Effect of electrical stimulation on chondrogenic differentiation of mesenchymal stem cells cultured in hyaluronic acid - Gelatin injectable hydrogels

[EN] Electrical stimulation (ES) has provided enhanced chondrogenesis of mesenchymal stem cells (MSCs) cultured in micro-mass without the addition of exogenous growth factors. In this study, we demonstrate for the first time that ES of MSCs encapsulated in an injectable hyaluronic acid (HA) - gelatin (GEL) mixture enhances the chondrogenic potential of the hydrogel. Samples were stimulated for 21 days with 10 mV/cm at 60 kHz, applied for 30 min every 6 h a day. Mechanical properties of hydrogels were higher if the precursors were dissolved in Calcium-Free Krebs Ringer Buffer (G' = 1141 +/- 23 Pa) compared to those diluted in culture media (G' = 213 +/- 19 Pa). Cells within stimulated hydrogels were rounder (55%) than non-stimulated cultures (32%) (p = 0.005). Chondrogenic markers such as SOX-9 and aggrecan were higher in stimulated hydrogels compared to controls. The ES demonstrated that normalized content of glycosaminoglycans and collagen to DNA was slightly higher in stimulated samples. Additionally, collagen type II normalized to total collagen was 2.43 times higher in stimulated hydrogels. These findings make ES a promising tool for enhancing articular cartilage tissue engineering outcomes by combining hydrogels and MSCs.The financial support received from COLCIENCIAS through Fellowship No. 647 and Grant 712-2015 No. 50457 is acknowledged, as is that from the Spain Ministry of Economy and Competitiveness through the MAT2016-76039-C4-1-R project (including the Feder Funds).Vaca-González, JJ.; Clara-Trujillo, S.; Guillot-Ferriols, MT.; Ródenas Rochina, J.; Sanchis Sánchez, MJ.; Gómez Ribelles, JL.; Garzón-Alvarado, DA.... (2020). Effect of electrical stimulation on chondrogenic differentiation of mesenchymal stem cells cultured in hyaluronic acid - Gelatin injectable hydrogels. Bioelectrochemistry. 134:1-11. https://doi.org/10.1016/j.bioelechem.2020.107536111134Vaca-González, J. J., Guevara, J. M., Moncayo, M. A., Castro-Abril, H., Hata, Y., & Garzón-Alvarado, D. A. (2017). Biophysical Stimuli: A Review of Electrical and Mechanical Stimulation in Hyaline Cartilage. CARTILAGE, 10(2), 157-172. doi:10.1177/1947603517730637Awad, H. A., Quinn Wickham, M., Leddy, H. A., Gimble, J. M., & Guilak, F. (2004). Chondrogenic differentiation of adipose-derived adult stem cells in agarose, alginate, and gelatin scaffolds. Biomaterials, 25(16), 3211-3222. doi:10.1016/j.biomaterials.2003.10.045Akanji, O. O., Lee, D. A., & Bader, D. A. (2008). The effects of direct current stimulation on isolated chondrocytes seeded in 3D agarose constructs. Biorheology, 45(3-4), 229-243. doi:10.3233/bir-2008-0473Burnsed, O. A., Schwartz, Z., Marchand, K. O., Hyzy, S. L., Olivares-Navarrete, R., & Boyan, B. D. (2016). Hydrogels derived from cartilage matrices promote induction of human mesenchymal stem cell chondrogenic differentiation. Acta Biomaterialia, 43, 139-149. doi:10.1016/j.actbio.2016.07.034Singh, D., Tripathi, A., Zo, S., Singh, D., & Han, S. S. (2014). Synthesis of composite gelatin-hyaluronic acid-alginate porous scaffold and evaluation for in vitro stem cell growth and in vivo tissue integration. Colloids and Surfaces B: Biointerfaces, 116, 502-509. doi:10.1016/j.colsurfb.2014.01.049Kim, I. L., Mauck, R. L., & Burdick, J. A. (2011). Hydrogel design for cartilage tissue engineering: A case study with hyaluronic acid. Biomaterials, 32(34), 8771-8782. doi:10.1016/j.biomaterials.2011.08.073Moulisová, V., Poveda-Reyes, S., Sanmartín-Masiá, E., Quintanilla-Sierra, L., Salmerón-Sánchez, M., & Gallego Ferrer, G. (2017). Hybrid Protein–Glycosaminoglycan Hydrogels Promote Chondrogenic Stem Cell Differentiation. ACS Omega, 2(11), 7609-7620. doi:10.1021/acsomega.7b01303Levett, P. A., Melchels, F. P. W., Schrobback, K., Hutmacher, D. W., Malda, J., & Klein, T. J. (2014). A biomimetic extracellular matrix for cartilage tissue engineering centered on photocurable gelatin, hyaluronic acid and chondroitin sulfate. Acta Biomaterialia, 10(1), 214-223. doi:10.1016/j.actbio.2013.10.005Chen, Y.-C., Su, W.-Y., Yang, S.-H., Gefen, A., & Lin, F.-H. (2013). In situ forming hydrogels composed of oxidized high molecular weight hyaluronic acid and gelatin for nucleus pulposus regeneration. Acta Biomaterialia, 9(2), 5181-5193. doi:10.1016/j.actbio.2012.09.039Murphy, C. M., Matsiko, A., Haugh, M. G., Gleeson, J. P., & O’Brien, F. J. (2012). Mesenchymal stem cell fate is regulated by the composition and mechanical properties of collagen–glycosaminoglycan scaffolds. Journal of the Mechanical Behavior of Biomedical Materials, 11, 53-62. doi:10.1016/j.jmbbm.2011.11.009Shu, X. Z., Liu, Y., Palumbo, F., & Prestwich, G. D. (2003). Disulfide-crosslinked hyaluronan-gelatin hydrogel films: a covalent mimic of the extracellular matrix for in vitro cell growth. Biomaterials, 24(21), 3825-3834. doi:10.1016/s0142-9612(03)00267-9Vanderhooft, J. L., Alcoutlabi, M., Magda, J. J., & Prestwich, G. D. (2009). Rheological Properties of Cross-Linked Hyaluronan-Gelatin Hydrogels for Tissue Engineering. Macromolecular Bioscience, 9(1), 20-28. doi:10.1002/mabi.200800141Camci-Unal, G., Cuttica, D., Annabi, N., Demarchi, D., & Khademhosseini, A. (2013). Synthesis and Characterization of Hybrid Hyaluronic Acid-Gelatin Hydrogels. Biomacromolecules, 14(4), 1085-1092. doi:10.1021/bm3019856Puetzer, J. L., Petitte, J. N., & Loboa, E. G. (2010). Comparative Review of Growth Factors for Induction of Three-DimensionalIn VitroChondrogenesis in Human Mesenchymal Stem Cells Isolated from Bone Marrow and Adipose Tissue. Tissue Engineering Part B: Reviews, 16(4), 435-444. doi:10.1089/ten.teb.2009.0705Hwang, N. S., Kim, M. S., Sampattavanich, S., Baek, J. H., Zhang, Z., & Elisseeff, J. (2006). Effects of Three-Dimensional Culture and Growth Factors on the Chondrogenic Differentiation of Murine Embryonic Stem Cells. Stem Cells, 24(2), 284-291. doi:10.1634/stemcells.2005-0024Balint, R., Cassidy, N. J., & Cartmell, S. H. (2013). Electrical Stimulation: A Novel Tool for Tissue Engineering. Tissue Engineering Part B: Reviews, 19(1), 48-57. doi:10.1089/ten.teb.2012.0183N. Tandon, et al., Alignment and elongation of human adipose-derived stem cells in response to direct-current electrical stimulation, in: 2009 Annual International Conference of the IEEE Engineering in Medicine and Biology Society, 2009, pp. 6517–6521.Zhao, Z., Watt, C., Karystinou, A., Roelofs, A., McCaig, C., … De Bari, C. (2011). Directed migration of human bone marrow mesenchymal stem cells in a physiological direct current electric field. European Cells and Materials, 22, 344-358. doi:10.22203/ecm.v022a26Banks, T. A., Luckman, P. S. B., Frith, J. E., & Cooper-White, J. J. (2015). Effects of electric fields on human mesenchymal stem cell behaviour and morphology using a novel multichannel device. Integrative Biology, 7(6), 693-712. doi:10.1039/c4ib00297kHernández-Bule, M. L., Paíno, C. L., Trillo, M. Á., & Úbeda, A. (2014). Electric Stimulation at 448 kHz Promotes Proliferation of Human Mesenchymal Stem Cells. Cellular Physiology and Biochemistry, 34(5), 1741-1755. doi:10.1159/000366375Xu, J., Wang, W., Clark, C. C., & Brighton, C. T. (2009). Signal transduction in electrically stimulated articular chondrocytes involves translocation of extracellular calcium through voltage-gated channels. Osteoarthritis and Cartilage, 17(3), 397-405. doi:10.1016/j.joca.2008.07.001Taghian, T., Narmoneva, D. A., & Kogan, A. B. (2015). Modulation of cell function by electric field: a high-resolution analysis. Journal of The Royal Society Interface, 12(107), 20150153. doi:10.1098/rsif.2015.0153Sundelacruz, S., Levin, M., & Kaplan, D. L. (2009). Role of Membrane Potential in the Regulation of Cell Proliferation and Differentiation. Stem Cell Reviews and Reports, 5(3), 231-246. doi:10.1007/s12015-009-9080-2Rodenas-Rochina, J., Kelly, D. J., Gómez Ribelles, J. L., & Lebourg, M. (2016). Compositional changes to synthetic biodegradable scaffolds modulate the influence of hydrostatic pressure on chondrogenesis of mesenchymal stem cells. Biomedical Physics & Engineering Express, 2(3), 035005. doi:10.1088/2057-1976/2/3/035005Poveda-Reyes, S., Moulisova, V., Sanmartín-Masiá, E., Quintanilla-Sierra, L., Salmerón-Sánchez, M., & Ferrer, G. G. (2016). Gelatin-Hyaluronic Acid Hydrogels with Tuned Stiffness to Counterbalance Cellular Forces and Promote Cell Differentiation. Macromolecular Bioscience, 16(9), 1311-1324. doi:10.1002/mabi.201500469Sanmartín-Masiá, E., Poveda-Reyes, S., & Gallego Ferrer, G. (2016). Extracellular matrix–inspired gelatin/hyaluronic acid injectable hydrogels. International Journal of Polymeric Materials and Polymeric Biomaterials, 66(6), 280-288. doi:10.1080/00914037.2016.1201828Shu, X. Z., Liu, Y., Luo, Y., Roberts, M. C., & Prestwich, G. D. (2002). Disulfide Cross-Linked Hyaluronan Hydrogels. Biomacromolecules, 3(6), 1304-1311. doi:10.1021/bm025603cThorpe, S. D., Buckley, C. T., Vinardell, T., O’Brien, F. J., Campbell, V. A., & Kelly, D. J. (2008). Dynamic compression can inhibit chondrogenesis of mesenchymal stem cells. Biochemical and Biophysical Research Communications, 377(2), 458-462. doi:10.1016/j.bbrc.2008.09.154Lennon, D. P., & Caplan, A. I. (2006). Isolation of human marrow-derived mesenchymal stem cells. Experimental Hematology, 34(11), 1604-1605. doi:10.1016/j.exphem.2006.07.014Vaca-González, J. J., Guevara, J. M., Vega, J. F., & Garzón-Alvarado, D. A. (2015). An In Vitro Chondrocyte Electrical Stimulation Framework: A Methodology to Calculate Electric Fields and Modulate Proliferation, Cell Death and Glycosaminoglycan Synthesis. Cellular and Molecular Bioengineering, 9(1), 116-126. doi:10.1007/s12195-015-0419-2Vaca-González, J. J., Escobar, J. F., Guevara, J. M., Hata, Y. A., Gallego Ferrer, G., & Garzón-Alvarado, D. A. (2019). Capacitively coupled electrical stimulation of rat chondroepiphysis explants: A histomorphometric analysis. Bioelectrochemistry, 126, 1-11. doi:10.1016/j.bioelechem.2018.11.004Casado, J. G., Gomez-Mauricio, G., Alvarez, V., Mijares, J., Tarazona, R., Bernad, A., & Sanchez-Margallo, F. M. (2012). Comparative phenotypic and molecular characterization of porcine mesenchymal stem cells from different sources for translational studies in a large animal model. Veterinary Immunology and Immunopathology, 147(1-2), 104-112. doi:10.1016/j.vetimm.2012.03.015Donnelly, P. E., Chen, T., Finch, A., Brial, C., Maher, S. A., & Torzilli, P. A. (2017). Photocrosslinked tyramine-substituted hyaluronate hydrogels with tunable mechanical properties improve immediate tissue-hydrogel interfacial strength in articular cartilage. Journal of Biomaterials Science, Polymer Edition, 28(6), 582-600. doi:10.1080/09205063.2017.1289035Pietrucha, K., & Marzec, E. (2005). Dielectric properties of the collagen–glycosaminoglycans scaffolds in the temperature range of thermal decomposition. Biophysical Chemistry, 118(1), 51-56. doi:10.1016/j.bpc.2005.07.006Dvořáková, J., Kučera, L., Kučera, J., Švík, K., Foglarová, M., Muthný, T., … Kubala, L. (2013). Chondrogenic differentiation of mesenchymal stem cells in a hydrogel system based on an enzymatically crosslinked tyramine derivative of hyaluronan. Journal of Biomedical Materials Research Part A, 102(10), 3523-3530. doi:10.1002/jbm.a.35033Chung, C., & Burdick, J. A. (2009). Influence of Three-Dimensional Hyaluronic Acid Microenvironments on Mesenchymal Stem Cell Chondrogenesis. Tissue Engineering Part A, 15(2), 243-254. doi:10.1089/ten.tea.2008.0067Salamon, A., van Vlierberghe, S., van Nieuwenhove, I., Baudisch, F., Graulus, G.-J., Benecke, V., … Peters, K. (2014). Gelatin-Based Hydrogels Promote Chondrogenic Differentiation of Human Adipose Tissue-Derived Mesenchymal Stem Cells In Vitro. Materials, 7(2), 1342-1359. doi:10.3390/ma7021342Ogawa, T., Akazawa, T., & Tabata, Y. (2010). In Vitro Proliferation and Chondrogenic Differentiation of Rat Bone Marrow Stem Cells Cultured with Gelatin Hydrogel Microspheres for TGF-β1 Release. Journal of Biomaterials Science, Polymer Edition, 21(5), 609-621. doi:10.1163/156856209x434638J.F. Escobar, Evaluación in vitro del efecto de una estimulación con campos magnéticos a condrocitos, Universidad Nacional de Colombia, 2019