溫感性甲殼素/明膠/甘油磷酸水膠做為阿魏酸持續釋放於髓核再生之應用

Thermosensitive Chitosan/Gelatin/Glycerol Phosphate Hydrogel as a Sustained Release System of Ferulic Acid for Nucleus Pulposus Regeneration

鄭詠馨(Yung-Hsin Cheng)

指導教授 ： 林峯輝

國立臺灣大學/工學院/醫學工程學研究所/博士(2012年)

https://doi.org/10.6342/NTU.2012.01000

摘要

椎間盤退化與椎間盤突出及背痛有高度的相關性，這些病徵增加了健康照護的支出。椎間盤退化過程可分為五個階段，在退化的第一至二階段時，並沒有明顯的病徵出現，但可透過核磁共振或電腦斷層掃描檢查來追蹤，臨床上一般並不會在此階段給予治療。近來的文獻指出，活性氧自由基不僅會加速椎間盤退化的過程，且會造成髓核細胞的凋亡和細胞外基質的降解。阿魏酸是一種抗氧化物並且可以較穩定地存在於空氣中；阿魏酸被證實對於活性氧自由基所引起的相關疾病具有預防的效果。本研究的目的除了評估阿魏酸對於雙氧水所引起的氧化壓力導致髓核細胞傷害的可能治療效果外，並評估利用溫感性甲殼素/明膠/甘油磷酸水膠做為阿魏酸持續釋放早期治療椎間盤退化的可行性。
在本研究中的試驗結果指出，500 μM為阿魏酸對紐西蘭兔的髓核細胞的安全閥值，利用阿魏酸治療被雙氧水所引起的氧化壓力所傷害的髓核細胞，其aggrecan, type II collagen和BMP-7 的基因表現可以有顯著的提升，而MMP-3的表現量有顯著的下降，而硫酸化葡萄胺聚醣含量有顯著的上升，細胞凋亡的情形也有顯著的抑制。而以甲殼素/明膠/甘油磷酸水膠做為阿魏酸持續釋放的試驗中，阿魏酸可以從水膠中緩釋，包覆阿魏酸的水膠除了能提升被雙氧水所引起的氧化壓力所傷害的髓核細胞中aggrecan和type II collagen基因的表現量外，並能抑制MMP-3的表現量，而在硫酸化葡萄胺聚醣生成量及alcian blue的染色的結果指出，包覆阿魏酸的水膠可以使受傷害的髓核細胞恢復到正常髓核細胞的表現量，另外在caspase-3和TUNEL的染色結果上，也指出其細胞凋亡的情形可以被顯著抑制。在本研究中證明，阿魏酸可成功藉由N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC)/ N-hydroxysuccinimide (NHS)固定於甲殼素/明膠/甘油磷酸水膠上；在中性環境下，該水膠之成膠溫度為攝氏31.8度，而以阿魏酸固定於甲殼素/明膠/甘油磷酸上的水膠治療被雙氧水所引起的氧化壓力所傷害的髓核細胞，可顯著的提升受傷害細胞中aggrecan和type II collage的表現量，並抑制其MMP-3的表現量；此外，硫酸化葡萄胺聚醣生成量也能恢復到正常的水平，另外在caspase-3和TUNEL的染色結果中顯示，細胞凋亡的情形可以被有效的抑制。
綜合上述，本研究的試驗結果指出阿魏酸可做為髓核再生的治療分子，而溫感性甲殼素/明膠/甘油磷酸水膠可做為阿魏酸長期釋放的良好載體，將阿魏酸固定於甲殼素/明膠/甘油磷酸水膠上可有效延長釋放的時間；結合阿魏酸及溫感性甲殼素/明膠/甘油磷酸水膠顯然可以有效治療因氧化壓力所傷害的髓核細胞，在未來更可應用於髓核再生的微創手術中。

關鍵字

髓核 ； 阿魏酸 ； 氧化壓力 ； 溫感性水膠 ； 抗氧化劑

並列摘要

Disc degeneration is strongly associated with back pain and herniation that increase the costs of health care. The degeneration of intervertebral disc (IVD) could be divided into 5 stages. In the first and second stages, there are no significant symptoms but could be traced by magnetic resonance imaging or computed tomography-scan. Generally, no aggressive treatment would be processed in the clinics. Recent studies indicated that overproduction of reactive oxygen species (ROS) may accelerate the degenerative process of IVD and associate with apoptosis of nucleus pulposus (NP) cells and degradation of extracellular matrix. Ferulic acid (FA) is an excellent antioxidant and relatively stable in air. FA has been proven to have ability to prevent ROS-induced diseases. The object of the study was aimed to evaluate the possible therapeutic effect of FA on hydrogen peroxide (H2O2)-induced oxidative stress NP cells and the feasibility of use the thermosensitive chitosan/gelatin/glycerophosphate (C/G/GP) hydrogel as a sustained release system of FA for early treatment in IVD degeneration.
In the study, NP cells were harvested from the IVD of New Zealand rabbits. The results showed that 500 μM of FA might be the threshold to treat NP cells without cytotoxicity. Post-treatment of FA on H2O2-induced oxidative stress NP cells significantly up regulated the expression of aggrecan, type II collagen and BMP-7 and down regulated the expression of MMP-3 in mRNA level. Post-treatment of FA on H2O2-induced oxidative stress NP cells could restore the production of sulfated glycosaminoglycans (GAGs) and inhibit the apoptosis caused by H2O2. The results showed that the release of FA from C/G/GP hydrogel could decrease the H2O2-induced oxidative stress. Post-treatment of FA-incorporated C/G/GP hydrogel on H2O2-induced oxidative stress NP cells showed up-regulation of aggrecan and type II collagen and down-regulation of MMP-3 in mRNA level. The results of sulfated GAGs to DNA ratio and alcian blue staining revealed that the GAGs production of H2O2-induced oxidative stress NP cells could reach to normal level. The results of caspase-3 activity and TUNEL staining indicated that FA-incorporated C/G/GP hydrogel decreased the apoptosis of H2O2-induced oxidative stress NP cells. The results showed that FA was successfully immobilized on C/G/GP hydrogel by N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC) and N-hydroxysuccinimide (NHS) crosslinking method. The gelation temperature of the FA-immobilized C/G/GP hydrogel was 31.80 degree celsius under neutral pH. Post-treatment of FA-immobilized C/G/GP hydrogel on H2O2-induced oxidative stress NP cells showed down-regulation of MMP-3 and up-regulation aggrecan and type II collagen in mRNA level. The sulfated GAGs production of H2O2-induced oxidative stress NP cells could be increased to the normal level in the post-treatment of FA-immobilized C/G/GP hydrogel group. The results of caspase-3 activity and TUNEL staining showed that the apoptosis of H2O2-induced oxidative stress NP cells could be inhibited by post-treatment of FA-immobilized C/G/GP hydrogel.
From the results of the study, FA could be used as a therapeutic molecule for NP regeneration and FA-incorporated C/G/GP hydrogel might be potentially applied as a long-term release system. The immobilization of FA on C/G/GP hydrogel could significantly prolong the release period of FA. These results suggest that combination of FA and thermosensitive C/G/GP hydrogel can treat NP cells from the damage caused by oxidative stress and may apply in minimally invasive surgery for NP regeneration in the future.

並列關鍵字

nucleus pulposus ； ferulic acid ； oxidative stress ； thermosensitive hydrogel ； antioxidant

參考文獻

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