Development Of Fast Degradable Citrate-based Bone Scaffold For Spinal Fusion

Part one:Introduce and evaluate an anterior interbody fusion model on rabbitIntroductionSince 1911,Hibbs firstly reported the spinal fusion(Spinal fusion)operation for more than 100 years,this kind of surgery method has become an important method of operation treatment of spinal surgical operation.With the application of advances in basic research and clinical practice,scholars found that spinal fusion operation still exist a series of problems have not been solved.For instance,fusion failure,pseudarthrosis,lack of source of autologous bone and the complications of autogenous bone donor site.In order to further explore the physiological process of spinal fusion,solve related complications caused by spinal fusion.In addition,now,as the development of bone tissue engineering and biological engineering,in order to test the effect and value of a variety of new synthetic bone biomaterials and biological factors,to establish a standard,stable,effective animal spinal fusion model is one of the key problems that scholars need to solve.In the study of spinal fusion,relative to the clinical research of human factors,not identify limitations of many factors,animal research animal can control effectively the same sex,feeding conditions and processing mode of the unity of the advantage,can obtain more accurate experimental data and results.In the aspect of the selection of animal,large animal have the similar bone structure with human,the acquired data is more reliable and practical,are mainly used for the study of biomechanics,new materials and operation apparatus studies,such as sheep,pigs and monkeys,etc.As to small animal,because its low price and convenient raise,we can use a lot of animal to put into a study.As a rusult,we can get large mount of experience data,it has a larger significance than the data getting from the large animal.The small animal usually are used to study the process of spinal fusion and the psychological mechanism,such as rabbits,mice and rats,even the transgenic mice.Rabbit is one of the small animal which has the similar lumbar spine anatomy and structure with human being.Since 1995,Boden had set establishment of New Zealand rabbit posterolateral spinal fusion model.The model has been widely used to test the effect of different bone tissue engineering biological factors and new materials in the process of spinal fusion.The model has the advantages of simple surgical operation,less postoperative complications,sufficient space for the graft filling,convenience to get adequate iliac bone of autogenous bone graft in control,similar fusion rate with human being,ect.However,the biggest disadvantage is the lower fusion rate than interbody fusion,as to the bad vascular conditions on the fusion position.Anotherdisadvantage is that now in clinic the interbody fusion operation has been used universally,the data getting from this model has some limitations.Interbody fusion model is less used by researchers,the main causes are the complex surgical operation and serious complications,such as injury of abdominal organs and infection.What's more is the operation has destroyed the structure of the intervertebral space.Then,because of the lose of stability during the interbodies,the implant are more likely to shift,causing the failure fusion.This model makes up the intertransverse fusion disadvantage,truly simulated clinical operation path and fusion methods to obtain more reliable data.But,now there has not yet been reported method to make a standard uinfied anterior interbody fusion model in rabbit such as the posterolateral fusion model.Objective:In order to provide a standardized model for tissue engineering research,we take advantage of the measurement of lumbar bodies on rabbit To establish a stable and standard anterior interbody fusion model in rabbit.Methods Forty-four healthy adult New Zealand white rabbit(2?2.5kg)were randomly divided into anatomical analysis group(group A n= 10),interbody fusion with fixation group(group B n=12),interbody fusion with no fixation group(group C n= 12)and blank control group(group D n= 10).Animal in group B?C?D enderwent x-ray examination at 4 weeks after operation.At 12 weeks all rabbits were killed,the fusion segments were tested by manual palpation testing.x-ray,micro-ct,biomechanical and histological examinations.Results Because of the fixed screw into the spinal canal,one case with spinal cord injury in group B was paralysis of the lower limbs.The result of manual palpation testing showed the fusion rate of group B is 100%(12/12),while the group C is 75%(9/12).There is no obvious destruction in intervertebral space in group D.results displayed that among 66.7%cases of C group the transplanted bone blocks were with different degree out the intervertebral disc spaces.The bone graft position of group B was superior to group C and the radiographic score of group B is significant higher than group C(p<0.05)as the imaging tests suggest.The histology indicated that at the fusion position between the interbodies we can see the new bone and at the failed fusion position only soft tissue existed.At last the biomechanics of group B showed the maximum load was higher than group C(p<0.05).ConclusionsMaking a lumbar fusion model on rabbit according to the lumbar anatomy is safe and reliable.The fixation can provide the stable space for the bone grafting fusion and prevent graft from extruding the fusion position,also do some help to improve the fusion effect.Part two:Development of fast degradable citrate-based bone scaffold for spinal fusionBackgroundAlong with the advancement of technology and people's daily lifestyle changes,in our country,incidence rate of spinal instability as a result of trauma and severe degenerative,tumor of spine and spinal infection diseases has increased year by year.This kind of disease if not timely intervention,easily lead to injury of spinal cord and nerve root,even lead to paralysis,incontinence and other irreversible consequences.At present,subtotal resection of vertebral body,discectomy and intervertebral bone grafting fusion has become a commonly used surgical technique in the treatment of spinal diseases.The intervertebral bone grafting material selection becomes one of the key factors in the success of operation.Because of autologous bone has good bone induction ability,also avoiding immune rejection,it is considered the gold standard for intervertebral bone grafting material.However,the shortcoming of source of autologous bone deficiency,donor site complications has limitted its wide application,at the same time it promotes the development of various biological bone alternative material.In the field of bone biomaterials,the study of allograft bone or animal bones have been studied in the early stage.This kind of bone have been applied to the human body after antigen processing and sterilization treatment.The nonunion leaded by immune rejection as a result of that the antigen processing is not completely.In addition,it also has the shortcomings of the propagation of diseases carried bone donor itself and poor quality of the bone,etc.With the progress of the study,mechanical metal materials,ceramic materials,hydroxyapatite bone like matrix,and scaffold composite material with various biological factors have been gradually developed and applied.But all kinds of materials have themselves certain deficiencies,such as:low mechanical strength,bad bone induction ability and biocompatibility.In order to improve the biological materials,scholars have been continuly seeking a kind of ideal alternative materials.The studies has found that citric acid content in the bones was significantly higher than that of other organs.Why and what's the role it play?As we know that Citric acid(also known as Citric Acid,citric acid)is a kind of organic acid and a natural preservatives,also used to add sour to food and soft drinks.In biochemistry,it is an important intermediate product of three carboxylic acid cycle,thus plays an important role in almost all biological metabolism.The research proved that the citric acid plays an important role in the process of bone metabolism,citrate complexes is a part of the crystal structure of nano apatite bone.In the human skeleton,the complex covers approximately 1/6 of the apatite crystals,its content is about the 5.5wt%of organic compounds,which is also very important for the physical combination of nano hydroxyapatite bone crystallization.Yang Jian team of research center of biological engineering and material in USA University of Pennsylvania,according to the important role of citric acid in bone formation,has compounded the bone biodegradable material using the citric acid in vitro synthesis crosslinking the hydroxyapatite.The high strength of biodegradable photoluminescent polymers-HA(BPLPs)has a very good repair for large segmental bone defect and Crosslinked urethane-doped polyesters-HA(CUPEs)in rabbit femoral epicondyle defect study and rat cranial defect repair test showed good biocompatibility,bone induction ability and very weak inflammatory reaction.Although the materials aboved have enough biological strength,however show defects in material degradation.To further improve the shortage of citrate hydroxyapatite,the team used N-methyldiethanolamine(MEDA)cross-linked citric acid hydroxyapatite(POC-HA)to prepare the POC-M-click-HA scaffold.This works have reduced the hydroxyapatite mechanical strength and are helpful to the material rapid degradation.In order to further test the biodegradability and the superiority of this material,we conducted a series of studies in vitro and in vivo.Object1?To test fusion effect of the match stick fast degradable hydroxyapatite scaffold composite citric acid based(POC-M-click-HA)material in rabbit anterior intervertebral fusion model;2?To evaluate the biocompatibilityof the material(POC-M-click-HA)with the biological tissue and cells,the ability of osteodegradation,osteogenesis,osteoinduction and osteoconduction.3?to summarize and evaluate the advantages and disadvantages of the new materials by comparing the"gold standard" clinical autologous bone and widely used biomaterials(PLLA-HA).Provide a preliminary scientific basis for the next step in the development of the material and the application in clinical.Methods1.In vitro,six healthy New Zealand rabbits rabbits(4-5W)were extracted for BMSCs cells,getting the purified third generation of rabbit BMSCs cells by adherent culture method,and cultured with POC-HA materials for 3 days.And to observe the compatibility of the POC-M-click-HA materials and mesenchymal stem cells by scanning electron microscope.2.In vivo,54 healthy New Zealand white rabbits(2-2.5kg)were randomly divided into three groups:group A for autologous bone fusion group(n=18),group B for polylactic acid hydroxyapatite composite(PLLA-HA)material fusion group(n=18),group C for the POC-M-click-HA material fusion group(N=18).At 4 weeks,8 weeks,12 weeks after operation,to get the preliminary judgment of gross specimen fusion according to the SUK system evaluation method,further testing of the biological degradation,material compatibility and bone induction using X-ray,micro-CT,biomechanics,histology.ResultsIn vitro,the results show that BMSCs cells were attached to the material surface and pore and grew well.In vivo,the results show that the fusion rate of POC-HA material is better than PLLA-HA material and has obvious difference(P<0.05),POC-M-click-HA scaffolds presented optimal degradation rates that facilitated faster new bone formation and higher spinal fusion rates(11.2±3.7,80±4.5 at week 4 and 8,respectively)than the PLLA-HA control group(9.3±2.4 and 71.1±4.4)(p<0.05)at 8?12 weeks.Although all animals in three groups had successful fusion at 12 weeks by manual palpation testing and radiography,histological staining results suggest there is more new bone area of group C than group B,it also shows that group C has a better degradation ability than group B at three points.Biomechanical testing results suggest maximum load and stiffness between the three groups were statistically significant(p<0.05).The POC-M-click-HA scaffold-fused vertebrates possessed a maximum load and stiffness of 880.8±14.5 N and 843.2±22.4 N/mm,respectively,which were also much higher than those of the PLLA-HA group(maximum:712.±37.5 N,stiffness:622.5±28.4 N/mm,p<0.05).The group C is better than group B and similar to group A.Conclusions1?Fastly degradable POC-M-click-HA materials had no obvious toxicity to bone marrow mesenchymal stem cells(BMSCs)cells and showed good compatibility with cells.2?POC-M-click-HA material has good bone induction ability and promoting intervertebral bone growth ability;3?POC-M-click-HA material has good histocompatibility and dose not show obvious toxicity and immune rejection;4?The rapid degradation properties of POC-M-click-HA provides ample space for new bone growth,while avoiding the foreign body reaction because the material long-standing.5?Biomechanical results showed that the strength and the stiffness of the material induced formation of intervertebral fusion was close to autologous bone fusion.Overall,the results suggest that POC-M-click-HA scaffolds could potentially serve as an ideal bone grafts for spinal fusion applications.