Fluid shear stress and endothelial cells synergistically promote osteogenesis of mesenchymal stem cells via integrin β1-FAK-ERK1/2 pathway

Fluid shear stress and endothelial cells synergistically promote osteogenesis of mesenchymal stem cells via integrin β1-FAK-ERK1/2 pathway

Prevascularization and mechanical stimulation have been reported as effective methods for the construction of functional bone tissue. However, their combined effects on osteogenic differentiation and its mechanism remain to be explored. Here, the effects of fluid shear stress (FSS) on osteogenic differentiation of rat bone-marrow-derived mesenchymal stem cells (BMSCs) when cocultured with human umbilical vein endothelial cells (HUVECs) were investigated, and underlying signaling mechanisms were further explored. FSS stimulation for 1–4 h/day increased alkaline phosphatase (ALP) activity and calcium deposition in coculture systems and promoted the proliferation of cocultured cells. FSS stimulation for 2 h/day was selected as the optimized protocol according to osteogenesis in the coculture. In this situation, the mRNA levels of ALP, runt-related transcriptional factor 2 (Runx2) and osteocalcin (OCN), and protein levels of OCN and osteopontin (OPN) in BMSCs were upregulated. Furthermore, FSS and coculture with HUVECs synergistically increased integrin β1 expression in BMSCs and further activated focal adhesion kinases (FAKs) and downstream extracellular signalrelated kinase (ERK), leading to the enhancement of Runx2 expression. Blocking the phosphorylation of FAK abrogated FSS-induced ERK phosphorylation and inhibited osteogenesis of cocultured BMSCs. These results revealed that FSS and coculture with HUVECs synergistically promotes the osteogenesis of BMSCs, which was mediated by the integrin β1-FAK-ERK signaling pathway.

___

  • Barron MJ, Goldman J, Tsai CJ, Donahue SW (2012). Perfusion flow enhances osteogenic gene expression and the infiltration of osteoblasts and endothelial cells into three-dimensional calcium phosphate scaffolds. International Journal of Biomaterials 2012: 915620.
  • Chen CA, Chang JM, Chang EE, Chen HC, Yang YL (2018). TGFbeta1 modulates podocyte migration by regulating the expression of integrin-beta1 and -beta3 through different signaling pathways. Biomedicine & Pharmacotherapy 105: 974-980.
  • Corrigan MA, Johnson GP, Stavenschi E, Riffault M, Labour MN et al. (2018). TRPV4-mediates oscillatory fluid shear mechanotransduction in mesenchymal stem cells in part via the primary cilium. Scientific Reports 8: 3824.
  • Dahlin RL, Gershovich JG, Kasper FK, Mikos AG (2014). Flow perfusion co-culture of human mesenchymal stem cells and endothelial cells on biodegradable polymer scaffolds. Annals of Biomedical Eengineering 42 (7): 1381-90.
  • Elashry MI, Gegnaw ST, Klymiuk MC, Wenisch S, Arnhold S (2019). Influence of mechanical fluid shear stress on the osteogenic differentiation protocols for Equine adipose tissue-derived mesenchymal stem cells. Acta Histochemica 121: 344-353.
  • Fan X, Teng Y, Ye Z, Zhou Y, Tan WS (2018). The effect of gap junction-mediated transfer of miR-200b on osteogenesis and angiogenesis in a co-culture of MSCs and HUVECs. Journal of Cell Science 131.
  • Gusmão CVBd, Belangero WD (2009). How Do Bone Cells Sense Mechanical Loading? Revista Brasileira de Ortopedia 44: 299- 305.
  • Heo DN, Hospodiuk M, Ozbolat IT (2019). Synergistic interplay between human MSCs and HUVECs in 3D spheroids laden in collagen/fibrin hydrogels for bone tissue engineering. Acta Biomaterialia 95: 348-356.
  • Huang X, Das R, Patel A, Nguyen TD (2018). Physical Stimulations for Bone and Cartilage Regeneration. Regenerative Engineering and Translational Medicine 4: 216-237.
  • James BD, Allen JB (2018). Vascular Endothelial Cell Behavior in Complex Mechanical Microenvironments. ACS Biomaterials Science & Engineering 4: 3818-3842.
  • Jiang Y, Wang Y, Tang G (2016). Cyclic tensile strain promotes the osteogenic differentiation of a bone marrow stromal cell and vascular endothelial cell co-culture system. Archives of Biochemistry and Biophysics 607: 37-43.
  • Jiang YN, Zhao J, Chu FT, Jiang YY, Tang GH (2018). Tension-loaded bone marrow stromal cells potentiate the paracrine osteogenic signaling of co-cultured vascular endothelial cells. Biology Open 7.
  • Jin C, Tian H, Li J, Jia S, Li S et al. (2018). Stem cell education for medical students at Tongji University: Primary cell culture and directional differentiation of rat bone marrow mesenchymal stem cells. Biochemistry and Molecular Biology Education 46: 151-154.
  • Kang Y, Ren L, Yang Y (2014). Engineering vascularized bone grafts by integrating a biomimetic periosteum and beta-TCP scaffold. ACS Applied Materials & Interfaces 6: 9622-9633.
  • Kanno T, Takahashi T, Tsujisawa T, Ariyoshi W, Nishihara T (2007). Mechanical stress-mediated Runx2 activation is dependent on Ras/ERK1/2 MAPK signaling in osteoblasts. Journal of Cellular Biochemistry 101: 1266-1277.
  • Kocherova I, Bryja A, Mozdziak P, Angelova Volponi A, DyszkiewiczKonwinska M et al. (2019). Human Umbilical Vein Endothelial Cells (HUVECs) Co-Culture with Osteogenic Cells: From Molecular Communication to Engineering Prevascularised Bone Grafts. Journal of Clinical Medicine 8.
  • Kreke MR, Huckle WR, Goldstein AS (2005). Fluid flow stimulates expression of osteopontin and bone sialoprotein by bone marrow stromal cells in a temporally dependent manner. Bone 36: 1047-1055.
  • Kumar NM, Sigurdson SL, Sheppard D, Lwebuga-Mukasa JS (1995). Differential modulation of integrin receptors and extracellular matrix laminin by transforming growth factor-beta 1 in rat alveolar epithelial cells. Experimental Cell Research 221 (2): 385-94.
  • Li YJ, Batra NN, You L, Meier SC, Coe IA et al. (2004). Oscillatory fluid flow affects human marrow stromal cell proliferation and differentiation. Journal of Orthopaedic Research 22: 1283– 1289.
  • Li YS, Haga JH, Chien S (2005). Molecular basis of the effects of shear stress on vascular endothelial cells. Journal of Biomechanics 38: 1949-1971.
  • Li X, Liu C, Li P, Li S, Zhao Z et al. (2013). Connexin 43 is a potential regulator in fluid shear stress-induced signal transduction in osteocytes. Journal of Orthopaedic Research 31: 1959-1965.
  • Lim KT, Kim J, Seonwoo H, Chang JU, Choi H et al. (2013). Enhanced osteogenesis of human alveolar bone-derived mesenchymal stem cells for tooth tissue engineering using fluid shear stress in a rocking culture method. Tissue Engineering Part C Methods 19: 128-145.
  • Litzenberger JB, Kim JB, Tummala P, Jacobs CR (2010). Beta1 integrins mediate mechanosensitive signaling pathways in osteocytes. Calcified Tissue International 86: 325-332.
  • Liu L, Zong C, Li B, Shen D, Tang Z et al. (2014). The interaction between beta1 integrins and ERK1/2 in osteogenic differentiation of human mesenchymal stem cells under fluid shear stress modelled by a perfusion system. Journal of Tissue Engineering and Regenerative Medicine 8: 85-96.
  • Luo W, Xiong W, Zhou J, Fang Z, Chen W et al. (2011). Laminar shear stress delivers cell cycle arrest and anti-apoptosis to mesenchymal stem cells. Acta Biochimica et Biophysica Sinica 43: 210-216.
  • Malone AM, Batra NN, Shivaram G, Kwon RY, You L et al. (2007). The role of actin cytoskeleton in oscillatory fluid flow-induced signaling in MC3T3-E1 osteoblasts. American Journal of Physiology 292: C1830-1836.
  • Mengistu M, Brotzman H, Ghadiali S, Lowe-Krentz L (2011). Fluid shear stress-induced JNK activity leads to actin remodeling for cell alignment. Journal of Cellular Physiology 226: 110-121.
  • Negishi M, Lu D, Zhang YQ (2001). Upregulatory expression of furin and transforming growth factor-beta by fluid shear stress in vascular endothelial cells. Arteriosclerosis, Thrombosis and Vascular Biology 21: 785–790.
  • Piard C, Jeyaram A, Liu Y, Caccamese J, Jay SM et al. (2019). 3D printed HUVECs/MSCs cocultures impact cellular interactions and angiogenesis depending on cell-cell distance. Biomaterials 222: 119423.
  • Salgado CL, Barrias CC, Monteiro FJM (2020). Clarifying the ToothDerived Stem Cells Behavior in a 3D Biomimetic Scaffold for Bone Tissue Engineering Applications. Frontiers in Bioengineering and Biotechnology 8: 724.
  • Schlaepfer DD, Hauck CR, Sieg DJ (1999). Signaling through focal adhesion kinase. Progress in Biophysics and Molecular Biology 71: 435-478.
  • Schreiber C, Saraswati S, Harkins S, Gruber A, Cremers N et al. (2019). Loss of ASAP1 in mice impairs adipogenic and osteogenic differentiation of mesenchymal progenitor cells through dysregulation of FAK/Src and AKT signaling. PLoS Genetics 15: e1008216.
  • Stavenschi E, Labour MN, Hoey DA (2017). Oscillatory fluid flow induces the osteogenic lineage commitment of mesenchymal stem cells: The effect of shear stress magnitude, frequency, and duration. Journal of Biomechanics 55: 99-106.
  • Takada Y, Ye X, Simon S (2007). The integrins. Genome Biology 8: 215.
  • Thompson WR, Guilluy C, Xie Z, Sen B, Brobst KE et al. (2013). Mechanically activated Fyn utilizes mTORC2 to regulate RhoA and adipogenesis in mesenchymal stem cells. Stem Cells 31: 2528-2537.
  • Wang N, Tytell JD, Ingber DE (2009). Mechanotransduction at a distance: mechanically coupling the extracellular matrix with the nucleus. Nature Reviews Molecular Cell Biology 10: 75-82.
  • Weinbaum S, Cowin SC, Zeng Y (1994). A model for the excitation of osteocytes by mechanical loading-induced bone fluid shear stresses. Journal of Biomechanics 27: 339–360.
  • Wittkowske C, Reilly GC, Lacroix D, Perrault CM (2016). In Vitro Bone Cell Models: Impact of Fluid Shear Stress on Bone Formation. Frontiers in Bioengineering and Biotechnology 4.
  • Yang Y, Wang BK, Chang ML, Wan ZQ, Han GL (2018). Cyclic Stretch Enhances Osteogenic Differentiation of Human Periodontal Ligament Cells via YAP Activation. BioMed Research International 2018: 2174824.
  • Zuo B, Zhu J, Li J, Wang C, Zhao X et al. (2015). microRNA-103a functions as a mechanosensitive microRNA to inhibit bone formation through targeting Runx2. Journal of Bone and Mineral Research 30: 330-345.
  • Zhou X, Liu D, You L, Wang L (2010). Quantifying fluid shear stress in a rocking culture dish. Journal of Biomechanics 43: 1598- 1602.