Osteoblastic Cell-Seeded Dense Collagen for Bone Tissue Engineering

Objectives: Collagen is a major component of bone and, as a natural polymer, is used as a biomaterial for tissue engineering and for basic studies of osteoblast biology. Collagen gel in hydrated form has poor mechanical properties due to the presence of excess fluid within the gel. We have used a technique of plastic compression to produce scaffolds that are dense and have improved handling properties. Based on the physical and biological nature of the dense collagen we hypothesised that this matrix would stimulate osteoblast differentiation and that this process would be dependent upon scaffold degradation.

Methods: Type I collagen gels were prepared by neutralisation of acidic collagen solution, seeded with MC3T3 cells (300,000/ml) and allowed to set. Dense collagen was produced by a process of plastic compression and cultured in osteogenic media (AA and â-GP) for up to 14 days. Mineralisation was assayed by measuring the calcium content. RNA was extracted using Trizol and mRNA levels of Col1a1, Mmp-13 and bone sialoprotein (BSP) were quantified by Q-PCR. Remodelling of the collagen was studied by examining the effects of the matrix metalloproteinase inhibitor Ilomastat.

Results: Cell-seeded dense collagen was heavily mineralised after 14 days in OS media as evaluated by the calcium content; we found that mineralisation did not occur in the absence of MMP activity. BSP expression was up-regulated 43-fold relative to zero hour cells in the absence or presence of inhibitor. In contrast Mmp-13 levels increased an additional 10-fold in the presence of the inhibitor over that in osteogenic media alone (a total increase over zero hour of 500-fold); whereas Col1a1 expression was slightly decreased (0.67-fold).

Conclusion: These results demonstrate the potential use of dense collagen seeded with osteoblastic cells for hard tissue regeneration in addition to providing insights into the reciprocity of bone cell differentiation within osteoid.