| BackgroundAs a common orthopedic disease, Bone tumor has the character of variety, malignancy diversity and high recurrence rate, which incidence has a tendency of annual increase. Following patients more emphasize the quality of life, the surgery combined chemical therapy become a major therapeutic means. Tumor resection, bone reestablishment and chemical therapy after operation is important process of the combined therapy and possible healing bone tumor well. Systemic chemical therapy has noticeable adverse reaction and can't use the maximum drug dose to kill tumor cell due to the restriction of drug dose and body toleration. However, local chemotherapeutics application can produce many times drug level at local organism than systemic therapy and avoid severe systemic toxic reaction. Nowadays, local chemotherapeutics means mainly include local chemotherapeutics pump insertion and slow-release chemotherapeutics implantation. But former drug carriers had much deficiency such as lacked of stability, limited by carrying dosage, persistence time was short and existed retarder foreign body reaction, and so on, which limited the application in treatment of bone tumor. After bone tumor resection, there are various available bone reconstruction and substitution material, however, few of those material can be use as ideal chemotherapeutics carrier. Therefore, it is urgent to find a kind of ideal bone reconstruction and substitution material for bone tumor therapy. Meanwhile, now lack of a local low-release chemotherapeutics system to treat bone tumor, developing an ideal local chemotherapeutics combined system become an available way for bone tumor therapy.ObjectiveTo develop an ideal local chemotherapeutics combined system for bone tumor therapy. This system not only has enough intensity and bone induction ability so that can restore the bone defect and reconstruct of bone integrity after bone tumor resection, but also can provide high drug level and long-term local chemotherapeutics, reduce systemic toxic reaction. It can resolve the problem of recurrence after tumor resection and bone reconstruction in surgery treatment of bone tumor, especial spinal tumor.Methods:1,Nanometer calcium phosphate powder was obtained from a thermal reaction between calcium carbonate and calcium phosphate dibasic. Nano-ZrO2 powder was obtained from ZrOCl2?8H2O and Y2O3 mixed with ammonia water of 3N levels by means of stirring, sediment, filtering, water, dry and cribration. Nano-zirconia particle diameter and calcium phosphate powder, added sufficient quantum compound SiO2 and Na2O adjunct, after added deionise water and grind 4h, select polyurethane scaffold (interval porosity of 75%,85% and 92%, pore diameter 300um) as mould to obtain nano-zirconia calcium phosphate scaffolds. Nanometer ZrO2 and calcium phosphate powders were observed and measured by transmission electron microscope. The interval porosity of nano-zirconia calcium phosphate scaffolds were measured by Archimedes's law. The aperture sizes of the scaffolds were observed by scanning electron microscope. The compressive strength, anti-tensile and rotation strength of the scaffolds were measured by almighty vitodynamics machine. 2,To make biologic safe test of nano-zirconia calcium phosphate scaffolds in vivo and vitro with polyethylene as a negative control and phenylic alcohol of 6.4% as positive control, include cell toxicity test, systemic acute toxic test, hemolysis test, the cytotoxicity and histocompatibility of the scaffolds were evaluated to offer valuable theoretical evidence for clinical safe application. Osteoblast strain MC-3T3 were inoculated to the nano-zirconia calcium phosphate scaffolds and observed the osteoblast characteristics such as adherence, growth and calcium nodums formation to study the biologic characteristics of the scaffolds.3,MTX-PLGA nanocapsules were obtained from MTX and PLGA with multiple emulsion method. The appearance of nanocapsules were observed by transmission electron microscope, the particle diameter were measured by laser light scattering particle size determination radiometer before and after freeze drying, the physical stability were measured by Zeta electric potential, the carried drug dosage and envelopment rate were measured by UV means, the characteristics of nanocapsules chemotherapeutics released in vitro were measured by simulating circumstances of drug released in vivo then draw the curve of drug released.4,The MG-63 osteosarcoma cells were used for the object of experiment. The MTX-PLGA nanocapsules were added to the 96 shadow mask where the osteosarcoma cells were cultivated, then observed the growth of cells by inverted microscope, the PLGA nanocapsules and MTX were control. The half number inhibition ratio and absorbance with different drug levels such as high, middle, low levels restraining tumor were measured by MTT means.5,The MTX-PLGA nanocapsules combined nano-zirconia calcium phosphate scaffolds were prepared. The muscle bags were prepared in both sides thighs of Newsland rabbits, the scaffolds were implanted and taken out of the muscle bags after two and four weeks separately, then compared two groups by observed the morphous and sliced with HE dyeing whether arise inflammatory reaction and new issue grow around the muscle issues. The iliac bone defect moulds were prepared and the compound scaffolds were implanted. The compound scaffolds were taken out after four and eight weeks separately, bone healing status and appearance of scaffolds were observed by eyes, the osteoplast growth status in compound scaffolds were viewed by HE and chinalizarin dyeing separately, the degradation of the compound scaffolds in vivo were observed by SEM. The MG-63 osteosarcoma cells were asepsis implanted subcutaneouly to nape of 4 weeks age athymic mouse, the cell density was 2×107, Implantations of the compound scaffolds were executed after one week, the weight of athymic mouse and the sizes of tumor were regularly observed. Two weeks after implantations, Tumor samples were obtained and the tumor cells death status were viewed by HE dyeing.Results1,Calcium phosphate powders are granulated, short rod-like crystals, the particle diameters are rang of 80 ~ 120nm and approximately uniform. The ZrO2 powders are granular, tetrahedron and polyhedron, the sizes are approximately uniform, the particle diameter distribution are rang of 40～70nm. The general shape of nano-zirconia/calcium phosphate porous scaffolds are massive, various-sized cylinder and granular, the macroscopic aperture sizes are rang of 200μm-450μm, the average pore rate of the synthetic porous scaffolds are range of 64.5%, 73.6% and 82.4%. The average crushing strength of nano-zirconia calcium phosphate porous scaffolds with different interval respectively are 20.3Mpa, 14.2Mpa, 7.6MPa.2,There weren't significant difference of cells growth and morphology among the cells cultivated in the leaching liquor of nano-zirconia calcium phosphate scaffolds group, the negative control group and the normal group. The cytotoxicity were measured by MTT method, the nano-zirconia calcium phosphate scaffolds group and the negative control group both were grade 0, 6.5% phenol solution group was grade 3-4 after two days. There weren't obvious toxicity representation in the detection of systemic toxic reaction. The hemolytic rate was 2.40% in detection of hemolytic reaction; the hemolytic reaction didn't presented in vitro experiments. In immunization and compatibility experiments, the allergic, inflammatory and rejection reaction didn't appeared by histological observation and surrounding tissue view after implantation. There weren't significant difference of blood T lymphocyte subsets scalar in the statistically results between post-implantation and pre-implantation at different time points. Cultured with osteoblasts in vitro and observed by SEM, the osteoblast adhered to bone scaffold pore wall and secreted calcium particles mineralized granulation.3,The MTX-PLGA nanocapsules colloidal disperse system was milky white, round, uniformity size. There weren't adhesion among the nanocapsules, the particle sizes of the nanocapsules present normal distribution and the average of diameter was 198.7±5.4 nm, the polydispersity index was 0.217. The average drug loading measured was 4.23%±0.77%, the average entrapment rate was 64.1%±4.8%, and the preparation process had a good reproducibility. One day RSD respectively were 1.6%, 0.8% and 2.3%, two days RSD were 0.8%, 0.5% and 4.3%. The precision RSD of samples in one day and tow days both were less than 5% that were in line with the requirements of the determination. The average particle size before freeze-dried was 161.2±6.7nm, the average particle size after freeze-dried and then dispersed was 152.4±40.6nm, by ANOVA analysis, the particle size before and after freeze-dried hadn't significant difference. The Zeta potential before freeze-dried was -41.57±0.87mv, the Zeta potential after freeze-dried and then dispersed was -30.84±5.52mv, by ANOVA analysis, there is significant difference between the Zeta potential before and after freeze-dried. The fitting release kinetic equation in vitro was: ln (1-Y) = - 0.0041t - 0.0648 (r = 0.9845), during the burst release period was only 6.75 %, and then release became more stable, stable drug releasing time started.4,The MTX-PLGA nanocapsules showed a good trait of restraining osteosarcoma cells growth in the chemotherapeutics nanocapsule anti-osteosarcoma detection in vitro, IC50=43.44μg/mL, it demonstrated that the MTX-PLGA nanocapsules had a good ability of restraint osteosarcoma cells. There was a significant difference of tumor control rate between MTX-PLGA nanocapsules and merely MTX when used the same quality of MTX.5,The degradation experiment in vivo showed that the specimens hadn't significant inflammatory reaction, destructure and necrosis, and there were fibrous connective tissue and new vessels grew. The experiment of using bone scaffold to repair bone defects showed that there were a great quantity of osteoblast hyperplasia, a large number of deeply stained granules of calcium salts could be seen by alizarin red staining, partial scaffolds decomposition was observed by the SEM after 16 weeks. The scaffold complex showed a good anti-tumor effect in anti-osteosarcoma experiments in vivo.Conclusions1,This experiment proved that nano-zirconia calcium phosphate scaffolds had good bone induction and bone conduction ability, can significantly improve the fusion rate of bone fusion and mechanical strength after tumor resection of, high porosity scaffolds can carry enough nano-scale chemotherapy drugs. It offered the theoretical basis for nanometer bone scaffolds further clinical application as a new bone graft substitutes and bone growth factor and drug carrier.2,the obtained MTX-PLGA nanocapsules could extend the MTX release time, made a smooth plasma concentration to avoid the peak-valley situation, were able to sustained inhibition of tumor cells, enhance bioavailability and effects of chemotherapy, to avoid chemotherapy drugs injury the important blood vessels and nerve around tumor, so as to achieve sustained drug treatment for the purpose of chemotherapy, it is a promising new chemotherapy drug.3,Nano-zirconia calcium phosphate scaffolds have good biocompatibility, high porosity, high mechanical strength and osteoinductive activity, combined with Nanocapsules which have controlled release of chemotherapeutic drugs, obtained the controlled release chemotherapeutic drugs scaffolds system be able to realize not only bone remodeling but also longer-term local chemotherapy, develop a new way for curing bone tumors.
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