以電紡絲法製備聚己內酯/動物明膠/透明質酸混合奈米纖維及其應用

Abstract

由於天然高分子擁有細胞相容性佳的特點，而人工合成高分子則是機械強度高，若將兩者混合，再製備成與人體細胞外間質相似的結構，即為奈米纖維狀，不但能夠維持纖維的機械強度，而且也能夠有利於細胞貼附及增殖。本研究選擇甲/乙酸做為溶劑，避免有機溶劑的高汙染問題，再搭配靜電紡絲法將聚己內酯/動物明膠/透明質酸(P/G/H)形成的高分子溶液製備成奈米纖維，另外同時製備聚己內酯/動物明膠(P/G)及聚己內酯(P)纖維膜做對照比較。進料流量分為0.005、0.0075及0.01 mL/min共3組、操作電壓則分為15、20及25 kV共3組，再嘗試改變溶劑種類、溶劑比例、高分子總濃度、聚己內酯與明膠間的比例及透明質酸分子量，最後選出甲/乙酸體積比例為7/3、高分子總濃度15 %(w/v)、聚己內酯/明膠重量比例為3/1、分子量為50 kDa的透明質酸佔1.5 %(w/v)，操作電壓為25 kV、進料流量為0.0075 mL/min，作為最佳實驗條件，可製成直徑約118.7 ± 20.0 nm的奈米纖維。 本研究在纖維膜的機械強度測定當中，發現經由戊二醛蒸氣交聯8小時後，P/G/H纖維膜的最大抗拉應力從3.02 ± 0.62 MPa升高到5.14 ± 0.7 MPa，和P纖維膜最大抗拉應力5.62 ± 0.72 MPa強度相近，因此選擇交聯8小時為最佳參數；藉由水接觸角測定發現添加明膠及透明質酸能夠改善聚己內酯疏水的問題，P纖維膜的接觸角：84.99° ± 1.39°，P/G纖維膜接觸角下降到42.3° ± 2.1°，P/G/H纖維膜則是下降至52.77° ± 1.28°；再由紅外線吸收光譜、X射線光電子能譜儀、X光繞射儀、熱重分析儀及示差掃描量熱分析儀證實P/G/H三成份均勻混合在一起，同時X光繞射儀也能夠證實聚己內酯的半結晶特性；在崩解性實驗裡，發現經由戊二醛蒸氣交聯8小時，能夠提升P/G及P/G/H纖維膜材料的穩定性，且P/G/H纖維膜較P/G穩定性佳；最後藉由蛋白質總量、MTT測定及光學、螢光顯微鏡觀察KP-hMSC細胞貼附及增殖結果顯示，P/G及P/G/H纖維膜具有良好的細胞相容性。希冀在未來能進一步發展P/G及P/G/H纖維膜在組織工程的應用。

關鍵字：奈米纖維、靜電紡絲法、聚己內酯、動物明膠、透明質酸、甲/乙酸、戊二醛

The advantage of natural polymers is good cell compatibility, while that of synthetic polymers is high mechanical strength. If natural and synthetic polymers are mixed, and fabricated into a nanofibrous structure similar to human extracellular matrix, it can maintain its mechanical strength and promote cell attachment and proliferation. To avoid pollution problems from organic solvents, formic acid/acetic acid was chosen to be the solvent of this research. Polycaprolactone/gelatin/hyaluronic acid (P/G/H), which was dissolved in formic acid/acetic acid, was fabricated into nanofibers by electrospinning. On the other hand, polycaprolactone/gelatin (P/G) and polycaprolactone (P) nanofibers were fabricated for comparison. The flow rates of the feed were 0.005, 0.0075, and 0.01 mL/min. The applied voltages of the experiment were 15, 20, 25 kV. Also, the influences of different kinds of solvents, ratios of solvents, total polymer concentrations, ratios of polycaprolactone to gelatin and the molecular weights of hyaluronic acid were explored. Finally, 7/3 formic acid/acetic acid ratio, 15 %(w/v) total polymer concentration, 3/1 polycaprolactone/gelatin ratio, 50 kDa molecular weight of hyaluronic acid with concentration of 1.5 %(w/v), 25 kV applied voltage and 0.0075 mL/min flow rate were selected as the final optimal experimental parameters for preparing nanofibers with diameters of 118.7 ± 20.0 nm. In the tensile strength test, after the P/G/H nanofibrous meshes were cross-linked by the vapor of glutaraldehyde for 8 hours, their maximum tensile stress, 3.02 ± 0.62 MPa, was elevated to 5.14 ± 0.7 MPa, which was close to the maximum tensile stress of P nanofibrous meshes (5.62 ± 0.72 MPa). Therefore, crosslinking for 8 hours was selected as the best parameter. Through the water contact angle measurements, it could be found that the addition of gelatin and hyaluronic acid could reduce the hydrophobicity of polycaprolactone. The water contact angle of P meshe was 84.99° ± 1.39°. On the other hand, the water contact angles of P/G and P/G/H meshes were reduced to 42.3° ± 2.1° and 52.77° ± 1.28°. The nanofibrous meshes were also analyzed by Fourier transform infrared (FT-IR) spectroscopy, X-ray photoelectron spectroscopy (XPS), X-ray diffraction (XRD), thermogravimetric (TGA) and differential scanning calorimetry (DSC). The results demonstrated that the three polymers (P/G/H) were mixed well. The XRD results also demonstrated the semi-crystalline properties of polycaprolactone. In the degradation experiments, after the meshes were crosslinked by the glutaraldehyde vapor for 8 hours, the stability of the P/G and P/G/H nanofibrous meshes was enhanced. Finally, through the total protein, MTT assays, and microscopic observation of KP-hMSC cell adhesion and proliferation, it was clear that P/G and P/G/H nanofibrous meshes had good cell compatibility. In the future, it is expected that these nanofibrous meshes can be used as tissue engineering scaffolds.

Keywords：Nanofibers, Electrospinning, Polycaprolactone, Gelatin, Hyaluronic acid, Formic acid/acetic acid, Glutaraldehyde