Enhanced osteoblastogenesis in three-dimensional collagen gels

Abstract

Growth and differentiation of osteoblasts are often studied in cell cultures. In vivo, however, osteoblasts are embedded within a complex three-dimensional (3D) microenvironment, which bears little relation to standard culture flasks. Our study characterizes osteoblast-like cells cultured in 3D collagen gels and compares them with cells in two-dimensional (2D) cultures. Primary rat osteoblasts and MC3T3-E1 cells were seeded within type I collagen gels, and differentiation was determined by mineral staining and gene expression analysis. Cells growing in 3D gels showed positive mineral staining and induction of osteoblast marker genes earlier than cells growing in 2D. A number of genes, including osteocalcin, bone sialoprotein, alkaline phosphatase and dentin matrix protein 1, were already highly upregulated in 3D cultures 24 h after seeding. The early expression of osteoblast genes was dependent on the 3D structure and was not induced in cells growing on collagen-coated dishes in 2D. Comparison of thymidine incorporation between cells in 3D and 2D cultures treated with agents that induce proliferation-transforming growth factor b, platelet-derived growth factor and lactoferrin-showed a much greater response in 3D gels. Cells in 3D cultures were also much more sensitive to inhibition of proliferation by the protein kinase inhibitor imatinib mesylate. The 3D collagen gels better represent the physiological bone environment and offer a number of technical advantages for the study of osteoblasts in vitro. These studies have additional practical implications as 3D collagen gels are considered as a scaffold material in regenerative medicine for the repair of bone defects.

Monosodium urate crystals reduce osteocyte viability and indirectly promote a shift in osteocyte function towards a proinflammatory and proresorptive state

“…For all experiments, MLO-Y4 cells or primary mouse osteocytes were seeded in three-dimensional (3D) collagen gels in 24-well plates (1 × 10 4 cells/50 μL gel; 1 gel/well) as previously described [ 14 ]. Briefly, rat collagen type I (Corning Inc., Corning, USA) was neutralized with 1 M NaOH and diluted to a final concentration of 3 mg/mL.…”

Local application of lactoferrin promotes bone regeneration in a rat critical-sized calvarial defect model as demonstrated by micro-CT and histological analysis

“…have previously utilized this type of collagen gel to assess its effect on osteoblastogenesis. In their study, the collagen gels remained intact after 24 days of culture (Matthews et al , ). In vivo , the degradation time of the type of collagen gels used in our study is unclear.…”

Fish Collagen Promotes the Expression of Genes Related to Osteoblastic Activity

“…The cells have well grown within a three-dimensional (3D) structure, as the conditions more closely resemble those of the in vivo condition of the osteoblast than the traditional two-dimensional (2D) cultures. Cells growth in 3D gels showed positive mineral staining and induction of osteoblastic marker genes earlier than cell growth in 2D cultures [8]. Although the present culture conditions were not the exact 3D gel culture system, the present environment was remarkably similar to a 3D culture environment, as a low-concentration TCA solution was gelled to mimic wet and elastic conditions [9].…”