Nerve growth factor-basic fibroblast growth factor poly-lactide co-glycolid sustained-release microspheres and the small gap sleeve bridging technique to repair peripheral nerve injury

We previously prepared nerve growth factor poly-lactide co-glycolid sustained-release microspheres to treat rat sciatic nerve injury using the small gap sleeve technique.Multiple growth factors play a synergistic role in promoting the repair of peripheral nerve injury;as a result,in this study,we added basic fibroblast growth factors to the microspheres to further promote nerve regeneration.First,in an in vitro biomimetic microenvironment,we developed and used a drug screening biomimetic microfluidic chip to screen the optimal combination of nerve growth factor/basic fibroblast growth factor to promote the regeneration of Schwann cells.We found that 22.56 ng/mL nerve growth factor combined with 4.29 ng/mL basic fibroblast growth factor exhibited optimal effects on the proliferation of primary rat Schwann cells.The successfully prepared nerve growth factor-basic fibroblast growth factor-poly-lactide-co-glycolid sustained-release microspheres were used to treat rat sciatic nerve transection injury using the small gap sleeve bridge technique.Compared with epithelium sutures and small gap sleeve bridging alone,the small gap sleeve bridging technique combined with drug-free sustained-release microspheres has a stronger effect on rat sciatic nerve transfection injury repair at the structural and functional level.

Preparation of Sustained-release Silybin Microspheres by Spherical Crystallization Technique

Aim To improve the dissolution rate and bioavailability of silybin. Methods Sustained-release silybin microspheres were prepared by the spherical crystallization technique with soliddispersing and release-retarding polymers. A differential scanning calorimeter and an X-ray diffractometer were used to investigate the dispersion state of silybin in the microspheres. The shape, surface morphology, and internal structure of the microspheres were observed using a scanning electron microscope. Characterization of the microspheres, such as average diameter, size distribution and bulk density of the microspheres was investigated. Results The particle size of the microspheres was determined mainly by the agitation speed. The dissolution rate of silybin from microspheres was enhanced by increasing the amount of the dispersing agents, and sustained by the retarding agents. The release rate of microspheres was controlled by adjusting the combination ratio of the dispersing agents to the retarding agents. The resuits of X-ray diffraction and differential scanning calorimetry analysis indicated that silybin was highly dispersed in the microspheres in amorphous state. The release profiles and content did not change after a three-month accelerated stability test at 40 ℃ and 75% relative humidity. Conclusion Sustained-release silybin microspheres with a solid dispersion structure were prepared successfully in one step by a spherical crystallization technique combined with solid dispersion technique. The preparation process is simple, reproducible and inexpensive. The method is efficient for designing sustained-release microspheres with water-insoluble drugs.

Release performance and sustained-release efficacy of emamectin benzoate-loaded polylactic acid microspheres

High-performance liquid chromatography （HPLC） was employed to determine drug release rates based on emamectin benzoate concentrations in the medium. Release kinetics equations were used to fit the drug release behavior. The effects of particle size and release medium pH on the release rate were also investigated. The indoor toxicity of emamectin benzoate-loaded polylactic acid microspheres on the diamondback moth larva （Plutella xylostella） was studied to explore drug sustained-release performance. In acidic and neutral media, the drug release behavior of the microspheres was in accord with the first-order kinetics equation. Increasing the spray dosage of emamectin benzoate-loaded polylactic acid microspheres initially resulted in an equivalent insecticidal efficacy with the conventional emamectin benzoate microemulsion. However, the drug persistence period was four-fold longer than that observed using the conventional formulation. The developed emamectin benzoate-loaded polylactic acid microspheres showed dramatic sustained-release performance. A treatment threshold of greater than 35 mg mL-1 was established for an efficient accumulated release concentration of emamectin benzoate-loaded microspheres.

In vitro release of 1,3-bis(2-chloroethyl)-1-nitrosourea sustained-release microspheres and the distribution in rat brain tissues

BACKGROUND: The implantation of released chemotherapeutic drugs, which takes biodegradable polymer as vector, into the tumor site can get high concentration and release the drug for a long time, it can directly act on the tumor cells, and reduce the general toxicity. OBJECTIVE: To explore the in vitro and in vivo course of 1,3-bis(2-chloroethyl)-1-nitrosourea (BCNU) sustained-release from BCNU-loaded polylactide (PLA) microspheres (MS) and location in rat brain tissue. DESIGN: A repetitive measurement. SETTING:Central Pharmacy, General Hospital of Chinese People’s Armed Police Forces. MATERIALS: Thirty male SD rats were used. PLA (Mr5000, batch number: KSL8377) was produced by Wako Pure Chemical Inc.,Ltd. (Japan); BCNU (batch number: 021121) by Tianjin Jinyao Amino Acid Co., Ltd.; BCNU-PLA-MS was prepared by the method of solvent evaporation and pressed into tablets (10 mg/tablet). High-performance liquid chromatography (HPLC) Agilent 1100 (USA); LS9800 liquid-scintillation radiometric apparatus (Beckman). Chromatographic conditions: Elite Hypersil ODS2 C18 chromatographic column (5 μm, 4.6 mm×150 mm); Mobile phase: methanol: water (50:50), flow rate was 1.0 mL per minute, wave length of ultraviolet detection was 237 nm, and the inlet amount of samples was 10 μL. METHODS: The experiments were carried out in the experimental animal center of the General Hospital of Chinese Armed Police from May 2004 to July 2005. ① In vitro BCNU-PLA-MS release test: BCNU-PLA-MS was prepared by the method of solvent evaporation, then placed in 0.1 mol/L phosphate buffered solution (PBS, pH 7.4, 37 ℃), part of MS were taken out at 1, 2, 3, 7, 10 and 15 days respectively, and the rest amount of BCNU in MS was determined by HPLC, then the curve of BCNU-PLA-MS release was drawn. ②In vivo BCNU-PLA-MS release and distribution test: The rats were anesthetized, then BCNU-PLA-MS were implanted to the site 1 mm inferior to the cortex of frontal lobe. Five rats were killed postoperatively at 4 hours, 1, 2, 3, 7 and 15 days, the residual MS was removed from the brain tissue. The rest amount of BCNU was determined with HLPC, and the curve of BCNU-PLA-MS release was drawn as compared with the amount of BCNU in the implanted tablets. Besides, brain tissues (1 g) at the implanted side and the contralateral one were obtained respectively, blood sample (0.5 mL) was also collected, 3H-BCNU was counted radioactively in radioactive liquid flash solution. The distributions of BCNU-PLA-MS in normal rat brain tissue and serum were detected. The analysis of variance was applied to compare the intergroup differences of the measurement data. MAIN OUTCOME MEASURES: ① Characteristics of BCNU-PLA-MS release in phosphate buffered solution (PBS) and rat brain tissue; ② Distributions of BCNU-PLA-MS in normal rat brain tissue and serum. RESULTS: ① Release of BCNU-PLA-MS in PBS and rat brain tissue: The BCNU released from BCNU-PLA-MS could be sustained for over 2 weeks both in PBS and brain tissue. In PBS, the released rate of BCNU was over 15% at 24 hours, nearly 50% at 72 hours and over 90% at 15 days. In brain tissue, the released rate was 8% at 4 hours, 16% at 24 hours, 60% at 72 hours, respectively, and BCNU could be sustained released for over 15 days. ② Distributions of BCNU-PLA-MS in normal rat brain tissue and serum: The concentrations of BCNU in the ipsilateral brain tissue were 6 to 70 times higher than those in the contralateral one. The concentrations of BCNU in the ipsilateral brain tissue were obviously higher than those in serum and contralateral brain tissue (F =103.47, P < 0.01). CONCLUSION: BCNU-PLA-MS can increase the drug concentration in targeted brain tissue, decrease that in the non-targeted brain tissue, reduce general toxic and side effects, and have good releasing function.

Effect of allelochemicals sustained-release microspheres on the ingestion, incorporation, and digestion abilities of Daphnia magna Straus

Allelochemicals sustained-release microspheres(ACs-SMs)exhibited great inhibition effect on algae,however,few studies have focused on ACs-SMs toxicity on invertebrate.In this study,the effects of single high-concentration ACs(15 mg/L,SH-ACs),repeated lowconcentration ACs(3×5 mg/L,RL-ACs)and ACs-SMs containing 15 mg/L ACs exposure on the ingestion,incorporation,and digestion of Daphniamagna Straus(DS)were investigated by stable isotope 15N labeling method.Meanwhile,the diversity and abundance of microflora in DS guts were determined by 16S rRNA genes and cloning methods.The results showed that SH-ACs exposure caused 50%and 33.3%death rates for newborn and adult DS,while RL-ACs exposure caused 10%death rate for newborn DS and no obvious effect on the activity of adult DS.And ACs-SMs exposure did not diminish the motility of both newborn and adult DS,indicating the lower acute toxicity of ACs-SMs.Furthermore,SH-ACs inhibited the ingestion(-6.45%),incorporation(-47.1%)and digestion(-53.8%)abilities of DS and reduced the microbial abundance(-27.7%)in DS guts.Compared with SH-ACs,RL-ACs showed relatively low impact on the ingestion(-3.23%),incorporation(-5.89%)and digestion(-23.9%)abilities of DS.Interestingly,ACs-SMs enhanced the ingestion(+9.68%),incorporation(+52.9%)and digestion(+51.3%)abilities of DS and increased the microbial abundance(+10.7%)in DS guts.Overall ACs and ACs-SMs reduced the diversity of microflora in DS guts.In conclusion,ACs-SMs can release ACs sustainably and prolong the sustained release time,which not only effectively reduce the toxicity of ACs,but also had positive effects on DS.

缓释万古霉素三维支架修复兔感染性骨软骨缺损

背景:大量研究证实组织工程支架几乎可完全修复骨软骨缺损,但当骨软骨缺损合并感染时,即使前期经过彻底的清创,单纯骨软骨组织工程支架的修复效果往往不理想。目的:制备盐酸万古霉素缓释微球丝素蛋白/壳聚糖/纳米羟基磷灰石支架,观察其对兔股骨远端感染性骨软骨缺损的修复效果。方法:①采用乳化溶剂挥发法制备盐酸万古霉素缓释微球;将不同质量(7.5,10,12.5 mg)的缓释微球分别与丝素蛋白-壳聚糖-纳米羟基磷灰石溶液混合,利用化学交联法制备盐酸万古霉素缓释微球丝素蛋白/壳聚糖/纳米羟基磷灰石支架,表征支架的孔隙率、吸水膨胀率、热水溶失率及体外药物缓释等。②将45只新西兰大白兔随机分为空白组、对照组、实验组,每组15只,均建立右后肢股骨远端骨软骨缺损并感染模型,空白组不作任何处理,对照组缺损处植入丝素蛋白-壳聚糖-纳米羟基磷灰石支架,实验组缺损处植入盐酸万古霉素缓释微球(10 mg)丝素蛋白/壳聚糖/纳米羟基磷灰石支架。术后1周,检测血液样本C-反应蛋白、白细胞水平;术后4,8,12周取术区组织,分别进行大体观察与病理学观察。结果与结论:①随着缓释微球含量的增加,支架的孔隙率降低,组间比较差异有显著性意义(P<0.05);3组支架的孔径大小、吸水膨胀率、热水溶失率比较差异均无显著性意义(P>0.05);3组支架体外均可持续释放盐酸万古霉素达30 d以上。②实验组兔血液样本C-反应蛋白、白细胞水平均低于空白组、对照组(P<0.05);实验组兔术后各时间点的大体软骨修复情况明显好于空白组、对照组;苏木精-伊红、Masson、阿利新蓝及Ⅱ型胶原免疫组化染色显示,实验组兔术后各时间点的骨软骨修复效果明显优于空白组、对照组。③结果表明,盐酸万古霉素缓释微球丝素蛋白/壳聚糖/纳米羟基磷灰石支架能有效促进开放性骨软骨缺损的修复。

万古霉素缓释微球的制备及其质量评价

目的制备质量稳定、安全有效的载万古霉素缓释微球,评价其质量特征。方法采用复乳溶剂挥发法将聚乳酸(polylactic acid,PLA)和聚乳酸-羟基乙酸共聚物[poly(lactic-co-glycolic acid),PLGA]按3种不同比例混合制备万古霉素微球进行筛选;利用单因素实验,研究不同浓度聚乙烯醇(polyvinyl alcohol,PVA)、内外水相比例对微球粒径、包封率、载药量的影响后并制备出优选的载万古霉素的PLA/PLGA缓释微球;对其体内外抗多重耐药菌耐甲氧西林金黄色葡萄球菌(methicillin-resistant Staphylococcus aureus,MRSA)进行药效学研究。采用单因素方差分析。结果内外水相比和PVA浓度对万古霉素的包封率和载药量影响不大(P>0.05),但微球中PLA和PLGA质量比对万古霉素的包封率和载药量差异有统计学意义(P<0.0001),纯PLA时微球平均粒径[(41.95μm)]、包封率[(28.87±0.32)%]和载药量[(17.80±0.21)%]最大。万古霉素微球对MRSA 252的抗菌活性高于其水溶液(最小抑菌浓度0.391 ng/L比0.781 ng/L)(P<0.0001)。万古霉素微球治疗受损皮肤感染MRSA 252比相同浓度的万古霉素水溶液具有更满意的杀菌活性(P<0.05)。万古霉素微球对感染MRSA 252小鼠的伤口治愈效果比万古霉素水溶液更好。结论通过优选获得的微球处方,制备的PLA/PLGA缓释微球可实现万古霉素药物的局部可控释放,通过调节PLA和PLGA在共混物中的比例提高药物包封率和载药量、定制降解速率、改变药物释放,是有效对抗金黄色葡萄球菌感染的制剂。

静电纺丝复合缓释抗菌微球的制备及性能分析

背景:临床静脉注射抗生素治疗骨髓炎时,由于骨骼血供较差,药物难以在骨髓炎局部病灶处达到有效治疗浓度,治疗效果不佳一直是临床瓶颈。目的:研制一种可局部缓释盐酸万古霉素、同时具备成骨作用的可降解生物材料植入物,在治疗骨髓炎的同时尽可能起到促进局部骨组织修复的作用。方法:将6,12,18 g/L的盐酸万古霉素-海藻酸钠溶液以电喷的方式制成微球,进行真空冷冻干燥处理;制备聚3-羟基丁酸酯4-羟基丁酸酯电纺溶液,电纺同时,向接收装置上加入载盐酸万古霉素微球,使其包裹于电纺丝中,制备了含6,12,18 g/L盐酸万古霉素微球的静电纺丝支架,分别记为P@VancoMs6、P@VancoMs12、P@VancoMs18。(1)体外实验:将MC3T3-E1细胞分别接种于3种支架上,检测细胞黏附、增殖,筛选适宜额盐酸万古霉素质量浓度,用于后续实验。将MC3T3-E1细胞接种于P@VancoMs6、P@VancoMs12上,加入成骨诱导培养基,通过Ⅰ型胶原和骨钙蛋白免疫荧光染色评价支架体外成骨性能。将P@VancoMs6、P@VancoMs12分别与金黄色葡萄球菌共培养,采用抑菌圈实验分析复合物的抗菌性能。(2)将聚3-羟基丁酸酯4-羟基丁酸酯支架与P@VancoMs6、P@VancoMs12分别植入SD大鼠皮下,组织学切片观察支架的生物相容性。结果与结论:(1)体外实验:细胞黏附与增殖实验显示,P@VancoMs6、P@VancoMs12对细胞增殖无影响,可用于后续实验。P@VancoMs6、P@VancoMs12可抑制金黄色葡萄球菌的生长,并且P@VancoMs12的抑制作用更明显。免疫荧光染色显示,P@VancoMs6、P@VancoMs12可促进细胞分泌Ⅰ型胶原和骨钙蛋白。(2)体内实验:皮下植入4 d后,3组植入物均可见炎细胞浸润,但未见坏死灶。(3)结果表明:含6,12 g/L载盐酸万古霉素微球的静电纺丝支架具有良好的抗菌作用与促成骨作用。

双重载药多层海藻酸盐-壳聚糖缓释微球的制备及体外释放

采用滴注法将海藻酸钠与钙离子交联,制成负载血管内皮生长因子（VEGF）的藻酸钙核心球,利用层层自组装技术在核心球的表面依次包覆壳聚糖、海藻酸和壳聚糖,壳聚糖中负载万古霉素（VAN）,形成多药载药缓控体系.采用正交实验考察海藻酸钠浓度、钙离子浓度及壳聚糖浓度对VEGF和VAN的药物包封率和载药量的影响,优化了制备工艺.采用扫描电子显微镜观察多层微球的表面、截面形貌及粒径,采用傅里叶变换红外光谱检测海藻酸盐与壳聚糖的自组装情况,分别采用酶联免疫吸附（ELISA）双抗体夹心法和紫外分光光度法检测VEGF和VAN的包封率、载药量及体外释放情况.结果表明,海藻酸钠最优浓度为0.04g/mL,氯化钙最优浓度为0.15g/mL,壳聚糖最优浓度为0.01g/mL.微球光滑圆整,均质实心,直径900~1100μm,VEGF的包封率达61.31%,VAN的包封率为3.48%.体外释放实验结果表明,VEGF缓释时间为15.5d,并出现2个释放高峰;VAN缓释时间为4.5d,释药情况平稳持续,无明显突释.双重载药多层包覆微球兼具控制感染和促进血管生成两种潜能,有望应用于组织工程骨的基础研究和临床实践.

载万古霉素缓释微球纳米羟基磷灰石/壳聚糖支架联合自体红骨髓可修复慢性骨髓炎兔的骨缺损

背景:前期研究证实,万古霉素缓释微球对兔慢性骨髓炎具有良好的治疗效果,纳米羟基磷灰石/壳聚糖具有良好的生物相容性、降解性及成骨诱导性,自体红骨髓联合纳米羟基磷灰石/壳聚糖支架可用于修复兔桡骨骨缺损。因此,3者联合修复兔慢性骨髓炎骨缺损的效果有待验证。目的:探讨载万古霉素/聚乳酸-羟基乙酸共聚物(polyactic-co-glycolicacid,PLGA)缓释微球的纳米羟基磷灰石/壳聚糖支架,联合自体红骨髓修复兔慢性骨髓炎骨缺损的治疗效果及可行性。方法:采用水-油-水乳相法制备万古霉素/PLGA缓释微球,将缓释微球与纳米羟基磷灰石/壳聚糖混合液充分混匀,采用真空冷冻干造法制备载万古霉素/PLGA缓释微球的纳米羟基磷灰石/壳聚糖支架,对支架进行力学性能测试及万古霉素释放量检测。在20只5月龄新西兰大白兔(解放军第三军医大学大坪医院实验动物中心提供)左侧胫骨髓腔内注射金黄色葡萄球菌,构建慢性骨髓炎骨缺损模型,随机分4组干预,A组仅作清创处理,B组清创后植入纳米羟基磷灰石/壳聚糖支架,C组清创后植入载万古霉素/PLGA缓释微球纳米羟基磷灰石/壳聚糖支架,D组清创后植入载万古霉素/PLGA缓释微球纳米羟基磷灰石/壳聚糖支架及自体红骨髓。治疗3个月后,通过X射线、病理苏木精-伊红染色评价骨缺损修复效果。结果与结论:(1)载万古霉素/PLGA缓释微球的纳米羟基磷灰石/壳聚糖支架,具有良好的生物力学性能及缓慢平稳释放万古霉素的能力;(2)治疗3个月后的X射线显示,A、B组胫骨近端仍见明显的缺损区、骨质破坏及骨膜反应;C组可见新生骨形成,缺损区稍模糊,但仍有缺损存在;D组可见新骨形成,缺损区模糊,密度稍增高,骨膜反应消失;(3)治疗3个月后的病理苏木精-伊红染色显示,A、B组骨小梁紊乱、破坏较多;C、D组骨小梁排列较为整齐,破坏较少,且D组骨小梁粗大,排列有序;(4)结果表明,载万古霉素/PLGA缓释微球的纳米羟基磷灰石/壳聚糖支架联合自体红骨髓修复兔慢性骨髓炎骨缺损效果良好。

万古霉素壳聚糖缓释微球的制备及其体外释药特性和抑菌作用研究

目的:制备万古霉素壳聚糖缓释微球,考察其体外释药特性和抑菌作用。方法:采用乳化交联法制备万古霉素壳聚糖缓释微球,观察微球的粒径分布及形态;应用紫外分光光度法测定微球药物含量,计算载药量及包封率;通过体外释放试验,比较其与盐酸万古霉素24 h内的体外释药情况,考察其7 d内的累积释放度(Q);通过体外抑菌试验观察其作用20 d对金黄色葡萄球菌抑菌圈直径的影响。结果:所制万古霉素壳聚糖缓释微球呈圆球形,表面圆整,无明显凹凸和孔隙,平均粒径为(165.6±19.7)μm,平均载药量为(32.6±3.2)%,平均包封率为(53.8±6.3)%,Q_(7d)为82.7%;其与盐酸万古霉素的Q_(2h)分别为18.6%、91.3%;其作用1 d时的抑菌圈直径>3 cm,随其作用时间的延长,抑菌圈直径逐渐缩小。结论:成功制得万古霉素壳聚糖缓释微球,其体外缓释效果明显,且具有良好的体外抑菌作用。

正交设计优化万古霉素/聚乳酸-羟基乙酸共聚物微球的制备及体外药物释放

背景:尽管现代抗生素和外科技术进步巨大,但在治疗如深部软组织感染或骨感染,特别是耐甲氧西林金黄色葡萄球菌感染时,全身长期应用抗生素治疗容易出现肾毒性、耳毒性和胃肠道等毒副作用,且费用昂贵,疗效不确定。而局部给药系统可以在感染局部实现持续释放高浓度的抗生素,从而更有效的控制和治疗感染,明显降低全身抗生素治疗的毒副作用。将万古霉素用聚乳酸-羟基乙酸共聚物包载成微球后,随着微球在体内逐渐降解、吸收,可在感染处长时间维持有效抑菌浓度,实现局部抗感染作用。目的:用正交设计实验对万古霉素/聚乳酸-羟基乙酸共聚物微球的制备工艺进行优选,制备出粒径均匀的万古霉素聚乳酸-羟基乙酸共聚物缓释微球,同时检测其体外药物释放及抑菌性能。方法:用复乳溶剂挥发法,以微球载药率和包封率为主要考察指标,对不同的聚乳酸-羟基乙酸共聚物溶液浓度、内水相药物浓度、外水相聚乙烯醇浓度、搅拌转速4个工艺条件进行四因素三水平的正交实验。结果与结论:最佳工艺条件为油相9 mL:聚乳酸-羟基乙酸共聚物500 mg、内水相1 mL:万古霉素300 mg、外水相:聚乙烯醇溶液浓度为3%、转速400r/min;其他三因素不变,搅拌速率为400,800,1200r/min时制备出了粒径(232±26)μm、(157±23)μm、(102±37)μm的微球;选用平均粒径为(102±37)μm的微球,测定其载药率为(17.40±1.87)%;包封率为(35.12±3.65)%,体外药物释放显示微球在第1天药物释放率为(17.91±2.41)%,有一定突释,2 d后释放率逐渐降低放缓,12 d时累计释放(58.78±1.54)%,12-24 d平均每天释放1.41%,于24d时累计释放(75.31±1.02)%。说明采用最佳工艺可制备粒径均匀药物释放平稳的万古霉素/聚乳酸-羟基乙酸共聚物缓释微球。

载万古霉素聚乳酸羟基乙酸微球的研制及体外释放研究

目的研制载万古霉素聚乳酸羟基乙酸(PLGA)缓释微球,考察微球的体外释放性能。方法应用复乳-固化法(w/o/w)制备载盐酸万古霉素PLGA微球(HV-PLGA MSs)。利用扫描电镜(SEM)及激光粒度分析仪进行形貌观测,采用紫外分光光度法测定盐酸万古霉素含量,计算载药率和包封率;通过体外药物释放实验考察微球化处理对盐酸万古霉素溶出度的影响,用抑菌实验检测盐酸万古霉素的抗菌能力。结果微球表面光滑、无孔隙,载药率达到(4.40±0.263 5)%,包封率达到(48.51±14.827 3)%,HV-PLGA MSs溶解7 d的累积释放率达到43.36%,有相对比较长的释放周期;在体外抑菌试验中,HV-PLGA MSs释放的万古霉素作用效果明显。结论载盐酸万古霉素PLGA微球性质稳定,释放周期比较长,抑菌效果明显,符合理想的局部抗感染应用要求。

盐酸万古霉素@聚乳酸-羟基乙酸共聚物-壳聚糖-透明质酸复合缓释微球的制备及体外评价

背景:局部抗生素缓释系统可解决全身应用抗生素时引发的全毒性反应与短期局部注射抗生素半衰期短的问题。目的:制备盐酸万古霉素@聚乳酸-羟基乙酸共聚物-壳聚糖-透明质酸[vaneomyeinhydroehlorid@poly(lactic acid glycolic acid)-chitosanhyaluronic acid,VA@PLGA-CS-HA]复合缓释微球,并对其性能进行评价。方法:采用乳液法制备VA@PLGA-CS-HA复合缓释微球与未载药PLGA-CS-HA复合微球,其中载药微球中万古霉素的质量浓度分别为25,50,100 g/L,检测载药微球的载药量、包封率与体外缓释性能。将3种载药微球分别与金黄色葡萄球菌菌液共培养,相应时间点内检测抑菌率。将4种微球浸提液分别与MC3T3-E1细胞和MG-63细胞共培养,培养1,3,7 d后采用CCK-8法检测细胞毒性。结果与结论:(1)含盐酸万古霉素25,50,100 g/L载药微球的包封率分别为(79.70±5.11)%,(86.41±3.91)%,(63.18±1.96)%,载药量分别为(3.98±0.26)%,(8.64±0.39)%,(12.63±0.39)%;50 g/L载药微球的包封率高于100 g/L载药微球(P<0.05),100 g/L载药微球的载药量高于其他两组(P <0.05);(2)3种载药微球在24 h内均无明显的突释,其中50 g/L载药微球不同时间点的药物释放率快于其他两组,100 g/L载药微球不同时间点的药物释放量高于其他两组,并且3组在56 d时释放的药物质量浓度均高于盐酸万古霉素最小抗菌浓度;(3)3种载药微球均能在一定时间内有效杀死金黄色葡萄球菌,在第14-28天期间3种微球的相对菌落率低于3%,说明3种载药微球能持续而有效杀灭金黄色葡萄球菌;(4)含盐酸万古霉素25,50 g/L载药微球对MC3T3-E1细胞和MG-63细胞无明显的细胞毒性,100 g/L载药微球具有一定的细胞毒性;(5)结果表明,VA@PLGA-CS-HA微球具有良好的缓释性能、抗菌能力与生物组织相容性。

缓释抗菌微球复合型组织工程材料水凝胶的制备及性能评价

背景:氧化羟丙基甲基纤维素-透明质酸钠水凝胶体系具有良好的力学性能、生物相容性与降解性,可作为组织工程支架对受损组织起保护支撑作用,也可作为药物载体实现局部缓释作用。目的:制备缓释抗菌微球复合型组织工程材料水凝胶,研究其理化性能、生物学性能、骨诱导性能和抗菌性能。方法:以聚乳酸乙醇酸共聚物、壳聚糖和透明质酸钠为材料,通过乳液法制备出具有多层结构的多孔微球载体,并对酸万古霉素进行负载,制备出抗菌缓释微球;以氧化羟丙基羧甲基纤维素和胺化透明质酸钠为基质制备成可注射水凝胶,并将不同质量浓度的抗菌缓释微球(0.1,0.2,0.3 g/m L)添加到水凝胶中,制备出载抗菌缓释微球的可注射水凝胶,对其理化性能(成胶时间测定、溶胀性能、降解性能)、生物学性能(细胞毒性、细胞溶血性)及成骨性能(碱性磷酸酶活性及诱导成骨基因RUN-x2、骨形态发生蛋白2、骨钙蛋白、Ⅰ型胶原蛋白表达水平)、抗菌性能进行检测及评价。结果与结论:(1)3种载抗菌缓释微球水凝胶具有良好的成胶性能与较小的溶胀比;体外降解扫描电镜结果显示,水凝胶具有稳定的自身降解性能;(2)细胞毒性实验显示,当抗菌缓释微球添加量≤0.2 g/m L时,水凝胶无明显的细胞毒性(7 d内细胞的相对增殖率均大于80%);溶血实验显示,3种载抗菌缓释微球水凝胶的细胞溶血率小于2%,无明显细胞溶血;(3)3种载抗菌缓释微球水凝胶均可提高MG-63细胞中成骨基因RUN-x2、骨形态发生蛋白2、Ⅰ型胶原、骨钙蛋白含量及碱性磷酸酶的生成;(4)3种水凝胶均可持续、长期地抑制金黄色葡萄球菌的生长,并且随着抗菌缓释微球添加量的增加水凝胶的抗菌能力增强;(5)结果表明,制备的可注射载抗菌微球水凝胶具有良好的缓释抗菌及成骨、骨诱导能力,同时具备较好的生物组织相容性、可降解性。

载药组织工程支架的构建及其性能研究

目的：构建含有万古霉素和重组人骨形态发生蛋白2（rhBMP-2）微球的组织工程支架，并研究其性能，为后期慢性骨髓炎的治疗奠定实验基础。方法取壳聚糖溶液、纳米羟基磷灰石适当比例混合，倒入准备好的铸模槽，通过冻干技术与化学交联方法制备成固体材料，并置于含有rhBMP-2/聚乳酸（PLA）和万古霉素/PLA缓释微球溶液中，-80℃冷冻2 h后放入真空冷冻干燥机内干燥12 h，用60Co辐照消毒备用，并检测支架性能及万古霉素和rhBMP-2的释放效率，绘制释放曲线。结果所构建的纳米羟基磷灰石/壳聚糖（n-HA/CS）支架孔径大小为80～270μm，平均146.53μm；rhBMP-2/PLA和万古霉素/PLA缓释微球成团状均匀分布于载药支架的孔隙中；载药支架能够较稳定的释放万古霉素和rhBMP-2，持续时间约18 d，随着时间的延长释放量逐渐减少。结论构建的载药组织工程支架具有良好的生物性能及释放效率，有望成为一种新型、优良的复合骨组织工程支架材料。

载VEGF/VAN多层海藻酸钠-壳聚糖微球的制备及其体外抗感染能力和稳定性研究

目的:制备载有血管内皮生长因子(VEGF)和万古霉素(VAN)的多层海藻酸钠(SA)-壳聚糖(CS)微球,并讨论其体外抗感染能力和稳定性。方法:采用滴注法和层层自组装技术制备多层载药微球;纸片法实验检测微球体外抗感染能力;分别采用ELISA法和紫外分光光度法测定微球中VEGF和VAN的载药量和包封率;微球经过不同条件处理后,通过SEM观察微球表面和截面形态,并分别测定不同条件处理后微球内VEGF和VAN的有效释放量的变化。结果:肉眼观制备出的微球呈类球形;VEGF和VAN的载药量分别为6.63×10-5%和1.39%,包封率分别为72.1%和3.37%;微球溶解后,溶液对金黄色葡萄球菌的抑菌环直径为(12.61±1.01)mm;不同条件处理后,微球直径约为900~1100μm,表面完整,存在少许褶皱,截面呈致密网状结构;不同条件处理后微球内VEGF和VAN的有效释放量均有一定下降。结论:本实验制备的载VEGF/VAN多层微球粒径均匀,对金黄色葡萄球菌具有一定抑菌效果,需低温(-80℃)且避光储存。

载VEGF/万古霉素的多层缓释微球对人胎盘源间充质干细胞增殖与成骨分化的影响

目的:观察载血管内皮生长因子(VEGF)/万古霉素的多层海藻酸盐壳聚糖缓释微球对人胎盘源间充质干细胞(HPMSCs)增殖与成骨分化的影响,为其在组织工程修复骨缺损中的临床应用提供理论基础。方法:根据前期基础制备缓释微球,将从人胎盘组织中分离培养的一部分HPMSCs分为载药微球组(载药微球+HPMSCs)、空载微球组(空载微球+HPMSCs)和无球组(仅HPMSCs)。采用CCK-8试剂盒检测HPMSCs增殖率。取另一部分HPMSCs分为载药微球诱导组(载药微球+HPMSCs+成骨诱导液)、空载微球诱导组(空载微球+HPMSCs+成骨诱导液)、无球诱导组(HPMSCs+成骨诱导液)和非诱导组(HPMSCs+PBS)。孵育21d后分别采用茜素红染色及碱性磷酸酶(ALP)试剂盒检测HPMSCs钙盐沉积与ALP活性。结果:与无球组比较,载药微球组和空载微球组共培养后HPMSCs增殖率均无明显改变(P>0.05)。成骨诱导后茜素红染色,载药微球诱导组钙盐沉积明显多于空载微球诱导组和无球诱导组;载药微球诱导组细胞内ALP活性明显高于空载微球诱导组和无球诱导组(P<0.05),空载微球诱导组细胞ALP活性高于无球诱导组(P<0.05)。结论:载VEGF/万古霉素的多层海藻酸盐壳聚糖缓释微球对HPMSCs增殖活性无明显影响,且能提高HPMSCs的成骨分化能力。

万古霉素缓释微球复合自体浓缩红骨髓对治疗兔桡骨骨缺损的临床研究

目的探讨万古霉素缓释微球复合自体浓缩红骨髓对治疗兔桡骨骨缺损的临床研究。方法选取2017年1~6月我院动物实验中心饲养的48只新西兰大白兔,雌雄皆可,体重2.5~3 kg,分别饲养,随机分为三组,每组16只。A组采用自体浓缩红骨髓治疗方式,B组采用自体浓缩红骨髓与纤维蛋白胶的治疗方式,C组采用万古霉素缓释微球复合自体浓缩红骨髓的治疗方式。比较三组的血清CRP水平、红细胞沉降率以及三组兔术后不同时间段X线的评分。结果术后3 d、7 d,B组兔血清的CRP水平与A组比较显著较低,而C组的CRP水平与B组比较显著降低(P<0.05);术后1 d、3 d及4周A组的ESR与B、C组比较显著较高;术后7 d A、B组的ESR与C组比较显著较高;术后2周B组的ESR与C组比较显著较高,而A组又显著高于B组(P<0.05);术后2周、4周以及8周B组及C组的X线评分显著高于A组(P<0.05),术后12周三组X线的评分比较无显著差异(P>0.05)。结论在对兔桡骨骨缺损进行治疗的过程中,采用万古霉素缓释微球复合自体浓缩红骨髓的治疗方式,有利于改善血清CRP水平以及红细胞沉降率,同时浓缩红骨髓主要取自自体,安全可靠,具有较好的组织相容性,对于骨缺损愈合的情况有积极的促进作用,因此在临床治疗中具有较高的应用价值。

基于Pickering乳液模板法制备载万古霉素PLGA多孔微球的研究

目的制备载万古霉素(VCM)的多孔微球,并对VCM多孔微球的物理性质进行表征。方法以羟基磷灰石(HA)稳定的Pickering乳液为模板,一步法制备聚乳酸-羟基乙酸共聚物(PLGA)多孔微球,采用吸附法制备载VCM的多孔微球,通过扫描电镜(SEM)、红外(FTIR)、差热分析(DSC)、BET、粒度分析方法,分别测定VCM多孔微球的形状、粒径、比表面积,最后测定VCM多孔微球的载药量和体外释放度。结果空白PLGA多孔微球的最佳制备工艺为:以5 ml二氯甲烷、0.25 g PLGA(LA:GA=50:50,黏度:0.38 dl/g)、0.0125g F-68 为油相,以 20 ml 水、0.1 g HA 为水相,两相混合振摇 10 min 得到 Pickering 乳液,置摇床(30℃水浴,120 r/min)振摇挥去二氯甲烷,滤过,干燥。该法制备的多孔微球平均粒径在10 μm左右,比表面积为22.1643 m^(2)/g,存在丰富的介孔结构,载VCM的PLGA多孔微球载药量可达7.56%±0.22%,在体外缓释作用可维持3 h。结论采用Pickering乳液模板法可通过一步乳化制备PLGA多孔微球,制备方法简便;该发制备的多孔微球具有丰富的介孔结构,可作为装载药物的主要场所;装载VCM的PLGA多孔微球具有缓释作用。