低頻率電磁場對於貼附在幾丁聚醣薄膜上之軟骨細胞之影響

低頻率電磁場目前在臨床使用上，能用來治療骨關節炎使病人之患部減輕發炎反應，以減低骨關節炎對於關節軟骨之損壞。有研究指出電磁場可以增進受損軟骨組織的修復，然而當體內軟骨組織缺損過大時，電磁場之治療無法有明顯之效果，因此須藉由導入載有細胞之組織工程材料來修補缺損。以幾丁聚醣所製作織細胞支架，具有細胞相容性、無毒性以及可分解之特性，因此具有刺激軟骨生長及痊癒後不需再經手術取出之優點。本實驗研究目的為測試以幾丁聚醣為支架來修復軟骨組織的情況下，若再以電磁場照射，是否能產生有如臨床之修復效果。實驗中將軟骨細胞培養在去乙醯基化程度96%之幾丁聚醣所製成之薄膜，並以每天2小時電磁場(電磁場參數：強度：18-33 G、頻率：75 Hz、脈衝寬：1.3 ms、振幅：3.5±1 mV)照射，照射時間達21天，並於第7、14、21天來測試軟骨細胞之反應。實驗主要測定項目為細胞活性(MTT assay)、細胞增殖狀況(dsDNA content)以及軟骨細胞特定胞外基質(GAG 及type II collagen content)之染色與分泌量。對照組為未經電磁場照射之細胞。從實驗結果得知軟骨細胞在照射完電磁波後其細胞活性及細胞增殖量和對照組相比雖有些微增加但無明顯差異；兩組之葡萄醣胺聚醣及第二型膠原蛋白染色結果均證實有正常分泌之情況，但因以肉眼觀察無法辨別明顯差異故藉由葡萄醣胺聚醣定量法(DMMB assay)以及第二型膠原蛋白免疫定量法(Type II collagen-ELISA assay)來定量探討實驗組與對照組胞外基質之分泌。由實驗結果得知實驗組之葡萄醣胺聚醣分泌量在21天，有明顯多於控制組；而實驗組之第二型膠原蛋白之分泌量於第14及21天，有明顯多於控制組。由本實驗之結果推測電磁場對於貼附於幾丁聚醣薄膜之豬軟骨細胞之細胞活性、細胞增殖雖有增加但並無統計學上之差異，然而對於胞外基質葡萄醣胺聚醣及第二型膠原蛋白之分泌量有明顯改善之情況。

關鍵字

幾丁聚醣薄膜；低頻率電磁波；軟骨細胞

並列摘要

Low Frequency Pulsed Electromagnetic Field (EMF) has been used in clinical application to treat osteoarthritis (OA). OA patients receiving EMF treatment have been shown to have reduced pain and inflammatory reaction. Literatures have also shown that EMF can improve articular cartilage repair. However, the healing effect on EMF on lesion larger than 1cm is unsatisfactory. Biocompatible tissue engineered (TE) constructs seeded with chondrocytes or mesenchymal stem cells are usually used to fill larger lesions and to accelerate the repair process. Among the biocompatible materials, chitosan resembles extracellular matrix of cartilage. It is biodegradable and easily available from nature. Chitosan TE scaffolds have been shown to enhance chondrocyte growth and extracellular matrix (ECM) deposition. This experiment exposed chondrocyte cells cultured on chitosan films to EMF to simulate in vitro tissue engineering condition. Tests were done on cells to assay whether EMF could reproduce its positive in vivo healing effects in an in vitro tissue engineering model. Primary porcine chondrocytes were seeded on chitosan (96% deacetylated) films and exposed to EMF (19-33 Gauss, 75Hz, duty cycle 1.3ms) two hours a day for 21 days. The cells were tested on day 7, 14, and 21 for their viability, proliferation, type II collagen and glycosaminoglycan (GAG) release. The cells were also stained for GAG and type II collagen and prepared for SEM imaging. The results showed EMF cell proliferation and viability were similar to those of controls in weeks 1, 2, 3. Both groups had normal GAG and type II collagen release according to results from immunohistological staining. The amount of GAG release by the EMF cells was significantly higher than that released by controls in week 3. Also the amount of type II collagen produced by EMF cells was higher than that by controls in weeks 2 and 3. Based on the results from this experimental setup, we found EMF could benefit articular cartilage repair when tissue engineered scaffolds are used. Though EMF simulated did not seem to stimulate cell growth, it accelerated cells’ ECM deposition.

並列關鍵字

chitosan ； low-frequency pulsed electromagnetic ； chondrocyte

參考文獻

[1] S. W. O’Driscoll, “The healing and regeneration of articular cartilage”, Journal of Bone and Joint Surgery - Series A, vol. 80, no. 12, 1998, pp. 1795-1812.

[2] R. M. Schulz, A. Bader, ” Cartilage tissue engineering and bioreactor systems for the cultivation and stimulation of chondrocytes,” European Biophysics Journal, vol. 36, no. 4-5, 2007, 539-568.

[4] V. C. Mow, C. C. Wang, C. T. Hung, “The extracellular matrix, interstitial fluid and ions as a mechanical signal transducer in articular cartilage,” Osteoarthritis and Cartilage, vol. 7, no. 1, 1999, pp. 41-58.

[5] M. P. Recht, D. Resnick,” MR Imaging of Articular Cartilage: Current Status and Future Directions,” American Journal of Roentgenology, vol. 163, no. 2, 1994, pp. 283-290.

[7] E. B. Hunziker,” Articular cartilage repair: basic science and clinical progress. A review of the current status and prospects,” Osteoarthritis and Cartilage, vol. 10, no. 6, 2002, pp. 432–463.

國際替代計量

低頻率電磁場對於貼附在幾丁聚醣薄膜上之軟骨細胞之影響

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