Immobilization of alkaline phosphatase on microporous chitosan membranes for bone tissue engineering

• Main Authors: Wen-chun Hsieh, 謝妏君
• Other Authors: Po-da Hong
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/7eky2r

碩士 === 國立臺灣科技大學 === 醫學工程研究所 === 99 === 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.