キトサン/ゼラチン足場上での骨髄間葉系幹細胞の骨形成分化。遺伝子発現プロファイルと力学的解析

Osteogenic differentiation of bone marrow mesenchymal stem cells on chitosan/gelatin scaffolds: Gene expression profile and mechanical analysis.

• Fotios Papadogiannis
• Aristea Batsali
• Michail E Klontzas
• Maria M Karabela
• Anthie Georgopoulou
• Athanasios Mantalaris
• Nikolaos E Zafeiropoulos
• Maria Chatzinikolaidou
• Charalampos G Pontikoglou

抄録

本研究では、多孔質キトサン/ゼラチン(CS:Gel)足場内で骨形成分化を誘導したヒト骨髄間葉系幹細胞(BM-MSCs)が産生する細胞外マトリックスを、複数の遺伝子発現プロファイルと力学的挙動を調べることで検討した。その結果、骨形成関連遺伝子DLX5, RUNX2, ALP, OSCの発現が有意に上昇し、BM-MSCsの骨形成分化が効率的であることが確認された。これらの知見に基づき、骨形成分化の14日目には、CS:Gel足場上のBM-MSCsでOSCとCol1A1タンパク質の発現も検出された。次に、PCRアレイを用いて84種類の細胞接着および細胞外マトリックス（ECM）分子の発現を初めてプロファイリングした。その結果、足場に播種した BM-MSC では、コラゲン、インテグリン、ラミニン、ECM プロテアーゼ、カテニン、トロンボスポンジン、ECM プロテアーゼ阻害剤、細胞接着分子を含む 49 の遺伝子が、組織培養ポリスチレン対照と比較して異なる発現を示した。さらに、細胞を担持した足場の動的力学解析を0,7,14日目に実施し、生物学的結果と構築物の力学的挙動の相関関係を調べた。その結果、保存弾性率が0.5MPaであったのに対し、7日目には2MPaに増加し、14日目には0.8MPaで剛性が低下した。

In the present study we explore the extracellular matrix produced by human bone marrow mesenchymal stem/stromal cells (BM-MSCs) induced to undergo osteogenic differentiation within porous chitosan/gelatin (CS:Gel) scaffolds by investigating their multiple gene expression profile and mechanical behavior. Initially, the efficiency of the BM-MSCs osteogenic differentiation within the constructs was confirmed by the significant rise in the expression of the osteogenesis associated genes DLX5, RUNX2, ALP and OSC. In line with these findings, OSC and Col1A1 protein expression was also detected in BM-MSCs on the CS:Gel scaffolds at day 14 of osteogenic differentiation. We then profiled, for the first time, the expression of 84 cell adhesion and extracellular matrix (ECM) molecules using PCR arrays. The arrays, which were conducted at day 14 of osteogenic differentiation, demonstrated that 49 genes including collagens, integrins, laminins, ECM proteases, catenins, thrombospondins, ECM protease inhibitors and cell-cell adhesion molecules were differentially expressed in BM-MSCs seeded on scaffolds compared to tissue culture polystyrene control. Moreover, we performed dynamic mechanical analysis of the cell-loaded scaffolds on days 0, 7 and 14 to investigate the correlation between the biological results and the mechanical behavior of the constructs. Our data demonstrate a significant increase in the stiffness of the constructs with storage modulus values of 2 MPa on day 7, compared to 0.5 MPa on day 0, following a drop of the stiffness at 0.8 MPa on day 14, that may be attributed to the significant increase of specific ECM protease gene expression such as MMP1, MMP9, MMP11 and MMP16 at this time period.

© 2020 IOP Publishing Ltd.