Preventing infections in joint replacements is a major ongoing challenge, with limited effective clinical technologies currently available for uncemented knee and hip prostheses. This research aims to develop a coating for titanium implants, consisting of a supported lipid bilayer (SLB) encapsulating an antimicrobial agent. The SLB will be robustly tethered to the titanium using self-assembled monolayers (SAMs) of octadecylphosphonic acid (ODPA). The chosen antimicrobial is Novobiocin, a coumarin-derived antibiotic known to be effective against resistant strains of ODPA SAMs were deposited on TiO2-coated quartz crystal microbalance (QCM) sensors using two environmentally friendly non-polar solvents (anisole and cyclopentyl methyl ether, CPME), two concentrations of ODPA (0.5mM and 1mM) and two processing temperatures (21°C and 60°C). QCM, water contact angle measurements, X-ray photoelectron spectroscopy (XPS), atomic force microscopy (AFM) and temperature-programmed desorption mass spectrometry (TPD-MS) were used to characterise the ODPA SAM. A SLB with encapsulated Novobiocin was subsequently developed on the surface of the ODPA SAM using fluorescent lipids and a solvent assisted method. The prototype implant surface was tested for antimicrobial activity against A well-ordered, uniform ODPA SAM was rapidly formed using 0.5 mM ODPA in CPME at 21°C during 10 min, as confirmed by high Sauerbrey mass (≍285-290 ng/cm2), high atomic percentage phosphorus (detected using XPS) and high water contact angles (117.6±2.5°). QCM measurements combined with fluorescence microscopy provided evidence of complete planar lipid bilayer formation on the titanium surface using a solvent assisted method. Incorporation of Novobiocin into the SLB resulted in reduced attachment and viability of Key parameters were established for the rapid, robust and uniform formation of an ODPA SAM on titanium (solvent, temperature and concentration). This allowed the successful formation of an antimicrobial SLB, which demonstrated potential for reducing attachment and viability of pathogens associated with joint replacement infections.
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 500nM to 1μM LPA were the optimum concentrations to significantly inhibit bacterial adhesion (ANOVA, p<0.001). These concentrations also reduced biofilm mass on the surface of the titanium. Proteomic analysis highlighted an increase in low molecular weight proteins as a result of optimal LPA surface concentrations. Fatty acid chains as found in LPA have previously been associated with causing leakage of low molecular weight proteins through increased cell membrane permeability. LPA coatings have the potential to enhance implant osseointegration whilst simultaneously reducing bacterial attachment. This technology may reduce both septic and aseptic failure of cementless joint prostheses, ultimately prolonging implant longevity and patient quality of life.
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.
Developing titanium (Ti) surfaces that are biocompatible yet serve as deterrents for bacterial attachment and growth are particularly appealing in tackling the ongoing problem of sepsis-induced implant failures. Realising this could include coating Ti with the bioactive lipid, lysophosphatidic acid. Surgical revision for failed total joint replacements costs a staggering £300m/yr and approximately 20% of this burden is attributed to implant failure through bacterial infection. Producing biomaterials that deter microbial attachment as well as securing robust osseointegration continues to be a significant research challenge in contemporary bone biomaterials design. Steps to realising novel improvements are further compounded by the concerns raised over resistance of bacteria to many antimicrobial agents. Clearly this is a major constraint necessitating an entirely novel approach to minimising implant infection risk. We therefore turned our attention to certain lysophosphatidic acids (LPAs) for Ti functionalisation. We have found LPA to enhance calcitriol-induced human osteoblast (hOB) maturation. Of further significance is the discovery that LPA can directly inhibit the growth of certain bacteria and even co-operate with some antibiotics to bring about their demise. Herein we describe the fabrication of a hOB-compatible Ti surface with palmitoyl-LPA (P-LPA) which we also find hinders bacterial attachment.Summary Statement
Introduction
Successful osseointegration requires the production of a mechanically competent collagenous matrix, by osteoblasts, at the implant site. Lysophosphatidic acid (LPA) is a bioactive lipid which we discovered interacts with vitamin D3 (D3) to secure human osteoblast (hOB) maturation on both titanium (Ti) and hydroxyapatite. We therefore covalently attached LPA and a related compound, (3S) 1-fluoro-3-hydroxy-4-butyl-1-phosphate (FHBP), to both solid and porous Ti discs and seeded them with hOBs to assess their ability to support D3-induced cell maturation. Solid functionalised discs were washed and reused a further two times, whilst other discs were stored for 6 months. Increased alkaline phosphatase (ALP) activity indicated that both LPA and FHBP-modified Ti serve as superior substrates for securing D3-induced hOB maturation compared to unmodified metal (p < 0.001). Although total ALP activity was less for cells on recycled discs and after storage, enzyme levels were still significantly greater compared to hOBs grown on control Ti. LPA and D3 co-treatment also resulted in an increase in osteocalcin (∼17ng/ml versus 6ng/ml for D3 alone, P < 0.001) and collagen synthesis (∼310pg/ml versus <10pg/ml for D3 alone, P < 0.001). Research is ongoing to evaluate the efficacy of our modified Ti surfaces to secure hOB formation from their stem cell progenitors.
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.
