Sefkow-Werner Julius, Machillot Paul, Sales Adria, Castro-Ramirez Elaine, Degardin Melissa, Boturyn Didier, Cavalcanti-Adam Elisabetta Ada, Albiges-Rizo Corinne, Picart Catherine, Migliorini Elisa
Grenoble Institute of Technology, Université Grenoble Alpes, LMGP UMR 5628, Grenoble, France; CEA, CNRS, UGA, BRM ERL 5000, Grenoble, France.
Université Grenoble Alpes, CNRS, DCM, Grenoble, France.
Acta Biomater. 2020 Sep 15;114:90-103. doi: 10.1016/j.actbio.2020.07.015. Epub 2020 Jul 13.
The chemical and physical properties of the extracellular matrix (ECM) are known to be fundamental for regulating growth factor bioactivity. The role of heparan sulfate (HS), a glycosaminoglycan, and of cell adhesion proteins (containing the cyclic RGD (cRGD) ligands) on bone morphogenetic protein 2 (BMP2)-mediated osteogenic differentiation has not been fully explored. In particular, it is not known whether and how their effects can be potentiated when they are presented in controlled close proximity, as in the ECM. Here, we developed streptavidin platforms to mimic selective aspects of the in vivo presentation of cRGD, HS and BMP2, with a nanoscale-control of their surface density and orientation to study cell adhesion and osteogenic differentiation. We showed that whereas a controlled increase in cRGD surface concentration upregulated BMP2 signaling due to β integrin recruitment, silencing either β or β integrins negatively affected BMP2-mediated phosphorylation of SMAD1/5/9 and alkaline phosphatase expression. Furthermore, the presence of adsorbed BMP2 promoted cellular adhesion at very low cRGD concentrations. Finally, we proved that HS co-immobilized with cRGD both sustained BMP2 signaling and enhanced osteogenic differentiation compared to BMP2 directly immobilized on streptavidin, even with a low cRGD surface concentration. Altogether, our results show that HS facilitated and sustained the synergy between BMP2 and integrin pathways and that the co-immobilization of HS and cRGD peptides optimised BMP2-mediated osteogenic differentiation. Statement of significance The growth factor BMP2 is used to treat large bone defects. Previous studies have shown that the presentation of BMP2 via extracellular matrix molecules, such as heparan sulfate (HS), can upregulate BMP2 signaling. The potential advantages of dose reduction and local specificity have stimulated interest in further investigations into biomimetic approaches. We designed a streptavidin model surface eligible for immobilizing tunable amounts of molecules from the extracellular space, such as HS, adhesion motifs (cyclic RGD) and BMP2. By studying cellular adhesion, BMP2 bioactivity and its osteogenic potential we reveal the combined effect of integrins, HS and BMP2, which contribute in answering fundamental questions regarding cell-matrix interaction.
细胞外基质（ECM）的化学和物理性质对于调节生长因子的生物活性至关重要。硫酸乙酰肝素（HS）（一种糖胺聚糖）和细胞粘附蛋白（含环状RGD（cRGD）配体）在骨形态发生蛋白2（BMP2）介导的成骨分化中的作用尚未得到充分研究。特别是，尚不清楚当它们如在ECM中那样以可控的紧密接近度呈现时，它们的作用是否以及如何能够增强。在此，我们开发了链霉亲和素平台，以模拟cRGD、HS和BMP2在体内呈现的选择性方面，并对它们的表面密度和取向进行纳米级控制，以研究细胞粘附和成骨分化。我们发现，虽然cRGD表面浓度的可控增加由于β整合素的募集而上调了BMP2信号传导，但沉默β1或β3整合素会对BMP2介导的SMAD1/5/9磷酸化和碱性磷酸酶表达产生负面影响。此外，吸附的BMP2的存在在非常低的cRGD浓度下促进了细胞粘附。最后，我们证明，与直接固定在链霉亲和素上的BMP2相比，即使在低cRGD表面浓度下，与cRGD共固定的HS既能维持BMP2信号传导，又能增强成骨分化。总之，我们的结果表明，HS促进并维持了BMP2和整合素途径之间的协同作用，并且HS和cRGD肽的共固定优化了BMP2介导的成骨分化。重要性声明生长因子BMP2用于治疗大的骨缺损。先前的研究表明，通过细胞外基质分子（如硫酸乙酰肝素（HS））呈现BMP2可以上调BMP2信号传导。剂量减少和局部特异性的潜在优势激发了人们对仿生方法进一步研究的兴趣。我们设计了一种链霉亲和素模型表面，适合固定来自细胞外空间的可调量分子，如HS、粘附基序（环状RGD）和BMP2。通过研究细胞粘附、BMP2生物活性及其成骨潜力，我们揭示了整合素、HS和BMP2的联合作用，这有助于回答有关细胞 - 基质相互作用的基本问题。
