Abstract
Collagen glycosaminoglycan (CG) scaffolds have been clinically approved as an application for skin regeneration. The goal of this study has been to examine whether a CG scaffold is a suitable biomaterial for generating human bone tissue. Specifically, we have asked the following questions: (1) can the scaffold support human osteoblast growth and differentiation and (2) how might recombinant human transforming growth factor-beta (TGF-β1) enhance long-term in vitro bone formation? We show human osteoblast attachment, infiltration and uniform distribution throughout the construct, reaching the centre within 14 days of seeding. We have identified the fully differentiated osteoblast phenotype categorised by the temporal expression of alkaline phosphatase, collagen type 1, osteonectin, bone sialo protein, biglycan and osteocalcin. Mineralised bone formation has been identified at 35 days post-seeding by using von Kossa and Alizarin S Red staining. Both gene expression and mineral staining suggest the benefit of introducing an initial high treatment of TGF-β1 (10 ng/ml) followed by a low continuous treatment (0.2 ng/ml) to enhance human osteogenesis on the scaffold. Osteogenesis coincides with a reduction in scaffold size and shape (up to 70% that of original). A notable finding is core degradation at the centre of the tissue-engineered construct after 49 days of culture. This is not observed at earlier time points. Therefore, a maximum of 35 days in culture is appropriate for in vitro studies of these scaffolds. We conclude that the CG scaffold shows excellent potential as a biomaterial for human bone tissue engineering.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Bilezikian JP (2001) Principles of bone biology, 2nd edn. Academic Press, London
Bokhari MA, Akay G, Zhang S, Birch MA (2005) The enhancement of osteoblast growth and differentiation in vitro on a peptide hydrogel-polyHIPE polymer hybrid material. Biomaterials 26:5198–5208
Byrne EM, Farrell E, McMahon LA, Haugh MG, O'Brien FJ, Campbell VA, et al (2008) Gene expression by marrow stromal cells in a porous collagen-glycosaminoglycan scaffold is affected by pore size and mechanical stimulation. J Mater Sci Mater Med 19:3455–3463
Compton CC, Butler CE, Yannas IV, Warland G, Orgill DP (1998) Organized skin structure is regenerated in vivo from collagen-GAG matrices seeded with autologous keratinocytes. J Invest Dermatol 110:908–916
Dhurjati R, Liu X, Gay CV, Mastro AM, Vogler EA (2006) Extended-term culture of bone cells in a compartmentalized bioreactor. Tissue Eng 12:3045–3054
Donahue HJ, Li Z, Zhou Z, Yellowley CE (2000) Differentiation of human fetal osteoblastic cells and gap junctional intercellular communication. Am J Physiol Cell Physiol 278:C315–C322
Farrell E, O'Brien FJ, Doyle P, Fischer J, Yannas I, Harley BA, et al (2006) A collagen-glycosaminoglycan scaffold supports adult rat mesenchymal stem cell differentiation along osteogenic and chondrogenic routes. Tissue Eng 12:459–468
Freyman TM, Yannas IV, Gibson LJ (2001a) Cellular materials as porous scaffolds for tissue engineering. Prog Mater Sci 46:273
Freyman TM, Yannas IV, Yokoo R, Gibson LJ (2001b) Fibroblast contraction of a collagen-GAG matrix. Biomaterials 22:2883–2891
Harris SA, Enger RJ, Riggs BL, Spelsberg TC (1995) Development and characterization of a conditionally immortalized human fetal osteoblastic cell line. J Bone Miner Res 10:178–186
Heng BC, Cao T, Stanton LW, Robson P, Olsen B (2004) Strategies for directing the differentiation of stem cells into the osteogenic lineage in vitro. J Bone Miner Res 19:1379–1394
Hsu WC, Spilker MH, Yannas IV, Rubin PA (2000) Inhibition of conjunctival scarring and contraction by a porous collagen-glycosaminoglycan implant. Invest Ophthalmol Vis Sci 41:2404–2411
Itthichaisri C, Wiedmann-Al-Ahmad M, Huebner U, Al-Ahmad A, Schoen R, Schmelzeisen R, et al (2007) Comparative in vitro study of the proliferation and growth of human osteoblast-like cells on various biomaterials. J Biomed Mater Res [A] 82:777–787
Jaasma MJ, O'Brien FJ (2008) Mechanical stimulation of osteoblasts using steady and dynamic fluid flow. Tissue Eng [A] 14:1213–1223
Jones JR, Tsigkou O, Coates EE, Stevens MM, Polak JM, Hench LL (2007) Extracellular matrix formation and mineralization on a phosphate-free porous bioactive glass scaffold using primary human osteoblast (HOB) cells. Biomaterials 28:1653–1663
Laurencin CT, Attawia MA, Elgendy HE, Herbert KM (1996) Tissue engineered bone-regeneration using degradable polymers: the formation of mineralized matrices. Bone 19 (1 Suppl):93S–99S
Lee CR, Grodzinsky AJ, Spector M (2001) The effects of cross-linking of collagen-glycosaminoglycan scaffolds on compressive stiffness, chondrocyte-mediated contraction, proliferation and biosynthesis. Biomaterials 22:3145
Lieb E, Vogel T, Milz S, Dauner M, Schulz MB (2004) Effects of transforming growth factor beta1 on bonelike tissue formation in three-dimensional cell culture. II. Osteoblastic differentiation. Tissue Eng 10:1414–1425
McMahon LA, Reid AJ, Campbell VA, Prendergast PJ (2008) Regulatory effects of mechanical strain on the chondrogenic differentiation of MSCs in a collagen-GAG scaffold: experimental and computational analysis. Ann Biomed Eng 36:185–194
O'Brien FJ, Harley BA, Yannas IV, Gibson LJ (2005) The effect of pore size on cell adhesion in collagen-GAG scaffolds. Biomaterials 26:433–441
Pieper JS, Oosterhof A, Dijkstra PJ, Veerkamp JH, Kuppevelt TH van (1999) Preparation and characterization of porous crosslinked collagenous matrices containing bioavailable chondroitin sulphate. Biomaterials 20:847
Setzer B, Bachle M, Metzger MC, Kohal RJ (2009) The gene-expression and phenotypic response of hFOB 1.19 osteoblasts to surface-modified titanium and zirconia. Biomaterials 30:979–990
Shea LD, Wang D, Franceschi RT, Mooney DJ (2000) Engineered bone development from a pre-osteoblast cell line on three-dimensional scaffolds. Tissue Eng 6:605–617
Stein GS, Lian JB (1993) Molecular mechanisms mediating proliferation/differentiation interrelationships during progressive development of the osteoblast phenotype. Endocr Rev 14:424–442
Subramaniam M, Jalal SM, Rickard DJ, Harris SA, Bolander ME, Spelsberg TC (2002) Further characterization of human fetal osteoblastic hFOB 1.19 and hFOB/ER alpha cells: bone formation in vivo and karyotype analysis using multicolor fluorescent in situ hybridization. J Cell Biochem 87:9–15
Sun H, Wu C, Dai K, Chang J, Tang T (2006) Proliferation and osteoblastic differentiation of human bone marrow-derived stromal cells on akermanite-bioactive ceramics. Biomaterials 27:5651
Ter Brugge PJ, Jansen JA (2002) In vitro osteogenic differentiation of rat bone marrow cells subcultured with and without dexamethasone. Tissue Eng 8:321–331
Tierney CM, Jaasma MJ, O'Brien FJ (2008) Osteoblast activity on collagen-GAG scaffolds is affected by collagen and GAG concentrations. J Biomed Mater Res [A] 2:202
Yannas I (2001) Tissue and organ regeneration in adults. Springer, New York
Zhang H, Ahmad M, Gronowicz G (2003) Effects of transforming growth factor-beta 1 (TGF-beta1) on in vitro mineralization of human osteoblasts on implant materials. Biomaterials 24:2013–2020
Author information
Authors and Affiliations
Corresponding author
Additional information
The authors acknowledge the RCSI Research Committee and President of Ireland Young Researcher Award (PIYRA) for funding.
Rights and permissions
About this article
Cite this article
Keogh, M.B., O’ Brien, F.J. & Daly, J.S. A novel collagen scaffold supports human osteogenesis—applications for bone tissue engineering. Cell Tissue Res 340, 169–177 (2010). https://doi.org/10.1007/s00441-010-0939-y
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s00441-010-0939-y