幾丁聚醣在很多領域被證實是一種生物相容性高的材料，不但自然界含量豐富，取得容易，也可以被製作成各種臨床所需的樣式，實為一值得發展之天然資源，常用來當作骨組織工程的支架，但幾丁聚醣本身並不具有骨引導性。鹼性磷酸酶活性是一個廣泛被大家知道用來當作造骨母細胞分化性指標，它最主要的作用機制為降解焦磷酸鹽 (氫氧基磷灰石抑制物)，產生無機磷酸鹽(氫氧基磷灰石促進物)，而氫氧基磷灰石更是自然骨的主要成分之一。所以我們利用冷凍乾燥幾丁聚醣膠體製造出幾丁聚醣孔洞膜，並藉由EDC/NHS化學交聯劑將鹼性磷酸酶固定於幾丁聚醣薄膜上，在細胞實驗方面探討此複合材料對於造骨母細胞的增生與鈣化是否有促進的效果。實驗結果發現，利用冷凍乾燥方法製備的幾丁聚醣為多孔性，孔洞大小約為80~100μm。利用化學交聯方法成功的讓鹼性磷酸酶固定於幾丁聚醣薄膜並在35天後仍保持40%活性。在電子顯微鏡下觀察大鼠頭蓋骨造骨母細胞貼附於不同幾丁聚醣薄膜的形態，發現有固定鹼性磷酸酶的組別在第三天時出現了細胞聚集並有分泌基質包圍的現象，而單純幾丁聚醣的組別則在第七天才開始有此現象產生。在造骨母細胞的增生表現，利用MTS觀察到，有固定鹼性磷酸酶的組別，在第三天與第七天時所偵測到的細胞活性較只有幾丁聚醣以及吸附鹼性磷酸酶的組別低；而DAPI螢光染色無法觀察到確切的細胞貼附數量，但也發現在第三天時，固定鹼性磷酸酶組別最先觀察到有細胞聚集的現象。而在誘導造骨母細胞鈣化方面，我們利用Von kossa染色以及測定鈣離子濃度的方法分別觀察到，有固定鹼性磷酸酶的幾丁聚醣組別都會提早偵測到鈣質的沉積以及較高的鈣離子表現。此外，我們也利用了半定量之RT-PCR方法測定細胞貼附於不同組別幾丁聚醣薄膜上，對於骨生成表現基因Cbfa-1以及OCN的影響 ，結果顯示，固定鹼性磷酸酶的組別在不同的時間點都展現了較高的骨生成表現mRNA表現量。在細胞實驗中，我們證明了此種鹼性磷酸酶/幾丁聚醣的複合材料對於頭蓋骨造骨母細胞的增生沒有明顯的促進效果，但對於誘導造骨母細胞鈣化的效果明顯優於幾丁聚醣薄膜。因此，此種鹼性磷酸酶/幾丁聚醣的複合材料為具有潛力成為骨組織工程之再生支架。

Chitosan (CS) has been proved as a biocompatible material in several fields. It’s abundant in nature and easy to get for clinical use. Although chitosan is frequently used as a scaffold for bone tissue engineering, it still exists some limitations with its non-osteoconduction. Alkaline phosphatase (ALP) is a well-known marker for osteoblast differentiation. One of its primary mechanism is to promote bone formation by degrading inorganic pyrophosphate (PPi), an inhibitor of hydroxyapatite formation, and generating inorganic phosphate (Pi), an inducer of hydroxyapatite formation. The hydroxyapatite is one of the main components in natural bone. In this study we fabricated microporous chitosan membranes by freeze-drying of chitosan gels and immobilized alkaline phosphatase on membranes by using EDC /NHS (a chemical cross-linking agent) to investigate that whether the chitosan/ALP composite materials would promote the proliferation and mineralization of osteoblasts in vitro. The results demonstrated that the chitosan membranes fabricated by the freeze-drying method have three-dimensional porous and interconnected structures with an average pore size of 80~120 μm. By using chemical cross-link methods for alkaline phosphatase immobilization on chitosan membranes, we found that 40% of ALP activity remained on membranes after 35 days. Scanning electron microscopic (SEM) observation showed that the primary calvarial osteoblasts attached, spread and formed multiple layers on the surface of the chitosan membranes. We observed that the cells attached on the ALP-immobilized scaffolds began to aggregate and be surrounded by matrix at day 3, but we found the similar phenomenon in non–ALP -immobilized group at day 7. We also observed calvarial osteoblasts attachment and proliferation by DAPI staining and MTS assay. The results indicated that the cells proliferating on CS/ALP membranes were weaker than the chitosan alone and chitosan absorbed ALP group. The DAPI staining, just as the SEM observation, confirmed that CS/ALP group would promote cells aggregation at day 3. In addition, the ALP immobilized scaffolds showed strong staining in Von kossa mineralization assays and high calcium ion concentration in quantitative calcium assay. Furthermore, cells seeded on immobilized CS/ALP exhibited higher osteoblast marker gene (Cbfa-1 and OCN) expression compared to the CS alone group by semi-quantitative RT-PCR technique. In conclusion, we proved that the chitosan/ALP composite materials can breakthrough the non-osteoconductive limitation of chitosan membrane and promote the rat calvarial osteoblasts mineralization; therefore, this CS/ALP scaffold is considered as a new promising vehicle for bone tissue engineering applications.

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