复合壳聚糖纳米微球聚乳酸-羟基乙酸/纳米羟基磷灰石缓释载体系统对蛋白的缓释作用

背景：聚乳酸-羟基乙酸支架材料具有良好的生物相容性、无毒、可以良好的塑性,并具有一定的强度和韧性。但其降解产物为酸性,会影响局部pH值变化,不利组织生长。 目的：制备能够良好缓释蛋白类药物的复合支架。 方法：以牛血清蛋白为模型药物,以离子凝胶法制备壳聚糖微球。将微球与纳米羟基磷灰石和聚乳酸-羟基乙酸按一定比例混合,以冰粒子为致孔剂,采用粒子沥虑-冷冻干燥复合工艺制备CMs/nHA/PLGA复合缓释支架。利用扫描电镜、透射电镜、压泵仪和力学性能测试仪检测复合支架的形态和性能,并考察其在体外对蛋白类药物释放的规律。 结果与结论：制备的壳聚糖纳米微球形态良好,呈规则球形或类球形,粒径分布在220~770 nm,以380~650 nm为多。微球对药物的载药量为39.2%,包封率为68.3%,两者均与牛血清蛋白的初始量相关,载药量随牛血清蛋白初始量的增加而增加,包封率则反之。复合支架呈白色多孔状,孔径为125~355 mm,孔与孔之间联通良好,孔隙率达83.4%,压缩强度为1.4~ 2.1 MPa,10周降解率为28.6%。PLGA/nHA支架对牛血清蛋白的2 d累积释放量为85%,而壳聚糖和CMs/nHA/PLGA复合支架对牛血清蛋白的9 d累积释放量分别是为48.9%和35.7%。提示制作的壳聚糖纳米微球和CMs/nHA/PLGA支架材料对牛血清蛋白有良好的缓释作用,复合支架材料形态好,强度和降解速率合适。     

胶原-壳聚糖载硫酸长春新碱微球缓释药膜的研究

目的本研究制备载硫酸长春新碱(vincristinesulfate,VCR)微球的胶原-壳聚糖缓释药膜。并考察加入壳聚糖对药膜性质的影响。选定适当的胶原壳聚糖比例制备药膜。方法采用W/O/O溶剂挥发法制备VCR的聚乳酸-羟基乙酸(poly(lactic-co-glycolicacid),PLGA)微球,并对微球性质表征,采用二次冻干法制备载VCR微球的胶原-壳聚糖药膜,对药膜的表面形态、降解性质、热力学性质及释放性质进行表征,并与释放2周后的药膜进行比较。采用高效液相法分析药物含量。结果VCR制成PLGA微球后再制备成药膜,可达到双重缓释的作用,明显减少药物突释,并延缓药物释放。添加了壳聚糖的药膜降解速度明显小于单纯的胶原药膜。在体外释放实验中,微球突释为(27.2±1.2)%,而胶原药膜的突释为(20.4±1.9)%,胶原与壳聚糖比例为9∶1、4∶1、3∶2的药膜突释分别为(20.2±2.1)%、(18.0±1.1)%和(16.3±1.8)%。结论胶原壳聚糖载VCR的缓释药膜能不同程度减少药物的突释,使药物释放更加平稳缓慢,优于单纯的胶原药膜。     

壳聚糖微球/纳米羟基磷灰石/聚乳酸-羟基乙酸复合支架制备及其蛋白缓释效果:与单纯纳米羟基磷灰石/聚乳酸-羟基乙酸支架、壳聚糖微球的比较

背景:骨组织工程骨构建中如何使生长因子持续高效发挥作用是影响成骨速度和质量的关键,现多以各种材料的微球或支架作为缓释载体,但缓释作用有待提高。目的:实验拟制备壳聚糖微球,然后复合到纳米羟基磷灰石/聚乳酸-羟基乙酸支架上,形成双重缓释作用,并测量对牛血清白蛋白的释放效果。方法:以牛血清白蛋白为模型药物,采用乳化交联法制备壳聚糖微球。将微球与纳米羟基磷灰石、聚乳酸-羟基乙酸按一定比例混合,以冰粒子为致孔剂,采用冷冻干燥法制备壳聚糖微球/纳米羟基磷灰石/聚乳酸-羟基乙酸复合支架。利用扫描电镜、激光粒度分析仪、压泵仪和力学性能测试仪检测复合支架的形态性能,考察药物在缓释支架上的体外释放规律。结果与结论:所制备的壳聚糖微球形态良好,呈规则圆球形,粒径集中分布在20～40μm,微球药物包封率为86.5%,载药量为0.8%,随牛血清白蛋白初始用量的增加,载药量可升高至2.6%,但包封率下降至74.1%。壳聚糖微球能均匀分布在聚乳酸-羟基乙酸支架上,形成壳聚糖微球/纳米羟基磷灰石/聚乳酸-羟基乙酸复合支架,孔径为100～400μm,孔隙率80%,压缩强度为1.1～2.3MPa,10周降解率为26.5%。单纯纳米羟基磷灰石/聚乳酸-羟基乙酸支架其牛血清白蛋白在36h累积释放量达85%以上,壳聚糖微球其牛血清白蛋白10d累积释放量为33.6%,复合支架其牛血清白蛋白40d累积释放量为81.5%。结果证实包埋壳聚糖微球的纳米羟基磷灰石/聚乳酸-羟基乙酸支架其压缩强度和降解速率合适,对蛋白类药物具有良好的缓释作用,有望作为组织工程的支架材料和生长因子的缓释载体。     

rhBMP-2和bFGF复合至壳聚糖-Ⅰ型胶原支架后在体外的降解和释放

目的： 探讨rhBMP-2和bFGF复合至壳聚糖-Ⅰ型胶原支架材料后在体外的释放规律。方法: 制备壳聚糖-Ⅰ型胶原支架及含rhBMP-2和bFGF的复合膜。扫描电镜观察测量支架孔径；体外观察降解情况,在不同时点收集浸出液，ELISA检测因子的释放浓度；观察浸出液对牙周膜细胞的影响。结果： 复合膜呈疏松多孔海绵状，高、中、低3种壳聚糖-Ⅰ型胶原支架平均孔径分别为（106±17）μm、（141±13）μm和（173±11）μm ；复合膜在含溶菌酶的PBS中可以降解，壳聚糖浓度越高支架降解越慢，rhBMP-2和bFGF的释放开始为“爆发性”，此后释放逐渐减慢，最后在低浓度可缓慢而持久地释放，壳聚糖浓度越高的复合支架，因子释放越慢越持久。与含溶菌酶的PBS间接接触的因子层随着其表面支架的降解而逐渐释放因子，释放延迟时间和表面支架的降解速度有关。复合bFGF因子支架的浸出液作用于HPDLCs，可以明显促进细胞的增殖。结论： 壳聚糖-Ⅰ型胶原复合因子支架体外可以降解并释放因子，降解和因子释放速度与壳聚糖的浓度密切相关；可以通过分层制作复合因子支架来控制因子有序释放，所释放的因子具有正常生物学功能。     

载ADM-PLGA纳米微球的ADM-PLGA-NHAC制备及其对人骨肉瘤MG63细胞的抑制作用

目的　研制载阿霉素(ADM)的聚乳酸-羟基乙酸共聚物(PLGA)纳米微球的纳米羟基磷灰石/胶原复合支架（ADM-PLGA-NHAC）,研究其性质及体外释药特点,探讨其体外抑制人骨肉瘤MG63细胞的作用,为骨肉瘤的治疗提供新策略。 方法　以纳米羟基磷灰石及胶原为原料制备纳米羟基磷灰石/胶原支架并在其中加载ADM-PLGA纳米微球, 通过扫描电子显微镜、体外释放行为等手段评价载药支架材料的性能。以CCK8法、活-死染色评价该复合支架的浸提液在体外对人骨肉瘤MG63细胞株的抗肿瘤活性。 结果　复合支架的孔径多在100~200 μm,孔隙率约为82%,微球与支架间结合较为紧密。复合支架具有良好的缓释特性,28 d内能持续缓慢释放阿霉素。复合支架的浸提液对骨肉瘤MG63细胞生长有明显的抑制作用。 结论　制备的ADM-PLGA-NHAC复合支架具有良好的药物缓释特性及抗肿瘤效应,是一种具有良好应用前景的抗骨肉瘤骨修复材料。     

成骨生长肽壳聚糖-海藻酸钠微球的制备及体外检测

背景：成骨生长肽体外注射可以刺激外周血和骨髓细胞数增加,增加动物的骨量,加速骨折愈合,但因多肽不稳定性及注射应用不方便,限制了其临床应用。 目的：应用乳化交联法制备成骨生长肽壳聚糖-海藻酸钠缓释微球,并对其粒径、载药、体外释药、理化特性进行检测。 方法：以戊二醛作为交联剂,应用乳化交联法制备具有控制释放功能的负载成骨生长肽壳聚糖-海藻酸钠微球,显微镜及扫描电镜观察微球的形态和粒径;利用酶联免疫吸附实验动态检测成骨生长肽壳聚糖-海藻酸钠微球的载药率、包封率和缓释规律。 结果与结论：乳化交联法制备的壳聚糖-海藻酸钠微球,球形良好,球体表面有较多微孔,具有较高的包封率(>72%)。体外药物释放实验表明,成骨生长肽可以从壳聚糖-海藻酸钠微球中缓慢释放,整个释放过程可达49 d,累积释放率>85%。提示应用乳化交联法制备的负载成骨生长肽壳聚糖-海藻酸钠缓释微球,具有很好的控制释放成骨生长肽的能力。     

硫酸长春新碱缓释药膜的制备及稳定性初步研究

目的制备出载硫酸长春新碱微球的胶原-壳聚糖缓释药膜,并考察该制剂的稳定性。方法采用W/O/O溶剂挥发法制备载硫酸长春新碱的聚乳酸/聚羟基乙酸共聚物(PLGA)微球,后把微球与壳聚糖、胶原溶液共混及二次冻干,制备出载硫酸长春新碱微球的胶原-壳聚糖药膜。对微球和药膜表面形态进行了电镜观察,测定了微球和药膜的包封率、载药量及药物释放,药物含量采用高效液相法检测。此外,还初步考察了缓释药膜的稳定性。结果制备的微球包封率达到79.0%±1.0%,微球药物的突释为27.2%±1.2%,制备成药膜后降低到18.0%±1.1%,采用该工艺流程制备出来的缓释药膜,药物突释明显减少。稳定性实验显示,该药膜在40℃条件下放置3个月药物含量下降到97.9%±0.1%,而高湿度或光照环境下放置10 d药物含量下降到91.4%±0.3%和91.2%±0.4%。结论药物包囊制成微球后与胶原、壳聚糖共混制备出的缓释药膜具有较好的释放特性和稳定性,有望成为一种实用的新型缓释抗肿瘤制剂。     

壳聚糖微球药物释放机制研究进展

近年来已有多种药物实现了以壳聚糖微球作为缓控释载体,并在生物医学领域展现出良好的应用前景,成为缓控释剂型研究的热点之一。目前对壳聚糖微球释放机制的研究进展落后于壳聚糖载药微球制备与应用的研究进展,而加强壳聚糖载药微球药物释放机制的研究,有利于更好地了解药物的释放行为和释放影响因素,并对深入研究壳聚糖缓释载药体系的制备与应用具有重要意义。主要从壳聚糖微球的药物释放机制、药物释放行为描述、药物释放影响因素等方面进行了综述。     

构建缓慢释放碱性成纤维细胞生长因子的脱细胞羊膜人工活性真皮**☆

背景：目前人工皮肤替代品的种类较多,各有优缺点,仍然没有一种理想的产品应用于临床。 目的：探讨构建一种可以缓慢释放碱性成纤维细胞生长因子的新型人工活性真皮的可行性。 方法：组织块法培养幼儿包皮成纤维细胞;采用酶-去垢剂法制备人脱细胞羊膜;双相法制备碱性成纤维细胞生长因子-明胶-壳聚糖缓释微球;缓释微球黏附于脱细胞羊膜上;三四代成纤维细胞培养于负载缓释微球的脱细胞羊膜上。 结果与结论：制备的脱细胞羊膜为白色半透明状薄膜有较高的孔隙率,空隙不规则,孔径大小为10~100 nm,无细胞毒性;碱性成纤维细胞生长因子-明胶-壳聚糖缓释微球分散较均匀,呈球形,粒径均匀,球体表面比较光滑,载药率为20 ng/g,包封率为80.5%,体外药物缓释曲线显示药物控释效果良好;成纤维细胞在支架表面爬行生长良好,层粘连蛋白表达较对照组高。表明将成纤维细胞种植于负载碱性成纤维细胞生长因子-明胶-壳聚糖缓释微球的脱细胞羊膜上,缓释微球能较好地黏附于脱细胞羊膜表面。     

bFGF缓释微球的制备及其促雪旺细胞分裂增殖的初步研究

研究碱性成纤维细胞生长因子(bFGF)缓释微球的制备方法及其对雪旺细胞的促分裂增殖作用。以聚乳酸-羟基乙酸共聚物(PLGA)为载体材料,采用复乳包囊法制备bFGF-PLGA缓释微球,并对微球的形态学、粒径分布、载药量和包封率、及体外释药进行研究。将bFGF、bFGF-PLGA微球分别加入不同组的雪旺细胞培养液中,分别测定雪旺细胞的数量、活力和细胞周期。结果显示,复乳包囊法制备的bFGF-PLGA缓释微球表面光滑圆整,球体均匀度好;微球平均粒径为1.552±0.015μm,平均径距为1.310±0.010;载药量和包封率分别为(27.18×10-3)%±(0.51×10-3)%、66.43%±1.24%;微球的体外释药过程较为稳定,11d释药率为72.47%。体外细胞试验中,培养1、2d时,bFGF组的细胞计数、吸光度明显高于bFGF缓释微球组;培养3、4d时,bFGF组和bFGF缓释微球组的细胞计数、吸光度无统计学差异;培养6、8d时,bFGF缓释微球组的细胞计数、吸光度明显高于bFGF组。流式细胞仪检测结果显示,培养2d后,bFGF组的G2/M S期百分数高于bFGF缓释微球组;培养4、8d后,bFGF缓释微球组的G2/M S期百分数高于bFGF组,差异具有统计学意义。说明采用复乳包囊法制备bFGF-PLGA缓释微球的工艺可行,微球中bFGF的生物活性保存良好,能缓慢持续释放活性bFGF,促进雪旺细胞的分裂增殖。     

Controlled release of bioactive doxorubicin from microspheres embedded within gelatin scaffolds

We have encapsulated the chemotherapeutic agent doxorubicin into biodegradable polymer microspheres, and incorporated these microspheres into gelatin scaffolds, resulting in a controlled delivery system. Doxorubicin was encapsulated in poly(D,L-lactide-co-glycolide) (PLGA) using a double emulsion/solvent extraction method. Characterization of the microspheres including diameter, surface morphology, and in vitro drug release was determined. The release of doxorubicin up to 30 days in phosphate buffered solution was assessed by measuring the absorbance of the releasate solution. Gelatin scaffolds were crosslinked using glutaraldehyde and microspheres were added to gelatin during gelation. The murine mammary mouse tumor cell line, 4T1, was treated with various doses of doxorubicin. A propidium iodide assay was utilized to visualize dead cells. Using a Transwell basket assay, PLGA microspheres and gelatin constructs were suspended above 4T1 cells for 48 h. Viable cells were determined using the CyQUANT cell proliferation assay. Results indicate that the release was controlled by the incorporation of PLGA microspheres into gelatin constructs. A significant difference was seen in the cumulative release over days 5-16 (p < 0.05). The bioactivity of doxorubicin released from the microspheres and scaffolds was maintained as proven by significant reduction in viable cells after treatment with PLGA microspheres as well as with the gelatin constructs (p < 0.001). The drug-polymer conjugate can be used as a controlled drug delivery system in a biocompatible scaffold that could potentially promote preservation of soft tissue contour.     

Modulation of protein delivery from modular polymer scaffolds

Growth factors are increasingly employed to promote tissue regeneration with various biomaterial scaffolds. In vitro release kinetics of protein growth factors from tissue engineering scaffolds are often investigated in aqueous environment, which is significantly different from in vivo environment. This study investigates the release of model proteins with net-positive (histone) and net-negative charge (bovine serum albumin, BSA) from various scaffolding surfaces and from encapsulated microspheres in the presence of ions, proteins, and cells. The release kinetics of proteins in media with varying concentrations of ions (NaCl) suggests stronger electrostatic interaction between the positively charged histone with the negatively charged substrates. While both proteins released slowly from hydrophobic PCL surfaces, plasma etching resulted in rapid release of BSA, but not histone. Interestingly, although negatively charged BSA released readily from negatively charged collagen (col), BSA released slowly from col-coated PCL scaffolds. Such electrostatic interaction effects were abolished in the presence of serum proteins and cells as evidenced by the rapid release of proteins from col-coated scaffolds. To achieve sustained release in the complex environment of serum proteins and cells, the model proteins were encapsulated into poly(D,L-lactic-co-glycolic acid) (PLGA) microspheres, which were embedded within col-coated PCL scaffolds. Protein release from microspheres was modulated by changing the lactide-to-glycolide ratio of PLGA polymer. BSA adsorbed to col released faster than histone encapsulated in microspheres in the presence of serum and cells. Collectively, the data suggest that growth factor release is highly influenced by scaffold surface and the presence of ions, proteins, and cells in the media. Strategies to deliver multiple growth factors and studies which investigate their release should consider these important variables.     

Controlled release of bioactive TGF-beta 1 from microspheres embedded within biodegradable hydrogels

Transforming growth factor-beta1 (TGF-beta1) is of great relevance to cartilage development and regeneration. A delivery system for controlled release of growth factors such as TGF-beta1 may be therapeutic for cartilage repair. We have encapsulated TGF-beta1 into poly(DL-lactide-co-glycolide) (PLGA) microspheres, and subsequently incorporated the microspheres into biodegradable hydrogels. The hydrogels are poly(ethylene glycol) based, and the degradation rate of the hydrogels is controlled by the non-toxic cross-linking reagent, genipin. Release kinetics of TGF-beta1 were assessed using ELISA and the bioactivity of the released TGF-beta1 was evaluated using a mink lung cell growth inhibition assay. The controlled release of TGF-beta1 encapsulated within microspheres embedded in scaffolds is better controlled when compared to delivery from microspheres alone. ELISA results indicated that TGF-beta1 was released over 21 days from the delivery system, and the burst release was decreased when the microspheres were embedded in the hydrogels. The concentration of TGF-beta1 released from the gels can be controlled by both the mass of microspheres embedded in the gel, and by the concentration of genipin. Additionally, the scaffold permits containment and conformation of the spheres to the defect shape. Based on these in vitro observations, we predict that we can develop a microsphere-loaded hydrogel for controlled release of TGF-beta1 to a cartilage wound site.     

Surface studies of coated polymer microspheres and protein release from tissue-engineered scaffolds

The controlled release of growth factors from porous, polymer scaffolds is being studied for potential use as tissue-engineered scaffolds. Biodegradable polymer microspheres were coated with a biocompatible polymer membrane to permit the incorporation of the microspheres into tissue-engineered scaffolds. Surface studies with poly(D,L-lactic-co-glycolic acid) [PLGA], and poly(vinyl alcohol) [PVA] were conducted. Polymer films were dip-coated onto glass slides and water contact angles were measured. The contact angles revealed an initially hydrophobic PLGA film, which became hydrophilic after PVA coating. After immersion in water, the PVA coating was removed and a hydrophobic PLGA film remained. Following optimization using these 2D contact angle studies, biodegradable PLGA microspheres were prepared, characterized, and coated with PVA. X-ray photoelectron spectroscopy was used to further characterize coated slides and microspheres. The release of the model protein bovine serum albumin from PVA-coated PLGA microspheres was studied over 8 days. The release of BSA from PVA-coated PLGA microspheres embedded in porous PLGA scaffolds over 24 days was also examined. Coating of the PLGA microspheres with PVA permitted their incorporation into tissue-engineered scaffolds and resulted in a controlled release of BSA.     

Surface studies of coated polymer microspheres and protein release from tissue-engineered scaffolds

The controlled release of growth factors from porous, polymer scaffolds is being studied for potential use as tissue-engineered scaffolds. Biodegradable polymer microspheres were coated with a biocompatible polymer membrane to permit the incorporation of the microspheres into tissueengineered scaffolds. Surface studies with poly(D,L-lactic-co-glycolic acid) [PLGA], and poly(vinyl alcohol) [PVA] were conducted. Polymer films were dip-coated onto glass slides and water contact angles were measured. The contact angles revealed an initially hydrophobic PLGA film, which became hydrophilic after PVA coating. After immersion in water, the PVA coating was removed and a hydrophobic PLGA film remained. Following optimization using these 2D contact angle studies, biodegradable PLGA microspheres were prepared, characterized, and coated with PVA. X-ray photoelectron spectroscopy was used to further characterize coated slides and microspheres. The release of the model protein bovine serum albumin from PVA-coated PLGA microspheres was studied over 8 days. The release of BSA from PVA-coated PLGA microspheres embedded in porous PLGA scaffolds over 24 days was also examined. Coating of the PLGA microspheres with PVA permitted their incorporation into tissue-engineered scaffolds and resulted in a controlled release of BSA.     

Effects of the controlled-released TGF-beta 1 from chitosan microspheres on chondrocytes cultured in a collagen/chitosan/glycosaminoglycan scaffold

The objectives of this study were (1) to develop a three-dimensional collagen/chitosan/glycosaminoglycan (GAG) scaffold in combination with transforming growth factor-beta1 (TGF-beta 1)-loaded chitosan microspheres, and (2) to evaluate the effect of released TGF-beta 1 on the chondrogenic potential of rabbit chondrocytes in such scaffolds. TGF-beta 1 was loaded into chitosan microspheres using an emulsion-crosslinking method. The controlled release of TGF-beta 1, as measured by enzyme-linked immunosorbent assay (ELISA), was monitored for 7 days. The porous scaffolds containing collagen and chitosan were fabricated by using a freeze drying technique and crosslinked using 1-ethyl-3-(3-dimethyl aminopropyl)carbodiimide (EDC) in the presence of chondroitin sulfate (CS), as a GAG component. The TGF-beta 1 microspheres were encapsulated into the scaffold at a concentration of 10 ng TGF-beta 1/scaffold and then chondrocytes were seeded in the scaffold and incubated in vitro for 3 weeks. Both proliferation rate and glycosaminoglycan (GAG) production were significantly higher in the TGF-beta 1 microsphere-incorporated scaffolds than in the control scaffolds without microspheres. Extracellular matrix staining by Safranin O and immunohistochemistry for type II collagen were elevated in the scaffold with TGF-beta 1 microspheres. These results suggest that TGF-beta 1 microspheres when incorporated into a scaffold have the potential to enhance cartilage formation.     

Cell affinity for bFGF immobilized heparin-containing poly(lactide-co-glycolide) scaffolds

In order to effectively and uniformly immobilize basic fibroblast growth factor (bFGF) to thick PLGA scaffold, the heparin-conjugated PLGA (H-PLGA) was synthesized at the first by reaction between heparin and a low molecular weight PLGA. Then heparin-containing PLGA (H-PLGA/PLGA) scaffold was fabricated by blending the H-PLGA with a high molecular weight PLGA. Finally, bFGF was immobilized on the H-PLGA/PLGA scaffold mainly by static electricity action between them. The effect of H-PLGA content on bFGF binding efficiency of the H-PLGA/PLGA scaffolds was investigated. It was found that bFGF binding efficiency increased with increasing H-PLGA content. The bound bFGF can release in vitro slowly from the H-PLGA/PLGA scaffolds and last over two weeks. The released bFGF has still preserved its bioactivity. The attachment and growth of mouse 3T3 fibroblasts on the H-PLGA/PLGA scaffolds were better than that on the PLGA scaffold, however bFGF immobilized H-PLGA/PLGA scaffolds showed much better cell affinity. Therefore, the method to use the H-PLGA/PLGA scaffold for immobilizing bFGF is not only effective for slow delivering bFGF with bioactivity, but also can be used for fabricating thick scaffold where bFGF could be combined and uniformly distributed.     

RhBMP-2 microspheres-loaded chitosan/collagen scaffold enhanced osseointegration: an experiment in dog

The purpose of this study is to develop a novel recombinant human bone morphogenetic protein-2 (rhBMP-2) sustained release scaffold for dental implant osseointegration, and to evaluate the effect of this scaffold on promoting bone formation. RhBMP-2 was encapsulated in the poly-D,L-lactide-co-glycolide (PLGA) biodegradable microspheres, which were subsequently dispersed in a chitosan/collagen composite scaffold. This rhBMP-2 microspheres-loaded scaffold (S-MB) was compared with a chitosan/collagen scaffold without microspheres that directly encapsulated rhBMP-2 (S-B) in vitro and in vivo. The microstructure of the new scaffold was examined with scanning electron microscopy. The release profile of rhBMP-2 in vitro was measured at interval periods. The effect of rhBMP-2 encapsulated scaffolds on enhancing bone formation through implantation in dogs' mandibles was identified by histological examination of the regenerated bone after 4 weeks of implantation. Due to PLGA microspheres being loaded, the S-MB exhibited lower values at porosity and swelling rate, as well as a higher effective release dose than that of the S-B. Bone density, bone-implant contact, and bone-fill values measured from dog experiments demonstrated that the S-MB induced bone regeneration more quickly and was timely substituted by new bone. It was concluded that this sustained carrier scaffold based on microspheres was more effective to induce implant osseointegration.     

Sequential release of bioactive IGF-I and TGF-beta 1 from PLGA microsphere-based scaffolds

Growth factors have become an important component for tissue engineering and regenerative medicine. Insulin-like growth factor-I (IGF-I) and transforming growth factor-beta1 (TGF-beta 1) in particular have great significance in cartilage tissue engineering. Here, we describe sequential release of IGF-I and TGF-beta 1 from modular designed poly(l,d-lactic-co-glycolic acid) (PLGA) scaffolds. Growth factors were encapsulated in PLGA microspheres using spontaneous emulsion, and in vitro release kinetics was characterized by ELISA. Incorporating BSA in the IGF-I formulations decreased the initial burst from 80% to 20%, while using uncapped PLGA rather than capped decreased the initial burst of TGF-beta 1 from 60% to 0% upon hydration. The bioactivity of released IGF-I and TGF-beta 1 was determined using MCF-7 proliferation assay and HT-2 inhibition assay, respectively. Both growth factors were released for up to 70 days in bioactive form. Scaffolds were fabricated by fusing bioactive IGF-I and TGF-beta 1 microspheres with dichloromethane vapor. Three scaffolds with tailored release kinetics were fabricated: IGF-I and TGF-beta 1 released continuously, TGF-beta 1 with IGF-I released sequentially after 10 days, and IGF-I with TGF-beta 1 released sequentially after 7 days. Scaffold swelling and degradation were characterized, indicating a peak swelling ratio of 4 after 7 days of incubation and showing 50% mass loss after 28 days, both consistent with scaffold release kinetics. The ability of these scaffolds to release IGF-I and TGF-beta 1 sequentially makes them very useful for cartilage tissue engineering applications.     

rhBMP-2／PLGA缓释支架的制备及对MSCs细胞成骨活性的体外研究

目的探讨骨形态发生蛋白（rhBMP-2）的聚乳酸聚乙醇酸共聚物（PLGA）体外缓释生物支架对人骨髓间充质干细胞（MSCs）细胞的影响。方法采用粒子沥滤-冷冻干燥复合工艺制备了附载rhBMP-2的PLGA生物支架,并检测了在PLGA的降解过程中rhBMP-2的释药规律;同时分离培养人骨髓间充质干细胞,体外培养后分别接种于附载和未附载rhBMP-2的PLGA支架上。扫描电镜观察不同时间段MSC在支架上的生长情况;MTT法测定细胞增殖情况。结果rhBMP-2能被包裹进PLGA支架中,而且可以在PLGA支架降解过程中持续释放出来并诱导骨发生。结论骨形态发生蛋白的PLGA复合载体是一种较为理想的新型生物支架。