組織工程被廣泛應用於再生醫學領域，H9c2 肌母細胞常被作為研究心肌細胞的替代模型，文獻中多藉由調控培養基中的胎牛血清濃度、或是添加全反式維甲酸 (all-trans retinoic acid, RA)，誘導 H9c2 分化為心肌細胞之方法。當 H9c2 分化為心肌細胞型態時會形成多核細胞，細胞增殖的能力下降。另一方面，膠原蛋白也可作為一種化學-物理方法誘導 H9c2 細胞分化，因為膠原蛋白為細胞外基質的主要成分，扮演細胞訊息傳導、貼附、和組織發育的關鍵作用 (化學方式)；此外，在特定的基材以及環境中，膠原蛋白具有特殊D週期性明暗帶 (D-banding) 的結構以導引細胞貼附的方向 (物理方式)。再者，近年來外源光生物調節法 (extrinsic photobiomodulation, EPM) 也被報導是一種簡單、且可有效誘導細胞分化的方法，只需照射特定波長的雷射光於細胞，就能促進細胞分化之效率。例如：在幹細胞培養於指定的膠原蛋白微圖案，同時結合 EPM 處理可以有效地增加脂肪細胞和成骨細胞的分化。
本研究目的是將膠原蛋白纖維作為基材，並探討結合胚胎幹細胞培養液 (embryonic stem cell medium, ESM) 與EPM之誘導分化方法，以發掘 H9c2 細胞分化為心肌細胞的潛力，同時驗證 ESM 培養基對於 H9c2 細胞分化為心肌細胞的重要性。為了製備具D-banding膠原蛋白纖維基材，將膠原蛋白沉積於雲母片上，形成D-banding 的膠原蛋白纖維 (collagen fibril)，再用 25 % 戊二醛 (glutaraldehyde, GA) 進行交聯以增強膠原蛋白的機械強度。另外，為了增強膠原蛋白纖維在基板上的附著性，將具 D-banding 膠原蛋白纖維轉印至官能基化的聚二甲基矽氧烷 (functionalized polydimethylsiloxane, f-PDMS) 上作為細胞貼附的基材。進而以50°C 的熱處理方式以暴露 RGD motif 在膠原蛋白纖維表面上 (CF)。將 H9c2 細胞種植於 CF 和聚乙烯培養皿 (tissue culture polystyrene, TCPS) 兩者基材上，並培養於 ESM 培養基中以 12 天為分化週期，控制組為 (1) ESM/TCPS、實驗組分別為 (2) EPM/TCPS、(3) 5-AZA (5-Azacytidine)/TCPS、(4) EPM+5-AZA/TCPS、(5) ESM/CF、(6) EPM/CF、(7) EPM+5-AZA/CF，以探討化學和物理方法對 H9c2 細胞的分化之影響。EPM則加入光敏劑 Verteporfin 以 690 nm 特定波長照射細胞以誘導 H9c2 細胞的分化為心肌細胞。
為鑑定 H9c2 細胞在 ESM 培養基中是否被誘導分化為心肌細胞，在培養時間為 12 天時，利用光學顯微鏡觀察心肌細胞之形態，以及使用即時定量聚合酶連鎖反應 (real-time PCR) 分析心肌細胞基因的表現量，例如：利用 ANP (atrial natriuretic peptide) 和 TNNT2 (troponin T type 2) 生物標記物以判斷心肌細胞的基因表現量。在相同 ESM 培養基條件環境下，比較 H9c2 細胞培養於兩者不同基材 (TCPS vs CF) 上對分化結果之影響。結果顯示，H9c2 細胞培養在膠原蛋白纖維基材 (CF) 上相較於控制組 (ESM/TCPS) 增加三倍 ANP 的基因表現量，確認 H9c2 細胞已開始進入早期心肌細胞分化階段。進一步地發現，當結合膠原蛋白纖維、5-AZA (化學因子)、和 EPM (物理因子) 等三種誘導因子，ANP 的基因表現量增加至 10 倍，由實驗結果顯示經三種誘導因子協同作用，可有效促進早期心肌細胞分化。此外，相關文獻指出 ESM 培養基可誘導纖維母細胞 (如 L929 和 3T3 fibroblasts) 分化為多潛能幹細胞 (induced pluripotent stem cells, iPSCs)，本研究發現ESM 培養基對誘導心肌細胞分化方面具有顯著的效果。因此，本研究利用化學 (5-AZA)、物理 (CF)、以及光照 (EPM) 的方式提供有效增進誘導H9c2細胞分化為心肌細胞的方法，可應用於再生醫學以及組織工程學相關重要議題。

Tissue engineering has been widely applied in the field of regenerative medicine. H9c2 cardiomyoblasts are often used as an alternative model for studying cardiac muscle cells. In the literature, various methods have been employed to induce the differentiation of H9c2 cells into cardiomyocytes, such as concentration adjustment of fetal bovine serum in the culture medium and the addition of all-trans retinoic acid (RA). When H9c2 cells differentiate into a cardiomyocytes phenotype, which becomes multinucleus, the proliferative capacity decreases. On the other hand, collagen can also be used as a chemo-physical method to induce H9c2 cell differentiation. Collagen is a major component of the extracellular matrix and plays a crucial role in cell signaling, adhesion, and tissue development (chemical factor). Additionally, in specific environments, collagen exhibits a distinct periodic banding structure (D-banding) that guides direction of cell attachment (physical factor). Moreover, extrinsic photobiomodulation (EPM) has been reported as a simple and effective method to induce cell differentiation. The combination of the photosensitizer (verteporfin) and laser light irradiation with specific wavelengths applied on cells, EPM can enhance the efficiency of cell differentiation. For example, when mesenchymal stem cells (MSCs) are cultured on specific collagen micro-patterns and treated by EPM, the conjunction of EPM with micro-patterns can effectively promote the differentiation of MSCs into adipocytes and osteocytes.
The aim of this study is to use collagen fibrils as a substrate and investigate the induction of H9c2 cell differentiation into cardiomyocytes by combining embryonic stem cell medium (ESM) with EPM. Meanwhile, the importance of ESM culture medium has been proven that could induce the differentiation of H9c2 cells into cardiomyocytes. To prepare collagen fibril substrate, collagen is deposited on mica to form D-banding structure. After that, glutaraldehyde (GA) is then used for cross-linking to enhance the mechanical strength of collagen fibrils. In addition, in order to enhance the adhesion of collagen fibrils on the substrate, collagen fibrils are transferred onto functionalized polydimethylsiloxane (f-PDMS) which is a cell attachment substrate. Subsequently, collagen fibril on f-PDMS is treated by heat treatment at 50°C to expose RGD motifs on the surface of collagen fibrils (CF). H9c2 cells are seeded on both CF and tissue culture polystyrene (TCPS) substrates and incubated in ESM medium for a differentiation period time within 12 days. The control group is cultured in ESM/TCPS, while the experimental groups are (1) EPM/TCPS, (2) 5-Azacytidine (5-AZA)/TCPS, (3) EPM+5-AZA/TCPS, (4) ESM/CF, (5) EPM/CF, and (6) EPM+5-AZA/CF. The effects of chemical and physical factors on H9c2 cell differentiation are investigated.
To determine whether H9c2 cells are differentiated into cardiomyocytes in ESM medium, the morphology of cardiomyocytes is observed by optical microscope after 12 days of culture time. Quantitative real-time polymerase chain reaction (qPCR) is performed to analyze the gene expression levels of cardiomyocytes biomarker, such as atrial natriuretic peptide (ANP) and troponin T type 2 (TNNT2). Under the same ESM culture conditions, the influence of different substrates (TCPS vs. CF) on the differentiation of H9c2 cells is compared. The results show that H9c2 cells cultured on CF exhibit a three-fold increase in the expression of ANP compared to the control group (ESM/TCPS), which confirmed the initiation of early-stage cardiomyocyte differentiation. Furthermore, when the three induction factors are combined with collagen fibril, 5-AZA (chemical factor), and EPM (physical factor), the expression of ANP gene increases up to ten-fold. The experimental results demonstrate the effective promotion of early-stage cardiomyocyte differentiation through the synergistic effects of the three induction factors. Moreover, previous studies have indicated that ESM culture medium can induce the differentiation of fibroblast cells (such as L929 and 3T3 fibroblasts) into induced pluripotent stem cells (iPSCs). This study found significant effects of ESM culture medium on the induction of cardiomyocyte differentiation. Therefore, this research provides an effective method for enhancing the differentiation of H9c2 cells into cardiomyocytes through the combination of chemical (5-AZA), (CF), and physical (EPM). The method has potential to apply in the fields of regenerative medicine and tissue engineering.

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