Acceleration Of Tendon-bone Healing In Anterior Cruciate Ligament Reconstruction Using Kartogenin-encapsulated Alginate Gelation In Rat Model

Background and Objectives:Anterior cruciate ligament(ACL)is the most frequently injured ligament of the knee and lead to about 400,000 ACL reconstructions per year worldwide.Because ACL can not heal spontaneously,surgical reconstruction using autograft or allograft tissue is frequently used to restore normal knee kinematics and to prevent degenerative osteoarthritis(OA).ACL attaches to bone across a functionally graded interface which is called “enthesis".The typical four-layered structure of enthesis is consist of four zones(tendon,fibrocartilage,mineralized fibrocartilage,and bone).It is believed to play an important role for dissipation of stress concentrations at mature fibrocartilaginous interfaces.So,the key part of ACL reconstruction is how to regenerate normal fibrocartilaginous enthesis structure efficiently.It has been reported that Scx+/Sox9+ progenitors contribute to the establishment of the junction between cartilage and tendon/ligament by lineage tracing.Sox9 is a high-mobility group box-containing transcription factor that functions as an important regulator of cartilage formation.Meanwhile,another study has reported that tenocytes can direct convert into chondrocytes by Sox9.All of this suggest that chondrogenic factor may play an important role in augmenting the healing of the tendon-bone interface.And what happened is that chondrogenic inducing factor,e.g BMP-2,TGF-β 3,PTH have been proved to own the ability to improves tendon-bone healing.Given cartilage importance during development,the overall objective of this dissertation was first to examine the role of cartilage in development and morphogene sis of the tendon-to-bone insertion,and then established a tendon-bone healing small animal model in ACL reconstruction and use this model to evaluate the potential therapeutic effects of chondrogenic molecular kartogenin(KGN)during enthesis healing.KGN is a small heterocyclic compound that has been proved to own strang ability of induction endogenous stem/progenitor cells such as mesenchymal stem cells(MSCs)to differentiate into chondrocytes.In this study,we attempted to accelerate the healing of tendon-bone in anterior cruciate ligament reconstruction using KGN-encapsulated alginate gelation in rat model.Methods:1.Six postnatal time points of SD rats were adopted to study the appearance of supraspinatus tendon enthesis.We used a rat model of supraspinatus tendon enthesis to study the formation of the tendon-to-bone insertion.Understanding the differences in extracellular matrix and structure change between the developing insertion.3,7,14,21,28,and 56-day-old SD rats were sacrificed and supraspinatus tendon enthesis complexes were isolated(3 rats per time point).Specimens were sectioned in 5μm thick and stained with safranin O and hematoxylin and eosin(H&E)staining to analyze the developing insertion.2.Establishing tendon-bone healing small animal models of ACL reconstruction in SD rat.Six male Sprague-Dawley rats(skeletally mature,weight range,340 to 360g;age,less than three months)underwent unilateral ACL reconstruction with a flexor digitorum longus(FDL)autograft.Three animals were killed on postoperative day 0 and were imaged with micro-computed tomography(Micro-CT).The remain animals were killed on postoperative day 28 for Micro-CT scanning,biomechanical testing,histomorphometric analysis.3.Using KGN-encapsulated alginate gelation to accelerate the healing of tendon-bone interface in our rat model.Fifteen male SD rats were divided into 2 groups: right knee was the KGN encapsulated alginate gelation group,and left knee was the control alginate gelation group.Each an imal underwent bilateral ACL reconstruction with a FDL autograft.The rats were euthanized at 2,4,8 weeks,and the tissue was harvested for Micro-CT scanning and histomorphometric analysis to determine bone tunnel change and the fibrocartilage formation at the tendon-bone interface.Results:1.The supraspinatus tendon enthesis of 3,7-day-old SD rats was proved as a condensation of chondrocytes.The interface between tendon and bone did not form into a mature fibrocartilaginous insertion until 21-days postnatally.The development of the graded mineralized interface is linked to endochondral bone formation near the tendon insertion.2.We successfully established a small animal research model of ACL reconstruction.All rats tolerated the surgery well and experienced almost no complications.The mean operative time from the harvest of FDL tendon autograft to completion of the wound was 35 min,with a standard deviation of 10 min.3.Micro-CT scanning and histomorphometric analysis showed that KGN encapsulated alginate gelation injected between the tendon-bone interface can stimulate the formation of cartilage-like tissue and promote osteogenesis.Conclusion:KGN can stimulate the formation of cartilage-like tissue between the tendon-bone interface.The formation of newly formed cartilage can promote osteogenesis and narrow the bone tunnel.