Ternary nanocomposite potentiates the lysophosphatidic acid effect on human osteoblast (MG63) maturation.

Aim: This study aimed to investigate the potential of ternary nanocomposite (TNC) to support MG63 osteoblast maturation to EB1089-(3S)1-fluoro-3-hydroxy-4-(oleoyloxy)butyl-1-phosphonate (FHBP) cotreatment. Materials & methods: Binary (P25/reduced graphene oxide [rGO]) nanocomposite was prepared, and silver (Ag) nanoparticles were loaded onto the surface to form TNC (P25/rGO/Ag). The influence of TNC on proliferation, alkaline phosphatase activity and osteogenic gene expression was evaluated in a model of osteoblast maturation wherein MG63 were costimulated with EB1089 and FHBP. Results: TNC had no cytotoxic effect on MG63. The addition of TNC to EB1089-FHBP cotreatment enhanced the maturation of MG63, as supported by the greater alkaline phosphatase activity and OPN and OCN gene expression. Conclusion: TNC could serve as a promising carrier for FHBP, opening up possibilities for its application in bone regeneration.

Nanoparticles (NPs) are often used in medicine because they have certain benefits over traditional drugs, such as increased delivery. Multiple NPs can be combined into hybrid NPs called nanocomplexes, which can have many positive effects. One application of nanomedicine is to encourage the repair of certain body tissues such as bones. Encouraging stem cells to differentiate into bone cells and immature bone cells to mature is key in this process. This study made a ternary nanocomplex (TNC), meaning it was comprised of three NPs. This TNC was designed to deliver a drug called (3S)1-fluoro-3-hydroxy-4-(oleoyloxy)butyl-1-phosphonate (FHBP), which has been shown to encourage the maturation and development of osteoblasts, a type of bone cell. The TNC was made up of silver NPs, which can kill bacteria; reduced graphene oxide, which enhances the production of bone cells; and titanium dioxide, which has shown effectiveness in wound healing and mixed results in bone tissue regeneration. This TNC was tested on a cell line that comes from a type of bone cancer called MG63. The TNC was found to not be toxic to these cells. TNC incorporation into FHBP treatment enhanced the maturation of MG63. This suggests that these TNCs could be an effective treatment to encourage bone repair following joint replacement surgeries.

Dual Action of Lysophosphatidate-Functionalised Titanium: Interactions with Human (MG63) Osteoblasts and Methicillin Resistant Staphylococcus aureus.

Titanium (Ti) is a widely used material for surgical implants; total joint replacements (TJRs), screws and plates for fixing bones and dental implants are forged from Ti. Whilst Ti integrates well into host tissue approximately 10% of TJRs will fail in the lifetime of the patient through a process known as aseptic loosening. These failures necessitate revision arthroplasties which are more complicated and costly than the initial procedure. Finding ways of enhancing early (osseo)integration of TJRs is therefore highly desirable and continues to represent a research priority in current biomaterial design. One way of realising improvements in implant quality is to coat the Ti surface with small biological agents known to support human osteoblast formation and maturation at Ti surfaces. Lysophosphatidic acid (LPA) and certain LPA analogues offer potential solutions as Ti coatings in reducing aseptic loosening. Herein we present evidence for the successful bio-functionalisation of Ti using LPA. This modified Ti surface heightened the maturation of human osteoblasts, as supported by increased expression of alkaline phosphatase. These functionalised surfaces also deterred the attachment and growth of Staphylococcus aureus, a bacterium often associated with implant failures through sepsis. Collectively we provide evidence for the fabrication of a dual-action Ti surface finish, a highly desirable feature towards the development of next-generation implantable devices.

24,25-Dihydroxyvitamin D3 cooperates with a stable, fluoromethylene LPA receptor agonist to secure human (MG63) osteoblast maturation.

Vitamin D receptor (VDR) agonists supporting human osteoblast (hOB) differentiation in the absence of bone resorption are attractive agents in a bone regenerative setting. One potential candidate fulfilling these roles is 24,25-dihydroxy vitamin D3 (24,25D). Over forty years ago it was reported that supraphysiological levels of 24,25D could stimulate intestinal calcium uptake and aid bone repair without causing bone calcium mobilisation. VDR agonists co-operate with certain growth factors to enhance hOB differentiation but whether 24,25D might act similarly in promoting cellular maturation has not been described. Given our discovery that lysophosphatidic acid (LPA) co-operated with VDR agonists to enhance hOB maturation, we co-treated MG63 hOBs with 24,25D and a phosphatase-resistant LPA analog. In isolation 24,25D inhibited proliferation and stimulated osteocalcin expression. When co-administered with the LPA analog there were synergistic increases in alkaline phosphatase (ALP). These are encouraging findings which may help realise the future application of 24,25D in promoting osseous repair.

Lysophosphatidic acid, human osteoblast formation, maturation and the role of 1α,25-Dihydroxyvitamin D3 (calcitriol).

The simplest signalling lipid Lysophosphatidic acid (LPA) elicits pleiotropic actions upon most mammalian cell types. Although LPA has an established role in many biological processes, particularly wound healing and cancer, the function of LPA for human osteoblast (hOB) biology is still unravelling. Early studies, identified in this review, gave a reliable indication that LPA, via binding to one of several transmembrane receptors, stimulated multiple intracellular signalling networks coupled to changes in cell growth, fibronectin binding, maturation and survival. The majority of studies exploring the actions of LPA on hOB responses have done so using the lipid in isolation. Our own research has focussed on the co-operation of LPA with the active vitamin D3 metabolite, 1α25,dihydroxycholecalciferol (calcitriol), in light of a serendipitous discovery that calcitriol, in a serum-free culture setting, was unable to promote hOB maturation. We subsequently learnt that the serum-borne factor co-operating with calcitriol to enhance hOB differentiation was LPA bound to the albumin fraction of whole serum. Recent studies from our laboratory have identified that LPA and calcitriol are a potent pairing for securing hOB formation from their stem cell progeny. Greater understanding of the ability of LPA to influence, for example, hOB growth, maturation and survival could be advantageous in developing novel strategies aimed at improving skeletal tissue repair and regeneration. Herein this review provides an insight into the diversity of studies exploring the actions of a small lipid on a major cell type key to bone tissue health and homeostasis. This article is part of a Special Issue entitled Advances in Lysophospholipid Research.

Lithocholate--a promising non-calcaemic calcitriol surrogate for promoting human osteoblast maturation upon biomaterials.

BACKGROUND AND AIMS: Calcitriol, an active vitamin D metabolite, has a limited application in bone repair because of its undesirable hypercalcaemic action. However it has emerged that lithocholic acid (LCA) is a non-calcaemic vitamin D receptor ligand but whether this steroid can support osteoblast maturation has not been reported. Using the human osteoblast cell line, MG63, we explored the potential of LCA and LCA derivatives to secure osteoblast maturation. RESULTS: The co-stimulation of cells with LCA, LCA acetate or LCA acetate methyl ester (0.5-5 microM) and lysophosphatidic acid (LPA, 20 microM) resulted in clear, synergistic increases in MG63 maturation that was both time and dose dependent. Cells grown upon both titanium and hydroxyapatite, two widely used implant materials, responded well to co-treatment with LCA acetate (5 microM) and LPA (20 microM) as demonstrated by stark, synergistic increases in ALP activity. Evidence of activator protein-1 (AP-1) stimulation by LCA acetate (30 microM) was demonstrated using an AP-1 luciferase reporter assay. Synergistic increases in ALP activity, and therefore osteoblast maturation, were observed for MG63 cells co-stimulated with LCA acetate (5 microM) and either epidermal growth factor (10 ng/ml) or transforming growth factor-beta (10 ng/ml). Ligands acting on either the farnesoid X receptor or pregnane X receptor could not substitute for the action of LCA acetate on MG63 maturation. CONCLUSIONS: Lithocholate is able to act as a calcitriol surrogate in generating mature osteoblasts. Given that LCA is non-calcaemic it is likely to find an application in bone repair/regeneration by aiding matrix calcification at implant sites.

Cytoskeletal reorganisation, 1alpha,25-dihydroxy vitamin D3 and human MG63 osteoblast maturation.

Bone tissue is especially receptive to physical stimulation and agents with the capacity to mimic the signalling incurred via mechanical loading on osteoblasts may find an application in a bone regenerative setting. Recently this laboratory revealed that the major serum lipid, lysophosphatidic acid (LPA), co-operated with 1alpha,25-dihydroxy vitamin D3 (D3) in stimulating human osteoblast maturation. Actin stress fiber accrual in LPA treated osteoblasts would have generated peripheral tension which in turn may have heightened the maturation response of these cells to D3. To test this hypothesis we examined if other agents known to trigger stress fiber accumulation co-operated with D3 in stimulating human osteoblast maturation. Colchicine, nocodazole and LPA all co-operated with D3 to promote MG63 maturation in a MEK dependent manner. In contrast, calpeptin, a direct activator of Rho kinase and stress fiber accumulation did not act with D3 to secure MG63 differentiation. Herein we describe how the signalling elicited via microtubule disruption cooperates with D3 in the development of mature osteoblasts.