Use Of Three Different Bone Grafts Loaded With BMP In Reconstruction Of Ligaments In Knee Trauma

The knee joint is among the most commonly injuried joints, and the anterior cruciate ligament(ACL) as a pivotal stabilizing mechanism in the knee joint is more vulnerable to trauma, with its rupture rate being as high as more than 70% in all cases. The poor healing capacity of the ACL observed clinically and confirmed in multiple in vitro and in vivo experiments has led orthopaedic surgeons to perform ACL reconstructions rather than repairs. Bone-patellar tendon-bone (BPTB)and combined semitendinosus and gracilis tendons are the most frequently used graft types for ACL reconstruction. In recent years, use of hamstring tendon grafts appears to have increased, perhaps related to a perception that hamstring tendon grafts are associated with lower postoperative morbidity compared with patellar tendon grafts.The firm healing of tendon grafts to bone is one of the most important factors for ACL reconstructive surgical procedures using hamstring tendon grafts. Early and enhanced graft-bone tunnel interface healing is obviously desirable. Though the precise mechanism of healing of a tendon graft in a bone tunnel is unclear, the researchers have reported that tendon-to-bone healing proceeds by bone ingrowth into the fibrous tissue interface that initially forms between the tendon and bone, and the progressive increase in strength of the bone-tendon interface appeared to correlate with the degree of bone ingrowth . It is advisable to enhance the rate and quality of osteointegration of grafted tendon at this site. Studies have demonstrated that the biologic response of the bone tunnel was"similar to the healing of a fracture."As we know, reconstruction of ACL involves the use of a tendon graft that is transplanted into bone tunnel at the femoral and tibial insertion sites, the marrow in bone tunnel provided a source of pluripotent cells that is the main principle of bone tissue engineering strategy. Various biologically active substances, such as bone morphogenetic protein (BMP) have been used to accelerate graft healing. However, it is not certain which is more suitable carrier biomaterial for BMP in ACL reconstruction. In this study, we used three different biomaterials with different physical characteristics combined with BMP to search for materials which are more suitable for ACL reconstruction and more effective in inducing new bone formation through histological, imaging and biomechanical studies. To our knowledge, there are few studies to compare the effect of accelerating tendon-to-bone healing after ACL reconstruction between injectable materials and solid materials combined with BMP. On the other hand, we explored the possibility of solid materials with three-dimensional fully interconnected porous structure being used in ACL reconstruction and discussed its effect on tendon-to-bone healing. Four main experiments were involved in this research as follows,1,An in vitro and in vivo analysis of the composites of three different biomaterials combined with BMPFor improving BMP efficacy, carriers are of paramount importance for retention of BMP at orthopaedic treatment sites. In order to investigate the different carriers with related difference combined BMP, injectable fibrin glue (FG), injectable calcium phosphate cement (CPC) and bovine cancellous bone (bCB) separately combined with BMP were examined for their in vitro characteristic features and in vivo ability to form new bone. The difference in initial setting time and final setting time between FG composite group and CPC composite group was not statistically significant compared with single FG and CPC groups. SEM analysis showed: single FG shows homogeneously smooth surface, FG composite shows roughness and irregularity, there are micro-pores among BMP powder; single CPC shows irregularity and/or roughness surface, crystals, granule crystals and some small rod-like crystals with micro-pores about 2-20μm, CPC composite shows it still consists the crystals like single CPC and BMP distributed in the micro-pores about 5-50μm; bovine cancellous bone shows a regular porous structure, with pores 300–500μm in diameter and walls 50–80μm thick between the pores, RBX shows a network-like appearance of BMPs fraction precipitated in the pores of the cancellous graft. The biomechanics study results demonstrsted that the values of maximum compression force of three different composites were slightly lower than those of single biomaterials, but there was no statistically significant difference between them. The three different composites were implanted into the thigh muscle pouches of mice. Bone formation was evaluated at 2 weeks. The ALP activity values of three different composites were all higher than those of single BMP group and the ALP activity in RBX group was the highest. Histological analysis also demonstrated that the effect of new bone formation among composite groups were all enhanced compared with single BMP group. The results suggest that the effect of BMP inducing new bone formation is not affected by the physical properties or biological behaviors of the three different biomaterials combined with BMP and the biomaterials themselves also are not affected by BMP.2,A comparative in vitro study of the proliferation and differentiation of bone marrow stem cells on various composite biomaterialsIn vitro studies about the growth and differentiation behaviors of stem cells onto biomaterials is a basic knowledge and a screening method for the development and application of scaffolds in vivo. In this in vitro study bone marrow stem cells (BMSC) were cultured on three different biomaterials used in orthopaedic surgery, respectively. The tested composite biomaterials were fibrin glue combined with BMP (FG composite), calcium phosphate cement combined with BMP (CPC composite) and bovine cancellous bone combined with BMP (RBX). The cell proliferation and cell differentiation were analyzed by MTT test, determination of ALP activity and type I collage expression level. Statistical analysis were performed. BMSC cultivated on RBX showed the highest proliferation rate (p<0.05). The expression level of type I collagen and ALP activity in RBX group were significantly higher than those in control group, FG composite group and CPC composite group. The results showed that RBX with favorable biodegradation and pores could significantly promote the cell differentiation into osteoblast and cell proliferation. Though the BMP in CPC composite was retained longer than that in FG composite group because of slow biodegradation, the release efficiency is yet to be improved for further studies. 3,A comparative in vivo study of two injectable biomaterials combined with BMP for enhancing tendon graft osteointegration in anterior cruciate ligament reconstructionTo biomechanically and histologically compare tendon-bone interface healing between fibrin sealant (FG) and calcium phosphate cement (CPC), both loaded with BMP, the two kinds of composite biomaterials were injected into the bone tunnel in a intra-articular rabbit ACL reconstruction model. The animals were killed at postoperative weeks 2, 6 and 12 for Micro-CT and subsequent histology staining. The remaining rabbits were sacrificed at weeks 6 and 12, and their femur-tendon graft-tibia complexes were harvested for mechanical testing. Micro-CT examination showed that CPC complex and FG complex all enhanced bone growth in the tunnel after ACL surgery. The values of BMD in the CPC composite group were lower than that in the FG composite group at 6 weeks while the values of BMD in the CPC composite group were higher than that in the FG composite group at 12 weeks. But there was no significant difference between the two groups at the 6-and 12-week time points. Histological findings of the interface between the tendon and bone tunnel were different in the two groups. 6 weeks after the operation,the ability of new bone formation between tendon and bone in FG composite group was stronger than that of CPC composite group, then 12 weeks after the operation, it shows little mature bone formation in tendon bone interface in FG composite group, whereas in CPC composite group the ability of new bone formation was gradually raised during the entire experiment. In biomechanical evaluation, at 6 weeks, the ultimate failure load of the CPC graft-tunnel complex appeared significantly greater than that of the FG graft-tunnel complex. At 12 weeks, there was no significant difference in the ultimate failure load between the 2 composites. In conclusion, the efficacy of BMP release in CPC would be improved.4,A comparative in vivo study comparing a three-dimensional porous structure biomaterial with two injected biomaterials both combined with BMP for enhancing tendon graft osteointegration in anterior cruciate ligament reconstructionTo investigate the effect of reconstituted bone xenografts (RBX) which possesses a three-dimensional fully interporous structure and more effective ability to induce bone formation in tendon-to-bone healing by means of imaging, histological and biomechanical studies. RBX was implanted into the treated knee of rabbits which were killed at postoperative weeks 2, 6 and 12 for Micro-CT and subsequent histological analysis. The remaining 16 rabbits were sacrificed at weeks 6 and 12, and their femur-tendon graft-tibia complexes were harvested for mechanical testing. 6 and 12 weeks after operation, the values of BMD in the RBX group were higher than those in the control group (p<0.05); histological findings showed that 2 weeks after the operation, large areas of chondrocyte-like cells that were fairly disorganized were noted between the tendon and bone in the treatment group. 6 weeks after the operation,more abundant bone formation around the tendon was observed and 12 weeks after operation an immature insertion structure was seen in the treatment group. In biomechanical evaluation, the maximum pull-out load of the tendon from the bone tunnel in the treatment group was significantly higher than that in the control group 6 and 12 weeks after the operation. RBX can augment the healing of tendon to bone after ACL reconstruction in a rabbit model.To compare tendon-bone interface healing among three different biomaterials combined with BMP in ACL reconstruction,a comparative study on accelerating tendon-bone interface healing was conducted by means of Micro-CT imaging, histological and biomechanical analysis on the basis of experimentⅠ,ⅡandⅢ. The results showed RBX not only offered the assiting initial fixation, but also enlarged the effect of BMP and accelerated the growth of new bone.The tendon-to-bone healing was stronger than those in FG composite and CPC composite groups. This study provides the possibility for the application of the solid biomaterials with three dimensional fully inter-connected porous structure in ACL reconstruction. .