The Osteoplastic Effect Of Demineralized Bone Matrix Collagen Composite Loading With Collagen-targeting Recombinant Human Bone Morphogenetic Protein-2 On Rabbit Metaphyseal Defect Model

Background:Bone grafting is a vital component in many surgical procedures to facilitate the repair of bone defects or fusions.Repair of bone defects is one of the major therapeutic goals in various clinical fields.Traditionally,autologous bone grafts have been considered as the "gold standard".At present,bone graft materials include natural and artificial synthesis.Natural bone substitutes are composed by autogenous bone,allograft bone,xenograft bone (such as bovine bone),heterologous plastic materials such as bone.Synthetic bone substitutes are mainly composed by polymers,including biologically active ceramics, polylactic acid,polyglycolic acid,etc.Osteogenesis,osteoinduction,and osteoconduction are three essential elements of bone regeneration along with the final bonding between host bone and grafting material which is called osteointegration.In general,some bone substitutes have only the properties of osteoconduction,and some of them can be absorbed and reconstructed.Bone graft materials which were used Commonly does not have the characteristics of osteoinduction and osteogenesis.Many types of bone filling materials have been developed and have played critical roles in bone repair.1.autologous bone graftAutologous bone grafts have the properties of Osteogenesis,osteoinduction, and osteoconduction.So autologous bone grafts have been considered as the "goldstandard".However the major drawbacks of this method are donor site morbidity and donor availabilities,autogenous bone will be absorbed easily,so they can not maintain an adequate bone scaffold for the bone formation.2.Simple bone graft substitutesIn general,some bone substitutes have only the properties of osteoconduction, and some of them can be absorbed and reconstructed.Bone graft materials which were used Commonly does not have the characteristics of osteoinduction and osteogenesis.So the Simple bone graft substitutes were restricted to use widely.It cannot stimulate bone formation in critical sized defects by itself.Therefore, growth factors such as TGF-b,bFGF,and bone morphogenetic proteins(BMPs) are often employed to promote bone formation.Among them,BMP-2 is a potent bone stimulator and it plays key roles in many steps during bone morphogenesis.Among them,BMP-2 is a potent bone stimulator and it plays key roles in many steps during bone morphogenesis。However,in practice,the therapeutic concentrations of BMP-2 are difficult to be maintained at wound sites due to its rapid diffusion by body fluid. The large amount and multiple administrations of BMP-2 may be clinically impractical and expensive as well.In addition,its excessive doses,especially to the normal tissues,may have undesirable systemic side effects.At present,a variety of carriers have been developed to restrict the free diffusion of BMP-2,and they often provide a slow releasing system.3.demineralized bone matrix loading with collagen-targeting bone morphogenetic protein-2 RhBMP-2 has the characteristics of osteoinduction.Our goal here is to construct acollagen-based BMP-2 targeting repair system,which will attach BMP-2 to the scaffolds not only to reduce the loading dose of BMP-2 but also to achieve a homogeneous repair in the whole defect site.In this work,a collagen-binding domain(CBD) of "TKKTLRT" was added to the N-terminal of recombinant human BMP-2 for its binding specially to collagen.This study evaluated bone formation and implant resorption of this bone graft substitutes in a standardized bilateral tibial defect model in New Zealand white rabbits.Objective:1.To set up the rabbit metaphyseal defect model.To approach the rabbit metaphyseal defect model,and it's advantages in the researches to evaluate marrow cavity restoration.2.To study the effect of osteogenesis with demineralized bone matrix loading with collagen-targeting bone morphogenetic protein-2(rhBMP-2 loaded DBM/collagen composite) on the rabbit metaphyseal defect model.Methods:1.To set up the rabbit metaphyseal defect model.Mature White New Zealand rabbits(3.0-3.5 kg) were used.All New Zealand white rabbits are passed ethical approval.Defects were created using a micro burr with a 3mm diameter tip under saline irrigation.Bilateral critical size defects(5 mm wide and 15 mm long) spanning,the metaphyseal and diaphyseal region were created 3 mm below the joint line in the anteromedial cortex of the proximal tibia in 32 skeletally mature(3.5 kg) New Zealand white rabbits.The skin was opened and the periosteum reflected using aperiosteal elevator.Defects were created using a microburr with a 3mm diameter tip under saline irrigation to minimise thermal damage.Creation of the defect included removal of the cancellous bone presentin the metaphyseal region of the proximal tibia.Marker pins(about 1 mm in diameter) were placed at the proximal and distal margins of the defect to identify the central portion for subsequent analysis.The bovine demineralized bone(DBM) were carefully placed in the defects.The periosteum was reflected back over the defect and the skin closed in using 3-0 Dexon.Animals were given post-operative analgesia (Temgesic,1 ml subcutaneously) and returned to their holding cages.The animals were free to mobilize and weight-bear immediately post-operatively as tolerated. During three days after the surgery,20 million units of penicillin was injected intramuscularly to prevent infection.At the 10^th,11^th day after the surgery,the rabbits would have subcutaneous injection of tetracycline.They also had subcutaneous injection of calcein at the 3^rd,4^th day before the executing.The tibiae were harvested at at the designated time points 2,4 and 6 weeks following surgery.2.48 New Zealand white rabbits were randomly divided into A,B,C,D four groups.A group of 12 rabbits was filled with rhBMP2-h loaded DBM/collagen composite.B group of 12 rabbits was filled with free BMP-2 on DBM/collagen composite.C group of 12 rabbits was filled with DBM/collagen composite.D group of 12 rabbits was filled with DBM.The tibiae were harvested at at the designated time points 2,4 and 6 weeks following surgery.At the 10^th,11^th day after the surgery, the rabbits would have subcutaneous injection of tetracycline.They also had subcutaneous injection of calcein at the 3^rd,4^th day before the executing.3.The specimen were dehydration in a graded series of alcohol and embedded in polymethlymethacrylate.The tibiae were cut longitudinally along the anteromedial cortex using the marker pins to identify the central portion of the defect using a diamond wire saw(Leica SP1600).Sections for histology were ground to approximately 25μm and with special staining,sections was observed through Backscatter electron imaging(BSEM),histology,histomorphometry and fluorescence observation.Results.The animals recovered well 2 hours later after the surgery.All animals tolerated the bilateral model without complication and were load bearing on the day after surgery.1.the animal model showed bone graft substitues could support bone formation in the marrow cavity.The restoration of the bone defect and marrow cavity varied with different bone graft substitutes,and the particle of them was observed differential effects too.2.Visual observation2 weeks later,the bone formation on the defect of the A group and B group were better than C group and D group.A and B group could be seen lots of cartilage uplifted on the wound.The periosteum could not be tom easily.These could not be seen at the C and D group.The particles of C group and D group could be seen outside of the wound.6 weeks later,the wound of the A Group and B group were healed completel,yet the wound of the C group and D group had only a partial healing.The particles of D group could be seen outside of the wound too.3.Radiology fluorescence observationThe inside of the model was scaned by the MORITA 3DX multi-image microCT. 2 weeks later,4 groud had nothing different.6 weeks later,the absorption of the particles of the A,B,C group were seen significantly.But the D group showed almost the same situation that was seen by 2 weeks.Trabecular bone could not be distinguished easily from the bone substitutes.Fluorescent area could be seen around the particles of the bone substitutes.But the doublt line could not be seen at all. 3.2 weeks later,the new bone area ratio of the A group was the best among four group(p＜0).the new bone area ratio of the B group was better than it of the C group(p＜0).The D group was the lowest among four groups(p＜0).4 weeks later,The new bone area ratio of the D group also was the lowest among four groups(p＜0).The new bone area ratio of the A group was the best among the four groups(p＜0).The group C was better than it of group B.6 weeks later,the absorption of the bone substitutes particles of the A,B,C group were seen significantly.The group A,B,C was showed the same phenomenon and non-statistically significant.The new bone area of group D was also the largest among them.The DBM provided a three-dimensional matrix to support the new bone to grow.Conclusion:1.The rabbit metaphyseal defect model can be served as a reliable model to evaluate osteogenic properties and their resorption or degradation of bone graft substitues,as well as marrow cavity restoration.2.This collagen-based BMP-2 targeting bone repair system showed a better boneinduction effect in the in vivo experiments.Group A was showed the greatest bone formation than that of the other groups.3.The effect of bone formation of the rhBMP-2 combined with the collagen was better than the effect of the free rhBMP-2.4.The rhBMP-2 loaded DBM/collagen composite benefits the early requirements of the bone formation.5.The absorption and degradation of bone substitutes are affected by the collagen contents of bone substitutes.