Abstract
Murine calvariae pre-osteoblasts (MC3T3-E1), grown in a novel bioreactor, proliferate into a mineralizing 3D osteoblastic tissue that undergoes progressive phenotypic maturation into osteocyte-like cells. Initially, the cells are closely packed (high cell/matrix ratio), but transform into a more mature phenotype (low cell/matrix ratio) after about 5 mo, a process that recapitulates stages of bone development observed in vivo. The cell morphology concomitantly evolves from spindle-shaped pre-osteoblasts through cobblestone-shaped osteoblasts to stellate-shaped osteocyte-like cells interconnected by many intercellular processes. Gene-expression profiles parallel cell morphological changes, up-to-and-including increased expression of osteocyte-associated genes such as E11, DMP1, and sclerostin. X-ray scattering and infrared spectroscopy of contiguous, square centimeter-scale macroscopic mineral deposits are consistent with bone hydroxyapatite, showing that bioreactor conditions can lead to ossification reminiscent of bone formation. Thus, extended-term osteoblast culture (≤10 mo) in a bioreactor based on the concept of simultaneous growth and dialysis captures the full continuum of bone development otherwise inaccessible with conventional cell culture, resulting in an in vitro model of osteogenesis and a source of terminally differentiated osteocytes that does not require demineralization of fully formed bone.
This is a preview of subscription content, log in via an institution to check access.
Similar content being viewed by others
References
Anderson H. C.; Garimella R. et al. The role of matrix vesicles in growth plate development and biomineralization. Front Biosci 10: 822–837; 2005.
Chaudhuri J.; Al-Rubeai M. Bioreactors for tissue engineering: Principles, design and operation. Springer, Berlin; 2005.
Cisar J. O.; Xu D. Q. et al. An alternative interpretation of nanobacteria-induced biomineralization. Proceedings of the National Academy of Sciences 97: 11511–11515; 2000.
Dhurjati R.; Krishnan V. et al. Metastatic breast cancer cells colonize and degrade three-dimensional osteoblastic tissue in vitro. Clin Exp Metastasis 25: 753–763; 2008.
Dhurjati R.; Liu X. et al. Extended-term culture of bone cells in a compartmentalized bioreactor. Tissue engineering 12: 3045–3054; 2006.
Doty S. B. Morphological evidence of gap junctions between bone cells. Calcif Tissue Int 33: 509–512; 1981.
Ducy P.; Schinke T. et al. The osteoblast: A sophisticated fibroblast under central surveillance. Science 289: 1501–1504; 2000.
Eriksen E. F.; Eghbali-Fatourechi G. Z. et al. Remodeling and vascular spaces in bone. J Bone Miner Res 22: 1–6; 2007.
Franz-Odendaal T. A.; Hall B. K. et al. Buried alive: How osteoblasts become osteocytes. Developmental Dynamics 235: 176–190; 2006.
Knothe Tate M. L.; Adamson J. R. et al. The osteocyte. The International Journal of Biochemistry & Cell Biology 36: 1–8; 2004.
Knothe Tate M. L.; Tami A. et al. Micropathoanatomy of osteoporosis—indications for a cellular basis for bone disease. Advances in Osteoporotic Fracture Management 2: 9–14; 2002.
Lian J. B.; Stein G. S. Concepts of osteoblast growth and differentiation: basis for modulation of bone cell development and tissue formation. Crit Rev Oral Biol Med 3: 269–305; 1992.
Mastro A. M.; Vogler E. A. A three-dimensional osteogenic tissue model for the study of metastatic tumor cell interactions with bone. Cancer Res 69: 4097–4100; 2009.
Mullender M. G.; van der Meer D. D. et al. Osteocyte density changes in aging and osteoporosis. Bone 18: 109–113; 1996.
Murshid S. A.; Kamioka H. et al. Actin and microtubule cytoskeletons of the processes of 3D-cultured MC3T3-E1 cells and osteocytes. J Bone Miner Metab 25: 151–158; 2007.
Nijweide P. J.; Burger E. H. Mechanisms of bone formation in vitro. In: Hall B. K. (ed) Bone. Volume 1: The osteoblast and osteocyte. The Teleford, Caldwell, pp 303–326; 1990.
Palumbo C.; Palazzini S. et al. Morphological study of intercellular junctions during osteocyte differentiation. Bone 11: 401–406; 1990.
Rodan G. A.; Martin T. J. Therapeutic approaches to bone diseases. Science 289: 1508–1514; 2000.
Rose G. G. Cytopathophysiology of tissue cultures growing under cellophane membranes. Int. Rev. Exp. Pathol. 5: 111–178; 1966.
Rubens R. D. Bone metastases—the clinical problem. European Journal of Cancer 34: 210–213; 1998.
Rubens R. D.; Mundy G. R. Cancer and the skeleton. Informa Health Care, London; 2000.
Service R. F. Tissue engineers build new bone. Science 289: 1498–1500; 2000.
Sugawara Y.; Kamioka H. et al. Three-dimensional reconstruction of chick calvarial osteocytes and their cell processes using confocal microscopy. Bone 36: 877–883; 2005.
Vogler E. A. A compartmentalized device for the culture of animal cells. J. Biomater. Artif. Cells Artif. Organs 17: 597–610; 1989.
Acknowledgments
This is supported by U.S. Army Medical and Material Command Breast Cancer Program WX81XWH-06-1-0432, Susan G. Komen for the Cure BCTR 0601044, with additional support from the National Foundation for Cancer Research. Authors appreciate the expert technical assistance of Ms. Donna Sosnoski and the Cytometry and the Electron Microscopy Facilities at Penn State. We thank Dr. Carol Gay for her thoughtful discussions and critique.
Author information
Authors and Affiliations
Corresponding author
Additional information
Editor: J. Denry Sato
Venkatesh Krishnan and Ravi Dhurjati contributed equally.
Electronic supplementary material
Below is the link to the electronic supplementary material.
Supplementary Table 1
(DOC 48 kb)
Supplementary Figure 1
Compartmentalized bioreactor design. (a) A cross-section diagram showing separation of the cell-growth space (A) from the basal-medium reservoir (B) by a dialysis membrane (C). Cells are grown on gas-permeable but liquid-impermeable film (E). The device is ventilated through film (D), which is the same material as (E). The whole device is brought together in a liquid-tight fashion using screws (b) and (c), which is an exploded view identifying separate components. Liquid access is through leur-taper ports (J, K), which mate to standard pipettes. Reproduced from Dhurjati et al. (2006) with permission.
Rights and permissions
About this article
Cite this article
Krishnan, V., Dhurjati, R., Vogler, E.A. et al. Osteogenesis in vitro: from pre-osteoblasts to osteocytes. In Vitro Cell.Dev.Biol.-Animal 46, 28–35 (2010). https://doi.org/10.1007/s11626-009-9238-x
Received:
Accepted:
Published:
Issue Date:
DOI: https://doi.org/10.1007/s11626-009-9238-x