Development Of A Injectable Calcium Sulphate/nano-hydroxyapatite Bone Substitute And Preliminary Study Of Its Biological Performance

BackgroundNowadays complex bone defect from many reasons has become a great challenge to Orthopedists. For many complications autogenous and ectogeneous bone graft having, people developed various kinds of artificial bone repairing materials(bone substitute) and which had become a hotspot discipline in traumatology and tissue engineering today. For quite a long time, these bone substitutes should be pre-molded outside the human bodies and then be planted into the defect sites by the operation. Injuries and surgical complications are inevitable. In recent years, some new injectable materials have changed the situation. Under the precise localizaion, surgeon could inject the material into the defect area through only a tiny wound. The injected material may set within a period of time and solids, which in turn stimulates the bone formation or serves as a scaffold for cell growing. These materials themselves are biodegradable and no side effect was left. On some special conditions, these materials can act as a control released device for bone repairing cytokines or drugs.As a classic bone repairing material, hydroxyapatite(HA) featured sound biocompatibility and low immunogenicity. Nano-sized hydroxyapatite(n-HA)is found to have better effect than traditional HA for its crystal size is close to those in human bone mineral. However, HA need much time to be absorbed in body and its shaping activity is unsatisfactory. Calcium sulphate, another bone substitute, is desirable for injection and absorption. In 1990's, a new hemihydrate crystallized Calcium sulphate(CSH) had been proved to have perfect degenerating rate, bone repairing effect and mechanical properties. Some authors had assemble the HA with CSH to form a new injectable artificial bone and yield good outcome. We replaced the HA with n-HA, coupled with CSH, also. To study the key factors in material preparing and the biological performance of the new injectable n-HA/CSH composite, following researches had been conducted:⑴n-HA crystal were prepared under different reactive conditions. Factors influencing or contributing to the injecting, setting, and mechanical properties were measured.⑵Study to evaluate the biological security of n-HA/CSH material.⑶Observation for the changes of n-HA/CSH material soaked in simulated body fluid.⑷Study to evaluate the biocompatibility, absorption rate and bone repairing compatibility of n-HA/CSH material in animals.Methods1. Preparation of n-HA crystal and the injection, hardening and mechanical properties of n-HA/CSH bone substitute: Precursor of n-HA was prepared through wet process, followed by water washing or ethanol washing. Aged under 20℃,30℃,40℃for 24h, then dispersed with DMA or glucose. Ultrasound treated for 3 hr and crystalized in 100℃water bath or 140℃oil bath. Three groups of CSH/n-HA samples (10%wtHA/CSH, 20%wtHA/CSH, 40%wtHA/CSH) were prepared. The injectability of composite material under varies liquid-to-solid ratio (L/S ratio) were tested. The hardening time and compressive strength of new material under different L/S ratio or Calcium sulphate dehydrate(CSD)amount were tested both at 25℃and 37℃, all compared with those of pure CSH.2. Biological Security of CSH/n-HA material: The 20%wtHA+CSH were subjected to acute whole body toxity test, endodermal stimulation test, anaphylactic test, and Ames test, compared with control groups. The samples of n-HA, CSH, 10%wtHA+CSH, 20%wtHA+CSH and 40%wtHA+CSH were subjected to MTT cytotoxity assay, compared with the control groups.3. In vitro study of CSH/n-HA material soaked in simulated body fluid: Sample 1 to 5 (CSD, n-HA and 10%HA+CSD, 20%HA+CSD, 40%HA+CSD, respectively )were soaked into SBF9# solutions. Daily losing mass of all samples, Calcium and Phosphorus element concentration of solutions were measured. Scaning electron microscope observation and XRD analysis were also performed.4. Biocompatibility, absorption rate and bone repairing compatibility of CSH/n-HA material in animals: Pillets of material(HA contained 20%wt)were implanted into dorsal muscle of rabbits and material powder were mixed and injected into drilled bone defect in right femoral condyles, with left dirlled sides as control. Observation for material changes, the general, pathological, TEM manifestation of tissues, and the X-ray and undecalcified pathological manifestation of rabbit distal femurs were conducted periodically followed the material implantation.Results1.The infra-red spectroscopic analysis and PCS showed that the average grain diameter of n-HA crystals prepared by wet process was 65.9nm, distributing within 40-120nm, close to those in human bone mineral. PCS and TEM showed the distribution of n-HA crystals were evener followed the water washing, ultrasound handling and scattering. The HA crystallized under 140℃were stronger than those under 140℃. With water as solidifying media, The injectability of new material was desired when L/S ratio more than 0.50. The hardening time of composite material were longer than those of CSH under whichever L/S ratio. The hardening time of all samples at 37℃were longer than those at 25℃. Whithin certain range, too much of too few CSD amount made the hardening time longer. The larger L/S ratio or CSD, n-HA content, the less compressive strength.2. After intravenous and intra-abdominal injection of soaked solution of material, there were no changes in breath, appetite, body weight of mice and no death. There were only red flecks or mild edema in rabbit skin 72h followed soaked solution endodermal injection and no allergic reaction were found in guinea pig after endodermal injection. MTT cytotoxicity assay indicated that the composite and it's main ingredients have cytotoxicity of 0~1(non-toxic). Ames test showed that the number of reversion mutation increased by bone substitute (40%wtHA) with diffrent doses/plate in any 4 strains with or without S9mix didn't exceed two times of those of negative controls.3.Sample of pure n-HA underwent a rapid weight losing within the first 24h after soaking and then kept stable. The higher content of HA, the more rapid degradation of composite material was observed. A sustaining lower Ca concentration was measured in solutions for HA. Phosphorus content was released from all samples and decreased when soaking time lengthened. XRD indicated that the diffractive waves of HA enhanced in composite material samples, while SEM showed new crystal formation on their surfaces.4.Early inflammative responses were found in muscle tissues, no infection and necrosis was observed. Material dissoluted in a layer-by-layer and cytophagic process. No cytotoxic reaction and cell injury were found in cytophagic process. The complete resorption of material was by 8 weeks in muscle and 12weeks in trabecular bone, with no fibrosis, heterotopic ossification and phymatoid change. New bone formation was observed at 6 weeks followed implantation.Conclusion1.Nano-sized hydroxyapatite crystals had been prepared successfully, and its diameter were close to those in human bone mineral. Appopriate proportion of n-HA, reasonable L/S ratio and CSD amount were key factors to new injectable nano-bone substitute.2.New material of n-HA/CSH does not cause acute whole body toxic reaction, endodermal stimulation, allergic reaction and no MTT cytotoxity. The material does not increase the number of reversion mutation in Ames test.3.Interaction between n-HA and CS may took place within composite material and a dynamic equilibrium of Ca/P releasing-adsorpsion thus occurred on material surfaces. Consequently a new apatite layer formed. The degradation rate of composite could be controlled by regulating it's component proportions.4.The CSH/n-HA bone substitute was bio-responsed well in vivo. It's absoption rate was compatible with bone regeneration and was adjustable. With fair bone repairing abilities, it may be served as a controlled release device for varies drugs.