富血小板血浆复合小牛脱蛋白骨重建前交叉韧带腱-骨愈合的组织学观察

HISTOLOGICAL OBSERVATION OF TENDON-BONE HEALING AFTER ANTERIOR CRUCIATE LIGAMENT RECONSTRUCTION BY PLATELET-RICH PLASMA COMBINED WITH DEPROTEINIZED BONE OF CALF

目的前交叉韧带(anterior cruciate ligament,ACL)重建术腱-骨愈合情况是手术成败的关键因素。将不同浓度富血小板血浆(platelet-rich plasma,PRP)/异种小牛脱蛋白骨(deproteinized bone,DPB)凝胶复合物作为骨隧道填充物,观察ACL重建后腱-骨愈合的组织学变化,探讨该复合物对腱-骨愈合的促进作用,并确定PRP的最佳浓度。方法取新西兰大白兔耳中央动脉血8mL,采用Landesberg方法制备PRP,并将上清液作为稀释剂,制备浓度分别为30%、60%、100%的PRP。取新鲜新生小牛股骨骺端松质骨,经30%H2O2、乙醚交替浸泡24h,连续6次,制备DPB。将DPB浸泡于不同浓度PRP中并加入激活剂制备PRP/DPB凝胶复合物。取54只8～12月龄新西兰大白兔,体重(2.5±0.4)kg,随机分成3组(n=18),分别为30%PRP/DPB凝胶复合物组(A组)、60%PRP/DPB凝胶复合物组(B组)、100%PRP/DPB凝胶复合物组(C组)。实验动物制备双侧ACL缺损模型。随机取一侧下肢以对应浓度的PRP/DPB凝胶复合物作为骨隧道填充物,以自体半腱肌肌腱重建ACL,作为实验侧;另一侧行自体半腱肌肌腱重建ACL,作为对照侧。术后观察各组动物一般情况,于术后3、6、12周取材行大体观察及HE染色,光镜下观察腱-骨愈合情况,并参照Demirag等标准对腱-骨界面愈合情况进行Ⅰ、Ⅱ、Ⅲ、Ⅳ级半定量评价。结果大体观察:术后3周各组关节滑液稍增多,股骨隧道内未见骨样组织;6周时各组无关节积液,隧道内口见瘢痕组织增生;12周时各组移植肌腱与骨隧道壁纤维组织连接致密。组织学观察:A组实验侧术后3周,腱-骨界面间形成新生肉芽组织界面;6周时腱-骨界面间出现宽度不一的Sharpey样纤维;12周时腱-骨界面Sharpey样纤维排列有序,形成不规则、模糊的"潮线"。B组实验侧在各时间点腱-骨界面胶原纤维连接均优于其他各组;术后3周腱-骨界面发现排列较规整的类软骨细胞;6周时腱-骨界面骨隧道类软骨细胞单位面积数量明显多于其他组;12周时腱-骨界面可见移植物类软骨细胞增生,分化成熟,Sharpey样纤维连接致密有序。C组实验侧3周时腱-骨界面胶原纤维连接与A组实验侧及对照侧相似,6、12周相对成熟的致密结缔组织分化较差。参照Demirag等标准对腱-骨界面愈合进行分级,术后3、6周B组实验侧和对照侧、术后12周B组实验侧与C组实验侧,比较差异均有统计学意义(P<0.05);其余各组实验侧之间以及同组实验侧与对照侧比较,差异均无统计学意义(P>0.05)。结论 PRP浓度为60%时,其与DPB的复合物形成凝胶后具有良好的生物相容性和骨诱导作用,可提高兔ACL重建术后腱-骨愈合。

Objective The tendon-bone healing is the key point to ensure the success of the anterior cruciate ligament（ACL） reconstruction.To observe the histological change in the tendon-bone healing after ACL reconstruction by different concentrations of platelet-rich plasma（PRP） combined with deproteinized bone（DPB） of calf as bone tunnel infilling and to investigate the active effect of the complex on tendon-bone healing and to define the optimal concentration of PRP. Methods Eight mL blood was drawn from central artery of New Zealand rabbit ears;PRP was prepared by Landesberg method,and liquid supernatant was used as thinner to prepare different concentrations of PRP（30%,60%,and 100%）.Fresh osteoepiphysis spongy bone was harvested from lower end of femur of newborn calf to prepare DPB by way of 30% H2O2 and ether alternating soaking for 24 hours continuous 6 times.DPB was soaked in different concentrations of PRP and mixed with activator to prepare the PRP/DPB complex.A total of 54 New Zealand white rabbits,aging 8-12 months,weighing（2.5 ± 0.4） kg,were divided randomly into 3 groups：group A（30%PRP/DPB complex,n=18）,group B（60%PRP/DPB complex,n=18）,and group C（100%PRP/DPB complex,n=18）.The legs of the rabbits were randomly divided into experimental side and the control side;ACL was reconstructed by semitendinosus and PRP/DPB complex in bone tunnel in the experimental side,and only by semitendinosus in the control side.The general conditions of the rabbits were observed postoperatively and HE staining was used to observe the tendon-bone healing,then I-IV levels of semi-quantitative analysis of the tendon-bone healing were evaluated according to Demirag standard at 3,6,and 12 weeks. Results General observation：Synovial fluid slightly increased in the specimens and no bony tissue was found in inner of femoral tunnel at 3 weeks;there was no synovial fluid in all the specimens and scar tissue was discovered in inner of femoral tunnel at 6 weeks;and there was no synovial fluid and the tendons became tighter with fibrous tissue at 12 weeks.Histological observation：New granulation tissue formed in the tendon-bone interface of group A experimental sides at 3 weeks;there was various widths of Sharpey type textile fiber in the tendon-bone interface at 6 weeks;Sharpey type textile fiber arranged regularly,which formed an irregular and blur ＂tidal line＂ at 12 weeks.Group B experimental sides were better than any other group at 3,6,and 12 weeks;chondrocyte-like arranged regularly in the tendon-bone interface at 3 weeks;the number of chondrocyte-like per unit area was more than that of the other groups at 6 weeks;and chondrocyte-like proliferated and matured in the tendon-bone interface,Sharpey type textile fiber became tighter and ordered.Group C experimental sides were similar to both sides of group A at 3 weeks,however,the proliferation of relatively mature dense connective tissue was worse than that of other groups at 6 and 12 weeks.According to Demirag grading,there were significant differences in tendon-bone healing between the experimental sides and the control sides of group B at 3 and 6 weeks,and between group B experimental sides and group C experimental sides at 12 weeks（P 0.05）. Conclusion The mixture of PRP/PRP has good biocompatibility and bone induction,so it can enhance tendon-bone healing after ACL reconstruction when the concentration of PRP is 60%.