Introduction: Bone marrow stromal cells (BMS) are fundamentally a heterogeneous population of cells with multilineage differentiation potential. When BMS cells were seeded onto a three-dimensional tubular scaffold fabricated from aligned type I collagen strands, and cultured in osteogenic media, they underwent simultaneous maturation and differentiation into osteoblastic and vascular cell lineages. In addition these cells produced mineralized matricellular deposits. This in vitro model provides an excellent model to study the concurrent temporal and spatial regulation of both osteogenesis and vasculogenesis during bone development. Methods: Rat BMS cells were isolated from Wister male rats maintained and expanded for two passages in basal medium. Then, these cells were seeded into aligned type I collagen tubes at density of 1x106 cells and cultured either in basal or osteogenic media for 3, 6, and 9 days. The cells were subsequently processed for RT-PCR, immunohistochemical and cytochemical analysis. Lineage specific proteins were localized by immunofluorescence using laser scanning confocal microscopy (Zeiss 510 Meta) and mRNA transcript analysis was performed by real-time PCR (RT-qPCR). Results: The expression pattern of key osteogenic markers significantly differed in response to basal and osteogenic media. Tubes containing BMS cells when cultured in basal media demonstrated an initial upregulation of osteopontin which returned to baseline level following 6 days. In contrast, tubes with BMS cells cultured in ostegenic media showed a sustained upregulation of osteocalcin beyond day 3. The Alkaline phosphatase activity and calcium content (alizarin red staining) significantly increased over the observed period of time in osteogenic medium. Surprisingly, sheets of abundant á-smooth muscle positive cells were observed in these tubes seeded and cultured with BMS cells. Moreover, nascent capillary-like vessels were also seen amidst the osteoblasts in osteogenic cultures. Conclusions: Aligned type I collagen tubes in osteogenic cultures augmented the maturation and differentiation of BMS cells into osteoblasts. Substantial upregulation of osteoblastic phenotypic markers is reminiscent of in vivo expression pattern. This system has potential for investigating the development of vascularized osteogenesis in vitro as well as for bone tissue engineering.

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