| ObjectiveBone is an important human organ, which is easily damaged by trauma, tumor, infection or age. These always cause large bone defect, which needs an implantation of bone repair material. Bone defect or delayed-union always followed by an osteomyelitis caused by batteries. As we known, osteomyelitis inhibits the union of bone. The interaction of bone defect and osteomyelitis brings a vicious circle, which makes a big challenge to orthopedic doctors. The situation of bone defect repair is urgent. Nowadays there are many kinds of bone repair materials, but each of them has its own limitation. With the rapid development of science and material technology, many new bone repair and implantation biomaterial were found or invented in this field, which shows a new direction of curing bone defect with osteomyelitis.An ideal bone replace biomaterial should have some condition:good biocompatibility, offers a local mild acidic micro-environment, helps the growths of vessels and osteoblasts, an 8-week completed biodegradation period, osteo-conductivity and osteo-inductivity. There are lots of artificial biomaterials in clinical use now, each of that has different advantages or disadvantages. Composite materials can take multiple advantages and overcome disadvantages of each single material, it can simulate natural bones more efficient. This study focused on developing two kinds of novel bone repair/replaced composite biomaterial, Baghdadite/Chitosan hydrogel, and strontium-doped a-calcium sulfate hemihydrate. And then their properties and biocompatibility were tested, in a purpose of offering scientific evidences for the clinical use in bone defect and osteomyelitis field.MethodsTwo main sections were included in the study:Baghdadite/Chitosan hydrogel, and strontium-doped a-calcium sulfate hemihydrate.Section one:The synthesis and physical, chemical and biological properties of Baghdadite/Chitosan hydrogel. The study used the sol-gel method to syntheses Ca3ZrSi2O9 (Baghdadite),used crosslinking method to make Baghdadite/Chitosan hydrogel. And then composite study and physical, chemical and biological properties were tested.1, composite study:use X-Ray Diffraction (XRD) method to test the constituents of Baghdadite/Chitosan hydrogel. Use Fourier transform infrared spectroscopy (FTIR) to test chemical functional groups of the material.2, physical and chemical properties study:(1) PH values and diameters test.7 groups were divided, the blank control group, the 0.05% Baghdadite/Chitosan hydrogel group, the 0.25% Baghdadite/Chitosan hydrogel group, the 0.5% Baghdadite/Chitosan hydrogel, the 1% Baghdadite/Chitosan hydrogel group and the 2.5% Baghdadite/Chitosan hydrogel group. PBS were add to every groups to soak for 7 days. PH values and materials diameters were checked every day and recorded. And then the changes of material volumes were calculated. (2) Gelation time.6 groups were divided according to be with or without crosslinking agent, Genipin:the Chitosan+Genipin group, the 0.5% Baghdadite/Chitosan and Genipin group, the 1% Baghdadite/Chitosan+ Genipin group, the 0.5% Baghdadite and Chitosan group, the 1% Baghdadite/Chitosan group. Add suspension of each group to 96 well plates, and then put the plates into a 37℃ for reaction. Use spectrophotometer to test the 450nm, 492nm and 570nm wavelength colorimeter to calculate the gelation times. (3) Scanning electron microscopy (SEM). Use SEM to observe the micro structure of materials, Chitosan hydrogel,0.5% Baghdadite/Chitosan hydrogel and 1% Baghdadite/Chitosan hydrogel. (4) Ion release test.4 groups were divided, the 1% Baghdadite/Chitosan in Milliq water group, the 1% Baghdadite/Chitosan in PBS group, the Baghdadite powder in Milliq water group and the Baghdadite powder in PBS group. Add relevant hydrogel or Baghdadite powder to 96 wells plates with Milliq water or PBS, and then 500ul extract solution were taken and tested the Ca, Si and Zr ion concentration in the 0,1,3,7,10,13,16,19,22,25,28 days. (5) Swelling ratio and evaporation ratio.1% Baghdadite/Chitosan hydrogel samples and Chitosan samples were dissolved in Milliq water. The samples weights were checked and recorded every day in 7 days. The weights in different time points were compared and divided by their original weights to calculate the swelling ratio. Similarly, sample of 0.5% Baghdadite/Chitosan hydrogel,1% Baghdadite/Chitosan hydrogel and Chitosan hydrogel were exploded to air in room temperature. The weights in different time points were compared and divided by their original weights to calculate the evaporation ratio. (6) Hydrophility test. Milliq water drops were dropped onto the surfaces of 0.5%,1% Baghdadite/Chitosan hydrogel and Chitosan hydrogel samples. Use camera to record and examine the contact angle in order to test the hydrophility.3, mechanical study.2 groups were divided, the 1% Baghdadite/Chitosan hydrogel group and the Chitosan hydrogel group. Each group has 5 samples. Materials were dissolved in milliq water over night for hydrated. Use rheology machine to record their storage modules, loss modules, elastic modules and yield strength.4, Biological study was detected by osteoblast proliferation.3 groups were divided, the 1% Baghdadite/Chitosan hydrogel group, the Chitosan hydrogel group and tissue culture plastic (TCP) group,4 samples in each group. Hydrogel samples were dissolved into PBS solution overnight. UV sterilization were performed before cell seeding. 600,000/ml osteoblast cells were seeded in the wells with the samples, lml for each. The proliferation of osteoblast cells were checked on the 1,3,7 day.Section two:the preparation and biocompatibility of strontium-doped a-calcium sulfate hemihydrate. Use coprecipitation method and hydrothermal reaction technology to make strontium-doped a-calcium sulfate hemihydrate. And the use Chitosan to parcel strontium-doped a-calcium sulfate hemihydrate to develop the new composite material, Chitosan parceled strontium-doped a-calcium sulfate hemihydrate. The new material was prepared and extracted according to the GB/T16886 Medical Apparatus and Instruments Biological Access Standard (Section 12). Biocompatibility experiments were designed and followed the standard. (1) Cytotoxicity test (MTT assay):Preparation of material extracts:Samples of CS-C-CSH(Sr) were immersed in normal saline at a ratio of 0.2g mL-1 for 72 hours at 37℃, and the supernatant was collected to prepare the material extracts. Each specimen was sterilized by 60Co irradiation before use. (2) Genetic toxicity test. Genetic toxicity of the microcapsules was tested using 30 Kunming mice (20-25g, Southern Medical University Experimental Animal Center). Three groups were tested:material extracts of CS-C-CSH(Sr) (experimental group),0.9% saline (negative control group), and cyclophosphamide (positive control group), with each group being tested in 5 male animals and 5 female animals. The samples were administered in two doses by intraperitoneal injection. All animals received injections of the first dose almost simultaneously and the second dose was administered after 24 hours. The same dosage was used for the two doses, which was 20mL/kg body weight for the experimental and negative control groups and 40mL/kg body weight for the positive control group. Injection volume was accurate to O.OlmL. The animals were sacrificed 18 hours after administration of the second dose.Preparation of bone marrow smears:The bilateral femurs of each animal were excised and attached muscles removed. The femoral head and femoral condyles were removed from each femur to expose both ends of the marrow cavity. The bone marrow was removed by suction using a syringe containing lmL FBS and made into a cell suspension inside a centrifuge tube by mixing with a dropper pipette. The cell suspension was centrifuged at 1000 rpm for 10 min, after which the supernatant was removed. One drop of the remaining suspension was smeared onto a microscope slide and dried over an alcohol lamp. The slide was fixed in methanol solution for 10 min and then stained with 10% Giemsa dye solution for 10 min. The slide was washed several times with PBS, rinsed with distilled water and dried.Smear observation:The slides were viewed using a light microscope (BX53F, Olympus, Japan) at low magnification to select areas showing evenly distributed and well-stained cells. The cells were then viewed and counted using the oil immersion lens, with polychromatic erythrocytes (PCE) appearing grayish blue and eosinophilic normochromatic erythrocytes (NCE) appearing orange. Toxicity assessment index: The frequency of micronucleated polychromatic erythrocytes (MPCE) was used as an indicator of genetic toxicity, which was calculated for each animal as the number of MPCE present among 1000 PCE observed. Genetic toxicity was also evaluated by calculating the PCE/NCE ratio from 200 erythrocytes in each animal, where the PCE/NCE ratio for the experimental group should not be lower than 20%of the value for the control groups. L929 mouse fibroblast cells were cultured in standard culture medium containing 10% fetal bovine serum (FBS; GE Healthcare, South Logan, UT, USA),200□mg mL-1 penicillin and 200□mg mL 1 streptomycin. Cell suspension at a concentration of 1 ×□105 mL-1 was seeded in 100μL aliquots into 96-well microtitre plates. The cells were incubated for 24 hours (37℃,5% CO2) until most of the cells have attached. The original culture medium was then discarded and each well was washed twice with phosphate buffered saline (PBS).6 groups of samples were added into the wells at 100μL per well with each group occupying 18 wells:4 experimental groups of 100%,75%,50% and 25% material extract of CS-C-CSH(Sr), positive control group of 0.64% phenol solution, and negative control group of fresh culture medium. MTT assay was performed in 6 wells of each group after culturing for 24,48 and 72 hours. At each time point, the original culture medium was discarded and 20μL of colorimetric reagent MTT (5 mg mL-1) was added to each well. After incubating for 4 hours at 37℃,150μL dimethyl sulfoxide (DMSO) was added to each well and the plate was shaken for 10 min. The absorbance of each well was measured at 490nm using a plate reader (Biotek, USA) and measurements were repeated 3 times. The relative growth rate of cells (RGR) was calculated as RGR= A/A0×100%, where A is the absorbance of the experimental group and A0 is the absorbance of the negative control group. Cytotoxicity assessment criteria:Class 0 for RGR≥100%, Class Ⅰ for RGR=75-99%, Class Ⅱ for RGR=50-74%, Class Ⅲ for RGR=25-49%, Class Ⅳ for RGR=1-25% and Class V for RGR=0%. (3) Intramuscular implantation test. The CS-C-CSH(Sr) microcapsules were processed into cylinders of 3mm diameter by 10mm height for intramuscular implantation in 9 SD rats (180-200g). Hair around the spine region of the animals was shaved 24 hours prior to operation. During surgery under general anaesthesia (induced by intraperitoneal injection of 3% sodium pentobartital at 30mg/kg body weight) and aseptic conditions, bilateral intramuscular pockets were created in each animal approximately 1cm from the midline of the spine. The experimental group was implanted into the left side while the right side was a sham control. After surgery, the animals were injected with gentamicin for 3 days to prevent infection. The animals were sacrificed at 1,4 and 12 weeks with 3 animals being sacrificed at each time point. Histological analysis:Tissue samples were harvested from each animal at the bilateral operation sites, embedded in paraffin and cut into 6μm thick sections using a microtome. The sections were stained with hematoxylin and eosin (H&E) and mounted to slides for observation using a light microscope. (4) Statistical analysis. Data for all experiments were obtained from at least 3 independent samples unless otherwise specified. All data were expressed as mean±standard deviation and analyzed using SPSS 13.0 statistical analysis software. ANOVA was used for comparisons between multiple groups. Differences were considered as significant for p<0.05.ResultsSection One:(1) Constituent research by XRD and Chemical functional groups analysis by FTIR confirm the successful production of Baghdadite/CS hydrogel. (2) 0.5% and 1% Baghdadite/CS hydrogel keep the PH value around 7, which is more stable than other samples of different concentration or pure Chitosan hydrogel. Moreover, shrinkage is less while the concentration of Baghdadite increase, which keeps a stability for the material. So we chose the 1% Baghdadite/CS hydrogel as the right concentration for following experiments. (3) Gelation time were confirm by colorimeter.0.5% Baghdadite/CS hydrogel was 240～260 minutes, and 1% Baghdadite/CS hydrogel was 245～260 minutes. (4)Observed by SEM, materials are rougher while Baghdadite concentration increased. Baghdadite particles are equably distributed inside the hydrogel. (5) Ion releasing test. Ca2+ of Baghdadite/CS hydrogel both in milliq water or PBS released faster than that in Baghdadite powder; But a reverse result was found for Si ion. (6) 1% Baghdadite/CS hydrogel swelled unobvious but dehydrated gradually for the PH reason. The evaporation rates in 15 minutes are:Baghdadite/CS hydrogel>0.5% Baghdadite/CS hydrogel>1% Baghdadite/CS hydrogel. This means 1% Baghdadite/CS hydrogel has a more stable swelling and evaporation ratio. (7) Hydrophily test demonstrated that 1% Baghdadite/CS hydrogel has a good hydrophily. Baghdadite has not change the material’s hydrophilic property. (8) Rheology and compression test showed that 1% Baghdadite/CS hydrogel has lower storage modulus, loss modulus, elastic modulus and yield strength. (9) Biological activity test by osteoblast proliferation experiment showed that more osteoblast cells are found in the 1%Baghdadite/CS hydrogel than in the pure Chitosan hydrogel in the seventh day.Section Two:1. Physical properties of the samples. The XRD patterns of CS-C-CSH(Sr) and CSH(Sr) showed characteristic peaks at 2θ=14.75,25.71,29.76 and 31.91 which corresponded to the (110), (310), (220) and (-114) crystal planes of a-calcium sulfate hemihydrate crystals. The peak intensity of CS-C-CSH(Sr) was weaker than that of CSH(Sr). XRD analysis indicated that the crystal structure of CSH(Sr) was preserved after formation of the CS-C-CSH(Sr) microcapsules. SEM examination showed that the CSH(Sr) crystals were rod-like in appearance with relatively uniform length averaging 50μm. In comparison, the CS-C-CSH(Sr) microcapsules were more spherical-shaped and averaged 60μm in diameter, also with a relatively uniform size distribution.2. Degradation and ion release test. The release of strontium ions from CSH(Sr) over 12 weeks followed a logarithmic relation, with rapid release of over 50% of the strontium ions into solution during the first week and progressively more limited amounts being released during the remaining 11 weeks. In contrast, the release of strontium ions from CS-C-CSH(Sr) over 12 weeks followed a linear relation, with the release rate remaining relatively constant over the entire 12 week period. No strontium was found in a-calcium sulfate hemihydrate (CSH) over the 12 weeks except error.3. Antibacterial test.The antibacterial properties of chitosan (CS), CS-C-CSH(Sr) and CSH(Sr) were demonstrated by comparing the amount of E. coli resulting from culture on the surface of each of the materials compared to the negative control (culture medium). CS exhibited the best antibacterial properties as shown by the complete absence of E. coli colonies in the agarose medium, indicating that chitosan could effectively inhibit bacterial growth. In contrast, CSH(Sr) had no effect in inhibiting bacterial growth as shown by the large amount of E. coli colonies, which had similar appearance to the negative control. The CS-C-CSH(Sr) exhibited reasonably good inhibition of bacterial growth as only a few isolated colonies were visible in the agarose medium, indicating that the beneficial antibacterial properties of chitosan were preserved in the CS-C-CSH(Sr) microcapsules.4. Evaluation of biocompatibility in vitro. After culturing for 72 hours, cells in the material extract groups and negative control group exhibited fibroblast-like morphology with good spreading and growth, in contrast to the positive control group where the cells appeared to have diminished volume, rounded morphology and lack of spreading and multiplication. At each time point, there were no significant differences in cell proliferation between any of the material extract groups and the negative control group, while the positive control group showed significantly lower cell proliferation compared to all other groups. The overall cytotoxicity at 72 hours was Class 0 for the material extract groups and negative control group, and Class III for the positive control group. The results indicated that material extracts of CS-C-CSH(Sr) had no cytotoxic effects and did not affect the normal proliferation of L-929 fibroblast cells.5. Evaluation of in vivo biocompatibility.(1)Genetic toxicity test.All animals survived until the time of sacrifice after both doses of the tested groups were administered. Genetic toxicity was evaluated by determining the frequency of MPCE and PCE/NCE ratio for the tested groups. In both male and female animals, the material extract group showed no significant differences in the frequency of MPCE when compared to the negative control group, and there was also no evidence of dose-dependent changes. The positive control group, consisting of cyclophosphamide which is known to cause chromosomal aberrations in mice, showed significantly higher frequency of MPCE compared to the other two groups. In both male and female animals, the PCE/NCE ratio of the material extract group showed no significant changes when compared to the control groups, and was also not lower than 20% of the values for the control groups. The results indicated that material extracts of CS-C-CSH(Sr) did not induce in vivo genetic toxicity in male and female mouse bone marrow. (2) Intramuscular implantation test.All animals survived until the time of sacrifice with no adverse events. The animals exhibited normal movement and feeding behaviour after operation, with no redness, effusion or implant extrusion observed at the wound site. Capsule formation was not noted at 1 week after operation but became visible at 4 and 12 weeks. Histological examination showed that at 1 week after operation, there were lymphocytic infiltration and the presence of macrophages around the implant but no visible fibrous capsule formation. At 4 weeks after operation, some lymphocytes and multinucleated giant cells were found surrounding the implant, with evidence of fibroblast proliferation and fibrous capsule formation. At 12 weeks after operation, few lymphocytes were visible around the implant and fibrous capsule formation was complete. The sham controls showed normal tissue structures at each time point (data not shown). The results indicated that CS-C-CSH(Sr) was well tolerated during intramuscular implantation in a rat model which confirmed its in vivo biocompatibility.Conclusions1, This study successful synthesis of bagadadite-chitosan hydrogel, which has good physical and chemical properties. And its biocompatibility needs further validation.2. Successful preparation of strontium-a-calcium sulfate chitosan complexes microsphere,with good biological compatibility, and in line with the national standards.So the composite material can be further validation in animal models in the therapy of bone defect. |
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