Use of biomimetic microtissue spheroids and specific growth factor supplementation to improve tenocyte differentiation and adaptation to a collagen-based scaffold in vitro

Tenocytes represent a valuable source of cells for the purposes of tendon tissue engineering and regenerative medicine and as such, should possess a high degree of tenogenic differentiation prior to their use in vivo in order to achieve maximal efficacy. In the current report, we identify an efficient means by which to maintain differentiated tenocytes in vitro by employing the hanging drop technique in combination with defined growth media supplements. Equine tenocytes retained a more differentiated state when cultured as scaffold-free microtissue spheroids in low serum-containing medium supplemented with l-ascorbic acid 2-phosphate, insulin and transforming growth factor (TGF)-β1. This was made evident by significant increases in the expression levels of pro-tenogenic markers collagen type I (COL1A2), collagen type III (COL3A1), scleraxis (SCX) and tenomodulin (TNMD), as well as by enhanced levels of collagen type I and tenomodulin protein. Furthermore, tenocytes cultured under these conditions demonstrated a typical spindle-like morphology and when embedded in collagen gels, became highly aligned with respect to the orientation of the collagen structure following their migration out from the microtissue spheroids. Our findings therefore provide evidence to support the use of a biomimetic microtissue approach to culturing tenocytes and that in combination with the defined growth media described, can improve their differentiation status and functional repopulation of collagen matrix.     

携载rhBMP-2微球的新型可注射自凝固复合人工骨的制备及特性研究

[目的]制备一种具有良好降解性和成骨活性、可注射的自凝固新型骨修复材料。[方法]制备携载rhBMP-2的聚乳酸与聚乙醇酸共聚物（PLGA）微球，并将其与rhBMP-2／磷酸钙骨水泥（CPC）复合，制备出rhBMP-2／PLGA微球／CPC复合人工骨。探讨了材料的特性，包括形貌、固化时间、抗压强度及反映材料体外降解速度的指标一体外降解液Ca、P浓度变化，测定复合材料rhBMP乏的释药速度及体外诱导MSCs细胞成骨分化的能力。[结果]与单纯CPC-rhBMP-2相比，复合材料的固化时间少量增加，抗压强度下降明显。体外降解速度及体外释药明显提高，释放的rhBMP-2具有骨诱导活性。[结论]rhBMP-2／PLGA微球／磷酸钙骨水泥新型复合人工骨是具有良好应用前景的骨修复材料。     

豚鼠耳蜗微循环的检测

应用生物微球技术,结合耳蜗连续切片,直接定量检测6只正常豚鼠耳蜗血流。结果耳蜗总血流为1.662±0.262μl/min·cochlea,蜗轴、外侧壁、耳蜗隔三处血流,分别为0.955±0.272μl/min、0.654±0.272μl/min和0.053±0.060μl/min。对本实验方法和结果进行了讨论,认为用该方法检测动物的耳蜗总血流和耳蜗局部血流是准确易行的。     

聚乳酸酮洛芬微球的制备及其体外释放度

制备了生物可降解的材料聚乳酸,用粘度法测定其分子量,并选用Ａ、Ｂ、Ｃ三种不同分子量的聚乳酸为药物的载体,制备了酮洛芬微球（ＡＳ,ＢＳ,ＣＳ）,测定其粒径和体外溶出速率。结果表明催化剂四苯基锡用量增加,所得的聚乳酸分子量增加,而制备酮洛芬微球的聚乳酸分子量越大,其微球粒径越大,溶出速率越小。     

霍乱弧菌O139菌毛微球疫苗制备初探

为寻求Ｏ１３９型霍乱弧菌菌毛疫苗研制的新途径,用高分子聚乳酸－聚乙二醇共聚物（ＤＬ－ＰＬＧ）包裹毒素共调菌毛（ＴＣＰ）抗的,进行免疫学研究,结果表明微轩比游离疫苗诱导的抗体水平高,其中小鼠血清ＩｇＧ以皮下ＭＳ－ＴＣＰ组最高,唾液中的ｓＩｇＡ以口服ＭＳ－ＴＣＰ组最高,口服组主要诱导粘膜免疫,皮下组主要导全身性免疫,ＴＣＰ是霍乱弧菌的共同抗原之一,可作为霍乱疫苗的候选抗原。     

复合三联抗结核药聚乳酸-羟基乙酸缓释微球的制备及体外释药特性

目的 :制备复合异烟肼(H)、利福平(R)、吡嗪酰胺(Z)的聚乳酸-羟基乙酸(HRZ/PLGA)缓释微球,观察其理化性质和体外缓释特性。方法:以PLGA(450mg)为载体,避光条件下称取H(40mg)、R(60mg)、Z(125mg),采用复乳-溶剂挥发法制备HRZ/PLGA缓释微球,应用扫描电镜观察微球的形态特征;应用高效液相色谱法(HPLC)测定其载药量、包封率;采用溶出法、HPLC于3h、6h、12h、1d、2d、3d、6d、9d、12d、15d、20d、25d、30d、40d、50d测定H、R、Z三种药物的浓度,观察其是否均大于10倍最低抑菌浓度(MIC),计算其日均释药率、累计释药率。结果:HRZ/PLGA微球在电镜下观察呈圆球形,平均粒径为10.3±4.7μm;H、R、Z三种药物的载药量分别为(18.02±0.36)%、(22.46±0.24)%、(21.68±0.37)%,包封率分别为(54.79±1.13)%、(72.35±0.39)%、(67.21±0.68)%;体外缓释试验显示微球缓释前12d左右,三种药物的累计缓释度均超过了50%,日均释药率分别为5.05%、4.89%、6.86%;第12天后三药的缓释基本趋于稳定,日均释药率分别为0.17%、0.26%、0.16%;三种药物缓释到50d时均大于10倍MIC。结论:HRZ/PLGA微球具有优良的载药及药物缓释效果,是一种理想的复合抗结核药物缓释系统。     

25羟基维生素D3-聚乳酸微球的制备及其对骨髓间充质干细胞增殖的影响

目的 制备25羟基维生素D3(25-dihydroxy-vitamin D3,25VD3)-聚乳酸微球,并探讨其对骨髓间充质干细胞(bone marrow mesenchymal stem cells,BMSC)增殖的影响,以期为研究牙槽骨缺损修复方法提供实验基础.方法 以W/O型乳化-溶剂挥发法制备25VD3-聚乳酸微球,检测微球的表面形貌、粒径分布、载药率、包封率及降解特性;与BMSC体外共培养,检测细胞形态、相对增殖率,以完全随机设计的单因素方差分析比较组间差异.结果 25VD3-聚乳酸微球呈球形,粒径主要为35 ～ 55 μm,载药率为(3.4±0.3)％,包封率为(71.5±2.8)％,降解6周pH值降低了(0.24 ±0.02)、累积释放量为(82.2±5.3)％;1.5×10-3 g/L微球培养基中BMSC相对增殖率最高,为(113.8±2.1)％.结论 W/O型乳化-溶剂挥发法制备的25VD3-聚乳酸微球粒径均一、包封率较高、缓释效能良好,可有效促进BMSC增殖.     

Time course study of the antigen-specific immune response to a PLGA microparticle vaccine formulation

Microparticle-based vaccine delivery systems are known to promote enhanced immune responses to protein antigens and can elicit T_H1-biased responses when used in combination with Toll-like receptor (TLR) agonists. It is important to understand the kinetics of the immune responses to microparticle-based protein vaccines in order to predict the duration of protective immunity and to optimize prime-boost vaccination regimens. We carried out a 10-week time course study to investigate the magnitude and kinetics of the antibody and cellular immune responses to poly(lactic-co-glycolic acid) (PLGA) microparticles containing 40 μg ovalbumin (OVA) protein and 16 μg CpG-ODN adjuvant (MP/OVA/CpG) in comparison to OVA-containing microparticles, soluble OVA plus CpG, or OVA formulated with Alhydrogel^® aluminum adjuvant. Mice vaccinated with MP/OVA/CpG developed the highest T_H1-associated IgG2b and IgG2c antibody titers, while also eliciting T_H2-associated IgG1 antibody titers on par with Alhydrogel^®-formulated OVA, with all IgG subtype titers peaking at day 56. The MP/OVA/CpG vaccine also induced the highest antigen-specific splenocyte IFN-γ responses, with high levels of IFN-γ responses persisting until day 42. Thus the MP/OVA/CpG formulation produced a sustained and heightened humoral and cellular immune response, with an overall T_H1 bias, while maintaining high levels of IgG1 antibody equivalent to that seen with Alhydrogel^® adjuvant. The time course kinetics study provides a useful baseline for designing vaccination regimens for microparticle-based protein vaccines.     

Phase-separated chitosan–fibrin microbeads for cell delivery

Matrix-enhanced delivery of cells is a promising approach to improving current cell therapies. Our objective was to create cell-laden composite microbeads that combine the attractive features of the natural polymers chitosan and fibrin. Liquid polydimethylsiloxane was used to emulsify a chitosan–fibrinogen solution containing suspended human fibroblast cells, followed by initiation of thrombin-mediated polymerization of fibrin and thermal/pH-mediated gelation of chitosan. Chitosan/fibrin weight percent (wt%) ratios of 100/0, 75/25, 50/50 and 25/75 were investigated. Microbead diameters ranged from 275?±?99?µm to 38?±?10?µm using impeller speeds from 600 to 1400?rpm. Fibroblasts remained viable on day 1 post-fabrication in all matrices, but cell viability was markedly higher in high-fibrin microbeads by day 8 post-fabrication. Cell spreading and interaction with the extracellular matrix was also markedly increased in high-fibrin matrices. Such composite microbeads containing viable entrapped cells have potential for minimally invasive delivery of cells for a variety of tissue repair applications.     

微球制剂的应用研究进展

微球制剂是一种极具潜力的剂型,近年来发展非常迅速,在药学领域有着广泛的应用。其在肿瘤治疗、疫苗免疫、蛋白给药方面都有独特的优势。从靶向和非靶向两个方面对微球制剂的应用进展进行综述,同时简要介绍了微球制剂的一些现有问题及解决办法。