幾丁聚醣/明膠/甘油磷酸之溫敏性水膠作為細胞載體於椎間盤髓核再生之研究

Abstract

椎間盤的退化與椎間盤突出和背痛有強烈的相關，而這些病徵會造成健康照護的支出，部分研究指出椎間盤退化源自於髓核，而近來的臨床的治療方式包括物理治療、藥物治療、椎間盤融合、人工椎間盤置換及椎間盤切除術，然而，這些現行的治療方式是以減輕病人疼痛為主要目的，而非以修復椎間盤為主要目的，而細胞治療即以修復椎間盤為目的，是相當具有潛力的治療方式，因此，本研究的目的即製備一溫度敏感性水膠作為髓核細胞的載體。 在本研究中，利用所製備的幾丁聚醣/明膠/甘油磷酸二鈉溫度敏感性水膠包覆紐西蘭白兔的椎間盤髓核細胞，先以流變儀來評估成膠溫度、成膠時間和成膠強度，和單使用幾丁聚醣/甘油磷酸二鈉相較，幾丁聚醣/明膠/甘油磷酸二鈉於37°C的成膠強度有明顯的提升，而隨著明膠濃度的提升，成膠溫度和成膠時間均會隨之下降，在降解測試的評估，提高明膠於水膠中的比例，降解的速度會略微提升，而單使用幾丁聚醣/甘油磷酸二鈉及幾丁聚醣/明膠/甘油磷酸二鈉水膠，均無明顯的細胞毒性，將水膠搭載椎間盤髓核細胞培養三週後，和單層培養之椎間盤髓核細胞相較下，硫酸基之葡萄胺聚醣含量對總DNA含量的比值有顯著的提升，而mRNA基因表現上，type II Collagen、Aggrecan、MMP-3、MMP-9、IGF-1、BMP-7和TGF-β均有顯著的提升。 總結實驗的結果，於本研究中所製備之幾丁聚醣/明膠/甘油磷酸二鈉溶液，在生理的pH值下，可在室溫維持液態並在似人體體溫形成膠態，所形成的水膠具有生物相容性及生分解性，並且適合作為椎間盤髓核細胞之載體，因此，此可注射之幾丁聚醣/明膠/甘油磷酸二鈉水膠，在未來在椎間盤的組織工程的應用上是相當具有潛力的。

Disc degeneration is strongly associated with back pain and herniation resulting in the cost of health care decreasing. Some evidence shows that disc degeneration originates in the nucleus pulposus (NP). However, current treatments such as physical therapy, pharmaceutical treatment, spinal fusion, artificial disc replacement and discectomy attempt to reduce pain rather than repair the degenerated disc. Cell–based therapies are aimed at repairing the degenerated disc, which is a potential treatment of disc degeneration. Therefore, the purpose of this study was to prepare the thermosensitive hydrogel as cell carrier of NP cells. In this study, the thermosensitive chitosan/gelatin/β- glycerol phosphate disodium salt hydrogels (C/G/GP hydrogels) was investigated. Nucleus pulposus cells which were harvested from the intervertebral discs of the adult New Zealand white rabbits were encapsulated in C/G/GP hydrogels. The gelation temperature, gelation time and gel strength were evaluated by rheometer. Compared with the formulations using only C/GP, the gel strength of C/G/GP was increased at 37°C. In the C/G/GP system, raising the concentration of gelatin resulted in a decrease in the gelation temperature and gelation time. The results of the in vitro cytotoxicity showed that the C/GP and the C/G/GP hydrogel are biocompatible. In the degradation test, raising the concentration of gelatin seems to increase the percentage of weight loss. The ratio of sulfated glycosaminoglycan (GAG) to DNA of NP cells cultured in hydrogels showed significantly higher than monolayer-cultured at the end of 3-week. Compared with monolayer-cultured, the mRNA expression of type II Collagen, Aggrecan, MMP-3, MMP-9, IGF-1, BMP-7 and TGF-β in hydrogel-cultured NP cells was significantly enhanced. The results showed that the C/G/GP solution remains liquid at room temperature but form the hydrogel at approximate body temperature under a neutral pH. The formed hydrogel is biocompatible and biodegradable. As three-dimensional carrier for NP cell culture, these results suggest the C/G/GP hydrogel is a suitable scaffold for the cell culture. These features make the C/G/GP hydrogel potential application as an injectable cell carrier for NP regeneration.