The Biocompatibility Of A Novel Strontium-containing Calcium Sulfate

The treatment of bone defects for open fractures, benign or malignant tumors and compression fractures of tibial plateau is the present major challenge for orthopedists. Bone grafting is a common procedure in orthopaedic surgery. Autograft bone is the gold standard graft materal. It is osteoinductive,osteoconductive, and osteogenic. However, it is not without morbidity, including infection, pain, large hematomas, sensory loss, and unsightly scars. The ideal bone graft substitute would be osteoconductive, osteoinductive, osteoconductive, and osteogenic and have the same or better biological potential compared with autogenous bone graft. Calcium sulfate is one of the common use bone graft substitutes. Currently, calcium sulfate is used for a wide variety of clinical problems.These include:1. treatment of nonunion; 2. filling contained bone defects after trauma, tumor, and vertebral body procedures; 3. antibiotic and antineoplastic delivery and so on. It is osteoinductive, osteoconductive, and osteogenic, however it lacks any intrinsic osteoinductive or osteogenic capacity. It is a hot topic that make it possess all of the properties above. It has been demonstrated that strontium has a benefit effect on bone. In vitro studies have showed that strontium enhances the replication of preosteoblastic cells, and stimulates bone formation in cell. The preparation of strontium-containing calcium sulfate has long been reported, but previous studies of strontium-containing compounds mixed with calcium sulfate in the physical, which can not guarantee the absolute uniformity at the micro level. In order to achieve this goal, hydrothermal reaction was used to develop a novel strontium-containing. Accroding to GB/T 16886.1-2011, delayed type hypersensitivity test, cytotoxicity test, intracutaneous stimulation test, acute systemic toxicity test, subacute systemic toxicity test, In vivo implantation test and red blood cells micronucleus test were used to study the biocompatibility of the materal.Chapter 1:The preparation of a novel strontium-containing calcium sulfatePurpose:To study the preparation of a novel strontium-containing calcium sulfate.Materals and methods:(1) Sr-CaSO4-2H2O powder was prepared by a coprecipitation method according to the following chemical formulas:Ca(OH)2+H2SO4= CaSO4↓+2H2O Sr(OH)2+H2SO4= SrSO4↓+2H2OTheoretically, the mole ratio of (Ca+Sr)/S is 1:1 and the mole ratio of Sr/Ca is 1:9. Firstly,0.05 mol Sr(OH)2·8H2O and 0.45 mol Ca(OH)2 were poured into 300 ml deionized water. Then 0.5 mol H2SO4 was diluted to 50 ml which was added dropwise to the basic suspension liquid while being stirred at 30℃. The reaction mixture was stirred for 3 hours(2)The elementary product was transferred into fine powder by planetary ball mill and 200 mesh. Then, the semi-finished was heated in Muffle furnace for 6 hours. The slurry was poured after a few minutes while hot and filtered subsequently. Then the material was analyzed with X-ray diffraction (XRD), Fourier transform infrared (FTIR) and thermogravimetric differential scanning calorimeter (TG-DSC) patterns.Results:The three strong characteristic peaks of α-CaSO4 at 14.63°,25.72° and 29.80° were shown from the Sr-CaS powder in the XRD spectra. The peaks of the material were also exhibited at 24.78°. The patterns of Sr-CaS were very similar to those of CaS in the FTIR patten. TG-DSC shown that the non-evaporable water content was 6.03% in the material. The in vitro cytotoxicity test:cytotoxicity of the cement was Class 1 in the test groups, indicating no cytotoxicity. Hemolysis test:haemolysis rate of the extract liquid was 4.3%, indicating no haemolysis. In vivo implantation test:a kind of steady state was observed between the material and tissue.Conclusion:We proved that the material was strontium-containing calcium sulfate.Chapter 2:Intracutaneous stimulation testPurpose:To evaluate whether the material has potential acute toxicity stimulation reactions.Materials and methods:In each rabbit five points on one side of the spine by injecting 0.2 mL polar solvent extracts. Also within the same side of the spine by injecting 0.2 mL polar solvent control extracts, in the other side of each rabbit spine by injecting 0.2 mL nonpolar solvent extracts and non-polar solvent control solution.Result:(1) Polar extract group and the control group showed no erythema, edema and ulceration reaction at 24h,48h,72h three time points, and the primary irritation score is 0. (2) Non-polar extract group and the control group both showed erythema, but extract group did not exceed the scope of the solvent control group at three time points. Two groups both show no edema.Conclusion:The intracutaneous stimulation test of the materal is negative.Chapter 3:Delayed type hypersensitivity testPurpose:To evaluate whether the material has the potential of delayed-type hypersensitivity reaction.Materials and methods:This experiment adopt the maximum dose sensitization test, which includes three stages:intradermal indution stage, local induction stage and activation stage.Result:Accroding to Magnusson and Kligman classification, the rating of the experimental group and control group is level 0.Conclusion:The delayed type hypersensitivity test of tthe materal is negative.Chapter 4:In vitro cytotoxicity testPurpose:Extract liquid was added in L-929 cells to evaluate the morphology and cytotoxicity.Materials and methods:L929 cells,were cultured in a standard culture medium containing 10% fetal bovine serum (FBS),200 mg/ml penicillin, and 200 mg/ml streptomycin. Once 1×105/ml cell suspension was achieved, the three 96-well microtitre plates were inoculated with prepared cells,100 uL per well. Then the plates were placed in a CO2 incubator with a humidified atmosphere and incubated for 24 h at 37℃. Next 36 wells on each plate were inoculated with 6 different contents and thus divided into 6 groups:fresh culture medium (negative control), 0.64% phenol solution (positive control),100% extract,75% extract,50% extract and 25% extract,100 uL respectively.Subsequently, the cells were incubated for another 72 h. Tetrazolium bromide (MTT) assay was performed in the 6 wells of each group on the three plates respectively at 24 h,48 h and 72 h. In all the wells the previous medium was exchanged with 20 uL colorimetric reagent MTT (5mg/mL) at each time point. Incubation was terminated after MTT staining for another 4 hours. The microtitre plates were shaken for 10 min after 150 uL DMSO was added into each well. Then, optical density of the medium was measured at 490 nm by a plate reader.Result:(1) The growth rate of the experimental groups at different time:with the time increasing, each experimental group’s A490 showing a tendency to increasing. There were no significant differences regarding the growth rate among the experimental and negative control groups (P>0.05). (2) The overall cytotoxicity of experimental groups and negative groups were Class I, indicating no cytotoxicity according to the criteria. However the positve groups were Class IV, indicating cytotoxicity.Conclusion:The in vitro cytotoxicity test of the materal shows no cytotoxicity.Chapter 5:Acute systemic toxiciry testPurpose:To evaluate whether the material has the potential of acute systemic toxicity.Materials and methods:12 SPF kunming mice were randomly divided into experimental group and control group,and the number of each group is 6. After anerdian abdominal routine disinfection, in the experimental group,extract was injected with the dose of 50 ml/kg body weight;while in the control group, saline was injected with the dose of 50 ml/kg body weight.Result:(1) There were no significant differences regarding the clinical symptom among the experimental and negative control groups. (2) The weight of all the mice are increased, and there is no significance at three time points (P> 0.05) when use statistical analysis (t test).Conclusion:The acute systemic toxicity test of the material shows no potential toxicity in short term.Chapter 6:Subacute systemic toxicity testPurpose:To evaluate whether the material has the potential of subacute systemic toxicity reaction.Materials and methods:Twenty mice (Guangzhou, Guangdong, China) 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 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 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.Result:(1) Weight change:the weight of all the mice are increased, and there is no significance between experimental group and control group (P> 0.05). (2) Clinical observation:the mice were generally normal during the experiment. No edema and erythema were observed on the skin. (3) Clincal pathology examination result:there is no statistical difference between experimental group and control group with regard to RBC, WBC, PLT, HGB, HCT, MCV, AST, ALT, BUN, TP, GLU, CRE, T-CHO and TG. (4) No other pathologic characteristics were observed. there were no significant differences between the experiment and control groups regarding organ weight. No inflammation or necrotic tissue was observed in the histological analysis of the heart, liver, spleen, lung and kidney.Conclusion:The subacute systemic toxicity test of the material shows no systemic toxicity in long stem.Chapter 7:Red blood cells micronucleus testPurpose:To evaluate whether the material has the potential of mutagenicity.Materials and methods:The extract solution was injected in the experiment group at an intraperitoneal dose of 20 ml/Kg. The 0.9% saline solution was injected in the negative group at the same dose. While the cyclophosphamide solution was injected in the positive group at an intraperitoneal dose of 40 ml/Kg. Then the steps were repeated once again after 24h.Result:(1) Clinical observation:no toxicity reaction was fund in both experimental and control group. (2) Polychromatic erythrocytes in the experimental group in the total proportion of red blood cells is not less than 20% in the control group. (3) There is no significance between experimental group and negative group with regard to the rate of micronucleus.Whereas, there is significance between experimental group and positive group with regard to the rate of micronucleus.Conclusion:The red blood cells micronucleus test of the material shows no potential of mutagenicity.Chapter 8:In vivo implantation testPurpose:To evaluate whether the material has the potential of local toxicity in vivo.Materials and methods:The 3% pentobarbital sodium solution was injected in the experiment group at an intraperitoneal dose of 2 ml/Kg. Each rat created a muscle compartment about 1 cm on both sides of the midline of the rat spinal, and each side was implanted with materials (10 mm,3 mm diameter cylinder). Right side was implanted with strontium sulfate, left as blank control group (cut but not implanted). All animals were injected with penicillin for 3 days to prevent infection after operation.Result:(1) No rejection reaction and systemic toxicity reaction were fund, and no animals died during the observation period. (2) The degree of inflammatory cell response is grade IV, cyst formation is Class 0 in 1 week after operation. The degree of inflammatory cell response is grade Ⅱ, cyst formation is Class Ⅱ in 4 week after operation. The degree of inflammatory cell response is grade Ⅰ, cyst formation is Class Ⅰ in 12 week after operation.Conclusion:In vivo implantation test of the material shows no potential of local muscle toxicity in vivo.