Characterisation of the constitutive over-expression of AJ18 in a novel rat stromal bone marrow cell line (D8-SBMC)

Abstract 

Objective

The elucidation of the molecular pathways involved in osteoblast proliferation and differentiation has been greatly enhanced by the availability of cell culture model systems. However, many of the current bone cell culture systems suffer from disadvantages such as the inability to generate mineralised bone-like nodules, a transformed genetic background, cell heterogeneity, and a relatively long time frame from cell seeding to mineralisation, often in the order of several weeks. Here we describe the establishment and characterisation of a novel bone cell line named D8-SBMC. As a first demonstration of their potential value, D8-SBMC was utilised to further support a role for AJ18 during osteogenesis.

Design

D8-SBMC was established from a single cell suspension of the previously characterised long term rat stromal bone marrow cells [Kotev-Emeth S, Pitaru S, Pri-Chen S, Savion N. Establishment of a rat long-term culture expressing the osteogenic phenotype: dependence on dexamethasone and FGF-2. Connect Tissue Res 2002;43(4):606–12; Pitaru S, Kotev-Emeth S, Noff D, Kaffuler S, Savion N. Effect of basic fibroblast growth factor on the growth and differentiation of adult stromal bone marrow cells: enhanced development of mineralized bone-like tissue in culture. J Bone Miner Res 1993;8(8):919–29]. AJ18 was constitutively and stably over-expressed in D8-SBMC and analysed.

Results

D8-SBMC possesses the ability to form robust mineralised bone-like nodules within 8 days proceeding cell confluency. Interestingly, a cement line-like matrix is also generated between the culture dish and a basal monolayer of cells. Constitutive and stable over-expression of AJ18 resulted in an increase in cell proliferation and mineralisation. Expression of bone marker genes, such as bone sialoprotein, osteopontin, osteocalcin, collage type 1, and osteonectin, was up-regulated by AJ18 over-expression.

Conclusion

A novel bone cell line, D8-SBMC, was established and characterised. D8-SBMC may be a valuable model system for biomineralisation studies. D8-SBMC was utilised to further understand the role of AJ18 in cell proliferation and differentiation during osteogenesis.

Abbreviations: AS, antisense, AA, ascorbic acid, β-GP, beta-glycerophosphate, BMP, bone morphogenetic protein, BSP, bone sialoprotein, CMV, cytomegalovirus, COL1, collagen type 1, D8-SBMC, D8-stromal bone marrow cell, Dex, dexamethasone, EV, empty vector, FRCC, foetal rat cavarial cell, FGF-2, fibroblast growth factor-2, HA, hydroxyapatite, KRAB, Krüppel-associated box, MEM, minimal essential medium, OSE2, osteoblast-specific element 1, OC, osteocalcin, OPN, osteopontin, Osx, osterix, RBMC, rat bone marrow cell, SPARC, secreted protein acidic and rich in cysteine, S, sense, TNAP, tissue non-specific alkaline phosphatase, TEM, transmission electron microscopy, WT, wild type

Keywords: Novel bone cell line, D8-SBMC, AJ18, Over-expression, Osteoblast differentiation