Investigation Of Platelet-rich Plasma/calcium Phosphate Cement Composite For Vertebral Bone Defect Repair In Goat

ObjectiveEvaluating the injectability and bioactivity of autologous platelet-rich plasma?PRP?and a self-setting calcium phosphate bone cement?CPC?composite material.Degradation of PRP/CPC and new bone formation in vivo,as well as the mechanical properties of the augmented vertebraes were observed by a novel goat lumbar vertebral critical-size bone defect model,then to evaluate whether if combined with PRP,CPC is more suitable for the repair of vertebral bone defect,the one of the load-bearing bone defects.Methods1.Establishment of a novel goat all lumbar vertebral critical size bone defect.18healthy adult black goats were selected for sex without limitation.Ketamine hydrochloride+propofol+pentobarbital sodium was used for anesthesia.After the anesthetic was successful,retroperitoneal approach was adopted to reach the lateral side of the vertebral body.A size of 6mm×10mm cylindrical bone defect was created in each of the lumbar vertebrae?L1-L6?.Every two vertebral voids were randomly filled with PRP/CPC,CPC or not filled with any materials according to the random number table.Therefore,the experiment was divided into three groups:the experimental group?PRP/CPC?,the control group?CPC?and the empty group.The changes of vital signs and blood oxygen saturation of experimental animals in each stage of anesthesia were observed.The size of bone defect was assessed 6 months after surgery through imaging examination.2.Preparation of PRP and the biological activity and injectability of PRP/CPC.5ml of jugular vein blood was drawn by a syringe containing 1 ml of compound sodium citrate,and made into PRP using a modified Landeasberg method.Platelet counts and growth factors levels were measured.PRP gel was got after be activated by CaCl₂ and thrombin.Combining CPC with 10wt%PRP and observing the setting time,injectable rate and anti-collapsibility of the compound.3.Material degradation and osteogenic evaluations.Vertebrae were harvested at 1,3and 6 months after surgery,the specimens were histologically observed by the decalcified tissue section HE staining and the undecalcified tissue section Van Gison staining.Calculating the material degradation rate of PRP/CPC and CPC by the unstained slices.4.Biomechanical study of vertebral body.At 1,3,and 6 months after operation,CT examination was performed to observe the growth and healing of cortical and cancellous bone in vertebral defects.The compressive strength and stiffness of the vertebral bodies at 1,3,and 6months after surgery among three groups were compared to evaluate the mechanical properties of the enhanced vertebral body.Results1.All animal modeling surgery were successfully completed;the excellent rate of anesthesia was 100%.The blood oxygen saturation,body temperature,respiration and other vital signs were stable during anesthesia stage.After the surgery,the animals did not have obvious struggles and restlessness when awakened.There were no adverse reactions such as bloating and vomiting occurred and all the animals survived for a long time until be sacrificed.No vertebral fractures were detected by CT examination at any time after operation.In the empty group,the bone defect did not heal spontaneously at 6 months after operation,reaching the critical bone defect standard.2.The platelet counts in PRP was more than 5 times in the whole blood?P<0.05?,and the contents of PDGF,TGF-?and VEGF in PRP were significantly higher than that in whole blood?P<0.05?.The initial coagulation time and the final coagulation time of PRP/CPC were longer than that of CPC?P<0.05?.Both groups of cements showed good viscosity during bolus injection.The injectable rate of PRP/CPC is higher than CPC?P<0.05?,and the anti-collapsibility of PRP/CPC also better than CPC.3.The HE staining showed that no inflammation and other immune responses occurred.At 1 month after surgery,only chondrogenesis was observed in the empty group.At 3 and 6months,a small amount of new fibrous bone and collagen bone along the edge of the defect were observed.In the experimental group and control group,a continuous new bone formation was observed within 6 months after surgery.At 6 months after operation in the control group,a"transformation zone"formed between the bone cement and the host bone tissue due to the formation of new bone,and this phenomenon was more obvious in the experimental group.Van Gison staining showed that the bone cement of the experimental group and the control group were absorbed in different degrees from 1 month after the operation,the absorbed space was replaced by new bone tissue.The osteogenic activity mainly occurred on the cement-bone interface.However,the new bone in the experimental group had a tendency to grow inside the cement.This difference is even more obvious at 3,6 months post-surgery.At 6 months,the experimental group had a greater area of bone cement absorption and more new bone formation.The degradation rate of cement in experimental group at 1,3 and 6 months was higher than the control group?P<0.05?.4.CT scan showed that the size of the bone defect in the empty group had no significant change within 6 months.At 6 months,although the cortical bone on the surface of the defect can be seen to grow,but the internal boundary of the defect was still clear,only the margin was rough.In the experimental group and the control group,the high-density shadow of the bone cement in the defect was observed at 1 month,and the filling of the material was good.At 3 and 6 months,the cements in both groups had been absorbed in different degrees,with the density decreased and the bone-cement boundary blurred,this phenomenon was more obvious in the experimental group.In the experimental group,bone defect could not be detected in some vertebraes,only unabsorbed bone cement could be seen,and the cortical part of the bone defect was completely bonyly connected at 6 months.There was no significant difference in stiffness between the CPC and empty group at 1month after surgery?P>0.05?,the strength and stiffness of the vertebral bodies filled with different materials at other times were higher than those in the empty group,and gradually increased with time?P<0.05?.There was no significant change in the empty group at each time point?P<0.05?.ConclusionPRP/CPC has good biocompatibility and does not cause inflammation and immune response in the host after implantation.Meanwhile,PRP/CPC has good injectability,it is not easy to leak due to its good viscosity,and the bone cement coagulation time of the compostie is extended.PRP,which is rich in a variety of growth factors,once combined with CPC for repairing vertebral bone defect,can accelerate the degradation and absorption of the cement and promote new bone formation,especially induce new bones to grow inside the cement,thus improve the mechanical properties of the vertebral body.PRP/CPC composite is more suitable for repairing the load-bearing bone defect,especially vertebral body.