Examination of Hydrogels and Mesenchymal Stem Cell Sources for Bioprinting of Artificial Osteogenic Tissues
Mesenchymal stem cells (MSCs) represent a very important cell source in the field of regenerative medicine and for bone and cartilage tissue engineering applications. Three-dimensional (3D) bioprinting has the potential to improve the classical tissue engineering concept as this technique allows the printing of cells with high spatial control of cell allocation within a 3D construct. In this study, we systematically compared different hydrogel blends for 3D bioprinting of MSCs by testing their cytocompatibility, ability to support osteogenic differentiation and their mechanical properties. In addition, we compared four different MSC populations isolated from different human tissues for their osteogenic differentiation capacity in combination with different hydrogels. The aim of this study was to identify the best MSC source and the most suitable hydrogel blend for extrusion-based bioprinting of 3D large-scaled osteogenic constructs.
Materials and Methods
MSCs were isolated from different tissues (umbilical cord, adipose tissue, bone marrow). MSCs were seed onto or into different hydrogels and analyzed for cell viability, proliferation and osteogenic differentiation. In addition, viscoelastic properties of the hydrogels were determined. MSC-containing cubes with the size of 1 cm3 were printed by means of 3D extrusion-based bioprinting and analyzed by (immuno)histology for cell survival and production of a calcified extracellular matrix.
Adipose tissue derived MSCs (ASCs) showed the highest osteogenic differentiation potential. A complex hydrogel blend consisting of fibrin, gelatin, hyaluronic acid, glycerol (F/G/H/Gl), tuned with hydroxyapatite, showed the best viscoelastic properties in combination with an excellent biocompatibility towards ASCs. This cell/hydrogel combination was used to bioprint 3D cubes. The cubes showed good mechanical stability and the printed ASCs were viable and able to calcify the hydrogel after bioprinting.
The combination of the HA-tuned F/G/H/Gl hydrogel blend along with ASCs can be considered as a very promising bioink for 3D bioprinting of artificial bone tissue equivalents for prospective applications in tissue engineering and regenerative medicine.
KeywordsBiomaterial 3D bioprinting Hydrogel Mesenchymal stem cell Osteogenesis Biocompatibility
This work was supported by funding through the Deutsche Forschungsgemeinschaft (FI 790/10-1 and KO 3910/1-1) and the Bundesministerium für Bildung und Forschung (03VNE1034C and 03VNE1034B). The authors would like to thank Brunhilde Baumer for excellent technical assistance, Matthias Schieker for providing immortalized mesenchymal stem cells and Anja Eisenhardt for critical reading of the manuscript.
Conflict of interest
Maximilian Wehrle, Fritz Koch, Stefan Zimmermann, Peter Koltay, Roland Zengerle, G. Björn Stark, Sandra Strassburg and Günter Finkenzeller declare that they have no conflicts of interest.
No human studies and no animal experiments were carried out by the authors of this article.
- 1.Alamdari, O. G., E. Seyedjafari, M. Soleimani, and N. Ghaemi. Micropatterning of ECM proteins on glass substrates to regulate cell attachment and proliferation. Avicenna J. Med. Biotechnol. 5(4):234–240, 2013.Google Scholar
- 2.Amati, E., S. Sella, O. Perbellini, A. Alghisi, M. Bernardi, K. Chieregato, C. Lievore, D. Peserico, M. Rigno, A. Zilio, M. Ruggeri, F. Rodeghiero, and G. Astori. Generation of mesenchymal stromal cells from cord blood: evaluation of in vitro quality parameters prior to clinical use. Stem Cell Res. Ther. 8(1):14, 2017.Google Scholar
- 3.Beeravolu, N., C. McKee, A. Alamri, S. Mikhael, C. Brown, M. Perez-Cruet, and G. R. Chaudhry. Isolation and characterization of mesenchymal stromal cells from human umbilical cord and fetal placenta. J. Vis. Exp. 122:e55224, 2017.Google Scholar
- 4.Benning, L., L. Gutzweiler, K. Tröndle, J. Riba, R. Zengerle, P. Koltay, S. Zimmermann, G. B. Stark, and G. Finkenzeller. Cytocompatibility testing of hydrogels toward bioprinting of mesenchymal stem cells. J. Biomed. Mater. Res. A 105(12):3231–3241, 2017.Google Scholar
- 5.Blum, J. S., R. H. Li, A. G. Mikos, and M. A. Barry. An optimized method for the chemiluminescent detection of alkaline phosphatase levels during osteodifferentiation by bone morphogenetic protein 2. J. Cell. Biochem. 80(4):532–537, 2001.Google Scholar
- 6.Böcker, W., Z. Yin, I. Drosse, F. Haasters, O. Rossmann, M. Wierer, C. Popov, M. Locher, W. Mutschler, D. Docheva, and M. Schieker. Introducing a single-cell-derived human mesenchymal stem cell line expressing hTERT after lentiviral gene transfer. J. Cell. Mol. Med. 12(4):1347–1359, 2008.Google Scholar
- 7.Dominici, M., K. Le Blanc, I. Mueller, I. Slaper-Cortenbach, F. Marini, D. Krause, R. Deans, A. Keating, D. Prockop, and E. Horwitz. Minimal criteria for defining multipotent mesenchymal stromal cells. The International Society for Cellular Therapy position statement. Cytotherapy 8(4):315–317, 2006.Google Scholar
- 8.Duarte Campos, D. F., A. Blaeser, K. Buellesbach, K. S. Sen, W. Xun, W. Tillmann, and H. Fischer. Bioprinting organotypic hydrogels with improved mesenchymal stem cell remodeling and mineralization properties for bone tissue engineering. Adv. Healthc. Mater. 5(11):1336–1345, 2016.Google Scholar
- 9.Dumbleton, J., P. Agarwal, H. Huang, N. Hogrebe, R. Han, K. J. Gooch, and X. He. The effect of RGD peptide on 2D and miniaturized 3D culture of HEPM cells, MSCs, and ADSCs with alginate hydrogel. Cell. Mol. Bioeng. 9(2):277–288, 2016.Google Scholar
- 10.Engebretson, B., Z. R. Mussett, and V. I. Sikavitsas. Tenocytic extract and mechanical stimulation in a tissue-engineered tendon construct increases cellular proliferation and ECM deposition. Biotechnol. J. 12(3):1600595, 2017.Google Scholar
- 11.Engler, A. J., S. Sen, H. L. Sweeney, and D. E. Discher. Matrix elasticity directs stem cell lineage specification. Cell 126(4):677–689, 2006.Google Scholar
- 12.Hu, L., J. Hu, J. Zhao, J. Liu, W. Ouyang, C. Yang, N. Gong, L. Du, A. Khanal, and L. Chen. Side-by-side comparison of the biological characteristics of human umbilical cord and adipose tissue-derived mesenchymal stem cells. BioMed Res. Int. 2013:438243, 2013.Google Scholar
- 13.Hung, B. P., D. L. Hutton, K. L. Kozielski, C. J. Bishop, B. Naved, J. J. Green, A. I. Caplan, J. M. Gimble, A. H. Dorafshar, and W. L. Grayson. Platelet-derived growth factor BB enhances osteogenesis of adipose-derived but not bone marrow-derived mesenchymal stromal/stem cells. Stem Cells 33(9):2773–2784, 2015.Google Scholar
- 14.Kang, H.-W., S. J. Lee, I. K. Ko, C. Kengla, J. J. Yoo, and A. Atala. A 3D bioprinting system to produce human-scale tissue constructs with structural integrity. Nat. Biotechnol. 34(3):312–319, 2016.Google Scholar
- 15.Kolesky, D. B., K. A. Homan, M. A. Skylar-Scott, and J. A. Lewis. Three-dimensional bioprinting of thick vascularized tissues. Proc. Natl. Acad. Sci. USA 113(12):3179–3184, 2016.Google Scholar
- 16.Kon, E., A. Muraglia, A. Corsi, P. Bianco, M. Marcacci, I. Martin, A. Boyde, I. Ruspantini, P. Chistolini, M. Rocca, R. Giardino, R. Cancedda, and R. Quarto. Autologous bone marrow stromal cells loaded onto porous hydroxyapatite ceramic accelerate bone repair in critical-size defects of sheep long bones. J. Biomed. Mater. Res. 49(3):328–337, 2000.Google Scholar
- 17.Lecoeur, L., and J. P. Ouhayoun. In vitro induction of osteogenic differentiation from non-osteogenic mesenchymal cells. Biomaterials 18(14):989–993, 1997.Google Scholar
- 18.Li, C.-Y., X.-Y. Wu, J.-B. Tong, X.-X. Yang, J.-L. Zhao, Q.-F. Zheng, G.-B. Zhao, and Z.-J. Ma. Comparative analysis of human mesenchymal stem cells from bone marrow and adipose tissue under xeno-free conditions for cell therapy. Stem Cell Res. Ther. 6:55, 2015.Google Scholar
- 19.Mehlhorn, A. T., H. Schmal, S. Kaiser, G. Lepski, G. Finkenzeller, G. B. Stark, and N. P. Südkamp. Mesenchymal stem cells maintain TGF-beta-mediated chondrogenic phenotype in alginate bead culture. Tissue Eng. 12(6):1393–1403, 2006.Google Scholar
- 20.Murphy, S. V., and A. Atala. 3D bioprinting of tissues and organs. Nat. Biotechnol. 32(8):773–785, 2014.Google Scholar
- 21.Murphy, S. V., A. Skardal, and A. Atala. Evaluation of hydrogels for bio-printing applications. J. Biomed. Mater. Res. A 101(1):272–284, 2013.Google Scholar
- 22.Olivares-Navarrete, R., E. M. Lee, K. Smith, S. L. Hyzy, M. Doroudi, J. K. Williams, K. Gall, B. D. Boyan, and Z. Schwartz. Substrate stiffness controls osteoblastic and chondrocytic differentiation of mesenchymal stem cells without exogenous stimuli. PLoS ONE 12(1):e0170312, 2017.Google Scholar
- 23.Oryan, A., A. Kamali, A. Moshiri, and M. Baghaban Eslaminejad. Role of mesenchymal stem cells in bone regenerative medicine: what is the evidence? Cells Tissues Organs 204(2):59–83, 2017.Google Scholar
- 24.Ozbolat, I. T., and Y. Yu. Bioprinting toward organ fabrication: challenges and future trends. IEEE Trans. Bio-med. Eng. 60(3):691–699, 2013.Google Scholar
- 25.Pavlin, D., S. B. Dove, R. Zadro, and J. Gluhak-Heinrich. Mechanical loading stimulates differentiation of periodontal osteoblasts in a mouse osteoinduction model: effect on type I collagen and alkaline phosphatase genes. Calcif. Tissue Int. 67(2):163–172, 2000.Google Scholar
- 26.Pittenger, M. F., A. M. Mackay, S. C. Beck, R. K. Jaiswal, R. Douglas, J. D. Mosca, M. A. Moorman, D. W. Simonetti, S. Craig, and D. R. Marshak. Multilineage potential of adult human mesenchymal stem cells. Science 284(5411):143–147, 1999.Google Scholar
- 27.Rao, R. R., A. W. Peterson, and J. P. Stegemann. Osteogenic differentiation of adipose-derived and marrow-derived mesenchymal stem cells in modular protein/ceramic microbeads. J. Biomed. Mater. Res. A 101(6):1531–1538, 2013.Google Scholar
- 28.Schneider, A. K., G. Cama, M. Ghuman, F. J. Hughes, and B. Gharibi. Sprouty 2, an early response gene regulator of FosB and mesenchymal stem cell proliferation during mechanical loading and osteogenic differentiation. J. Cell. Biochem. 118(9):2606–2614, 2017.Google Scholar
- 29.Strassburg, S., N. Torio-Padron, G. Finkenzeller, A. Frankenschmidt, and G. B. Stark. Adipose-derived stem cells support lymphangiogenic parameters in vitro. J. Cell. Biochem. 117(11):2620–2629, 2016.Google Scholar
- 30.Sunthar, P. Polymer rheology. In: Rheology of Complex Fluids, edited by J. Krishnan, A. Deshpande, and P. Kumar. New York: Springer, 2010.Google Scholar
- 31.Tijore, A., J.-M. Behr, S. A. Irvine, V. Baisane, and S. Venkatraman. Bioprinted gelatin hydrogel platform promotes smooth muscle cell contractile phenotype maintenance. Biomed. Microdevices 20(2):32, 2018.Google Scholar
- 32.Wakitani, S., T. Goto, S. J. Pineda, R. G. Young, J. M. Mansour, A. I. Caplan, and V. M. Goldberg. Mesenchymal cell-based repair of large, full-thickness defects of articular cartilage. J. Bone Joint Surg. 76(4):579–592, 1994.Google Scholar
- 33.Witt, R., A. Weigand, A. M. Boos, A. Cai, D. Dippold, A. R. Boccaccini, D. W. Schubert, M. Hardt, C. Lange, A. Arkudas, R. E. Horch, and J. P. Beier. Mesenchymal stem cells and myoblast differentiation under HGF and IGF-1 stimulation for 3D skeletal muscle tissue engineering. BMC Cell Biol. 18(1):15, 2017.Google Scholar
- 34.Zajdel, A., M. Kałucka, E. Kokoszka-Mikołaj, and A. Wilczok. Osteogenic differentiation of human mesenchymal stem cells from adipose tissue and Wharton’s jelly of the umbilical cord. Acta Biochim Pol. 64(2):365–369, 2017.Google Scholar