組織工程研究中多孔性細胞支架是常用的細胞載體，而醣胺素及幾丁聚醣材料被認為是極具開發潛力的支架材料。本研究的主旨在利用實驗設計法設計供軟骨組織工程使用的醣胺素/幾丁聚醣(chitosan)生物模擬材料，並探討醣胺素(glycosaminoglycan; GAGs)對軟骨細胞增生、細胞外間質(extracellular matrix; ECM)產生及相關基因表現的影響，以進一步了解醣胺素調節軟骨細胞生理行為的作用機制。軟骨細胞於體外增生3代後即容易去分化，因此本研究首先利用部份因子實驗設計法評估醣胺素/幾丁聚醣薄膜中chondroitin-4-sulfate (CSA), chondroitin-6-sulfate (CSC), dermatan sulfate (DS)及heparin 等醣胺素對軟骨細胞分化、增生及ECM產生之影響，並探討何種醣胺素組成有利於體外培養軟骨細胞。結果顯示在實驗的劑量範圍，低劑量CSA (code -1、實際添加量2.6 mg)有利於軟骨細胞分泌膠原蛋白但不利細胞增生；高劑量CSC (code +1、實際添加量1.3 mg)有利於GAGs合成且不利細胞增生；高劑量DS (code +1、實際添加量0.13 mg)及heparin (code +1、實際添加量0.33 mg)有利於軟骨細胞增生但不利軟骨細胞產生膠原蛋白及GAGs產生。此外，不同種類的醣胺素間的交互作用亦是影響軟骨細胞的生理行為的原因之一。在8組醣胺素/幾丁聚醣薄膜中，含低劑量CSA、heparin(code -1、實際添加量0.033 mg)的N1及N4組醣胺素配方由於有利於維持軟骨細胞的形態、膠原蛋白及GAGs產生及可刺激軟骨細胞特殊標的基因的表現，因此顯示最具有應用於體外培養軟骨細胞的潛力。
第二階段的研究將使用的醣胺素種類由原本的4種減少為2種(CSC、DS)，希望藉由回應曲面實驗設計法的幫助，設計最適化的CSC/DS幾丁聚醣支架，並探討CSC及DS對軟骨組織再生的影響。結果顯示CSC/DS的組成並不會影響細胞支架的孔隙度及孔徑等物理性質及細胞貼附，但會影響軟骨細胞的形態、膠原蛋白及GAGs產生量及軟骨細胞的基因表現。此外，回應曲面分析顯示高劑量CSC及低劑量DS有利於膠原蛋白及GAGs產生，且最適合軟骨組織再生的CSC/DS/幾丁聚醣細胞支架其CSC、DS含量分別為2.8 mg及0.01 mg。另外，本研究亦證實在最適化CSC/DS組成中添加微量DS對軟骨組織再生有其必要性。最重要的是，本研究發現同時添加CSC/DS於幾丁聚醣細胞支架具刺激BMP2、TGF-β1、Ihh、PTHrP、Sox 9、aggrecan、collagen II、TIMP3及MMP13基因表現的效果、但卻抑制decorin及BMP4基因的表現。因此CSC/DS可能藉由調控BMP2、TGF-β1表現，影響BMP、TGF-β及Hedgehog等訊息傳遞路徑，進而影響ECM含量及相關基因表現。

Generally, scaffold is used as cell carrier for tissue engineering. Co-conjugating CSC/DS to chitosan is a potential scaffold material for cartilage tissue engineering. The aim of this study is to co-conjugate multiple glycosaminoglycans (GAGs) to 2D chitosan membranes and 3D porous chitosan scaffolds, hoping to more intimately mimic the natural extracellular matrix (ECM) for culturing chondrocyte in vitro and cartilage tissue engineering and further investigating the roles of GAGs in regulating ECM production and related gene expression. The chondroitin-4-sulfate (CSA), chondroitin-6-sulfate (CSC), dermatan sulfate (DS), and heparin were co-immobilized to chitosan membranes for maintaining the differentiation of monolayer chondrocytes culture and improving the ECM production in vitro. With the aid of 4-factor, 2-level 24-1 fractional factorial design, we formulated 8 GAG/chitosan compositions (groups N1-N8) which were found to profoundly influence chondrocyte behavior. Within the level range between -1 and +1, low levels of CSA (code -1; 2.6 mg), was desired for collagen production but undesired for cell proliferation. High level CSC (code +1; 1.3 mg) was favorable for GAG production but not for cell proliferation. Conversely high level DS (code +1; 0.13 mg) and heparin (code +1; 0.33 mg) were desired for cell proliferation but undesired for GAG and collagen production. Additionally, the interactions between GAG species affected collagen and GAG production too. Among the 8 GAGs/chitosan membranes, the ones with low CSA and heparin levels (N1 and N4) led to proper chondrocyte phenotype, as judged by chondrocyte-like morphology, modest cell expansion, higher GAG and collagen production and proper cartilage marker gene expression. This result indicates the potential of N1 and N4 GAGs formulation for culturing chondrocyte in vitro.
Since simultaneous quantification of 4 different GAGs in the 3D scaffolds is technically difficult, the number of GAG species was reduced to 2 (CSC and DS) to simplify the experimental design. To optimize the CSC/DS formulation and investigate the roles of CSC and DS in cartilage formation, Response Surface Methodology (RSM) was employed to design CSC/DS/chitosan scaffolds of various formulations. Conjugating CSC or DS did not affect the physical properties of scaffolds, cell adhesion and proliferation, but impacted collagen and GAGs production. Within the experimental range, the GAGs and collagen production were found to positively correlate with amount of CSC on scaffold, but were negatively correlated with that of DS. According to the analysis by RSM, co-conjugating CSC 2.8 mg/scaffold and DS 10 μg/scaffold to chitosan scaffold is optimal formulation for cartilage tissue engineering. Further result illustrates that the minor DS on the CSC/DS/chitosan scaffolds was needed for improving the collagen and GAGs production. Moreover, the optimal formulation of CSC/DS/chitosan scaffolds up-regulated the gene expression of BMP2、TGF-β1、Ihh、PTHrP、Sox9、aggrecan、collagen II、TIMP3 and MMP13, but down-regulated the decorin and BMP4 expression. These results indicate the co-conjugating CSC/DS to chitosan scaffold modulated ECM production and related gene expression in vitro by regulating the BMP、TGF-β and Hedgehog signaling transduction pathway.

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