二階段藥物釋放於脂肪組織工程之應用

Abstract

近年的脂肪組織工程研究中指出欲重建脂肪組織需結合細胞、支架和生長因子三大要素。脂肪前驅細胞(preadipocytes)是在脂肪組織工程中最常用的細胞來源，因為此細胞可在體外大量增生後，經過特定生長因子誘導分化成為脂肪細胞。細胞一旦分化後就不會再增生，所以欲得到大量的成熟脂肪細胞，必須在分化前先加入生長因子促使脂肪前驅細胞快速增生。 增生和分化都需要加入特定的生長因子。在本實驗中發現0.5 ng/mL bFGF可以促使3T3-L1在體外增生，而根據實驗室過去的研究也發現insulin 和dexamethasone可以誘導細胞分化。為了要讓增生的藥物和分生的藥物分別在不同階段釋放，我們所製備的核殼粒子(core-shell particles)的內殼為PDLLA微球體可包覆insulin和dexamethasone;外殼為明膠可吸附bFGF。因此，核殼粒子會先釋放bFGF，再釋放insulin和dexamethasone。實驗結果發現核殼粒子的外殼可控制釋放bFGF，並且在MTS的結果發現控制釋放bFGF可更有效促使細胞增生。在動物實驗中，將核殼粒子和細胞分別與兩種支架:支架膠原蛋白/玻尿酸混合膠體和明膠微球體打入Balb/c老鼠背部，10天後組織切片觀察。 總結，我們可以成功製備明膠鍍附PDLLA的核殼粒子並且可做為二階段藥物釋放系統應用於脂肪組織工程。第一階段的控制釋放bFGF可促使3T3-L1增生in vitro。結合脂肪前驅細胞、支架和二階段藥物系統最為軟組織重建的替代物是有潛力的。

Recent studies in adipose tissue engineering have indicated that in vivo adipogensis requires an appopriate cell source, scaffold and growth factors. Preadipocytes, precursor cells committed to the adipocyte lineage are the ideal cell source. They are capable of proliferating to obtain extensive cell numbers in vitro and differenting into mature fat cells. Once cells differentiate, they are not capable of replication. Therefore, in order to induce the formation of larger fat tissue, it is essential to increase the numbers of preadipocytes before differentiation. Preadipocytes proliferate and differentiate in the presence of the growth factors. In this study, we found that 0.5 ng/mL bFGF is able to accelerate cell proliferation in vitro, and insulin and dexamethasone enable preadipocytes to differentiate according to the previous study. In order to release two types of growth factor in two-stage, we develop a method to obtain the core-shell particles with insulin/dex in the PDLLA core and gelatin shell layer for bFGF sorption. Core-shell particles are prepared for the controlled release of bFGF and followed by insulin/dex. The proliferation ability of 3T3-L1 incorporated by core-shell particles containing bFGF in vitro is assessed by the MTS assay. The MTS result shows that the controlled release of bFGF increases the number of preadipocytes. By injecting two types of scaffold, collagen/HA and gelatin microspheres respectively, in combination with core-shell particles and preadipocytes into the back of the Balb/c mice, the adipogenesis at the subcutasneous implantation sites is evalutated histologically. In conclusion, PDLLA particles (core) coating with gelatin (shell) are successfully acheived and ued as the two-step controlled release system in the application of adipose tissue engineering. The first step controlled release of bFGF increases the proliferation rate of 3T3-L1. The combination of preadipocytes and two-step drug release system with scaffold has the potential for augmentation of adipose tissue.