Medium Perfusion Enhances Osteogenesis by Murine Osteosarcoma Cells in Three-Dimensional Collagen Sponges

Abstract

In this study, we examined in vitro histogenesis by murine K8 osteosarcoma cells maintained in three-dimensional (3D) collagen sponges. We tested the hypothesis that perfusion of medium enhances cell viability and their biosynthetic activity as assessed by expression of the osteoblastic phenotype and mineral deposition. At intervals, samples were harvested and analyzed histologically, biochemically, and by Northern hybridization for type I collagen, osteopontin (OPN), osteocalcin (OC), and core binding factor alpha 1 (Cbfa1). Histologic evaluation showed greater viability, more alkaline phosphatase (ALP)-positive cells, and more mineralized tissue in the perfused sponges after 21 days. Immunohistological assessment of proliferating cell nuclear antigen revealed 5-fold more proliferating cells in the perfused sponges compared with the controls (p = 0.0201). There was 3-fold more ALP activity in the perfused sponges than the controls at 6 days and 14 days (p = 0.0053). The perfused sponges contained twice the DNA and eight times more calcium than the nonperfused controls after 21 days (p < 0.0001 for both). Northern hybridization analysis revealed more mRNA for collagen type I (2-fold) and 50% more for OC at 14 days and 21 days, whereas OPN and Cbfa1 mRNA expression remained unaffected by the medium perfusion. These results show that medium perfusion had beneficial effects on the proliferation and biosynthetic activity of this osteosarcoma cell line. This system mimics the 3D geometry of bone tissue and has the potential for revealing mechanisms of regulation of osteogenesis. (J Bone Miner Res 1999;14:2118-2126)

Collagen scaffolds for tissue engineering

“…Low flow (1.3 ml/min) perfusion of the medium through collagen sponges seeded with adherent (stromal) and nonadherent (hematopoietic) cells from mouse bone marrow resulted in greater cell viability and more hematopoiesis, in comparison with static cultures 29 (Figure 2). Likewise, perfusion enhanced viability and osteogenesis by murine osteosarcoma cells 30 and mucosa formation by oral keratinocytes. 27 In sharp contrast to the positive effects on other cell types, perfusion of medium inhibited chondrocyte gene expression and matrix synthesis and accumulation, 32 probably due to chondrocytes' preference for low pH and oxygen concentration.…”

“…30 Because porous collagen sponges lack rigidity that could be required for weight-bearing applications, composites can be developed with additives such as a bone-like calcium phosphate. 31 One of the known limiting factors of traditional high-density 3D culture is that cells in the center of the mass do not survive because of poor exchange of nutrients and dissolved gases.…”

Influence of Shear Stress in Perfusion Bioreactor Cultures for the Development of Three-Dimensional Bone Tissue Constructs: A Review

“…For 3D culture, fluid shear stress experiments have been almost exclusively nonhuman 43,[74][75][76]81,85,86,97,98 cell line orientated, with only a single study using a human 87 cell source (Fig. 3) RunX2, 98 Col1, 97 and mineralized matrix production, 43,85,87,97 can be achieved with shear stresses in the range 1Â10 À4 to 1.2 Pa.…”

“…3) RunX2, 98 Col1, 97 and mineralized matrix production, 43,85,87,97 can be achieved with shear stresses in the range 1Â10 À4 to 1.2 Pa. The majority of work was focused in the 1 to 5Â10 À2 Pa range, which is at least an order of magnitude below the average for 2D culture and up to two orders of magnitude lower in some cases (other groups [99][100][101] have observed osteogenic differentiation within 3D systems but shear stresses were not quantified making comparison of the data unfeasible).…”

Computational modeling of the fluid mechanical environment of regular and irregular scaffolds

“…Others have used computer-aided design approaches where scaffolds were built as a repeat pattern of unit cells [11,19] . This method allows proper actual estimation results of fluid flow velocity, wall shear stress and pressure within the model which can be used for easy manufacture.…”