富血小板血浆复合物对腱-骨愈合界面力学性能影响的组织学研究

目的通过对健康成年新西兰大白兔行生物力学拉伸试验后标本的腱骨界面、移植物及其断裂层面进行组织学及组织化学观察,评价富血小板血浆(platelet rich plasma,PRP)复合异体脱蛋白骨(deproteined bone,DPB)对前交叉韧带(anterior cruciate ligament,ACL)重建后腱骨愈合的影响。方法 36只成年新西兰大白兔,随机分为3组,每组12只:富血小板血浆复合异体脱蛋白骨组(PRP+DPB组),异体脱蛋白骨组(DPB组),空白对照组。建立双侧自体单股半腱肌肌腱重建ACL模型,前2组骨隧道内分别植入PRP凝胶与DPB复合物、DPB,空白对照组行单纯ACL重建。术后4、8、12、24周取材行生物力学测试(单一轴向的拉伸试验)后,采用HE、Alcian blue、Masson染色及VEGF免疫组织化学观察各组腱骨愈合、移植物及其断裂层面组织学特点,分析各自的力学薄弱点。结果行拉伸试验后,术后4、8周各组标本肌腱移植物均从股骨隧道内断裂拉出,但PRP+DPB组术后8周时标本肌腱断裂处靠近股骨隧道内口,DPB组及空白对照组断裂部位位于隧道中段。术后12周时PRP+DPB组6个标本中有5个肌腱断裂部位在关节腔内部分,另2组仍从隧道内断裂拉出。术后24周时各组标本均在关节腔内部分断裂。组织学观察:术后4周时PRP+DPB组HE染色见断裂层面在腱骨结合部,可见少许疏松的纤维组织,Alcian blue染色未见细胞异染,Masson染色见肌腱断端纤维排列紊乱,VEGF免疫组化染色见较多阳性细胞表达;DPB组及空白对照组断裂层面在腱骨界面,断端见瘢痕组织。术后8周时PRP+DPB组HE染色见断裂层面在隧道内口处腱骨结合部,可见较致密的纤维结缔组织,Alcian blue染色未见细胞异染,Masson染色见肌腱断端纤维排列较前规则,VEGF免疫组化染色仍有较多阳性细胞表达,但较之前少;DPB组及空白对照组在靠近隧道中段处腱骨界面断裂,断端见疏松纤维组织。术后12周时PRP+DPB组肌腱断端见胶原纤维排列较前有序,梭形细胞散在分布,Alcian blue染色见断端有少量细胞异染,VEGF免疫组化阳性细胞数量少;DPB组及空白对照组在靠近隧道内口处腱骨界面断裂,断端见较致密的纤维组织,肌腱断端纤维排列紊乱,Alcian blue染色未见异染。术后24周时各组断端纤维排列整齐,PRP+DPB组可见椭圆形细胞,Alcian blue染色呈异染,较另2组明显,VEGF免疫组化3组均难以检出。术后4、8、12周,PRP+DPB组VEGF表达与DPB组、空白对照组有显著性差异(P<0.05),即表达增强;DPB组与空白对照组各时间点VEGF表达差异无统计学意义(P>0.05);术后24周3组标本的VEGF表达差异无统计学意义(P>0.05)。结论 ACL重建术后早期(8周以内)的薄弱点在腱骨界面,晚期在于移植肌腱。PRP可以提高腱骨间骨向肌腱内长入,从而增加腱骨愈合后的抗拉伸力。

Effect of Platelet-rich Plasma on the Mechanical Property of Healing Tendon-bone Interface: Histological Study

Objective To observe the histological changes at the tendon-bone interface,graft and ruptured locations after tensile fracture test of the anterior cruciate ligament reconstruction,and to evaluate effect of platelet-rich plasma(PRP) combined with deproteinized bone(DPB) of the calf as a bone tunnel infilling on the healing tendon-bone interface.Methods A total of 36 healthy,skeletally ature New Zealand white rabbits were randomly divided into PRP+DPB,DPB,and control groups,with 12 rabbits in each.An ACL reconstruction model was established with semitendinosus tendon autograft.In the PRP+DPB and DPB groups,PRP and/or DPB were placed into the bone tunnel.At week 4,8,12,and 24,specimens were obtained for tensile fracture test,and then HE,Alcian blue,Masson,and VEGF immunohistochemistry staining were used to observe the tendon-bone healing,graft,and the rupture locations.Results After the tensile fracture test,the tendon grafts were ruptured and pulled out from the femoral tunnel in all the groups at weeks 4 and 8;however in the PRP+DPB group,the rupture was close to the inner end of the femoral tunnel at week 8,while in the DPB and control groups,it was located in the middle of the tunnel.At week 12,five of the six specimens from the PRP-DPB group showed the rupture site in the articular cavity,while in the other groups,it was still in the femoral tunnels.At week 24,in all the groups,the rupture was located in the articular cavity.At week 4,the rupture in the PRP+DPB group was shown at the tendon-bone interface,and a little loose fibrous tissue were detected with HE staining at 4 weeks;irregular collagen fibers were seen in the stump with Masson staining and VEGF.In the other two groups,the rupture was also at the tendon-bone interface,and scar tissues were discovered there.At week 8,the rupture in the PRP+DPB group was at the tendon-bone interface,but closer to the cavumarticulare;there were dense fibrillar connective tissues shown with HE staining;collagen fibers became more regular with Masson staining,and expression of VEGF were still strong but less then that at week 4.Whereas,in the DPB and control groups,the rupture was located at the tendon-bone interface close to the mid-tunnel,and loose fibrillar connective tissues were seen in the stump.At week 12,in the PRP+DPB group,the collagenous fibers were arranged regularly at the rupture site,and some spindly cells interspersed in it;the oblong cells presented dielectial dyeing around them with Alcian blue staining,and the expression of VEGF was weak.At week 24,in the stump,there were compact fibrous tissues and irregular collagenous fiber in all the groups;in the PRP+DPB group,oblong cells were detected,and Alcian Blue staining showed dielectial dyeing,which was more obvious than that in the DPB and control groups;VEGF was not detectable in any of the groups.The expression of VEGF in the PRP+DPB group was significantly higher than that in the other two groups at weeks 4,8,and 12(all P<0.05).However,at week 24,no difference was detected among the three groups(P>0.05).Between the DPB and control groups,no significant difference was detected in the level of VEGF expression at any of the time points(P>0.05).Conclusions The weak spot in the tendon-bone healing after anterior cruciate ligament reconstruction is at the tendon-bone interface in early stage(8 week),and then moves to the tendon graft later.PRP can enhance new born tissues growing into the graft and promote the healing process at the tendon-bone junction,and thus strengthen the anti-tensile force in tendon-bone healing.