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Orthopaedic Proceedings
Vol. 102-B, Issue SUPP_5 | Pages 42 - 42
1 Jul 2020
Lotfi N Hughes E McCulloch R Horner C Shepherd D Grover L Nightingale P Davis E
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Polymethylmethacetate (PMMA) is a bone cement used in over 725,000 primary hip arthroplasties in 2018. Cement integrity is affected by external factors, including temperature, mixing technique and moisture uptake, which can influence cement microstructure. Changes in the cement microstructure may ultimately threaten the survivorship of the implant.

The introduction of enhanced recovery and various local anaesthetic infiltration techniques have been adopted in an attempt to facilitate early mobilisation and reduce length of stay. Our study aims to investigate if the mechanical properties of PMMA are altered with exposure to Ropivacaine LA.

Cements were cured in three separate states (air, serum and serum with LA) and the mechanical properties tested at 24 hours and 28 days. Using Refobacin bone cement provided by ZimmerBIOMET, cylindrical molds (12×6mm) were constructed with a split-mold. The LA used was 2mg/ml Ropivacaine hydrochloride solution. Using pilot data, this study was powered to 80% and a sample size of 10 per group (n=60) was calculated.

Cement samples were subjected to compressive loading using a universal testing apparatus (Zwick/Roell). Yield-strength and modulus values were extracted from the respective stress versus strain curves. Significant differences were determined by one-way anova for each time point, and Bonferroni post-hoc testing to determine significance between actual groups.

At 24-hours there were no significant differences in strength or modulus between groups. At 28-day strength and modulus increased in all groups. Compared to the air group, both serum and LA groups show a significant decrease in compressive strength. The modulus for the LA group is significantly less stiff compared to the air group.

The results suggest that the initial exposure to LA has a significant impact on the physical properties of the PMMA. We propose increased awareness of the potential effects this may have on the longevity and survivorship of cemented implants.


Orthopaedic Proceedings
Vol. 101-B, Issue SUPP_5 | Pages 129 - 129
1 Apr 2019
Lowther M Cox SC Grover L
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Introduction

Implant associated infections are responsible for over 10 % of recorded orthopaedic revision surgeries across the UK, with higher infection rates commonly observed for other endoprostheses such as cranioplasties. To prevent colonization and biofilm formation on implant surfaces, the use of silver coatings has shown positive results in clinical setting due to its synergistic function with conventional antibiotic prophylaxes. Additive manufacturing allows manufacture of entirely new implant geometries such as lattice structures to enhance osseointegration, however this limits the ability to uniformly coat implants. Direct integration of silver into the powder feedstock for selective laser melting (SLM) may allow manufacture of a biomedical alloy with innate, long lasting antimicrobial properties without compromising possible geometries and with no coating process necessary.

Methods

Feedstock powders of 15–45 micron Grade 5 Ti-64 (Renishaw Plc) and Ag-999 powder (CooksonGold) were characterized using laser particle size analysis, ICP-OES, LECO-ONH, and morphological analysis in SEM. A blend of Ti-64 with 3 wt% Ag-999 powder (Ti-643) was produced by tumble blending, and validated by SEM and EDS. Parameters for manufacture were established using a 17 point design of experiment (DoE) exploring a 2D parameter space of applied laser power and laser scanning speed. Samples were manufactured using a ConceptLaser M2 LaserCusing SLM. Density was assessed by He pycnometry, and cross-sections analysed for defects by optical microscopy. Silver distribution was mapped by micro X-Ray Fluoroscopy (µXRF) and energy-dispersive X-ray spectroscopy (EDS). Optimum parameters were identified and used to manufacture all subsequent samples.

Cylindrical Ti-643 samples were manufactured for further physical characterization and bacterial investigation, alongside control Ti-64 samples manufactured using existing optimum parameters. Samples were polished using silicon carbide papers to a 4000-grit surface finish. Contact angle measurements were made by goniometry. Silver elution characteristics were assessed by immersion in water refreshed on a daily basis, and sampled over a 14 day period using ICP-OES. Viability of S. aureus was compared to control samples according to the Japanese standard test method, JIS Z 2801:2000.


Orthopaedic Proceedings
Vol. 100-B, Issue SUPP_14 | Pages 100 - 100
1 Nov 2018
Grover L
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Phosphate metabolism is central to the bone formation pathway. Phosphate is shuttled through the cell membrane to the mitochondria, where it is polymerised to form adenosine triphosphate. Once exocytosed the ATP may then be cleaved to form pyro and orthophosphates, the balance of which can determine whether mineralisation occurs or not. We are developing a range of materials at the University of Birmingham that have been formulated so that they can influence this balance, with the potential either to drive or prevent mineralisation from occurring. This talk will describe how we have used this process to develop materials that can be used to stimulate bone formation around an implant or to prevent the formation of pathological bone. It will also talk about the steps that we have taken to move these therapies towards clinical trial.


Orthopaedic Proceedings
Vol. 100-B, Issue SUPP_7 | Pages 23 - 23
1 May 2018
Eisenstein N Williams R Cox S Stapley S Grover L
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Heterotopic ossification is the formation of lamellar bone in soft tissues and is a common complication of high-energy combat injury. This disabling condition can cause pain, joint ankylosis, and skin ulceration in the residua of amputees. This project is aimed at developing a novel treatment to dissolve hydroxyapatite in heterotopic ossification and prevent the crystallisation of this this mineral at sites of ectopic bone formation. Previously reported results demonstrated that hexametaphosphate could dissolve hydroxyapatite at physiological pH. Further work has been undertaken to investigate the mechanism of this dissolution and establish a means of temporal control of action. In addition, physicochemical analyses of samples of human heterotopic ossification have yielded important insights into the nature of this pathological tissue. Techniques include mapped micro X-ray fluorescence, mapped Raman spectroscopy, scanning electron microscopy, and micro computed tomography. Formulation engineering work has begun in order to develop an appropriate delivery vehicle for this agent. This includes rheological testing and hexametaphosphate elution profiles. Finally, micro CT analysis has shown that hexametaphosphate is able to dissolve human heterotopic ossification tissue. In summary, this work has moved us closer towards our goal of a novel injectable agent for the treatment and prevention of heterotopic ossification.


Orthopaedic Proceedings
Vol. 100-B, Issue SUPP_4 | Pages 47 - 47
1 Apr 2018
Hughes E Williams R Cooke M Hall T Cox S Grover L
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Background

Bone is a hierarchically structured hard tissue that consists of approximately 70 wt% low-crystallinity hydroxyapatite. Intricate tubular channels, such as Haversian canals, Volkman's canals, and canaliculi are a preserved feature of bone microstructure. These structures provide pathways for vasculature and facilitate cell-to-cell communication processes, together supporting viability of cellular components and aiding in remodeling processes. Unfortunately, many commercial bone augmentation materials consist of highly crystalline phases that are absent of the structuring present within the native tissue they are replacing. This work reports on a the development of a novel bone augmentation material that is able to generate biologically analogous tubular calcium phosphate mineral structures from hydrogel-based spheres that can be packed into defects similar to those encountered in vivo.

Experimental

Calcium loaded spheres were made by adding 5 wt% agar powder to 1 M calcium nitrate solutions, before heating the mixture to 80–90 oC and feeding droplets of gel into a reservoir of liquid nitrogen. Deposition of tubular mineral was initiated by exposure to ammonium phosphate solutions at concentrations between 500 mM and 1 M, and was characterized by micro-XRF mapping, XRD and SEM techniques. For an ex vivo model, human bone tissue was collected from patients undergoing elective knee replacement surgery. The United Kingdom National Research Ethics Service (East of Scotland Research Ethics Service) provided ethical approval (11/ES/1044). The augmented defect of the model was characterised by micro-XRF mapping and micro-CT techniques.


Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_8 | Pages 46 - 46
1 Apr 2017
Floyd H Lord J Davies E Addison O Grover L
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Background

Investigations into the response to implant debris tend to concentrate on how a population of cells proliferate in the presence of implant material, and how the regulation of cytokines change. For the problem of cobalt-chromium (CoCr) implants this has been done for osteoblasts and osteoclasts to understand how bone resorption, leading to aseptic loosening, is occurring. However, investigating the formation of the extracellular-matrix (ECM) may give a better indication of the mechanisms occurring. ECM is excreted from cells and is important for adhesion, structure, signaling and growth. Type I collagen is the most abundant protein in the ECM and is known to direct tissue development and is therefore a key part of understanding the mechanism behind aseptic loosening.

Methods

3T3-fibroblasts were seeded in Dulbecco's Modified Eagle Medium (DMEM) and supplemented with 100mM ascorbic acid. Every 48hours cells were fed with DMEM and doped with Co and Cr ions until fixation. Sirius Red dye was used to bind to the type I collagen, then removed using NaOH and analysed using UV absorption to show relative amounts of collagen. Type I collagen gel was formed in the presence of Co and Cr ions with and without DMEM and the fibers were imaged using AFM.


Orthopaedic Proceedings
Vol. 99-B, Issue SUPP_8 | Pages 85 - 85
1 Apr 2017
Hughes E Williams R Chouhan G Jamshidi P Grover L
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Background

Calcium orthophosphates, such as hydroxyapatite (Ca5(PO4)3OH) (HA), have long been employed as bone graft materials. Recent work has suggested that calcium pyrophosphate (Ca2P2O7) (CaPy) may strongly stimulate bone deposition. In this study we compare calcium orthophosphate and pyrophosphate precipitates as suitable bone regeneration materials. As well as HA, two forms of pyrophosphate precipitate were compared in this work: amorphous calcium pyrophosphate (amCaPy) and star particle calcium pyrophosphate (stCaPy).

Methods

Briefly, 0.15M Na4P2O7·10H2O and 0.3M Ca2Cl·2H2O solutions of equivalent volume were combined and left to age before performing a series of filtration and re-suspension steps upon the precipitate. Drying yielded amCaPy powder. stAmPy was produced by the same procedure however the pH of the starting solutions were altered to pH7 before combination.


Orthopaedic Proceedings
Vol. 97-B, Issue SUPP_8 | Pages 9 - 9
1 Jun 2015
Eisenstein N Grover L Stapley S
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Heterotopic ossification (HO) is the formation of bone in extraskeletal sites. It is a major problem for combat-related casualties with 64% of such patients showing radiological evidence of the disease. Of these, 19% require surgical excision. Current prophylaxis is problematic due to poor efficacy and unsuitability in a military setting. Our novel anti-HO strategy is to use an inorganic reagent to inhibit the deposition of HA and disperse any pre-formed mineral. Literature review identified several potentially effective agents. These were tested for their ability to disperse solid monoliths of HA. In addition, a standard HA synthetic reaction was performed in the presence of each agent to establish their inhibiting activity. One reagent (a condensed phosphate) dispersed a solid monolith of HA by 38% (mass loss) over 30 days. This reagent was also shown to inhibit HA crystal synthesis yield by 28%. Early work on a hydrogel delivery system has produced favourable results. These preliminary data demonstrate proof of concept that HA may be dispersed and its formation inhibited by a non-toxic polyphosphate. This work will form the justification for development into in vitro osteogenic cell culture models and animal HO models.


Orthopaedic Proceedings
Vol. 96-B, Issue SUPP_11 | Pages 294 - 294
1 Jul 2014
Williams R Salimi N Leeke G Bridson R Grover L
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Summary Statement

Calcium phosphate (CaP) particles have attracted great interest as transfection reagents, yet little is known about their mechanism of internalisation. We report live cell time-course tracking of CaP particles during internalisation and the influence of Ca:P ratio on transfection efficiency.

Introduction

Relatively recent work has seen calcium phosphate (CaP) salts used for the delivery of biological materials into cells in the form of peptides, polymers and DNA sequences. Calcium phosphate salts have a critical safety advantage over other vectors such as viruses in that they pose no risk of pathogenicity due to mutation and show no apparent cytotoxicity. Previous work within the group showed that Ca:P ratio influenced the transfection efficiency, but the fate of the particles on internalisation is yet unknown. The difficulty in tracking the particles can be related to the visual similarity to granulation within the cells. Using a surface modification method that enables the fluorescent labeling of silicon-substituted hydroxyapatite (SiHA) particles, we have tracked the internalisation of the particles to understand their mechanism of entry and how particle composition may influence transfection efficiency.