Simvastatin is a 3-Hydroxy-3-methylglutaryl Co-enzyme inhibitor, widely used to reduce lipid levels. Recent studies have demonstrated pleiotropic beneficial effects the skeleton. We aim to demonstrate the effect of Simvastatin on the osteogenic differentiation and proliferation of murine embryonic stem cells.

Tg2a cells were cultured in maintenance medium until confluence and passaged twice into tissue culture flasks. They were then seeded into 6 well and 24 well plates at a density of 10.000 cells/cm2 and cultured for 3 days in maintenance medium mixed at 1:1 with HepG2 conditioned medium. The culture then continued using osteogenic medium with different concentrations of simvastatin for another 16 days. Measurements included Alizarin Red quantification for calcified matrix, ALP assay, RT-PCR for genes expressed during osteogenic differentiation (osteocalcin, Runx2, osterix, Col1a1).

Simvastatin has dose dependent effect on mineralized matrix formation. Alizarin Red quantification assays demonstrated that simvastatin (all dose groups) induced a statistically significant increase in calcified matrix formation on day 11 (P< 0.05) and 16 (P< 0.01) compared to the control group. ALP activity was significantly higher on day 8 in the groups that had a simvastatin concentration of 1nM, 10nM and 100nM (P< 0.05). RT-PCR has demonstrated that simvastatin caused increased expression of all genes measured on differentiation. Statins can induce bone formation when combined with embryonic stem cells.

Impaction allografting is a bone reconstruction technique currently used in lower limb revision arthroplasty. Demineralisation and addition of osteogenic protein-1 (OP-1) can improve the osteoinductivity of the allograft however recent reports indicate significant allograft resorption when it is combined with OP-1 during impaction. Our hypothesis was that hydroxyapatite (HA) and OP-1 could effectively replace demineralised allograft. The objective was to evaluate human mesenchymal stem cell (h-MSC) proliferation (tritiated thymidine incorporation, total DNA Hoechst 33258 and scanning electron microscopy) and osteogenic differentiation (alkaline phosphatase activity) in human demineralised bone matrix (h-DBM) and HA, with or without OP-1. Cell proliferation on HA+OP-1 was significantly higher compared to HA at all time points (p< 0.05) and to DBM alone (day 1, p=0.042; day 14, p< 0.001). Cell proliferation was higher in DBM+OP-1, at all time points compared to HA+OP-1 but only in absolute values. Cell differentiation was significantly higher in HA+OP-1 compared to HA (p< 0.05) but comparable to DBM alone. Differentiation was significantly higher on DBM+OP-1 at all time points compared to HA (p< 0.05) and to HA+OP-1 (p< 0.05). HA is a potential graft expander in impaction allografting. When combined with OP-1 is comparable to DBM alone and being non absorbable may support the impacted graft in the early stages after the administration of OP-1.

Impaction allografting is a bone tissue engineering technique currently used in lower limb reconstruction orthopaedic surgery. Our hypothesis was that biological optimisation can be achieved by demineralisation and addition of osteogenic protein-1(OP-1) to the allograft. The objective of our in vitro study was to evaluate human mesenchymal stem cell (MSC) proliferation (Alamar Blue assay, titrated thymidine assay, total DNA Hoechst 33258 and scanning electron microscopy) and osteogenic differentiation (alkaline phosphatase assay) in two types of impacted carrier, namely demineralised bone matrix (DBM) and insoluble collagenous bone matrix (ICBM), with or without OP-1. The objective in vivo was to compare the osteogenic potential of impacted DBM with or without OP-1, with that of impacted fresh frozen allograft (FFA), again with or without OP-1. DBM+OP-1 optimized osteoinduction and significantly improved (p< 0.05) proliferation and differentiation in comparison to the majority of all other graft preparation in vitro. In addition DBM+OP-1 was significantly superior, with regard to osteogenesis, compared to the impacted FFA alone (p< 0.001), FFA+OP-1 (p=0.01) and DBM alone (p=0.02) in vivo. We propose that partial demineralisation and addition of OP-1 provides a good method for improving the osteoinductive properties of fresh allograft currently used in the impaction grafting technique.