Department of Orthopedics, University Medical Center Utrecht, Utrecht, The Netherlands.
Institute of Biological Chemistry, Biophysics and Bioengineering, School of Engineering and Physical Sciences, Heriot-Watt University, Edinburgh, UK.
Acta Biomater. 2018 Jan 15;66:238-247. doi: 10.1016/j.actbio.2017.11.029. Epub 2017 Nov 23.
Hydrogel-based 3D cell cultures are an emerging strategy for the regeneration of cartilage. In an attempt to regenerate dysfunctional intervertebral discs, nucleus pulposus (NP) cells can be cultured in hydrogels of various kinds and physical properties. Stiffness sensing through focal adhesions is believed to direct chondrogenesis, but the mechanisms by which this works are largely unknown. In this study we compared focal adhesion formation and glycosaminoglycan (GAG) deposition by NP cells in a range of hydrogels. Using a focal adhesion kinase (FAK) inhibitor, we demonstrated that focal adhesion signaling is involved in the response of NP cells in hydrogels that contain integrin binding sites (i.e. methacrylated gelatin (gelMA) and type II collagen), but not in hydrogels deplete from integrin binding sites such as alginate and agarose, or CD44-binding hydrogels based on hyaluronic acid. As a result of FAK inhibition we observedenhanced proteoglycan production in gelMA, but decreased production in type II collagen hydrogels, which could be explained by alteration in cell fate as supported by the increase in the adipogenic marker peroxisome proliferator-activated receptor gamma (PPARy). Furthermore, GAG deposition was inversely proportional to polymer concentration in integrin-binding gelMA, while no direct relationship was found for the non-integrin binding gels alginate and agarose. This corroborates our finding that focal adhesion formation plays an important role in NP cell response to its surrounding matrix.
Biomaterials are increasingly being investigated for regenerative medicine applications, including regeneration of the nucleus pulposus. Cells interact with their environment and are influenced by extracellular matrix or polymer properties. Insight in these interactions can improve regeneration and helps to understand degeneration processes. The role of focal adhesion formation in the regenerative response of nucleus pulposus cells is largely unknown. Therefore, the relation between materials, stiffness and focal adhesion formation is studied here.
水凝胶基 3D 细胞培养是软骨再生的一种新兴策略。为了尝试再生功能失调的椎间盘，核髓细胞（NP）可以在各种物理性质的水凝胶中培养。通过粘着斑感知硬度被认为可以指导软骨生成，但其中的机制在很大程度上尚不清楚。在这项研究中，我们比较了 NP 细胞在一系列水凝胶中的粘着斑形成和糖胺聚糖（GAG）沉积。使用粘着斑激酶（FAK）抑制剂，我们证明粘着斑信号参与了含有整合素结合位点的水凝胶（即甲基丙烯酰化明胶（gelMA）和 II 型胶原）中 NP 细胞的反应，但不参与缺乏整合素结合位点的水凝胶，如藻酸盐和琼脂糖，或基于透明质酸的 CD44 结合水凝胶。由于 FAK 抑制，我们观察到在 gelMA 中增强了蛋白聚糖的产生，但在 II 型胶原水凝胶中减少了产生，这可以通过细胞命运的改变来解释，这得到了脂肪生成标记物过氧化物酶体增殖物激活受体γ（PPARγ）增加的支持。此外，GAG 沉积与整合素结合的 gelMA 中的聚合物浓度成反比，而对于非整合素结合的藻酸盐和琼脂糖凝胶则没有发现直接关系。这证实了我们的发现，即粘着斑形成在 NP 细胞对周围基质的反应中起着重要作用。
生物材料越来越多地被用于再生医学应用，包括椎间盘的再生。细胞与其环境相互作用，并受到细胞外基质或聚合物特性的影响。了解这些相互作用可以改善再生并有助于理解退化过程。粘着斑形成在核髓细胞再生反应中的作用在很大程度上尚不清楚。因此，这里研究了材料、刚度和粘着斑形成之间的关系。
