Clinical And Fundamental Studies On Using A Hybrid Graft For Anterior Cruciate Ligament Reconstruction

Anterior cruciate ligament(ACL)injury is one of the most common knee ligament injuries.An injured ACL cannot heal naturally due to the lack of clot providing a stable scaffold and is associated with an increased risk of meniscal injury and osteoarthritis.Thus,ACL reconstruction is recommended to restore knee stability and allow patients to return to sports.The most commonly used tissue for ACL reconstruction is a hamstring tendon autograft.Hamstring tendon autografts have gained popularity because of a perception of less donor-site morbidity as compared with bone–patellar tendon–bone autografts.Some patients have a comparatively small hamstring tendon,however,which may compromise the mechanical properties of the autograft.Another common tissue used for primary ACL reconstruction is an allograft.Major benefits of using allografts for ACL reconstruction include a predictable graft size and no donor-site morbidity.As such,augmenting the autograft with an allograft to create a hybrid graft is theoretically an ideal solution to inadequate graft diameter.Up to now,the published studies on hybrid grafts is limited to the evaluation of partial clinical outcomes,and the results are not uniform,lacking more comprehensive basic studies,clinical studies and evidence-based medical studies on this field.Meanwhile,the role of tendon-derived stem cells(TDSCs)in tendon-bone healing process was gradually revealed,which provided a new idea for promoting hybrid graft healing after ACL reconstruction.Thus,this study was to analyze the practical effect and application prospect of using hybrid grafts for ACL reconstruction from the perspective of basic study,clinical study and evidence-based medical study,and attempt to promote the graft healing with TDSCs conditioned medium to explore a new strategy to promote hybrid graft healing ability.Part one: Comparison of Clinical Outcomes after Anterior Cruciate Ligament Reconstruction Using a Hybrid Graft versus a Hamstring AutograftPurpose: This study aimed to compare the clinical outcomes of patients who underwent anterior cruciate ligament(ACL)reconstruction with a hybrid graft versus an autograft after 3 years of follow-up.Methods: Among 57 patients with an ACL injury who underwent ACL reconstruction,28 patients received a hybrid graft(gracilis and semitendinosus tendon autograft plus a soft tissue allograft)and 29 patients received an autograft(gracilis and semitendinosus tendon autograft).The two groups were compared after a minimum 3-year follow-up regarding IKDC assessment of knee function and stability,pivot-shift test,Lachman test,and KT-1000 side-to-side differences.The patient-reported Tegner activity score,Lysholm score,and subjective IKDC score were also compared.Graft failures were identified by patient-reported outcomes,physical examinations,or MRI,and were confirmed on second-look arthroscopy;failure rate was compared between groups.Results: At final follow-up,the two groups significantly differed in pivot-shift test result(P=0.013)and Lachman test result(P =0.027).The failure rate tended to be greater in the hybrid graft group(14.3%)than in the autograft group(3.4%)(P =0.328).All five patients with failed graft reconstruction were revised after second-look arthroscopy.The KT-1000side-to-side differences at final follow-up were significantly inferior in the hybrid graft group(3.5±2.0)compared with the autograft group(2.5±1.0,P=0.024).The hybrid graft group also had a lower mean Lysholm score(P=.000)and subjective IKDC score(P =.006)than the autograft group.The mean Tegner activity score was 6.8±0.8 in the hybrid graft group and 6.9±0.6in the autograft group(P =.436).Conclusion: The knee stability and patient-reported scores in the autograft-irradiated allograft hybrid graft ACL reconstruction group were significantly inferior compared with those in the autograft ACL reconstruction group.Part two: Hybrid graft versus autograft for anterior cruciate ligament reconstruction in a rat modelPurpose: This study aimed to compare the outcomes of using an autograft versus a hybrid graft for ACL reconstruction in biomechanical testing,radiographic analysis,and histologic evaluation in a rat model.Methods: Sixty-six adult male Sprague–Dawley rats underwent unilateral ACL reconstruction with an autograft(AT group,n = 33)or a hybrid graft(HB group,n = 33).Samples were harvested for biomechanical testing,micro-computed tomography(CT)quantification,and histologic evaluation at4,8,and 12 weeks postoperatively.Bone tunnels on femoral and tibial sides were divided into three subregions;intra-articular(IA),mid-tunnel(MT),and extra articular(EA).New bone formation in bone tunnels and bone remodeling around bone tunnels were measured via micro-CT.Results: In the AT group there were significantly higher failure loads and stiffness at 8 weeks(failure load P =0.006,stiffness P <0.001)and at 12weeks(failure load P =0.008,stiffness P =0.008)than in the HB group.With regard to new bone formation in the bone tunnel,in the AT group bone volume/tissue volume(BV/TV)was significantly higher on the tibial side at 8weeks(IA P <.001,EA P =0.030)and at 12 weeks(IA P =0.010,MT P=0.041,EA P =0.003).With regard to bone remodeling around the tunnel,BV/TV was also significantly higher on the tibial side at 8 weeks(MT P=0.007,EA P =0.030)and at 12 weeks(IA P <0.001,MT P <0.001)in the AT group.Sharpey-like fibers were formed at 8 weeks in the AT group.A large number of fibroblasts withdrew at 12 weeks.In the AT group the widths of the interface were significantly narrower at 4 weeks(P <0.001),8 weeks(P=0.002),and 12 weeks(P =0.001)than in the HB group.Conclusion: Using an autograft for ACL reconstruction may result in better graft biomechanical performance and tendon-bone incorporation than using a hybrid graft.Part three: Hamstring autograft versus hybrid graft for anterior cruciate ligament reconstruction: a meta-analysis and systematic reviewPurpose: The purpose of this systematic review was to compare the clinical outcomes of primary ACL reconstructions performed using hamstring autografts with those of primary ACL reconstructions performed using hybrid grafts in terms of patient-reported evaluation,failure rate,and knee stability.Methods: A systematic review was performed to identify prospective and retrospective comparative studies and cohort studies(evidence levels 1,2,or 3)comparing outcomes of primary ACL reconstructions performed using hamstring autografting alone and hybrid grafting.Outcomes included patient-reported evaluation,failure rate,and knee stability.Results: Ten studies were included,one of level 2 and nine of level 3.Collectively they included 398 autografts and 341 hybrid grafts.The mean respective follow-up durations ranged from 24.0 to 69.6 months and 24.0 to70.8 months.Patient-reported evaluations including Lysholm scores,Tegner scores,and subjective International Knee Documentation Committee(IKDC)scores were reported in 8/10 studies.Failure rates were reported in all 10 studies.Results of knee stability examinations including KT-1000 measurements,the pivot-shift test,Lachman test,and overall IKDC results were reported in 4/10 studies.In this review there were no statistically significant differences between the patient-reported evaluations,failure rates,or KT-1000 measurements associated with autografts and hybrid grafts.Conclusion: In this systematic review there were no significant differences in patient-reported evaluation or failure rate between primary ACL reconstructions performed using autografting alone and those performed using hybrid grafting.Whether there is a substantial difference in knee stability examination results between autografts and hybrid grafts remains unknown due to a relative lack of reports on knee stability.Part four: The effect of tendon-derived stem cells-conditioned medium on NIH/3T3 cells: an in vitro studyPurpose: This study focused on the effect of tendon-derived stem cell-conditioned medium(TDSCs-CM)on fibroblasts,simulating the early effects of TDSCs-CM on intra-articular ligamentization,in order to explore a new strategy of TDSCs promoting the healing of ACL grafts with patients.Methods: TDSCs were isolated by type I collagenase digestion and identified by surface markers and multiple differentiation potentials.The TDSCs-CM was prepared by DMEM culture medium of the third generation of TDSCs with non-antibiotics and serum after passage and purification.By comparing the growth of NIH/3T3 cells under DMEM medium(control group)and TDSCs-CM(TDSCs-CM group),the expression of m RNA related to proliferation and differentiation of mouse fibroblasts was detected.Results: All TDSCs expressed CD44 and CD90,with positive rates of80.02% and 99.92%,respectively.The expression of hematopoietic stem cell markers(CD34 and CD45)and endothelial cell markers(CD31)was less than1%.After the induction of the lipid induction solution,the cells accumulated lipid,the lipid droplets became larger and merged into beads,and the oil red O staining presented a bright red color.After the induction by chondroblast induction fluid,hard chondro-like tissue was formed,and the acid mucopolysaccharide in the cartilage tissue was stained blue by alisin blue solution.After the induction by osteogenic induction solution,calcification deposition was found in the cells,and the calcification nodules were red after alizarin red staining.The expression level of Mki67 m RNA in fibroblasts of TDSCs-CM group was 2.27±0.39,significantly higher than that in the control group(P =0.010).The expression level of Col1a1 m RNA in fibroblasts of TDSCs-CM group was 1.09±0.21,significantly higher than that in the control group(P =0.012).The expression level of Acta2 m RNA in fibroblasts of TDSCs-CM group was 1.41±0.25,significantly higher than that in the control group(P =0.011).Conclusion: High purity rat TDSCs can be obtained by digestion,which have the potential of self-renewal and multiple differentiation.Tendon stem cell conditioned medium has the potential to promote the proliferation,differentiation and collagen synthesis of fibroblasts,which provides a new therapeutic strategy for promoting the ligamentization of grafts after ACL reconstruction.