BACKGROUND & AIMS:
BACKGROUND:Natural articular cartilage has a limited capacity for spontaneous regeneration. Controlled release of transforming growth factor-beta1 (TGF-beta1) to cartilage defects can enhance chondrogenesis. In this study, we assessed the feasibility of using biodegradable chitosan microspheres as carriers for controlled TGF-beta1 delivery and the effect of released TGF-beta1 on the chondrogenic potential of chondrocytes.
METHODS:Chitosan scaffolds and chitosan microspheres loaded with TGF-beta1 were prepared by the freeze-drying and the emulsion-crosslinking method respectively. In vitro drug release kinetics, as measured by enzyme-linked immunosorbent assay, was monitored for 7 days. Lysozyme degradation was performed for 4 weeks to detect in vitro degradability of the scaffolds and the microspheres. Rabbit chondrocytes were seeded on the scaffolds containing TGF-beta1 microspheres and incubated in vitro for 3 weeks. Histological examination and type II collagen immunohistochemical staining was performed to evaluate the effects of released TGF-beta1 on cell adhesivity, proliferation and synthesis of the extracellular matrix.
RESULTS:TGF-beta1 was encapsulated into chitosan microspheres and the encapsulation efficiency of TGF-beta1 was high (90.1%). During 4 weeks of incubation in lysozyme solution for in vitro degradation, the mass of both the scaffolds and the microspheres decreased continuously and significant morphological changes was noticed. From the release experiments, it was found that TGF-beta1 could be released from the microspheres in a multiphasic fashion including an initial burst phase, a slow linear release phase and a plateau phase. The release amount of TGF-beta1 was 37.4%, 50.7%, 61.3%, and 63.5% for 1, 3, 5, and 7 days respectively. At 21 days after cultivation, type II collagen immunohistochemical staining was performed. The mean percentage of positive cells for collagen type II in control group (32.7% +/- 10.4%) was significantly lower than that in the controlled TGF-beta1 release group (92.4% +/- 4.8%, P < 0.05). Both the proliferation rate and production of collagen type II in the transforming growth factor-beta1 microsphere incorporated scaffolds were significantly higher than those in the scaffolds without microspheres, indicating that the activity of TGF-beta1 was retained during microsphere fabrication and after growth factor release.
CONCLUSION:Chitosan microspheres can serve as delivery vehicles for controlled release of TGF-beta1, and the released growth factor can augment chondrocytes proliferation and synthesis of extracellular matrix. Chitosan scaffolds incorporated with chitosan microspheres loaded with TGF-beta1 possess a promising potential to be applied for controlled cytokine delivery and cartilage tissue engineering.
背景与目标:
背景:天然关节软骨的自发再生能力有限。转化生长因子-beta1(TGF-beta1)向软骨缺损的控制释放可以增强软骨生成。在这项研究中,我们评估了使用可生物降解的壳聚糖微球作为载体控制TGF-β1传递的可行性以及释放的TGF-β1对软骨细胞软骨形成潜能的影响。
方法:采用冻干法和乳液交联法分别制备载有TGF-β1的壳聚糖支架和壳聚糖微球。通过酶联免疫吸附测定法测量的体外药物释放动力学被监测7天。进行溶菌酶降解4周以检测支架和微球的体外降解性。将兔软骨细胞接种在含有TGF-β1微球的支架上,并在体外孵育3周。进行组织学检查和II型胶原免疫组织化学染色,以评估释放的TGF-β1对细胞粘附性,细胞外基质增殖和合成的影响。
结果:TGF-β1被包埋在壳聚糖微球中,TGF-β1的包封率较高(90.1%)。在溶菌酶溶液中温育4周以进行体外降解的过程中,支架和微球的质量持续下降,并且形态发生了显着变化。从释放实验中,发现TGF-β1可以以多相方式从微球释放,包括初始爆发阶段,缓慢的线性释放阶段和平稳阶段。 TGF-beta1在1、3、5和7天的释放量分别为37.4%,50.7%,61.3%和63.5%。培养后21天,进行II型胶原免疫组织化学染色。对照组中II型胶原蛋白阳性细胞的平均百分比(32.7%/-10.4%)显着低于对照TGF-beta1释放组(92.4%/-4.8%,P <0.05)。掺入了转化生长因子-β1微球的支架中的II型胶原的增殖速率和产生均显着高于不含微球的支架中的II型胶原的生长,这表明TGF-β1的活性在微球制备过程中和生长因子释放后得以保留。
结论:壳聚糖微球可以作为TGF-β1控释的载体,释放的生长因子可以促进软骨细胞的增殖和细胞外基质的合成。壳聚糖支架与负载TGF-β1的壳聚糖微球相结合,具有可用于控制细胞因子递送和软骨组织工程的潜力。