Animal Experimental Study On The Histological Outcome Of Anterior Cruciate Ligament Reconstruction With Allogenic Lyophilized Tendons

Allogeneic tendon is one of the options for ACL reconstruction graft.This experiment compares autologous tendons,allogeneic deep-frozen tendons and allogenic lyophilized tendons on the histological outcome and tendon bone healing process of animals after ACL reconstruction.It also provides a histological basis for allograft selection and postoperative rehabilitation.Out of a total of 80 New Zealand rabbits,20 were randomly selected to obtain a total of 40 flexor digitorum longus tendons of the hind leg,out of which,20 were lyophilised and 20 were deep-frozen.The remaining 60 rabbits were randomly divided into autograft group,deep-frozen allograft group,and lyophilized allograft group,with 20 in each group,and the corresponding grafts were used for ACL reconstruction.Five rabbits were selected from the three groups at 3 weeks,8 weeks,12 weeks and 24 weeks after surgery,the relevant tissue was harvested and stained using two stains(hematoxylin-eosin stain and safranin O-fast green stain)for histological observation.The fibroblasts in the intra-articular segment(outside the bone tunnel),the fibroblasts in the bone tunnel,and the chondrocytes at the tendon-bone interface were counted,statistical analysis of the data in each group at each time point was performed using a univariate analysis of variance and LSD-t test.The fibroblast counts of the grafts inside and outside the bone tunnel were statistically analysed by a paired t-test.Histological results: Intra-articular section of the graft(outside the bone tunnel): 3 weeks after surgery,the surface of the graft is covered with synovial tissue and the cellular structure in the graft disappears.8 weeks after surgery,fibroblasts proliferated in the superficial layer,but acellular fibres were deep in the graft.12 weeks after surgery,the number of cells in the superficial layer of the graft continued to increase in each group,and cells gradually proliferated from superficial to the deep layer of the graft.24 weeks after surgery,the density of the superficial and deep cells in each group was uniform and the deep acellular areas disappeared.Graft bone tunnel: 3 weeks after surgery,the surface of the graft is covered with granulation tissue and the cellular structure in the grafts disappeared.The histological results at 8 weeks,12 weeks,and 24 weeks after surgery were similar as those of the intra-articular section of the graft.Graft tendon-bone interface: 3 weeks after surgery,hyperplasia of granulation tissue at tendon-bone interface without chondrocytes.8 weeks after surgery,chondrocytes appeared at the tendon-bone interface and oblique Sharpey’s fibres were seen;the density and range of Sharpey’s fibres in the lyophilized allograft group were lower than those in the deep-frozen allograft and autograft group.12 weeks after surgery,chondrocytes increased in each group,and the number of oblique Sharpey’s fibres increased in each group;vertical Sharpey’s fibres appeared in the autograft group,with a continuous “tidal line” structure.24 weeks after surgery,the number of chondrocytes continued to increase in each group,the proportion of vertical Sharpey’s fibres increased,and the scope of the continuous“tidal line” structure expanded.Graft vascularization: Vascularisation is concentrated on the surface of the graft or within the superficial loose connective tissue.At 8 weeks,12 weeks and 24 weeks after surgery,the number of blood vessels gradually increased,the diameter of the vessels increased,and the degree of vascularization in the outside bone tunnel of the graft was lower than that inside the bone tunnel.Statistical results: 1.Over time,the number of graft fibroblasts and chondrocytes at the tendon-bone interface increased significantly within and outside the bone tunnel(P<0.05);the number of cells present in each group was ranked as autologous>deep-frozen>lyophilized.2.At 8 weeks and 12 weeks after surgery,the difference in combined fibroblast count(within the bone tunnel and outside the bone tunnel)for each group was statistically significant(P<0.05);8 weeks after surgery the groups were ranked as autologous>deep-frozen>lyophilized(P<0.05);12 weeks after surgery,the groups were ranked as deep-frozen>lyophilized(P<0.05),there was no difference between the deep-frozen and autologous group(P>0.05);24 weeks after surgery,the differences between the groups were not statistically significant(P>0.05).3.At 8 weeks and 12 weeks after surgery,for each group,the fibroblast count within bone tunnel>outside bone tunnel,P<0.05,therefore showing statistical significance;24 weeks after surgery,the difference between the fibroblast count within bone tunnel and outside bone tunnel had no statistical significance(P>0.05).4.At 8 weeks and 12 weeks after surgery,the difference in chondrocyte count at the graft tendon-bone interface for each group was statistically significant(P<0.05);8 weeks after surgery,the groups were ranked as autologous>deep-frozen>lyophilized(P<0.05);12 weeks after surgery,the groups were ranked as autologous>deep-frozen,there was no difference between the lyophilized and deep-frozen group(P>0.05);24 weeks after surgery,the differences between the groups were not statistically significant(P>0.05).Based on the results of this experiment: 1.The cell proliferation of ACL reconstruction graft is gradually completed from superficial to deep.From 8 to 12 weeks,areas of graft necrosis and cell proliferation existed simultaneously.2.The early biological transformation and tendon healingprocess of ACL reconstruction after allogenic lyophilized tendon lags behind allogenic deep-frozen tendon and autologous tendon.24 weeks after surgery the transformation and healing process tend to be synchronized.3.Tendon bone healing of ACL reconstruction grafts started from 8 weeks after surgery.The tendon-bone interface gradually transitioned from indirect healing to direct healing.The transformation process was still in progress 24 weeks after surgery.4.Differences are present in cell proliferation and vascularization on the surface of internal and external bone tunnel grafts.Fibroblasts are present outside the bone tunnel.In addition to fibroblasts,chondrocytes appear in the bone tunnel at 8 weeks and gradually increase;and the degree of vascularization of the graft outside the bone tunnel is significantly lower than that inside the bone tunnel.