Antibacterial bone biomaterial coatings appeal to orthopaedics, dentistry and veterinary medicine. Achieving the successful, stable conjugation of suitable compounds to biomaterial surfaces is a major challenge. A pragmatic starting point is to make use of existing, approved antibiotics which are known to remain functional in a stationary, immobilised state. This includes the macrocyclic glycopeptide, teicoplanin, following the discovery, in the 1990’s, that it could be used as a chiral selector in chromatographic enantiomeric separations. Importantly teicoplanin works at the level of the bacterial cell wall making it a potential candidate for biomaterial functionalisations. We initially sought to functionalise titanium (Ti) with polydopamine and use this platform to capture teicoplanin, however we were unable to avoid the natural affinity of the antibiotic to the oxide surface of the metal. Whilst the interaction between teicoplanin and Ti was robust, we found that phosphate resulted in antibiotic loss. Before contemplating the covalent attachment of teicoplanin to Ti we examined whether a commercial teicoplanin stationary phase could kill staphylococci. Whilst this commercially available material could bind N-Acetyl-L-Lys-D-Ala-D-Ala it was unable to kill bacteria. We therefore strongly discourage attempts at covalently immobilising teicoplanin and/or other glycopeptide antibiotics in the pursuit of novel antibacterial bone biomaterials.

In England and Wales in 2012 over 160,000 primary total hip and knee replacements were performed with 57% of hip replacements utilising uncemented prostheses. The main cause of failure, affecting approximately 10% of patients, is aseptic loosening. Previous research has found that functionalising titanium with lysophosphatidic acid (LPA) induces an increase in human osteoblast maturation on the implant surface through co-operation with active metabolites of vitamin D3. This feature, the small size of the LPS molecule and its affinity to readily bind to titanium and hydroxylapatite makes it an especially desirable molecule for bone biomaterials. Nevertheless biomaterials that also demonstrate anti-microbial properties are highly desirable.To test the antimicrobial efficacy of the LPA-functionalised titanium, a clinical isolate of Staphylococcus aureus, obtained from an infected revision surgery, was cultured on the surface of titanium discs functionalised with 0, 0.1. 0.5, 1, 2 and 5μM LPA. Bacterial adhes...

Enhancing the performance and longevity of titanium (Ti) implants continues to be a significant developmental theme in contemporary biomaterials design. Our specific focus pertains to the surface functionalisation of Ti using the bioactive lipid, lysophosphatidic acid (LPA) and certain phosphatase-resistant analogues of LPA. Coating survivorship to a plethora of testing regimens is required to align with due regulatory process before novel biomaterials can enter clinical trials. One of the key acceptance criteria is coating retention to the physical stresses experienced during implantation. In assessing coating stability to insertion into porcine bone we found that a subsequent in vitro assessment to confirm coating persistence was masked by abundant alkaline phosphatase (ALP) contamination adsorbed to the metal surface. Herein we report that ALP can bind to Ti in a matter of minutes by simply immersing Ti samples in aqueous solutions of the enzyme. We strongly discourage the in vitro monitoring of osteoblast and stromal cell ALP expression when assessing bioactive coating survivorship following Ti implant retrieval form native bone tissue.

Chitinase 3-like 1, otherwise known as YKL-40, is a secreted glycoprotein purported to have a role in extracellular matrix metabolism. The first mammalian cell type found to express YKL-40 was the human osteosarcoma-derived osteoblast, MG63. In that first study the active vitamin D3 metabolite, 1,25-dihydroxycholecalciferol (1,25D), stimulated YKL-40 expression, thereby indicating that a vital factor for skeletal health promoted YKL-40 synthesis by bone forming cells. However, when these MG63 cells were exposed to 1,25D they were also exposed to serum, a rich source of the pleiotropic lipid mediator, lysophosphatidic acid (LPA). Given that 1,25D is now known to co-operate with selected growth factors, including LPA, to influence human osteoblast differentiation we hypothesised that 1,25D and LPA may work together to stimulate osteoblast YKL-40 expression. Herein we report that 1,25D and LPA synergistically promote YKL-40 expression by MG63 cells. Inhibitors targeting AP1, MEK, Sp1 and STAT3 blunted the expression of both alkaline phosphatase and YKL-40 by MG63 cells in response to co-stimulation with 1,25D and LPA. Other ligands of the vitamin D receptor also co-operated with LPA in driving YKL-40 mobilisation. Collectively our findings highlight another important role of 1,25D and LPA in the regulation of human osteoblast function.

The physiological effects of 1,25 vitamin D3 (1,25D) are well known and the previously held dogma was that this was the only active vitamin D metabolite. A number of methods have been employed to demonstrate the effects of 24,25-dihydroxyvitamin D3 (24,25D) on osteoblast maturation responses, in the presence of FHBP, ((3S) 1-Fluoro-3-hydroxy-4-(oleoyloxy)butyl-1-phosphonate), an agonist of lysophosphatidic acid (LPA). These include alkaline phosphatase (ALP) expression and investigation of the role of CYP27B1, which is the enzyme responsible for converting 24,25D to 1,24,25D. Ketoconazole, which inhibits the actions of CYP27B1, as well as an enzyme-linked immunosorbant assay (ELISA) for CYP27B1 were used. The results clearly demonstrate that 24,25D stimulates maturation of MG63 cells when combined with FHBP. It has also been shown that the metabolite is not converted to another active form (for example, 1,24,25D) within osteoblasts, due to the absence of CYP27B1. 24,25D is an active vitamin D metabolite and exerts its effects in a bone fide manner, rather than following conversion to another active metabolite in osteoblasts. Given it is non-calcaemic, this metabolite has the exciting potential of being used in a bone regenerative setting in orthopaedic applications.

Small molecules with the potential to promote human osteoblast maturation are desirable in a bone regenerative context, particularly if they have proven efficacy in disease management or treatment; finding novel uses for existing drugs saves time and costs for drug development and is a proven short cut between laboratory and clinic.Indeed James Black (pharmacologist and Nobel laureate) reminds us that "the most fruitful basis for the discovery of a new drug is to start with an old drug" [1,2].A potentially interesting candidate that might fulfil this role is the biguanide metformin (Met).Originally sold as Glucophage, Met is a first-line drug of choice for the treatment of type 2 diabetes [3] but there is emerging evidence that Met may have beneficial effects on bone by supporting osteoblast maturation.Thus far all the studies exploring the action of Met on osteoblast activity have looked at murine or rodent cells sourced from ectodermally derived calvarial tissue, Met has been found to promote collagen and alkaline phosphatase (ALP) expression in rat and mouse osteoblasts [4] and to enhance murine osteoblast differentiation, as supported by raised matrix calcification in extended cell cultures [5][6][7].In addition, Met has been reported to attenuate osteopaenia in ovariectomised rats [8] and to support an osteogenic response in rodent bone marrow derived stromal cells [9].The largely held view from these collected works is that Met influences osteoblast activity via its ability to stimulate adenosine monophosphate-activated protein kinase (AMPK), a heterotrimeric protein central to maintaining cellular energy homeostasis [10,11].Suffice it to say conditions leading to energy depletion, such as exercise, activate AMPK [12] resulting in the suppression of energy consumption, e.g., gluconeogenesis, but activation of energy generation, e.g., glycolysis.

The impedance of normal osteoblast function by microorganisms is at least in part responsible for the failure of dental or orthopedic implants. Staphylococcus aureus is a major pathogen of bone, and exhibits high levels of adhesion and invasion of osteoblasts. In this article we show that the commensal oral bacterium Streptococcus gordonii also adheres to and is internalized by osteoblasts. Entry of S. gordonii cells had typical features of phagocytosis, similar to S. aureus, with membrane protrusions characterizing initial uptake, and closure of the osteoblast membrane leading to engulfment. The sensitivities of S. gordonii internalization to inhibitors cytochalasin D, colchicine and monensin indicated uptake through endocytosis, with requirement for actin accumulation. Internalization levels of S. gordonii were enhanced by expression of S. aureus fibronectin-binding protein A (FnBPA) on the S. gordonii cell surface. Lysosomal-associated membrane protein-1 phagosomal membrane marker accumulated with intracellular S. aureus and S. gordonii FnBPA, indicating trafficking of bacteria into the late endosomal/lysosomal compartment. Streptococcus gordonii cells did not survive intracellularly for more than 12 h, unless expressing FnBPA, whereas S. aureus showed extended survival times (>48 h). Both S. aureus and S. gordonii DL-1 elicited a rapid interleukin-8 response by osteoblasts, whereas S. gordonii FnBPA was slower. Only S. aureus elicited an interleukin-6 response. Hence, S. gordonii invades osteoblasts by a mechanism similar to that exhibited by S. aureus, and elicits a proinflammatory response that may promote bone resorption.

Lysophosphatidic acid (LPA), a pleiotropic signalling lipid is assuming growing significance in osteoblast biology. Although committed osteoblasts from several mammalian species are receptive to LPA far less is known about the potential for LPA to influence osteoblast formation from their mesenchymal progenitors. An essential factor for both bone development and post-natal bone growth and homeostasis is the active metabolite of vitamin D3, calcitriol (D3). Previously we reported how a combination of LPA and D3 synergistically co-operated to enhance the differentiation of immature human osteoblasts. Herein we provide evidence for the formation of human osteoblasts from multiple, primary human bone marrow derived stromal (stem) cells (hBMSCs). Importantly osteoblast development from hBMSCs only occurred when LPA was administered as a complex with albumin, its natural carrier. Collectively our findings support a co-operative role of LPA and D3 in osteoblastogenesis, findings which may aid the development of novel treatment strategies for bone repair.

Oral squamous cell carcinoma (OSCC) is the sixth most common cancer worldwide, accounting for more than 250,000 new cases each year. The 5-year survival rate is approximately 50% and new, targeted approaches for therapy are urgently needed. Lysophosphatidic acid (LPA) is a bioactive lipid that regulates many biological processes associated with cancer development and progression, such as proliferation, migration and apoptosis and deregulated LPA signaling has been demonstrated in a limited number of tumour types. The role of LPA in the pathogenesis of OSCC warrants investigation, not least because LPA is present in significant amounts in saliva. Lysophospholipid levels are elevated in vivo in a number of pathophysiological states and we demonstrate that 6/7 OSCC cell lines secreted LPA into the culture medium, as determined by ELISA. Using quantitative real-time PCR we show that OSCC cell lines express LPA receptors 1-3 and that the LPA3 receptor is over-expressed in 6/7 OSCC cell lines relative to non-tumour derived control cells. Treatment of OSCC cell lines with LPA (1-5µM) had little effect on cell proliferation, but greatly stimulated cell motility in colony dispersal assays, causing colonies to disperse into single, highly migratory cells with an elongated morphology and lamellipodia. Similarly, LPA stimulated OSCC cell migration in Boyden chamber-style chemotactic assays. LPA-induced cell migration was dependent on the MEK, ROCK and Rac-1 signaling pathways. Ultraviolet B (UVB) radiation was used to examine the effect of LPA on mitochondrial-mediated apoptosis. With 50 mJ/cm2 irradiation there was a significant reduction in cell death in OSCC cells treated with 5µM LPA compared to untreated controls (t-test, p = 0.002). By contrast, LPA had no significant effect on non-tumour derived keratinocytes (t-test, p = 1.00). The results of the present study indicate that de-regulated LPA production and/or signaling may contribute to a more aggressive phenotype in OSCC. Work is ongoing to examine the precise role of the LPA3 receptor in this context and to determine whether LPA and its receptors may be used as targets for therapeutic intervention. Citation Information: In: Proc Am Assoc Cancer Res; 2009 Apr 18-22; Denver, CO. Philadelphia (PA): AACR; 2009. Abstract nr 2511.

Zirconia toughened alumina (ZTA) has been regarded as the next generation orthopedic graft material due to its excellent mechanical properties and biocompatibility. Porous ZTA ceramics with good interconnectivity can potentially be used as bone grafts for load-bearing applications. In this work, three-dimensional (3D) interconnected porous ZTA ceramics were fabricated using a direct foaming method with egg white protein as binder and foaming agent. The results showed that the porous ZTA ceramics possessed a bimodal pore size distribution. Their mechanical properties were comparable to those of cancellous bone. Due to the bio-inertness of alumina and zirconia ceramics, surface bioactivation of the ZTA foams was carried out in order to improve their bioactivity. A simple NaOH soaking method was employed to change the surface chemistry of ZTA through hydroxylation. Treated samples were tested by conducting osteoblast-like cell culture in vitro. Improvement on cells response was observed and the strength of porous ZTA has not been deteriorated after the NaOH treatment. The porous 'bioactivated' ZTA ceramics produced here could be potentially used as non-degradable bone grafts for load-bearing applications.

An environmentally friendly direct foaming method was investigated to produce porous alumina ceramics. Egg white protein was used as a binder and foaming agent. The microstructures show that pores are interconnected with pore size of a few hundreds μm and pore window size of ca. 50 μm. The compressive strength of alumina foam is up to 100 MPa depending on porosity. Bioactivation of alumina was carried out using an alkaline solution treatment. Hydroxylation of alumina was achieved using 5M NaOH at 80°C for 4 days. In vitro assessments of the alumina in a human osteoblast cell-like cell (MG63) culture showed that the bioactivated alumina foams exhibited better cellularity and alkaline phosphatase (ALP) activity compared to untreated alumina foams. The results indicate that it is possible to improve the osseointgration of alumina ceramics by structural and surface modifications and to extend the applications of biocompatible alumina ceramics in biomedical implants and tissue engineering scaffolds.

Osteoblast maturation is partly controlled by the interaction of 1α,25(OH)2D3 (D3), an active metabolite of Vitamin D, with other growth factors. The first reports describing the in vitro effect of D3 on human osteoblast differentiation performed experiments in the presence of serum. One potentially exciting candidate that might help explain the D3 responses observed for osteoblasts cultured with serum is lysophosphatidic acid (LPA). Drawn to the possibility that D3 and serum borne LPA might interact to induce osteoblast maturation we co-treated human cells with D3 and serum in the presence of Ki16425, an LPA receptor antagonist. Ki16425 inhibited osteoblast maturation as determined by markedly reduced alkaline phosphatase (ALP) expression. We subsequently found that LPA and D3 acted synergistically in generating mature osteoblasts and that this differentiation response could be inhibited using pertussis toxin, implying an important role of Gαi signal transduction. Furthermore, we found evidence for a dependency on both mitogen activated protein kinase kinase (MEK) and Rho associated coiled kinase (ROCK) for LPA and D3 stimulated maturation.

Osteoblast growth and differentiation are central to the formation and maintenance of healthy bone tissue. The search for novel mechanisms resulting in osteoblast maturation are highly desirable on several fronts. Firstly they provide potentially important information on the normal development of bone, in addition they may offer alternative therapies for bone diseases like osteoporosis and finally they may facilitate ex-vivo manipulation of cells for the subsequent improvement of oseointegration in transplantation/tissue engineering regimens. Recently we have been addressing how calcitriol, an active metabolite of vitamin D3, integrates with the signalling of epidermal growth factor (EGF) following reports that calcitriol can influence EGF receptor trafficking, expression and ligand binding. We have also extended our studies to investigating how other growth factors known to signal via receptor tyrosine kinases (RTKs) interact with calcitriol in controlling osteoblast growth and differentiation. The co-treatment of human pre-osteoblasts (MG63) with EGF and calcitriol resulted in the synergistic induction of their differentiation as supported by demonstrable increases in alkaline phosphatase activity and osteocalcin. The intracellular components responsible for eliciting the maturation response included protein kinase C and MEK 1/2 since the addition of calphostin C or UO126, respectively, blocked the differentiation response. Other ligands known to signal via RTKs, namely IGF1, VEGF and FGF1 could not induce differentiation in the presence of calcitriol. These findings support the specific integration of calcitriol/EGF signalling in osteoblast maturation. Collectively we have identified a novel, integrated, signalling pathway that drives terminal differentiation of osteoblasts. Our findings support earlier predictions (Yoneda 1996) in identifying novel actions of EGF in bone that will lead to advances in the field. Yoneda, T. 1996. Local regulators of bone: Epidermal growth factor – transforming growth factor-α. In Principles of bone biology (ed. J.P. Bilezikian, L.G. Raisz and G.A. Rodan.), pp. 729–738. Academic press Ltd.

Objective: Several genome-wide scans have revealed an osteoarthritis (OA)-susceptibility locus on chromosome 11q in close proximity to the low-density lipoprotein receptor-related protein 5 (LRP5) gene. The regulation of bone mass is under the control of LRP5 and since increased bone mass is thought to play a role in the pathology of OA we examined LRP5 polymorphisms and haplotypes to determine if variants of this locus may predispose to OA.Methods: A UK control population of 187 individuals was examined for five commonly occurring polymorphisms against a cohort of 158 DNAs from patients with knee OA. An additional UK cohort was also examined to confirm the findings of the first study; this second group consisted of 110 knee OA patients. Haplotype analysis was also performed on patient and control DNAs.Results: A study of individual polymorphisms revealed no association with disease. However, haplotype analysis of the initial two populations revealed a common haplotype (C-G-C-C-A) that provided a 1.6-fold increased risk of OA (P = 0.021). The data obtained from the second cohort confirmed the initial findings, with a 1.6-fold increased risk observed within this cohort for the risk haplotype (P = 0.012).Conclusions: A closer investigation of LRP5 and associated Wnt signalling molecules in OA will help determine disease aetiology and the development of novel treatment strategies that specifically target the bone compartment. (C) 2005 OsteoArthritis Research Society International. Published by Elsevier Ltd. All rights reserved.

The subchondral bone has long been known to thicken in osteoarthritis. However, recent evidence has demonstrated that the turnover of the bone is increased several fold, and further suggests that the thickening occurs prior to degradation of the articular cartilage, indicating that it plays a role in the pathogenesis of osteoarthritis. The mechanical and biochemical properties of the subchondral bone are therefore of particular interest in any attempt to determine the nature of the factors initiating osteoarthritis. We have shown that the subchondral bone collagen of the femoral head possessed a 20-fold increase in turnover, as assessed by procollagen rate of synthesis and metalloproteinase degradation, and a 25% decrease in mineralisation. This increased metabolism and high lysyl hydroxylation leads to narrower and weaker fibres. Additionally the phenotypic expression of the osteoblasts is modified to produce increasing proportions of type I homotrimer in addition to the normal type I heterotrimer, which further reduces the mechanical strength of the bone. Overall, the narrow immature collagen fibres, the reduction in pyrrole cross-linking, decreased mineralisation, and increased amounts of type I homotrimer, all contribute to a weakening of the mechanical properties of the subchondral bone.

Calcitriol (1alpha,25(OH)(2)D(3)) plays a key role in the differentiation of osteoblasts, the cells responsible for the formation and maintenance of healthy bone matrix. Recently it has emerged that calcitriol influences the trafficking or stability of epidermal growth factor (EGF) receptors. However, how these agents might work together in regulating growth and differentiation has not been examined. Using the human osteoblast cell line, MG63, we were able to induce a profound differentiation response by treating these cells with a combination of calcitriol (100 nM) and EGF (10 ng/ml). Co-stimulation of MG63 osteoblasts with calcitriol and EGF led to synergistic increases in osteocalcin and alkaline phosphatase (ALP), proteins expressed by differentiating cells. Inhibition of differentiation was accomplished by MEK and protein kinase C (PKC) inhibitors. Other ligands known to signal via receptor tyrosine kinases could not substitute for EGF in the maturation response. These novel findings may help identify new processes that drive osteoblast differentiation.

To investigate placental protein 13 (PP13) localization in relation to cytoskeleton and lipid rafts in preeclampsia and HELLP syndrome.Placental cryosections from patients with preeclampsia and HELLP, and controls were stained for PP13, actin, PLAP (lipid raft marker), and CD71 (nonraft marker). BeWo cells exposed to stress conditions were stained for PP13 and actin. Protein localizations were investigated by confocal microscopy, PP13 concentrations by ELISA.PP13-actin colocalization was increased in syncytiotrophoblast juxtamembrane regions in term/preterm preeclampsia and HELLP. PP13-CD71 colocalization was decreased and PP13-PLAP proximity was increased in preterm but not term preeclampsia and HELLP. PP13-release from BeWo cells was inhibited by cytoskeleton disruption, and augmented by Ca2+-influx and ischemic stress.The actin cytoskeleton, probably in connection with lipid rafts, controls trophoblastic "nonclassical" PP13 export. PP13 is released from the syncytiotrophoblast in preterm preeclampsia and HELLP, mimicked in BeWo cells by ischemic stress, suggesting PP13 is a placental alarmin.