意者为4%(7/156),非常不满意为3%(4/156)。68例患者股四头肌峰力矩患侧占健侧下肢的百分比为93.6±10.7,等速胭绳肌峰力矩患侧占健侧下肢的百分比为95.8±12.0。与LARS人工韧带重建术直接相关的失败病例11例,失败率为7%,其中3例为固定螺钉松动,4例因为定位错误导致关节松动,3例为LARS人工韧带关节内断裂,伴1例膝关节滑膜炎,1例韧带残端处理不当。
结论对于保留有ACL残端的急性和慢性ACL损伤病例应用LARS人工韧带重建ACL,术后随访50个月的主观和客观临床疗效尚好,骨道扩大发生率为51%。术后4年的失败率为7%,包括了螺钉松动、韧带断裂等。LARS人工韧带两端与骨隧道之间愈合不够牢固。临床相关提示,必须探索促进骨组织PET人工韧带两端愈合的有效方法。
第二部分LARS人工韧带重建前交叉韧带术后膝关节稳定性的三维步态分析
目的评价并比较LARS人工韧带和自体腘绳肌腱移植重建ACL术后,患者在步行状态下的动态膝关节旋转稳定性和步态结果。
方法LARS人工韧带和自体腘绳肌腱(HT)移植重建ACL术后1年以上已经恢复运动的患者各8例,正常对照8名。在步态分析实验室,应用装置8台高速红外摄像机的Vicon三维步态分析系统和测力台,采集实验对象步行时的运动学和动力学数据。分别采集每位实验对象在正常速度步行时3次符合要求的测试数据,取平均值,作为每位实验对象的结果。应用Visual 3D软件对采集的运动学和动力学数据进行分析。步态分析指标：运动学,包括时间-距离参数：步速、步长,患侧下肢的站立期时间占步态周期时间的百分比；患侧膝关节屈伸和旋转角度；步态动力学指标：患侧下肢矢状面垂直地面反作用力,并与正常对照组比较。
结果步速、步长、步频,站立期时间占步态周期的百分比,LARS组、HT组与对照组比较,差异无统计学意义(p>0.05)。站立期的矢状面膝关节角度：足跟着地时的屈膝角度,LARS组、HT组和对照组分别为3.0±1.4度、2.8±1.6度和2.74±2.1度；站立期最大屈膝角度和最小屈膝角度,LARS组、HT组、对照组分别为：33.2±9.6度和7.0±3.1度,33.5±10.8度和7.7±3.8度,32.8±5.7度和7.2±3.4度；三组间比较,差异无统计学意义(p>0.05)。膝关节最大内旋角度,LARS组、HT组和对照组分别为6.2±1.6度、5.8±1.3度和5.2±1.8,三组间差异无统计学意义(p>0.05)。膝关节最大外旋角度,LARS组、HT组和对照组分别为7.8+1.1度、8.2±1.4度和5.6±1.9度,HT组和LARS组均显著大于对照组(p<0.001)；HT组和LARS组比较,组间差异无统计学意义(p>0.05)。动力学,三组的矢状面垂直地面反作用力均呈正常的M形曲线；地面反作用力第1峰值,LARS组、HT组和对照组分别为120.8±15.3、119.7±12.8和121.1±19.0；地面反作用力谷值,LARS组、HT组和对照组分别为73.0±12.9、72.6±13.7和68.9±13.0；地面反作用力第2峰值,站立中期力的变化,地面反作用力达到第1峰值的时间,LARS组、HT组和对照组间的差异均无统计学意义(p>0.05)。
结论LARS人工韧带重建和自体HT移植单束重建ACL术后1年步行时的时间-距离参数和矢状面垂直地面反作用力恢复至正常水平。步行时的矢状面膝关节活动度恢复正常,但2组患者的水平面膝关节旋转稳定性未恢复到正常水平。
第三部分PET韧带材料纳米化对骨髓基质干细胞黏附、增殖以及分化影响的实验研究
目的观察人骨髓基质干细胞(human Bone Marrow Mesenchymal Stem Cells, hBMSCs)在经过表面纳米化处理的PET材料表面的黏附、增殖及成骨分化的能力。
方法观察hBMSCs在以下3组PET膜片上的粘附、增殖和分化：PET膜片表面经壳聚糖、透明质酸纳米自组装涂层处理(LBL组)；PET膜片表面经壳聚糖、透明质酸纳米自组装涂层处理后,表面再加纳米羟基磷灰石涂层(HA组),PET膜片未经表面处理(PET组)。扫描电镜(SEM)观察hBMSCs在材料上培养24小时和7天时的细胞形态。检测hBMSCs培养7天后的增殖和碱性磷酸酶(ALP)活性。Real Time PCR检测培养3天时hBMSCs的整合素β1 mRNA表达。
结果电镜下hBMSCs在纳米羟基磷灰石和透明质酸、壳聚糖涂层的PET表面能更好地伸展和黏附,形态明显优于普通PET组。培养7天的细胞增殖、ALP活性定量,HA组和LBL组均显著高于PET组(p<0.001)。HA组细胞增殖,显著高于LBL组(p=0.01)。HA组的ALP活性,显著高于LBL组(p<.001)。hBMSC培养3天的整合素β1 mRNA表达,HA组和LBL组显著高于PET组(p<.001)；HA组显著高于LBL组(p=.003)。
结论PET表面经壳聚糖、透明质酸纳米自组装涂层和纳米羟基磷灰石涂层处理后,能明显促进人hBMSCs在PET膜表面的粘附、增殖和分化。纳米羟基磷灰石涂层的作用最明显。
Anterior cruciate ligament (ACL) injury results in instability of the knee and damage of its function. ACL reconstruction is the main treatment option for ACL injury, and it is always the research focus in sports medicine area. ACL reconstruction grafts include auto graft, allograft and artificial ligament. The previous artificial ligaments have been abandoned because of the high failure rate. As both of autograft and allograft have some drawbacks that could not be overcome, artificial ligament has been recently reconsidered for ACL reconstruction choice. The modified polyethylene terephthalate (PET) artificial ligament has been used for ACL reconstruction clinically, but its clinical outcome and safety over two years after surgery are still unknown and less reported. Currently, basic research of artificial ligament focus on how to improve the biocompatibility of PET material, but the advancement is too minimal to meet the need of clinical practice. Some drawbacks of the modified PET artificial ligament used in clinic have been found with the poor biocompatibility of PET material. The healing between the both ends of the PET artificial ligament with the bone tunnel is not firm, and the fixation of the artificial ligament is dependent on the interference screw. Also, the tissue growth into the free bibers of intra-articular part of the artificial ligament is poor, so the long-term clinical outcome could not be guaranted. If the nano technology surface modification improve the biocompatibility of the PET artificial ligament material, and make for the raise of the long-term outcome of artificial ligament?
In order to understand the current situation and true fact after ACL reconstruction using PET artificial ligament and to solve the questions above-mentioned, a multicenter study was underwent to evaluate the clinical outcome of ACL reconstruction using the Ligament Advanced Reinforcement System (LARS) artificial ligament with 3-to 5-year follow up. The rotational stability of the knee and function of the lower limb during walking were quantitatively evaluated by use of three-dimensional gait analysis technology on patients who underwent ACL reconstruction using LARS artificial ligament and hamstring autograft. Human bone mesenchymal stem cells (hBMSCs) were cultured on PET artificial ligament materials which have been coated with chitosan and hyaluronic acid or nanohydroxyapatite by use of layer-by-layer nano self-assemble, and the adhesion, proliferation and differentiation of the hBMSCs were observed. This study included three parts as follows.
PartⅠAnterior Cruciate Ligament Reconstruction with LARS Artificial Ligment:A Multi-center Study
Purpose Multicenter study to evaluate the clinical outcome of anterior cruciate ligament (ACL) reconstruction by use of the Ligament Advanced Reinforcement System (LARS) artificial ligament with 3-to 5-year follow up.
Methods From August 2004 to July 2006,159 patients with ACL rupture underwent arthroscopic ACL reconstruction with LARS artificial ligament at 4 orthopaedic sports medicine centers. They were retrospectively followed up for 50 months (36 to 62 months). Outcome assessment included physical examination, KT-1000 arthrometer testing, magnetic resonance imaging, radiography, Lysholm score, Tegner score, International Knee Documentation Committee score, and subjective satisfaction rate. Quadriceps and hamstring isokinetic strength was evaluated in 68 patients.
Results The side-to-side difference in anterior translation (injured side-uninjured side) measured by KT-1000 arthrometer was 1.5±1.6 mm postoperatively, and knee stability was significantly improved compared with preoperative data (P<.0001). The Lysholm score improved from 65.1±12.3 points preoperatively to 94.5±7.0 points postoperatively (P<.0001). The Tegner score improved from 3.1±1.6 preoperatively to 6.1±1.5 postoperatively (P<.0001). According to the International Knee Documentation Committee score,94%patients were graded A or B at the last follow up. The rate of bone tunnel enlargement was 51%(80/156). Ninety-three percent of patients were very satisfied or satisfied with their outcome. Quadriceps and hamstring isokinetic peak torque of the injured limb expressed as a percentage of the contralateral limb was 93.6±10.7 and 95.8±12.0, respectively. Total 11 cases underwent the failure with the rate of 7%, including 3 cases with interference screw loose,4 cases with knee laxity due to surgical technical mal-position of bone tunnel,3 cases with artificial ligament rupture and one case accompanied synovitis.
Conclusions ACL reconstruction with LARS artificial ligament used in patients with the ACL stump preserved in the acute and chronic phases has a very good outcome at mean of 50 months'follow up. The overall failure rate for ACL reconstruction with LARS artificial ligament was 7%, and one case with knee synovitis.
PartⅡThree-Dimensional Gait Analysis for Knee Stability After ACL Reconstruction with LARS Artificial Ligament
Purpose To evaluate the dynamic rotation stability of the knee and lower limb function in patients who had undergone anterior cruciate ligament reconstruction by use of LARS artificial ligament and hamstring autograft using single bundle technique.
Methods Three groups were compared:eight LARS artificial ligament reconstruction patients, eight hamstring autograft anterior cruciate ligament reconstruction patients, and 8 matched control.All patients received the operation before 12 months when the gait analysis was performed and they all returned to the sports activity before injury. A 3-dimension motion analysis and force plate system was used to determine sagittal plane kinematics and kinetics of the lower limb during walking. Each subject underwent 3 trials and the mean were as their results. Visual 3D software was used to resolve the data. Variable analysis included step length, step speed, and stance of gait cycle percent, the angle of knee flexion, extension, and rotation. The sagittal vertical ground reaction force was also analyzed.
Results There was no statistically significant difference between the three groups with respect to the step length, step speed, and stance of gait cycle percent (p>0.05).The knee flexion and extension at heel contact in LARS group, HT group and control were 3.0±1.4,2.8±1.6 and 2.7±2.1, respectively. The maximal and minimal knee flexion and extension during stance in LARS group, HT group and control were 33.2±9.6 and 7.0±3.1,33.5±10.8 and 7.7±3.8,32.8±5.7 and 7.2±3.4, respectively; and there was no significant difference between the three groups (p>0.05).The maximal knee internal rotation in LARS group, HT group and control were 6.2±1.6,5.8±1.3, and 5.2±1.8, respectively, and there was no significant difference between groups (p>0.05). The maximal knee external rotation in LARS group, HT group and control were 7.8±1.1,8.2±1.4, and 5.6±1.9, respectively. The values of LARS group and the HT group were more larger than the control, and indicating a significant difference (p<0.001); there was no significant difference between the HT group and the LARS group (p>0.05).The sagittal vertical ground reaction force:the first peak force of LARS group, HT group and control were 120.8±15.3、119.7±12.8 and 121.1±19.0, respectively; the valley force of LARS group, HT group and control were 73.0±12.9、72.6±13.7 and 68.9±13.0, respectively; there was no significant difference between the three groups (p>0.05). As for the second peak force, the force change in the midfoot, and the first peak time, there was no significant difference between the three groups (p>0.05).
Conclusions The time-spatial data, sagittal knee angle, and sagittal vertical ground reaction force return to normal level during walking at 1 year after anterior cruciate ligament reconstruction by use of LARS artificial ligament or hamstring autograft using single bundle technique, but the knee rotation does not resume to normal level.
Part III Effect of Nanoscale Coating for PET Materials on Adhesion, Proliferation and Differentiation of Mesenchymal Stem Cells
Purpose To observe the effect of chitosan, hyaluronic acid and hydroxyapatite nanoscale coating for polyethylene terephthalate (PET) material on adhesion, proliferation and differentiation of human Bone Marrow mesenchymal stem cells (hBMSCs).
Methods The hBMSCs were cultured on the PET coating of nanoscale chitosan, hyaluronic acid and hydroxyapatite, and the adhesion, proliferation and differentiation of hBMSCs were observed. The PET film were divided into three groups based on the surface modification using layer-by-layer of chitosan and hyaluronic acid (LBL group), LBL plus nano hydroxyapatite coating (HA group), or without treatment (PET group). The morphology of hBMSC were observed by scanning electron microscope (SEM) at 24 hours and 7 days after seeding. The proliferation and the alkali phosphatase (ALP) activity of hBMSCs were detected when cultured for 7 days. Real Time PCR was used to measure the mRNA expression of integrinβ1 at 3 days culture.
Results The hBMSCs showed better spreading in the HA group and LBL group than the PET group. The proliferation and ALP activity of hBMSCs of the HA group and LBL group were significantly higher than the PET group (<.001). The proliferation of the HA group was significantly higher than the LBL group (p=0.01), and the ALP activity of the HA group was also higeher than the LBL group (p<.001).As for the mRNA expression of integrinβ1 at 3 days after seeding, the HA group and LBL group were significantly higher than the PET group (<.001), and the HA group showed significantly higher than the LBL group (p=.003).
Conclusions The chitosan, hyaluronic acid and hydroxyapatite nanoscale coating for PET material significantly improve the adhesion, proliferation and differentiation of hBMSCs. The effect of nano hydroxyapatite is more notable.
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