| Summary: | 博士 === 國立陽明大學 === 醫學工程研究所 === 93 === Abstract
Both hyaluronic acid (HA) and collagen are major components of extracellular matrix (ECM) in the connective tissues and are of great value for the applications of tissue engineering. HA, a negatively charged polysacharride, has been used as a major component for fabricating anti-adhesion membrane. It is also used as the material to assist the regeneration of cartilage and skin. The aims of this study are (1) use of polygalacturonic acid (PGA) to replace HA for anti-adhesion membrane and (2) coupling of HA to collagen and used as the carrier for bone marrow mesenchymal stem cells.
Several commercial anti-adhesion membranes are currently available but not fully satisfied for clinical practice. In this work, three anti-adhesion films were prepared via (1) crosslinking of PGA with EDC (PGA-EDC film), (2) photo crosslinking of PGA with cinnamyl bromide (PGA-Cin film), (3) photo crosslinking of the mixture of PGA and HA (PGA/HA-Cin film). The PGA-EDC film has gel and water contents of 86 and 47%, respectively. Based on the data of 1H-NMR, PGA-Cin polymers had 5-14% of its D-galacturonic residues reacted with cinnamate groups, and it exhibited maximal absorption at 252 nm. When irradiated with UV light, PGA-Cin undergoes photoconversion and the reaction extent increases with irradiation time reaching about 80% in 12 min. On the other hand, the HA-Cin had 7% of its β-D glucose residues reacted with cinnamate group and displayed an absorption peak at 255 nm. The extent of crosslinking reached about 82.1% after 12 min of UV irradiation. The photo crosslinked films were evaluated for their anti-adhesion capabilities both in vitro and in vivo. All tested polymeric films did not show any evidence of cytotoxic effects. It was found that about 45-55% of the inoculated L929 cells attached to PGA-EDC film and 25-40% to PGA-Cin and PGA/HA-Cin film. When each membrane implanted in 21 rats, tissue adhesion occurred in 1 rat for PGA-EDC and 4 rats for PGA-CIN films, both are much lower than 11 rats out of 21 rats for the commercial product SeprafilmTM. The anti-adhesion potential of PGA/HA-Cin, with tissue adhesion in 14 rats out of 21 rats operated, was the worst in all membranes evaluated.
For constructing the materials for cartilage regeneration, we have prepared reconstituted type II collagen fibrils and modified with HA. We have synthesized (1) type II collagen fibril with diameter of about 150-250nm, (2) type II collagen fibrils with long chain HA (LHA, Mw 180 KD) covalently bound to the surface, and (3) type II collagen fibrils with short chain HA (SHA, Mw 14 KD) covalently bound to the surface. By changing the feed ratio of HA to collagen, type II collagen fibrils with different surface coverage of HA were obtained. The HA molecules immobilized on fibrillar surface of collagen are confirmed by the presence of surface bound nano gold particles tagged with wheat germ agglutinin (WGA) specific for HA. The number of nano gold-WGA particles bound to each 1 �慆2 LHA-collagen increases from 1420, 877 to 152 when the nominal surface HA coverage increases from 10%, 50% to 100%. With similar trend, the number of nano gold observed for SHA-collagen increases from 945, 884 to 221 for 10%, 50% and 100% coverage of SHA-collagen. The denatured temperature (Td) of HA-collagen is about 74℃ which is about 2℃ higher than the 72℃ of EDC crosslinked collagen. Collagen after modified with HA is more resistant to collagenase as shown by the decrease of digestion rate on HA-collagen. The extent of collagen released into the medium after 12 hr of collagenase treatment are: 90% (unmodified collagen), 53％ (nominal 10% LHA coverage), 33％ (nominal 50% LHA coverage) and 27％ (nominal 100% LHA coverage). With a similar trend for SHA-collagen, the extent of digestion in 12 hr decreases from 90% to 52％, 30％, and 27％. When treated with collagenase and HAase simultaneously for 12 hours, the digestion extents of collagen are 89％ for unmodified collagen, and 58％, 45％, 38％ for 10%, 50% and 100% of LHA coverage, respectively. With the same treatment on SHA-collagen, the digestion extents are 52%, 43% and 42%. These results indicate that both LHA and SHA have successfully coupled to type II collagen, and the resulted HA-collagen is more resistant to collagenase. Therefore, we can adjust the feed ratio of HA to collagen to synthesize various HA-collagen coupling products with different HA contents to control the loss and degradation of HA and collagen. The HA-collagen fibrils were used as carriers of chondrogenic cells and cultured in vitro. Human bone marrow stem cells (hBMSCs) were cultured with these three different carriers in a pellet system and induced into chondrogenesis by 10 ng/ml of TGF-β. The hBMSCs in each of the culture system recapitulated the sequential expression of genes that characterized chondrocyte differentiation, including Aggrecan, Col 2A1, Sox 9 and Col 1A1. The m-RNA of two chondrocyte specific markers - Aggrecan and Col 2A1 increased with the culturing time. The histological data indicate that the ECM organization obtained from culturing in type II collagen fibrils, LHA- type collagen II fibrils and SHA-type II collagen fibrils are all better than that without material groups.
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