Buxton P G, Bitar M, Gellynck K, Parkar M, Brown R A, Young A M, Knowles J C, Nazhat S N
Biomaterials and Tissue Engineering, UCL Eastman Dental Institute, 256 Gray's Inn Road, London, WC1X 8LD, UK.
Biomaterials and Tissue Engineering, UCL Eastman Dental Institute, 256 Gray's Inn Road, London, WC1X 8LD, UK; Materials Biology Interactions Group, Swiss Federal Laboratories for Materials Testing and Research (EMPA), Lerchenfeldstr 5, CH-9014 St. Gallen, Switzerland.
Bone. 2008 Aug;43(2):377-385. doi: 10.1016/j.bone.2008.03.028. Epub 2008 Apr 15.
Bone is distinguished from other tissues by its mechanical properties, in particular stiffness. However, we know little of how osteoblasts react to the stiffness of their microenvironment; in this study we describe their response to a dense (>10 wt.%) collagenous 3D environment. Primary pre-osteoblasts were seeded within a novel form of native collagen, dense collagen, and cultured for up to 14 days in the presence and absence of osteogenic supplements: analysis was via Q-PCR, histology, fluorescent in situ zymography, MMP loss-of-function and tensile testing. Differentiation as measured through the up-regulation of Bsp (247-fold), Alp (14.2-fold), Col1A1 (4.5-fold), Mmp-13 (8.0-fold) and Runx2 (1.2-fold) transcripts was greatly accelerated compared to 2D plastic at 7 and 14 days in the same medium. The scale of this enhancement was confirmed through the use of growth factor stimulation on 2D via the addition of BMP-6 and the Hedgehog agonist purmorphamine. In concert, these molecules were capable of the same level of osteo-induction (measured by Bsp and Alp expression) as the dense collagen alone. Mineralisation was initially localised to remodelled pericellular regions, but by 14 days embedded cells were discernible within regions of apatite (confirmed by MicroRaman). Tensile testing of the matrices showed that this had resulted in a significant increase in Young's modulus at low strain values, consistent with a stiffening of the matrix. To determine the need for matrix remodelling in the mineralisation event the broad spectrum MMP Inhibitor Ilomastat was used. It was found that in its presence mineralisation could still occur (though serum-specific) and the apoptosis associated with MMP inhibition in hydrated collagen gels was abrogated. Analysis of gene expression indicated that this was due to the up-regulation of Mmp-13 in the presence of Ilomastat in dense collagen (400-fold), demonstrating a powerful feedback loop and a potential mechanism for the rescue from apoptosis. Osteoid-like matrix (dense collagen) is therefore a potent stimulant of osteoblast differentiation in vitro and provides an environment that enables survival and differentiation in the presence of MMP inhibition.
骨骼因其机械性能，尤其是硬度，而与其他组织有所不同。然而，我们对成骨细胞如何响应其微环境的硬度知之甚少；在本研究中，我们描述了它们对致密（>10 wt.%）胶原三维环境的反应。将原代前成骨细胞接种于一种新型天然胶原——致密胶原中，并在添加和不添加成骨补充剂的情况下培养长达14天：通过定量聚合酶链反应（Q-PCR）、组织学、荧光原位酶谱分析、基质金属蛋白酶（MMP）功能丧失实验和拉伸试验进行分析。与在相同培养基中二维塑料上培养的情况相比，在第7天和第14天，通过骨唾液蛋白（Bsp，上调247倍）、碱性磷酸酶（Alp，上调14.2倍）、I型胶原α1链（Col1A1，上调4.5倍）、基质金属蛋白酶-13（Mmp-13，上调8.0倍）和Runx2（上调1.2倍）转录本上调所衡量的分化显著加速。通过在二维条件下添加骨形态发生蛋白-6（BMP-6）和刺猬因子激动剂嘌呤霉素进行生长因子刺激，证实了这种增强的程度。这些分子共同作用，能够产生与单独的致密胶原相同水平的骨诱导作用（通过Bsp和Alp表达来衡量）。矿化最初定位于重塑的细胞周围区域，但到第14天，在磷灰石区域内可辨别出包埋的细胞（通过显微拉曼光谱证实）。对基质的拉伸试验表明，这导致在低应变值下杨氏模量显著增加，这与基质变硬一致。为了确定矿化过程中基质重塑的必要性，使用了广谱MMP抑制剂异洛卡星。结果发现，在其存在的情况下矿化仍然可以发生（尽管具有血清特异性），并且与水合胶原凝胶中MMP抑制相关的细胞凋亡被消除。基因表达分析表明，这是由于在致密胶原中存在异洛卡星时Mmp-13上调（400倍），这证明了一个强大的反馈回路以及一种从细胞凋亡中挽救细胞的潜在机制。因此，类骨质样基质（致密胶原）是体外成骨细胞分化的有效刺激物，并提供了一种在MMP抑制存在的情况下能够实现存活和分化的环境。
