利用匍枝根黴菌(Rhizopus stolonifer)細胞壁組成 RHIZOCHITIN
作為生物支架探討生物降解性及生物相容性
Biodegradability and Biocompatibility of the Scaffold Derived from Cell Wall Component of Rhizopus stolonifer
中文摘要
本研究利用延緩孢子產生的突變Rhizopus stolonifer F6 菌株，經液體培養基 培養得到海綿狀的菌絲墊。此菌絲利用1N NaOH 在 121oC 處理 20 分鐘後凍 乾得到的物質稱為RHIZOCHITIN (RC)。RC 再經 95%酒精沖洗，以去除非 皂化的酯類後得到的物質稱為alcohol-treated RHIZOCHITIN (ARC)。市售 的正向控制組BESCHITIN-W (BC)與 RC 和 ARC 作為生物支架分別進行體外 和體內的生物相容性和生物降解性測試。體外測試包括溶菌酶分解和纖維母細胞 (L-929)培養，分別看生物支架前後重量改變與顯微鏡觀察(光學顯微鏡與電子 顯微鏡)結構前後是否不同，得知溶菌酶在前三週 RC 和 ARC 材料重量減少明顯 比BC 還要少，表示 RC 和 ARC 不會很明顯的被溶菌酶分解，反之 BC 就明顯
的被分解，在SEM 中也可看到三者結構的改變。RC 和 ARC 不會影響纖維母細
胞的存活，並且在細胞培養中也可以發現，RC 和 ARC 材料重量減少，可知 L-929 會對RC 和 ARC 產生分解作用，但是 BC 不僅會降低 L-929 存活率外，而且只
可以分解少許的材料。體內研究證明將RC 和 ARC 植入 SD 大鼠皮下組織發現
在兩週已完全被分解，然而BC 在四週時還可被取出，表示 BC 在體內分解速度
慢於RC 和 ARC。實驗中 RC、ARC 和 BC 植入皮下組織均不會造成皮膚過敏和
刺激性現象產生。最後，在植入後四週，利用直徑為8 mm 大小的鑽孔器，在
植入的區域取出組織，進行HE 和 PAS 染色，兩者結果均顯示 RC 和 ARC 在體 內被分解的速度遠大於BC。本研究得知從 R.stolonifer F6 細胞壁組成成分的 物質，是具有生物相容性與生物降解性。
英文摘要
In the present study, the sporangia-formation-delayed mutant strain Rhizopus
stolonifer F6 was used to obtain mycelium mattress by liquid culture. The mycelium mattress was treated with 1N NaOH at 121oC for 20 minutes and followed by lyophilization to have RHIZOCHITIN (RC). RC was then again washed with 95%
alcohol to remove non-saponification lipid to get alcohol-treated RHIZOCHITIN (ARC). RC and ARC together with a commercial product BESCHITIN-W (BC) for positive control were employed as scaffold for the tests of biocompatibility and biodegradability in vitro and in vivo. In vitro tests included lysozyme degradation and co-culture to fibroblast cell line (L-929) with the weight change and microscopy (light
and scanning electron microscopy). Lysozyme did not digest RC and ARC at the first three weeks but significant degradation was observed in the group of BC by the decrease in weight and the observation under SEM. RC and ARC did not affect the survival of fibroblast cell line in culture condition with an obvious decrease in weight.
However BC significantly decreased the survival rate of the fibroblast cell with only slightly degradation of the scaffold. In vivo study demonstrated that RC and ARC had totally degraded after 2 weeks imbedded in subcutaneous layer of SD rats and BC showed a pattern of slow degradation through a four-week observation. No allergic reaction or irritation on the skin of the rats was observed through the experiment for RC ARC and BC. Finally, the tissues of the embedded area were sampled by a punch of 8 mm in diameter after 4 weeks after imbedding of the scaffolds and section of the tissues revealed that RC and ARC were far more degradable than BC in both HE and PAS stains. The study suggested that the cell wall component derived from Rhizopus stolonifer F6 was biocompatible and biodegradable.
