摘要
背景
骨肿瘤作为骨科中的常见疾病,种类多,恶性程度差异大,复发率高,其发病率有逐年增加趋势。随着人们对生活质量要求的提高,以手术治疗为中心结合化疗药物的综合治疗已经成为治疗的主要手段。骨肿瘤切除重建及术后化疗是骨肿瘤综合治疗中十分重要的部分,是有望治愈肿瘤的重要手段。全身化疗副作用多,受到药物剂量和人体耐受性的限制,往往不能达到杀死肿瘤细胞的最大药物剂量,而局部应用化疗药物能在局部产生高于全身化疗数倍甚至十几倍的药物浓度,而且避免严重的全身毒性反应。目前局部化疗方法主要有局部植入化疗泵或者局部植入缓释化疗药物的方法,然而传统的载药缓释体系稳定性较差、载药量有限、持续时间短、存在缓释剂异物反应等缺陷,从而限制了其在骨肿瘤中的应用。骨肿瘤切除后骨重建替代材料繁多,但作为理想的化疗药物载体材料甚少。因此,理想的骨重建替代材料已经成为骨肿瘤治疗十分迫切的需要,同时,目前用于治疗骨肿瘤的局部控释化疗药物系统几乎还是空白,开发理想的骨肿瘤局部化疗药物复合材料能够为骨肿瘤治疗开辟一条十分有效的途径。
目的
研制理想的骨肿瘤局部化疗药物复合材料,该系统既能够实现骨肿瘤切除后修补骨缺损,具有一定的骨诱导能力和强度,重建骨骼完整性,又能提供局部高浓度长时间的化疗作用,减少全身毒性反应,解决骨肿瘤尤其脊柱肿瘤外科中最棘手的肿瘤切除术后复发和重建困难的问题。
方法
1、以碳酸钙和磷酸氢钙为原料,经过热反应,制得纳米磷酸钙粉体。以ZrOCl2?8H2O和Y2O3混合物为原料,与浓度为3N的氨水同时混合搅拌,沉淀、过滤、水洗、干燥、过筛,制得的纳米ZrO2粉体。以纳米氧化锆颗粒和磷酸钙粉体为原料,加入适量含SiO2和Na2O的复合添加剂制成复合粉体,加入去离子水后混磨4小时,分别选用孔隙率75%、85%和92%,孔径为300um的聚氨酯支架为模板,制成纳米氧化锆/磷酸钙骨支架。透射电镜下观察并测量纳米磷酸钙粉体及纳米ZrO2粉体形貌和大小。采用阿基米德法测定纳米氧化锆/磷酸钙骨支架的孔隙率。扫描电镜观察高孔隙率骨支架孔径大小。万能生物力学机测定骨支架的压缩强度、抗拉伸强度和旋转强度。
2、以聚乙烯做阴性参照材料、6.4%苯酚溶液做阳性对照材料,对纳米氧化锆/磷酸钙骨支架进行了体内、外生物安全性试验研究,包括细胞毒性试验、全身急性毒性试验、溶血试验,评价其细胞毒性、组织相容性,为确保该材料临床应用的安全性提供有价值的理论依据。将成骨细胞株MC-3T3细胞接种到纳米氧化锆/磷酸钙骨支架上,通过研究MC-3T3细胞在纳米氧化锆/磷酸钙支架上的粘附、生长繁殖以及形成钙结节等成骨细胞的特性,研究支架材料的生物学特性。
3、以MTX、PLGA为原材料,采用复乳法制备MTX-PLGA纳米囊,透射电镜及激光散射粒度测定仪观察纳米囊形态和粒径检测,测定冻干前后粒径和Zeta电位比较测定纳米囊的物理稳定性;UV法测定化疗药物纳米囊载药量和包封率;通过模拟体内释药环境,拟和释放曲线,测定化疗药物纳米囊体外药物释放特点。
4、以MG-63骨肉瘤细胞作为实验对象,将MTX-PLGA纳米囊加入到培养的骨肉瘤细胞96孔板中,倒置显微镜下观察细胞的生长情况。分别以空白PLGA微球纳米囊和单纯MTX作为对照,MTT法测定MTX-PLGA纳米囊的半数抑制率及高、中、低不同药物浓度抑制肿瘤的吸光度以及与游离MTX对MG-63的抑制率的比较。
5、制备MTX-PLGA纳米囊复合纳米氧化锆/磷酸钙骨支架。制作新西兰大白兔股部肌袋,在双侧肌袋内植入骨支架,分别在术后2、4周取材,大体肉眼及HE染色切片观察两组材料的形态,肌肉组织周围有无明显的炎性反应及有无新生组织生长。制作髂骨骨缺损模型,在骨缺损处植入骨支架复合体,植入后4周、8周取材大体观察材料与周围骨愈合情况,HE染色及茜素红染色观察支架复合体内成骨细胞生长及扫描电镜观察支架体内降解情况。将MG-63骨肉瘤细胞在无菌条件下接种4周龄裸鼠颈背部皮下,细胞浓度为2×107, 1周后行骨支架植入术,定期观察裸鼠体重、肿瘤大小,术后2周取肿瘤标本,HE染色观察肿瘤细胞坏死程度,比较各组肿瘤抑制率。
结果
1、磷酸钙粉体呈颗粒状、短棒状结晶体,颗粒粒径为80~120nm,粒径分布基本均匀。ZrO2粉体呈颗粒状,四面体和多面体样,大小基本均匀,粒径分布为40~70nm。纳米磷酸钙/氧化锆骨支架为多孔状,大体形状有块状、各种大小的圆柱状、圆筒状和颗粒状,宏观孔径大小有200um-450um之间,合成的多孔支架的平均孔隙率有64.5%、73.6%和82.4%不等,各种不同孔隙率的多孔纳米磷酸钙/氧化锆骨支架的平均抗压强度分别为20.3Mpa、14.2Mpa、7.6MPa。
2、纳米氧化锆/磷酸钙骨支架浸提液的细胞生长、形态与阴性对照组和正常培养组无明显差别。MTT法测定纳米氧化锆/磷酸钙骨支架材料组和阴性对照组细胞毒性均为0级,6.5%苯酚液毒性级别在2d时为3-4级。全身毒性反应检测中未见明显毒性表现。溶血反应检测中溶血率为2.40%,体外实验不引起溶血反应。免疫与相容性实验中材料植入后周围组织大体和组织学观察未见明显的过敏、炎性及排斥反应,植入后血液内T淋巴细胞亚群各时间点与植入前血液中数值统计学结果无明显差别。体外与成骨细胞共同培养后扫描电镜观察见成骨细胞黏附于骨支架的孔壁上并有钙结节矿化颗粒分泌。
3、MTX-PLGA纳米囊胶体分散体系外观呈乳白色状态。形态较圆整,大小均匀,纳米囊之间无黏连,粒径分布呈正态分布,平均粒径198.7±5.4 nm,多分散性指数为0.217。测得平均载药量为4.23%±0.77%,平均包封率为64.1%±4.8%,制备工艺的重复性良好。日内RSD分别为1.6%、0.8%、2.3%;日间RSD分别为0.8%、0.5%、4.3%。样品日内、日间精密度RSD均﹤5%,符合测定要求。冻干前平均粒径161.2±6.7nm,冻干后再分散平均粒径为152.4±40.6nm,经ANOVA分析,冻干前后粒径无显著性差异。冻干前Zeta电位为-41.57±0.87mv,冻干后再分散Zeta电位为-30.84±5.52 mv,经ANOVA分析,冻干前后Zeta电位有显著性差异。体外释放的拟合释药动力学方程为:ln(1-Y) =-0.0041t– 0.0648 (r=0.9845),突释期内释放度仅为6.75%,以后释放较平稳,进入稳定药物释放时间。
4、化疗药物纳米囊体外抗骨肉瘤细胞检测中MTX-PLGA纳米囊具有良好的抑制骨肉瘤细胞生长的特性,IC50=43.44μg/mL,显示MTX-PLGA纳米囊具有良好的抑制骨肉瘤细胞的能力。载有相同MTX质量的MTX-PLGA纳米囊与游离MTX在肿瘤抑制率上有显著的差异性。
5、体内降解实验结果显示标本未见明显炎性反应及变性坏死,并有纤维结缔组织及新生血管生长。骨支架修复骨缺损的实验结果显示支架内有大量成骨细胞增生,茜素红染色可见大量深染的钙盐颗粒,16周后电镜扫描可见支架部分分解。体内抗骨肉瘤实验中骨支架复合体具有良好的抗骨肿瘤效果,
结论
1、本实验通过纳米技术处理后的纳米氧化锆/磷酸钙支架具有良好的骨诱导和骨传导能力,可以明显提高肿瘤切除术后骨融合术的融合率和力学强度,高孔隙率能够携带足量的纳米级化疗药物,为纳米骨支架作为一种新的骨移植替代物及骨生长因子载体及药物载体进一步在临床推广应用提供理论基础。
2、制备的MTX-PLGA纳米囊能够延长MTX释放时间,使血药浓度平稳,避免峰谷现象,能够对肿瘤细胞持续抑制作用,提高生物利用度及其化疗疗效,避免化疗药物对肿瘤周围重要血管、神经的损伤,从而达到药物持续化疗的治疗目的,是具有应用前景的新型化疗药物。
3、把具有良好生物相容性、高孔隙率、高力学强度和有骨诱导活性的纳米氧化锆/磷酸钙骨支架和具有可控释的化疗药物纳米囊复合构建的化疗药物控释骨支架系统能够既有骨重建功能又有较长期局部化疗作用,为治愈骨肿瘤开辟了一条新的途径。
Background
As 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.
Objective
To 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.
Results
1、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.
Conclusions
1、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.
引文
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