接枝層黏蛋白之海藻膠球包覆前脂肪細胞應用於自體脂肪乳房重建之組織工程研究

Abstract

本研究試圖以發展接枝層黏蛋白之海藻膠球(Laminin-alginate beads, LABs) 結合細胞治療來解決自體脂肪移植存活率的問題，做為改善以自體脂肪移植乳房重建效果或，使隆乳效果維持長久的脂肪組織工程之先驅研究。前脂肪細胞（pre-adipocyte）則經誘導後包覆於LABs，為進一步提升層黏蛋白海藻膠球LABs脂肪分化效果，生長因子FGF-2亦添加於LABs作為評估。包覆細胞的LABs則稱為“cell-laden LABs” 。 材料經儀器分析，傅里葉轉換紅外光譜（FT-IR）、掃描式電子顯微鏡（SEM）、微量天秤以及可見光紫外光分光光譜儀分別用來分析其官能基、膠球微結構、降解測試、和澎潤測試。從降解測試、和澎潤測試結果顯示LABs其生物穩定性以及降解度均良好。透過FT-IR可觀察到氧化海藻膠的二醛基以及與層黏蛋白laminin交聯之官能基。 在前脂肪細胞的體外培養試驗，乳酸脫氫酶毒性分析LDH assay 與水溶性四唑鹽比色法WST-1 assay用來評估生物相容性，脂肪分化與細胞存活則在之後進行評估，本研究使用五種CD markers用於前脂肪細胞在流式細胞技術之分析。實驗顯示CD44表現於多種細胞包含脂肪細胞;CD105在前脂肪細胞表現明顯減少; CD73與CD90表現顯示出前脂肪細胞開始進行脂肪分化。以相對螢光單位定量（R.F.U.）之脂肪分化分析指出LABs除了可提供保護細胞的基本功能外，並能讓包覆其中之前脂肪細胞有顯著脂肪分化效果，而FGF-2的存在亦有此分化效果。相對螢光單位在實驗組與對照組可達數倍。LABs海藻膠球可維持初期前脂肪細胞之分化效果與良好存活率，且因接枝laminin而更具長效顯著性。 在動物實驗，於ㄧ、二、四周取出NOD SCID老鼠身上的LABs移植物秤重，一周後LABs重量約原來的一半，而四周後因LABs海藻膠球逐漸水解、重量則不到原來一半。移植LABs實驗組老鼠之血液生化分析包括肝功能GOP、GPT;腎功能BUN、creatinine之數值皆與對照組相似。蘇木素-伊紅染色（H/E stain）切片ㄧ周仍可見膠球剖面，並於二周後即有脂肪細胞與組織生成，並於四周後觀察有血管新生的現象。顯示接枝層黏蛋白之海藻膠球具備提供前脂肪細胞存活、脂肪分化、以及血管新生的環境。

It is advantageous via autologous fat grafting in breast reconstruction not only breast augmentation but also more natural body shape. The problem is that the variant survival rate of autologous fat grafting after implantation of 10 to 12 months. The absorption of autologous fat grafting is from 30 % to 60 %, leading to fat death, calcification, and pseudo-cyst detected in the patients’ breast. In this study, we aimed to develop laminin-alginate beads (LABs) based on the concept of cell therapy, solving the low survival rate and relative problem for autolougous fat grafting. We partially oxidized the alginate to graft the extra-cellular matrix protein－laminin. Committed pre-adipocyte cells were firstly underwent differentiation for two day via chemical induction before LABs encapsulation. In order to further promote adipogenesis with growth factor, FGF-2 would be added into modified laminin–alginate during gelation as well. The beads without pre-adipocyte incorporation were abbreviated as “LABs” and the beads with pre-adipocyte encapsulation were further referred to as “cell-laden LABs”. Material characteristic analysis of laminin–alginate was performed. Fourier transform infrared spectrometer (FT-IR), scanning electron microscope (SEM), micro-balance and UV-Vis spectrophotometer were used for material characterization to analyze the functional groups, bead microstructure, swelling/degradation, and laminin concentration, respectively. Laminin–alginate beads were stable from the statistics of degradation and swelling test. The characteristic di-aldehyde group of partial oxidized alginate and functional group crosslinking with laminin would be shown in FT-IR. In pre-adipocyte, 3T3-L1 culture, adipogenesis and cell survival were investigated. Before the mentioned performance and in animal studies, the in vitro biocompatibility LDH assay and WST-1 assay would be carried out. We evaluated five CD markers by flowcytometry for the differentiation of chemically induced pre-adipocytes. CD44 (or its isoforms) showed a wide range of cell types, including adipocytes and others specific cells. CD44 was significantly separately from undifferentiated pre-adipocytes. Furthermore, the expression of the cell surface proteins CD105 was going to significantly decrease. Two CD markers of stromal cell-associated markers CD73, CD90 showed differentiating tendency. AdipoRed assay indicated a significant difference lipid accumulation for LABs and the addition of FGF-2 existence as well. The relative fluorescence unit has several folds between control and experimental groups. The existence of LABs maintained the adipogenesis of pre-adipocytes at initial stage and showed significance in long-term culture with the combination of laminin-grafting. In animal study, implant weight were calculated by 1st week, 2nd week, and 4th week. Implant weight was half the original after one week. Weight loss was over half implant after one month due to hydrolysis of LABs. The blood biochemistry evaluation of implantation animal contained lots of important information of biocompatibility. All the NOD SCID nude mice in each group have similar GOP, GPT, BUN and creatinine levels compared to the level of normal mice. Hematoxylin and eosin staining suggested newly adipose tissue formation and blood vessels present for angiogenesis.