Novel approaches to the development of PMMA bone cement

With an ageing population on the increase, there is a growing need for more effective treatments to enhance the quality of life of patients. Biomaterials employed in such treatments are therefore required to last longer and function more effectively. A biomaterial of particular interest is polymethyl methacrylate (PMMA) bone cement, which is widely employed in joint replacement surgery. Although this replacement procedure reduces pain and restores joint function, it is associated with a failure rate of approximately 10% after 15 years usually as a consequence of cement functional deterioration. Failure usually requires a complicated revision surgery, which is a burden on both the patient and health care services. This study has therefore applied novel interdisciplinary approaches to the design of PMMA bone cements in an effort to reduce failure in cemented joint replacements. Failure of PMMA bone cements has been previously linked to agglomerations of the radiopacifier employed, which create stress concentrations and initiate cracks. A model cement was therefore developed, with compositional, mechanical, fatigue and rheological properties similar to commercial cements, which enabled two novel radiopacifiers to be tested (anatase TiO2 and yttria-stabilised ZrO2). Regardless of the material employed, agglomerations of the radiopacifiers were found to be a significant problem. Silane treating the radiopacifiers enhanced their dispersion, improving the mechanical and fatigue properties of the cement. Furthermore, anatase TiO2 and silane-treated anatase TiO2 were found to induce hydroxylapatite mineralisation in vitro and enhance the adhesion of MC3T3-E1 osteoblast precursor cells on the surface of the cement. The silane treatment however, was found to decrease the rate of osteoblast proliferation. Ageing effects and moisture uptake in PMMA bone cements were also examined as cement is known to fail predominantly after long periods of use. Ageing cements in isotonic fluid resulted in a maximum moisture uptake of approximately 2%w/w, which was found to induce structural changes over time and caused degradation in the mechanical properties of the cement, potentially contributing to cement failure. A major obstacle with joint replacements is the likelihood of post-operative infections. In an attempt to prevent this, many commercial cements incorporate large amounts of powdered antibiotic to achieve a local therapeutic release. The powdered antibiotic was found to be poorly dispersed and resulted in an uncontrolled initial release from surface agglomerations within the first 6 hours, with potentially sub-inhibitory resistance-inducing levels thereafter. Furthermore, only a small percentage (2-9%) of the antibiotic was released, the commercial cements demonstrated poor bacterial inhibition and incorporating powdered antibiotics was detrimental to the mechanical and fatigue performance of the cement. To overcome these limitations a novel delivery system was developed based on drug-entrapped liposome vesicles. A block co-polymer coating was applied to phospholipid liposomes (100nm diameter) to achieve a uniform dispersion in a commercial bone cement (Palacos R). When antibiotic-loaded liposomes (gentamicin sulphate) were dispersed in the cement, greater levels of antibiotic were released in a more prolonged manner, with enhanced antimicrobial, mechanical, fracture toughness and fatigue properties. Techniques from a variety of disciplines were employed in this study and this inter-disciplinary approach has allowed many features of PMMA bone cement to be investigated. The experiments have offered an insight into cement failure while novel techniques and formulations have been developed, which have the potential to reduce failure and infection in cemented implants and may have wider application in a variety of biomaterials

The effectiveness of adhesives on the retention of mandibular free end saddle partial dentures: an in vitro study

Objectives: Existing in vitro methods for testing denture adhesives do not fully replicate the complex oral geometries and environment; and in vivo methods are qualitative, prone to bias and not easily reproducible. The purpose of this study was to develop a novel, quantitative and more accurate model to test the effect of adhesives on the retentive force of mandibular free end saddle partial dentures. Methods: An in vitro model was developed based on an anatomically accurate cast of a clinical case. Conclusions: An in vitro denture adhesive model was developed, which demonstrated that mass of adhesive plays a significant role in enhancing denture retention and supported the design principle of placing as few teeth as clinically necessary on the distal end of the free end saddles. Experimentally, the amount of adhesive was varied (0.2g-1g) and the tensile force required for displacement was measured. Different commercially available adhesives were then tested at the optimum volume using the in vitro model. A 3D finite element model of the denture was used to assess how the forces to induce denture displacement varied according to the position of the force along the saddle length. Results: The mass of adhesive was found to significantly alter retention forces, with 0.4-0.7g being the optimum range for this particular scenario. Use of adhesives significantly improved mandibular free end saddle partial denture retention with the worst performing adhesive increasing retention ninefold whilst the best performing adhesive increased retention twenty three-fold. The finite element model revealed that 77% more force was required to displace the denture by positioning forces towards the mesial end of the saddle compared to the distal end

Nanoparticle and nanotopography-induced activation of the Wnt pathway in bone regeneration

Background and Aims: Recent research has focused on developing nanoparticle and nanotopography-based technologies for bone regeneration. The Wnt signalling pathway has been shown to play a vital role in this process, in particular in osteogenic differentiation and proliferation. The exact mechanisms by which nanoparticles and nanotopographies activate the Wnt signalling pathway however are not fully understood. This review aimed to elucidate the mechanisms by which nano-scale technologies activate the Wnt signalling pathway during bone regeneration. Methods: The terms "Wnt", "bone", and "nano*" were searched on PubMed and Ovid with no date limit. Only original research articles related to Wnt signalling and bone regeneration in the context of nanotopographies, nanoparticles or scaffolds with nanotopographies or nanoparticles were reviewed. Results: The primary mechanism by which nanoparticles activated the Wnt pathway was by internalisation via the endocytic pathway or diffusion through the cell membrane, leading to accumulation of non-phosphorylated β-catenin in the cytoplasm and subsequently downstream osteogenic signalling (e.g. upregulation of RUNX2). The specific size of the nanoparticles and the process of endocytosis itself has been shown to modulate the Wnt-β-catenin pathway. Nanotopographies were shown to directly activate frizzled receptors, initiating Wnt/β-catenin pathway signalling. Additional studies showed nanotopographies to activate the Wnt/Ca2+ dependent and Wnt/planar cell polarity pathways via nuclear factor of activated T-cells, and α5β1 integrin stimulation. Finally, scaffolds containing nanotopographies/nanoparticles were found to induce Wnt signalling via a combination of ion release (e.g. lithium, boron, lanthanum and icariin) which inhibited GSK-3β activity, and via similar mechanisms to the nanotopographies. Conclusion: This review concludes that nanoparticles and nanotopographies cause Wnt activation via several different mechanisms, specific to the size, shape and structure of the nanoparticles or nanotopographies. Endocytosis-related mechanisms, integrin signalling and ion release were the major mechanisms identified across nanoparticles, nanotopographies and scaffolds respectively. Knowledge of these mechanisms will help develop more effective targeted nanoscale technologies for bone regeneration

Ageing and moisture uptake in polymethyl methacrylate (PMMA) bone cements

Bone cements are extensively employed in orthopaedics for joint arthroplasty, however implant failure in the form of aseptic loosening is known to occur after long-term use. The exact mechanism causing this is not well understood, however it is thought to arise from a combination of fatigue and chemical degradation resulting from the hostile in vivo environment. In this study, two commercial bone cements were aged in an isotonic fluid at physiological temperatures and changes in moisture uptake, microstructure and mechanical and fatigue properties were studied. Initial penetration of water into the cement followed Fickian diffusion and was thought to be caused by vacancies created by leaching monomer. An increase in weight of approximately 2% was experienced after 30 days ageing and was accompanied by hydrolysis of poly(methyl methacrylate) (PMMA) in the outermost layers of the cement. This molecular change and the plasticising effect of water resulted in reduced mechanical and fatigue properties over time. Cement ageing is therefore thought to be a key contributor in the long-term failure of cemented joint replacements. The results from this study have highlighted the need to develop cements capable of withstanding long-term degradation and for more accurate test methods, which fully account for physiological ageing

Initial Investigations of the cranial size and shape of adult Eurasian otters (Lutra lutra) in Great Britain

Three-dimensional (3D) surface scans were carried out in order to determine the shapes of the upper sections of (skeletal) crania of adult Eurasian otters (Lutra lutra) from Great Britain. Landmark points were placed on these shapes using a graphical user interface (GUI) and distance measurements (i.e., the length, height, and width of the crania) were found by using the landmark points. Male otters had significantly larger skulls than females (P < 0.001). Differences in size also occurred by geographical area in Great Britain (P < 0.05). Multilevel Principal Components Analysis (mPCA) indicated that sex and geographical area explained 31.1% and 9.6% of shape variation in “unscaled” shape data and that they explained 17.2% and 9.7% of variation in “scaled” data. The first mode of variation at level 1 (sex) correctly reflected size changes between males and females for “unscaled” shape data. Modes at level 2 (geographical area) also showed possible changes in size and shape. Clustering by sex and geographical area was observed in standardized component scores. Such clustering in a cranial shape by geographical area might reflect genetic differences in otter populations in Great Britain, although other potentially confounding factors (e.g., population age-structure, diet, etc.) might also drive regional differences. This work provides a successful first test of the effectiveness of 3D surface scans and multivariate methods, such as mPCA, to study the cranial morphology of otters

Interrogating the osteogenic potential of implant surfaces in vitro: a review of current assays

The success of implantable devices relies heavily on their interaction with the host cells facilitating the osseointegration process. However, with so many new surface modifications, with subtly varying design parameters, in vitro assays can, with proper interpretation, provide valuable information for understanding cellular behavior. This review brings together pertinent in vitro experimental protocols available to researchers and discusses them in relationship to the development of the osteoblast phenotype during bone repair. Consideration is also paid to the influence of endothelial and macrophage cells that can substantially change osteogenic cell activity and thus can provide added value for predicting the osseointegration potential in vivo. Due to the diverse and heterogeneous nature of cell types available for culture use, this review concludes that there is no “gold standard” series of assays. Rather, we present guidance in the experimental design of in vitro assays to better identify those surfaces with promising osteogenic potential

Growth Factor release and dental pulp stem cell attachment following dentine conditioning- an in vitro study

Aim To investigate the effect of dentine conditioning agents on growth factor liberation and settlement of Dental Pulp Progenitor Cells (DPSCs) on dentine surfaces. Methodology The agents used included ethylenediaminetetraacetic acid (EDTA; 10%, pH 7.2), phosphoric acid (37%, pH<1), citric acid (10%, pH 1.5) and polyacrylic acid (25%, pH 3.9). Human dentine slices were conditioned for exaggerated conditioning times of 5 and 10 minutes, so that the growth factor liberation reached quantifiable levels above the limit of detection of the laboratory methods employed. Transforming growth factor beta1 (TGF-β1) release and surface exposure were quantified by Enzyme-linked immunosorbent assay (ELISA) and immunogold labelling. Scanning electron microscopy (SEM) was used to assess the morphology of cells and coverage by DPSCs cultured on dentine surfaces for 8 days. Results After 5-minutes conditioning of dentine slices, citric acid was the most effective agent for growth factor release into the aqueous environment as measured by ELISA (Mann Whitney U with Bonferroni correction, P<0.01 compared with phosphoric and polyacrylic acid). As well as this, dentine slices treated with phosphoric acid for the same period, displayed significantly less TGF-β1 on the surface compared with the other agents used, as measured by immunogold labelling (MWU with Bonferroni correction, P<0.05). After 8 days, widespread coverage by DPSCs on dentine surfaces conditioned with citric acid and EDTA were evident under SEM. On dentine surfaces conditioned with phosphoric and polyacrylic acid respectively, less spread cells and inconsistent cell coverage were observed. In conclusion Based on the findings of this in vitro study, a desirable biological growth factor mediated effect may be gained when conditioning dentine by milder acidic or chelating agents such as citric acid and EDTA. The results must be interpreted in the context that the potential of the applied materials in inducing a desirable biological response in DPSCs, is only one consideration amongst other important ones in a clinical setting. However, it is crucial to look beyond the mere physical effects of materials and move towards biologically based treatment approaches as far as the restorative management of teeth with viable dental pulps are concerned

Liposomal delivery of demineralised dentine matrix for dental tissue regeneration

Current dental restorations have short longevity, consequently there is a need for novel tissue engineering strategies that aim to regenerate the dentine-pulp complex. Dentine matrix contains a myriad of bioactive growth factors and extracellular matrix proteins associated with the recruitment, proliferation and differentiation of dental pulp progenitor cells. Here, we show that demineralised dentine matrix (DDM), from non-carious dentine, can be encapsulated into liposomes for delivery to dental tissue to promote regeneration. Liposomes were formulated to encapsulate 0 - 100 μg/mL DDM, lysed with Triton X and used in VEGF and TGF-β1 ELISAs to quantify release. The encapsulation efficiency was calculated to be 25.9% and 28.8% (VEGF/TGF-β1) for 50 μg/mL DDM liposomes and 39% and 146.7% (VEGF/TGF-β1) for 100 μg/mL DDM liposomes. All liposome formulations had no cytotoxic effects on a dental pulp stem cell (DPSC) clone, as shown by MTT, Caspase 3/7 assays and cell counts. The ability of the liposomes to stimulate DPSC chemotactic recruitment was tested by Boyden chamber chemotaxis assays. Unloaded liposomes alone stimulated significant progenitor cell recruitment, while DDM loaded liposomes further promoted chemotactic recruitment in a dose dependent manner. DDM liposomes promoted the upregulation of 'osteodentine' markers osteocalcin and RUNX2 in DPSCs after 9 days of treatment, determined by Real Time quantitative PCR. Furthermore, Alizarin Red S staining showed that unloaded liposomes alone induced biomineralisation of DPSCs and DDM liposomes further increased the amount of mineralization observed. DDM liposomes were more effective than free DDM (10 μg/mL) at activating recruitment and osteogenic differentiation of DPSC, which are key events in the endogenous repair of the dentine-pulp complex. The study has highlighted the therapeutic potential of bioactive DDM liposomes in activating dental tissue repair in vitro, suggesting that liposomal delivery from biomaterials could be a valuable tool for reparative dentistry and hard tissue engineering applications