Mechanical loading is a potent stimulator of bone formation. A screen for genes associated with mechanically-induced osteogenesis implicated the glutamate transporter GLAST-1 (1), in the mechanoresponse. We are investigating whether modulation of glutamate transporters represents a potential anabolic therapy in bone. Bone cells express functional components from each stage of the glutamate signalling pathway and activation of ionotropic glutamate receptors on osteoblasts can increase bone forming activity (2). Five high affinity Na+-dependant excitatory amino acid transporters (EAATs 1-5) regulate glutamatergic signalling. EAAT1 (GLAST-1) is expressed by osteocytes and bone-forming osteoblasts in vivo.

We quantified transcripts for EAATs 1-3 and two splice variants (EAAT1a and EAAT1ex9skip) in human osteoblasts (MG63, SaOS-2 and primary) using real time-PCR. EAAT1a expression was very low whilst levels of the dominant negative EAAT1ex9skip were much higher in all cell types. EAAT1 and EAAT3 proteins were detected by immunofluorescence. We also demonstrated that glutamate transporters function in human osteoblasts. Sodium-dependent 14C-labelled glutamate uptake, sensitive to pharmacological EAAT inhibitors (t-PDC, TBOA) and extracellular glutamate concentration (10-500μM) was detected in MG63 and SaOS-2 cells.

To determine whether modulation of EAATs can influence bone formation, we used pharmacological inhibitors of EAATs 1-5 (t-PDC and TBOA) and also over-expressed EAAT1exon9skip using antisense oligonucleotides (AONs) targeted to splice donor sequence of exon 9. Experiments were performed in 0-500μM glutamate. Pharmacological inhibition of EAATs over 5-21 days increased alkaline phosphatase activity and mineralisation of SaOS-2 cells and human primary osteoblasts. Over-expression of EAAT1ex9skip significantly increased cell number and decreased cell death as well as significantly increasing PCNA, Osteonectin and Type I collagen mRNAs in MG63 cells. Furthermore, over-expression of EAAT1ex9skip increased mean alkaline phosphatase activity over 48hrs in SaOS-2 cells.

These data show that EAATs are expressed and functional in osteoblasts and that pharmaceutical and genetic inhibition of their activity increases bone formation. These mechanically regulated glutamate transporters are important in regulating bone homeostasis and their manipulation may represent a new anabolic therapy for the treatment of disorders such as osteoporosis or non-union fractures.

Introduction: There is controversy regarding the effectiveness of PRC for bone healing. A possible explanation is the different bone graft substitutes (BGSs) used with PRC. Here we investigated the effect of combining different BGSs with PRC on hBMSCs differentiation and growth factor release from the BGS/PRC composites.

Method: hBMSCs, DBM and allograft were prepared from femoral heads donated by patients undergoing total hip replacement. Growth factor release (TGF-â, VEGF, PDGF-AB, BMP-2) was measured by ELISA. The effect of PRC on hBMSC differentiation was determined by ALP activity and mineralisation. PRC was produced using the CAPTION device (S& N) from 10 healthy volunteers.

Results: Combining PRC with BGSs increased hBMSC proliferation (p< 0.05) and decreased ALP activity (p< 0.05) compared to DBM or â-TCP (GenOS, S& N) alone, but had no effect on allograft following 3 and 5 days treatment. After 21 days PRC enhanced mineralisation compared to all BGSs alone (16%–56%). Compared to PRC alone addition of DBM and allograft increased proliferation (p< 0.05), decreased ALP activity (p< 0.005) and decreased mineralisation (p< 0.005). TGF-â, VEGF and BMP-2 release from PRC was unaffected when combined with DBM but PDGF-AB release was reduced by 50%.

Conclusions: Combining PRC with the majority of BGSs enhanced cell proliferation and decreased osteoblastic differentiation at early time points but increased total mineralisation compared to the BGSs alone. However, compared to PRC alone combining DBM or allograft with PRC reduced mineralisation. One potential explanation for the effects of combining PRC with DBM is altered growth factor release profiles compared to the components alone.

Autologous platelet rich plasma (PRP) has an established history of clinical use in dental and orthopaedic procedures. However, there is little scientific data demonstrating a mode of action and conflicting clinical data to support its use. The aim of this study was to determine the cellular and metabolic pathways by which PRP modulates the osteogenic response. PRP is a concentrate of platelets in a small volume of plasma derived from whole blood. Platelets contain pre-packaged growth factors in & #61537;-granules that are released during clotting at the trauma site and are an essential requirement for the hard (bone) and soft tissue healing process.

S& N’s Caption ™ device, a standalone disposable device that prepares autologous PRP in 15minutes, was used to prepare human PRP. We determined a platelet concentration factor of 3.4& #61617;1.2 fold and significant increases in the concentration of platelet derived growth factor–AB (PDGF-AB), transforming growth factor-& #61538; (TGF-& #61538;) and vascular endothelial growth factor (VEGF). A 5.9 fold increase in VEGF, 4 fold increase in TGF-& #61538; and 1.5 fold increase in PDGF-AB indicate that PRP has the potential to enhance bone repair as each of these growth factors individually and synergistically affect multiple cell responses essential for tissue repair.

An in vitro study was then undertaken to investigate the effect of human PRP compared to human serum on the proliferation and differentiation of human primary osteoblasts (hOBs) and human mesenchymal stem cells (hMSCs). A significant proliferative effect of PRP compared to serum was observed in both cell types. In hMSCs, PRP treatment significantly increased proliferation after 24 hours as determined by Pico green analysis. However, in osteoblasts a proliferative effect of PRP over and above that of serum was not observed until 72 hours. These data indicate that PRP may have specific differing stimulatory effects on each cell type. Quantitative RT-PCR analysis also determined that PRP significantly increased the expression of BMP 2 over and above that of serum in human osteoblasts at both 6 and 12 hour time points. Furthermore, in hMSCs, PRP increased both BMP-2 and alkaline phosphatase gene expression at early time points suggesting the commitment of these cells to the osteoblastic lineage. This hypothesis was consistent with alkaline phosphatase protein expression which was significantly increased at 72hrs in hMSCs and was further confirmed by increased alizarin red staining, indicative of calcium deposition, in long term cultures of hMCSs treated with PRP.

In summary, these data demonstrate that PRP initiates proliferation in hMSCs and osteoblasts, enhances BMP-2 mRNA expression and induces osteoblast differentiation and maturation in human MSC cultures. Together these data demonstrate a positive effect of PRP on osteogenesis and highlight the potential for Caption™ derived PRP to enhance bone repair.