| Background:The bone defect often result from trauma,infection,tumors,congenital defects,and so on.The purpose of the bone defect reconstruction is to completely regain the normal construction and function as soon as possible.Autogenous bone graft is the golden standard for the treatment of bone defects,as its osteoconductive,osteoinductive and osteogenic activity.But autogenous bone graft has some apparent limitations,such as extra surgical trauma,limited sources and complications due to bone harvested area,such as infection,huge hematoma and sensory disorders.Although extensive source of allogeneic bone,there are obvious shortcomings in immunogenicity and pathogenicity,so it is not ideal material for bone repair.To solve these problems,new bone substitute material(BSM)was designed to repair bone defects.The first study about bone substitute material was reported in 1895.The essential characteristics of ideal bone substitute material is listed as follows:? good biocompatibility;?biodegradability;?osteoconduction;?osteoinduction;?structure similar to normal bone;? easy to use;? good cost performance.Approximately 2.2 million bone graft procedures are performed each year worldwide to repair bone defects in orthopaedics,neuro-surgery and oral and maxillofacial surgery with a yearly estimated costs of $2.5 billion.An increasing number of bone graft materials with completely different origins are commercially available for many applications throughout the human body.They are variable in their composition,their mechanism of action and,therefore,their indications.Synthetic bone substitute material is the focus in biomaterial field,which can be roughly divided into:?ceramics:biological glasses,tricalcium phosphate and hydroxyapatite;?metal:titanium alloy,titanium coating and other metal coating;?polymers:polymethylmethacrylate,polylactides/polyglycolides and copolymers;?cements:calcium phosphate and calcium sulfate cement.To attach a novel material to simulate autogenous bone which could synthesize all the advantages of various materials,some authors developed a variety of composited bone substitute materials that contained inorganic and organic materials.Composited biosynthetic transplants consist of a carrier as an osteoconductive scaffold combined with osteogenic cells(osteoblast or preosteoblast)and/or growth factors which could promote bone formation.Hydroxyapatite and calcium phosphate both have good biocompatibility,but neither has osteoinductive.Because of this cell-based approaches have increasingly become a focus of interest,so composite materials based on the cellular level has become a hot topic,which involve the implantation of:?unfractionated fresh bone marrow;?purified,culture-expanded MSCs;?differentiated osteoblasts;?cells that have been modified genetically to express rhBMP.The addition of osteoinductive agents to osteoconductive materials will mimic the osteogenic ability of autogenous bone without the morbidity associated with harvest surgery and complications.In the future BSM will convert from a simple filling substance to an innovative biomaterial in the sense of a scaffold,which will play an important role in BSM applications.Calcium sulfate due to good biocompatibility and biodegradability,it became the most historic use in clinical bone substitute material.The biodegradability of calcium sulfate is in accordance with local vascular ingrowth and bone formation.Furthermore,calcium sulfate has plasticity so that it could be self-setting into various kinds of shape.Moreover,it has a certain mechanical strength and no immunogenicity,which is suitable for bone defect repair as a bone graft substitute material.However,the weakness of calcium sulfate is that as a BSM,it only has osteoconduction,with no osteoinduction.Calcium sulfate can be used as a simple bone substitute,bone-graft expander,demineralized bone matrix additives and a release system for antibiotic delivery.Used as a bone-graft expander,calcium sulfate is mixed with autogenous bone in a ratio of 50:50 so that the effect is equal to pure autogenous bone.To improve the osteoinduction of calcium sulfate,we introduced strontium in the preparation in order to develop a novel bone substitute material with both osteoconduction and osteoinduction.Nowadays,topical application of strontium in BSM mainly focused on hydroxyapatite,Sr-HA was demonstrated not only that it could activate the Ras/MAPK Runx2 transcription factor and its downstream signaling pathway to promote the osteogenic differentiation of BMSCs to at the molecular level,further to promote bone formation,but also the strontium ions released from Sr-HA can promote osteogenic differentiation of bone precursor cells,and can improve the expression of alkaline phosphatase(ALP)and osteopontin(OPN).Based on the above two points,strontium containing-calcium sulfate(Sr-CaS)has the both advantages in theory.Based on the above ideas and the previous study of our team,the co-precipitation and hydrothermal technique were conducted to prepare the novel BSM,Sr-CaS.The physicochemical properties,mechanical properties and biocompatibility of Sr-CaS were all evaluated,besides,the osteogenic effect was demonstrated by the critical tibia bone defect model in SD rats.All the above provide experimental evidence for clinical application of Sr-CaS.Objective:1 To explore the preparation methods and the properties of novel bone substitute material,Sr-CaS.2 To explore the biocompatibility and safety in vitro and in vivo of Sr-CaS.3 Sr-CaS was implanted in the critical tibia bone defect model(size:15mm2)in SD rats.Methods:1 The preparation and material characterization of Sr-CaS1.1 Calcium sulfate dehydrate was prepared through co-precipitation methodCompounds containing Ca2+and Sr2+ were mixed at a certain molar ratio of Sr/Sr+Ca,aqueous solution was prepared with the pour of deionized water.Then corresponding volumetric H2SO4 was diluted which was added dropwise to the basic suspension liquid while being stirred at 30?.The pH value of the slurry across the whole reaction process was controlled at 8.5 by pH electrode before H2SO4 was finished.The reaction mixture was stirred for 3 hours.After incubation,the slurry was filtered through filter paper and dried at 50? for 4 hours in a stove.Finally,the stoved gypsum was grinded into powder.1.2 a-calcium sulfate dehydrate hemihydrates was prepared through hydrothermal reactionThe elementary product was transferred into fine powder by planetary ball mill and 200 mesh.Then,the semi-finished was heated in Muffle furnace(F46240CM,Thermolyne,USA)at a 15%mass ratio of product/deionized water at 130? for 6 hours.The slurry was poured after a few minutes while hot and filtered subsequently.The product was stoved at 50? for 4 hours and pulverized again.The powder samples were examined by X-ray diffraction(XRD),fourier transform infrared(FTIR)and thermogravimetric differential scanning calorimeter(TG-DSC).Three types of Sr-CaS were made at a molar ratio of Sr/Sr+Ca(1%,5%and 10%).Compressive strength and degradation testing in vitro were conducted to explore the effect of strontium containing ratio.2 Cytocompatibility of Sr-CaSExtracts of 10%Sr-CaS was acquired according to International Standards Organization(ISO)10993-1-2009.The experiment groups were divided into 100%,75%,50%and 25%concentration ratio of extracts,the negative control group was normal saline and the positive control group was 0.64%phenol solution.L929 cell adhesion was observed under inverted phase contrast microscope.The absorbance(A490)was measured with a microplate reader at 490nm wavelength and calculated the relative growth rate(RGR).Finally,cytotoxicity was evaluated.3 Histocompatibility of Sr-CaS3.1 100%extracts of 10%Sr-CaS were selected to conduct hemolysis and delayed-type hypersensitivity tests.3.2 Systemic subacute toxicity testTwenty Kunming mice were randomized into an experiment group and a control group,sex in half.The extract solution was injected in the experiment group at an intraperitoneal dose of 50 ml/Kg abiding by the maximum dosage criteria of ISO 10993-11-2009 while normal saline was injected in the control group.Injections were conducted in both groups each per day during the whole experiment for 2 weeks.The specification of the injection syringe was 2 mL and the accuracy was 0.1 mL.The results of common clinical signs and observations in ISO 10993-11-2009 were regarded as an evaluation.Besides,body weight changes were also recorded at pre-experiment,1 week and 2 weeks post-experiment.Furthermore,clinical pathology,organ weights and histopathology at 2 weeks were also evaluated.4 Implantation in rats4.1 Experiment designA group,empty control group:no material was implanted in bone defectsB group,CaS group:CaS was implanted in bone defectsC group,5%Sr-CaS group:5%Sr-CaS was implanted in bone defectsD group,10%Sr-CaS group:10%Sr-CaS was implanted in bone defects30 male SD rats,60 lateral tibia,were divided into four groups,with 15 scaffolds each group.Rats were sacrificed at postoperative 4 weeks,8weeks and 12 weeks.4.2 The critical tibia bone defect model in SD rats.Twenty eight-week-old male SD rats weighing 200-250g were used to create bone defect models as described previously.The general anesthesia was induced by intraperitoneal injection of Nembutal at 40 mg/kg body weight.The skin over the proximal tibia was incised and periosteum was cleared using a periosteal elevator.A defect 3 mm wide and 5 mm long was created using a micro-burr with a 2.5 mm tip,starting 10 mm below the articular surface in the anteromedial cortex at both tibias.The defects and intramedullary canals were flushed with physiological saline to remove any residual bone and bone marrow.The 60 legs of the 30 rats were randomized into 4 even groups(15 legs each group)to fill the defects with different materials:nothing(empty control),CaS,5%Sr-Cas and 10%Sr-CaS.The defects were filled flush to the anterior cortex manually with paste-form material before allowed to set in situ.After surgery,the skin was carefully sutured and injected with antibiotics(3%Penicillin).All mice recovered well from surgery,and were housed separately in plastic cages for 12 weeks.Food and water were supplied ad libitum.4.3 Evaluations4.3.1 Generally observed and recorded postoperative animal spirit,diet,activity,wound drainage,swelling and so on.4.3.2 X ray digital radiographs were taken at immediate,4,8 and 12 weeks post-operation respectively under anesthesia to examine the bone defects repair.4.3.3 Tibial specimens were taken at 4,8 and 12 weeks postoperatively for general observation.4.3.4 Tibial specimens 12 week postoperatively were taken for micro-computed tomography scan.Consecutive Micro-CT cross images of region of interest were achieved and the relative measurements were calculated by related software.4.3.5 Rats were sacrificed at postoperative 2weeks,4 weeks,8 weeks and 12 weeks.Osteotylus was reserved in the bone defects,and then decalcified specimens was prepared for routine sections.Finally,HE and Masson trichrome staining were conducted.Results:1 XRD and FTIR patterns of Sr-CaS powder are presented in Fig.lA,B.The three strong characteristic peaks of ?-CaSO4 at 14.63°,25.72° and 29.80° were shown from the Sr-CaS powder in the XRD spectra.Furthermore,the peaks of strontium salt were also exhibited at 24.78°.The both patterns of Sr-CaS were very similar to those of CaS.These indicated the strontium distribution in Sr-CaS did not appear to affect the diffraction patterns of CaS.The results of thermal analysis showed that the prepared products exhibited that non-evaporable water content was 6.03%(the theoretical value is 6.04%).An endothermic peak at 146.37?and an exothermic peak at 174.64? demonstrated the products had a characteristic comparable to a-CaS.2 There were significant differences between 1%Sr-CaS group and both 5%Sr-CaS and 10%Sr-CaS groups in the compressive strength(P=0.019,P=0.000).Furthermore,there was significant difference between 5%Sr-CaS group and 10%Sr-CaS group as to the compressive strength(P=0.003).The rising trend were observed in the weightlessness rate curves of three different materials with prolongation of exposure time.Besides that,the weightlessness trendency was more and more clear with the rising of strontium content.3 The rising trend were observed in the absorbance(A490)of experimental groups with prolongation of exposure time.There were no significant differences between negative control group and all experimental groups at three time points in the A490 value(P>0.05).All experimental groups and negative control group did not exhibit cytotoxicity,demonstrated by ? level toxic reaction.Nevertheless,positive control group exhibited cytotoxicity,demonstrated by ? level toxic reaction.Based on above results,extracts of testing material could be defined as no cytotoxicity.4 The hemolysis ratio of experimental group was 4.3%<5%,indicating that Sr-CaS could not lead to hemolysis.The results showed that no sensitization occurred at the two time points after three trigger periods,which means that the testing material did not cause delayed hypersensitivity.There were no significant differences between the two groups in body weight,blood routine examination,blood chemistry,and the weight of vital organs(P>0.05).HE staining of vital organs showed that normal morphologic cells and no oedematous,congestive and necrotic tissues,indicating no subacute toxicity.In summary,Sr-CaS has good histocompatibility.5 All animals reached their sacrifice time without any adverse events and especially no effusion was encountered.The tracked X-ray radiographs of two typical models are listed.The CaS and Sr-CaS were radiopaque so that the resorption of the bone substitutes was visible.At 4 weeks postoperatively,all implants showed resorption to some extent because the images became blurred at the filling area and the CaS was hard to note.The bone absorption at the edge was seen in the empty group.Subsequently,most of the substitutes degraded and were hard to observe 8 weeks after operation.Besides,repair started in the empty cavity as well.At 12 weeks postoperatively,no CaS or Sr-CaS was noted radiographically and the cortex closed in both groups.In the empty group,some bone repair was observed but the cortex was thin by scrutiny.The radiography obviously showed remarkably slower resorption rates and more compact bone repair in the two Sr-CaS groups than in the CaS group.The morphology of the newly formed bone was reconstructed using micro-CT.The results were almost inconsistent with those the X-ray images.The micro-CT cross sections of the defect area showed more obvious new bone formation in the CaS and Sr-CaS groups than in the empty group.Besides,many cavities scattered over the area in the empty group.Compared with the empty group,the CaS group exhibited better new bone formation but incomplete cortex and voids beneath the cortex.By comparison with the CaS group,the Sr-CaS groups resulted in better new cortex formation.In all groups,sporadic trabecular bone was observed in the marrow canal.The quantity of the newly formed bone in the defect sites was calculated by morphometrical analysis.Evidently,the empty group exhibited the lowest levels of both BMDs and BV/TV of the defects when compared to all other groups(P=0.000).The data concerning BMDs revealed that Sr-CaS groups resulted in significantly more BMDs of the defect compared to the CaS group(n=10,5%Sr-CaS vs CaS,P=0.001,10%Sr-CaS vs CaS,P=0.000).However,there was no difference between the two Sr-CaS groups(P=0.055).The ?CT data on BV/TV showed that the same tendency like BMDs.The percentage in 10%Sr-CaS group was significantly higher than that in CaS group(n=10,P=0.000),but there was no difference compared with 5%Sr-CaS group(P=0.099).There was significant difference between CaS group and 5%Sr-CaS group(P=0.001)in BV/TV.Conclusions:1 With the rising of strontium content,the mechanical strength of novel bone substitute material,strontium-containing a-calcium sulfate hemihydrate,declined significantly.The same tendency was observed in the weightloss rate.2 The novel bone substitute material,strontium-containing a-calcium sulfate hemihydrate is non-cytotoxic biomaterial with good cytocompatibility.3 The novel bone substitute material,strontium-containing a-calcium sulfate hemihydrate showed no hematologic toxicity,no systemic toxicity.Besides,it did not cause skin sensibility,with good biological safety.So it was in line with the basic conditions as a bone substitute material.4 Good bone repair performance of novel bone substitute material,strontium-containing a-calcium sulfate hemihydrate were demonstrated through the critical tibia bone defect model in SD rats. |
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