Abstract
Chitosan/β-glycerophosphate/collagen (C/GP/Co) is a promising injectable scaffold in the bone tissue engineering. In this study, we prepared this scaffold and evaluated its biocompatibility and effects on the osteogenic differentiation of mesenchymal stem cells (MSCs). After fabrication, the C/GP/Co hydrogel was examined in a scanning electron microscope (SEM) and showed a porous microstructure. Its biocompatibility was assessed by cell morphology and cell viability assays. Cell morphological observations were performed by fluorescent microscope in 2D cultivation and by laser confocal scanning microscope (LCSM) in 3D cultivation, respectively. Cell viability in 2D and that in 3D cultivation were both evaluated by the Cell Counting Kit-8 (CCK-8) assay. Its effect on osteogenic differentiation of MSCs in vitro was clarified by alkaline phosphatase (ALP) activity, Alizarin Red staining, and real-time polymerase chain reaction (Real-time PCR). An additional experiment of the ectopic bone formation in nude mice was conducted to investigate its effects on osteogenic differentiation of MSCs after subcutaneous injection. The results proved that C/GP/Co hydrogel exhibited good biocompatibility and enhanced the in vitro osteogenic differentiation of MSCs. In the experiment of ectopic bone formation, this hydrogel demonstrated its capability of supporting neovascularization and differentiation of MSCs toward osteogenic lineage. Therefore, C/GP/Co hydrogel scaffold holds a great promise for the bone tissue engineering applications.
Similar content being viewed by others
References
Chenite, A., C. Chaput, D. Wang, C. Combes, M. D. Buschmann, C. D. Hoemann, J. C. Leroux, B. L. Atkinson, F. Binette, and A. Selmani (2000) Novel injectable neutral solutions of chitosan form biodegradable gels in situ. Biomaterials 21: 2155–2161.
Ahmadi, R. and J. D. de Bruijn (2008) Biocompatibility and gelation of chitosan-glycerol phosphate hydrogels. J. Biomed. Mater. Res. A 86: 824–832.
Cho, M. H., K. S. Kim, H. H. Ahn, M. S. Kim, S. H. Kim, G. Khang, B. Lee, and H. B. Lee (2008) Chitosan gel as an in situforming scaffold for rat bone marrow mesenchymal stem cells in vivo. Tissue Eng. Part A 14: 1099–1108.
Richardson, S. M., N. Hughes, J. A. Hunt, A. J. Freemont, and J. A. Hoyland (2008) Human mesenchymal stem cell differentiation to NP-like cells in chitosan-glycerophosphate hydrogels. Biomaterials 29: 85–93.
Kim, K. S., J. H. Lee, H. H. Ahn, J. Y. Lee, G. Khang, B. Lee, H. B. Lee, and M. S. Kim (2008) The osteogenic differentiation of rat muscle-derived stem cells in vivo within in situ-forming chitosan scaffolds. Biomaterials 29: 4420–4428.
Wang, L. and J. P. Stegemann (2010) Thermogelling chitosan and collagen composite hydrogels initiated with beta-glycerophosphate for bone tissue engineering. Biomaterials 31: 3976–3985.
Song, K., M. Qiao, T. Liu, B. Jiang, H. M. Macedo, X. Ma, and Z. Cui (2010) Preparation, fabrication and biocompatibility of novel injectable temperature-sensitive chitosan/glycerophosphate/collagen hydrogels. J. Mater. Sci. Mater. Med. 21: 2835–2842.
Sun, B., W. Ma, F. Su, Y. Wang, J. Liu, D. Wang, and H. Liu (2011) The osteogenic differentiation of dog bone marrow mesenchymal stem cells in a thermo-sensitive injectable chitosan/collagen/beta-glycerophosphate hydrogel: In vitro and in vivo. J. Mater. Sci. Mater. Med. 22: 2111–2118.
Glowacki, J. and S. Mizuno (2008) Collagen scaffolds for tissue engineering. Biopolymers 89: 338–344.
Wang, G., L. Zheng, H. Zhao, J. Miao, C. Sun, N. Ren, J. Wang, H. Liu, and X. Tao (2011) In vitro assessment of the differentiation potential of bone marrow-derived mesenchymal stem cells on genipin-chitosan conjugation scaffold with surface hydroxyapatite nanostructure for bone tissue engineering. Tissue Eng. Part A 17: 1341–1349.
Wang, H., Y. Li, Y. Zuo, J. Li, S. Ma, and L. Cheng (2007) Biocompatibility and osteogenesis of biomimetic nano-hydroxyapatite/polyamide composite scaffolds for bone tissue engineering. Biomaterials 28: 3338–3348.
Zhi, L., C. Chen, X. Pang, H. Uludag, and H. Jiang (2011) Synergistic effect of recombinant human bone morphogenic protein-7 and osteogenic differentiation medium on human bone-marrow-derived mesenchymal stem cells in vitro. Int. Orthop. 35: 1889–1895.
Rafat, M., F. Li, P. Fagerholm, N. S. Lagali, M. A. Watsky, R. Munger, T. Matsuura, and M. Griffith (2008) PEG-stabilized carbodiimide crosslinked collagen-chitosan hydrogels for corneal tissue engineering. Biomaterials 29: 3960–3972.
Raghunath, J., J. Rollo, K. M. Sales, P. E. Butler, and A. M. Seifalian (2007) Biomaterials and scaffold design: Key to tissue-engineering cartilage. Biotechnol. Appl. Biochem. 46: 73–84.
Liao, H. T., C. T. Chen, and J. P. Chen (2011) Osteogenic differentiation and ectopic bone formation of canine bone marrowderived mesenchymal stem cells in injectable thermo-responsive polymer hydrogel. Tissue Eng. Part C Methods 17: 1139–1149.
Sun, H., K. Feng, J. Hu, S. Soker, A. Atala, and P. X. Ma (2010) Osteogenic differentiation of human amniotic fluid-derived stem cells induced by bone morphogenetic protein-7 and enhanced by nanofibrous scaffolds. Biomaterials 31: 1133–1139.
Gauthaman, K., J. R. Venugopal, F. C. Yee, A. Biswas, S. Ramakrishna, and A. Bongso (2011) Osteogenic differentiation of human Wharton’s jelly stem cells on nanofibrous substrates in vitro. Tissue Eng. Part A 17: 71–81.
Kim, I. Y., S. J. Seo, H. S. Moon, M. K. Yoo, I. Y. Park, B. C. Kim, and C. S. Cho (2008) Chitosan and its derivatives for tissue engineering applications. Biotechnol. Adv. 26: 1–21.
Zhang, Y. and M. Zhang (2001) Synthesis and characterization of macroporous chitosan/calcium phosphate composite scaffolds for tissue engineering. J. Biomed. Mater. Res. 55: 304–312.
Kawakami, T., M. Antoh, H. Hasegawa, T. Yamagishi, M. Ito, and S. Eda (1992) Experimental study on osteoconductive properties of a chitosan-bonded hydroxyapatite self-hardening paste. Biomaterials 13: 759–763.
Di, M. A., M. Sittinger, and M. V. Risbud (2005) Chitosan: A versatile biopolymer for orthopaedic tissue-engineering. Biomaterials 26: 5983–5990.
Bear, M., M. Butcher, and S. G. Shaughnessy (2008) Oxidized low-density lipoprotein acts synergistically with beta-glycerophosphate to induce osteoblast differentiation in primary cultures of vascular smooth muscle cells. J. Cell. Biochem. 105: 185–193.
Alonso, M., S. Claros, J. Becerra, and J. A. Andrades (2008) The effect of type I collagen on osteochondrogenic differentiation in adipose-derived stromal cells in vivo. Cytotherapy 10: 597–610.
Kihara, T., M. Hirose, A. Oshima, and H. Ohgushi (2006) Exogenous type I collagen facilitates osteogenic differentiation and acts as a substrate for mineralization of rat marrow mesenchymal stem cells in vitro. Biochem. Biophys. Res. Commun. 341: 1029–1035.
Kruger, E. A., D. D. Im, D. S. Bischoff, C. T. Pereira, W. Huang, G. H. Rudkin, D. T. Yamaguchi, and T. A. Miller (2011) In vitro mineralization of human mesenchymal stem cells on threedimensional type I collagen versus PLGA scaffolds: a comparative analysis. Plast. Reconstr. Surg. 127: 2301–2311.
Ryu, Y. M., Y. S. Hah, B. W. Park, D. R. Kim, G. S. Roh, J. R. Kim, U. K. Kim, G. J. Rho, G. H. Maeng, and J. H. Byun (2011) Osteogenic differentiation of human periosteal-derived cells in a three-dimensional collagen scaffold. Mol. Biol. Rep. 38: 2887–2894.
Tsai, K. S., S. Y. Kao, C. Y. Wang, Y. J. Wang, J. P. Wang, and S. C. Hung (2010) Type I collagen promotes proliferation and osteogenesis of human mesenchymal stem cells via activation of ERK and Akt pathways. J. Biomed. Mater. Res. A 94: 673–682.
Mathews, S., R. Bhonde, P. K. Gupta, and S. Totey (2011) A novel tripolymer coating demonstrating the synergistic effect of chitosan, collagen type 1 and hyaluronic acid on osteogenic differentiation of human bone marrow derived mesenchymal stem cells. Biochem. Biophys. Res. Commun. 414: 270–276.
Ma, L., C. Gao, Z. Mao, J. Zhou, J. Shen, X. Hu, and C. Han (2003) Collagen/chitosan porous scaffolds with improved biostability for skin tissue engineering. Biomaterials 24: 4833–4841.
Kadler, K. E., D. F. Holmes, J. A. Trotter, and J. A. Chapman (1996) Collagen fibril formation. Biochem. J. 316: 1–11.
Hu, X., S. H. Park, E. S. Gil, X. X. Xia, A. S. Weiss, and D. L. Kaplan (2011) The influence of elasticity and surface roughness on myogenic and osteogenic-differentiation of cells on silk-elastin biomaterials. Biomaterials 32: 8979–8989.
Lutolf, M. P., P. M. Gilbert, and H. M. Blau (2009) Designing materials to direct stem-cell fate. Nature 462: 433–441.
Gilbert, P. M., K. L. Havenstrite, K. E. Magnusson, A. Sacco, N. A. Leonardi, P. Kraft, N. K. Nguyen, S. Thrun, M. P. Lutolf, and H. M. Blau (2010) Substrate elasticity regulates skeletal muscle stem cell self-renewal in culture. Science 329: 1078–1081.
Peault, B., M. Rudnicki, Y. Torrente, G. Cossu, J. P. Tremblay, T. Partridge, E. Gussoni, L. M. Kunkel, and J. Huard (2007) Stem and progenitor cells in skeletal muscle development, maintenance, and therapy. Mol. Ther. 15: 867–877.
Author information
Authors and Affiliations
Corresponding authors
Rights and permissions
About this article
Cite this article
Ding, K., Zhang, Y.L., Yang, Z. et al. A promising injectable scaffold: The biocompatibility and effect on osteogenic differentiation of mesenchymal stem cells. Biotechnol Bioproc E 18, 155–163 (2013). https://doi.org/10.1007/s12257-012-0429-z
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s12257-012-0429-z