Introduction

The incidences of fragility fractures, often because of osteoporosis, are increasing. Research has moved towards bioresorbable scaffolds that provide temporary mechanical stability and promote osteogenesis. This research aims to fabricate a 3D printed composite Poly (l-lactic-co-glycolic acid)-strontium doped tricalcium phosphate (PLGA-SrTCP) scaffold and evaluate in an in vitro co culture study containing osteoporotic donor cells.

Abstract

With an ever-increasing aging population, total hip and knee arthroplasty is projected to increase by 137% and 601%, respectively, between the period; 2005–2030. Prosthetic Join Infection (PJI) occurs in approximately 2% of total joint replacements (TJRs) in the U.S. PJI is primarily caused by adherence of bacteria to the surface of the prosthesis, ultimately forming an irreversibly attached community of sessile bacteria, known as a biofilm, highly tolerant to antibiotic treatment. Often the only resolution if the ensuing chronic infection is surgical removal of the implant – at high cost for the patient (increased morbidity), and for healthcare resources. Strategies to prevent bacterial adherence have significant potential for medical impact. Laser surface treatment using an automated continuous wave (CW) fiber laser system has shown promise in producing anti-adherent and bactericidal surfaces. Work presented here aims to investigate the effect of this approach on orthopaedic metals as a proof of concept, specifically Ti-6Al-4V (kindly supplied by Stryker Orthopaedics, Limerick). A coupon was surface treated using a laser (MLS-4030; Micro Lasersystems BV, Driel). Samples were incubated in Müller Hinton Broth (MHB) inoculated with methicillin resistant Staphylococcus aureus (MRSA; ATCC 43300) for 24h before Live/Dead staining (BacLight™ solution; Molecular Probes) and inspection by fluorescence microscopy (GXM-L3201 LED; GX Optical). Images were analysed using ImageJ software (NIH) and a significant reduction (p > 0.05, n=24) in total biofilm coverage and Live/Dead ratio was observed between the laser treated and as received surfaces. This data demonstrates the anti-adherent, and indeed bactericidal, effect of Laser-surface treatment.

Diatoms are unicellular microalgae whose cell walls are composed of remarkably uniform, hierarchical micro/nanopatterned, amorphous biosilica that cannot be replicated synthetically. Each species hosts its own unique morphology which is identically replicated generation-to-generation. There are currently estimated to be over 200,000 different diatom species, each with their own unique shape and morphology. This offers a huge array of surface topographies, particle sizes and shapes, each with the same silica precursor. Our research to date has shown that diatom-biosilica is non-cyctotoxic to J774.2 macrophages and hBMSC cells and does not invoke an immunological response or organ toxicity (kidney, spleen and liver) when tested in a murine model. Before testing diatom-biosilica in vivo in an animal fracture model, methods to incorporate the frustules into the defect are being investigated. Two methods have been developed 1) using a bioresorbable hydrogel and 2) 3-D printed polymer-biosilica scaffolds. Both methods have shown promising results with enhanced mechanical properties with the addition of the diatom-biosilica. Work is ongoing to further map and quantify the role of surface topography and chemical cues on cell fate through the systematic in vitro studies of different species of diatom-biosilica.

Research in orthopaedics is now moving away from permanent metallic implants, and looking towards the use of bioresorbable polymers (e.g. PLLA, PGA and related co-polymers) that, when implanted into the injured site, bioresorb as the tissue heals. However, reports of a delayed inflammatory reponse occurring in the late stages of polymer degradation has limited the wide scale use of these polymers. Few studies assess the long-term biocompatibility of these polymers and with an increasing market for bioresorbable materials it is anticipated that this will be a future issue. This work aims to develop a predictive tool that can be used to assess the delayed inflammatory response of poly(D,L-lactide-co-glycolide) (PDLGA) using in vitro tests. An elevated temperature accelerated test (47^oC) was developed and utilitised to induce predetermined amounts of degradation in PDLGA. This was used to mimic a range of clinically relevant in vivo implantation times up to 5–6 months. All pre-degradion work was performed under sterile conditions, in PBS solution. At predetermined time intervals, indicators of late stage inflammation will be assessed using an MTT cytotoxicity assay, an inflammation antibody array and an ELISA analysis for inflammatory factors, with mouse L929 fibroblasts, RAW264.7 and primary BMDM macrophages. It is hypothosised that at the later degradation time intervals signs of inflammatory factors will be observed. The methodologies developed in this work can be applied to the optimisation of polymer degradation profiles to minimise late-stage inflammatory repsonse and identification of beneficial additives in this regard.

Vertebroplasty is a minimal invasive surgical procedure for treatment of vertebral compressive fractures, whereby cement is injected percutaneously into a vertebral body. Cement viscosity is believed to influence injectability, cement wash-out and leakage. Altering the liquid to powder ratio can affect the viscosity, level of cohesion and extent cement fill within the vertebral body and the ultimately strength and stiffness of the cement-vertebra composite. The association of these combined factors remains unclear. The aim of this study was to determine the relationship between cement viscosity and the potential augmentation of strength and stiffness in a model simulating in-vitro prophylactic vertebroplasty of osteoporotic vertebral bodies.

Samples of synthetic bone (Sawbone) representing osteoporotic bone were manually injected with 1mL of calcium phosphate cement using a 11G cannulated needle. Calcium phosphate cement was produced by mixing alpha-tricalcium phosphate, calcium carbonate and hydroxyapatite with an aqueous solution of 5 wt% disodium hydrogen phosphate. Three liquid to powder ratio (LPR) representing different viscosity levels were used; i.e. 0.5mL/g (low viscosity), 0.45mL/g (medium viscosity) and 0.35mL/g (high viscosity). Cement filled samples were then placed in an oven (37oC) for 20 min and then immersed in Ringer's solution (37oC) for 3 days. Samples of synthetic bone without cement injection were used as controls.

Potential for leakage and wash-out was determined using gravimetric analysis. Extent of cement fill was determined using computer tomography (CT).

Samples were tested under axial compression at a rate of 1 mm/min and the strength and stiffness determined. Statistical significance against controls was determined using a one-way analysis of variance (p<0.05).

Low viscosity cement showed more cement leakage (p=0.512) and increased cement wash-out after 3 days in Ringer's solution (p=0.476). Qualitative assessment of cement fill within the vertebral body using CT imaging supported the wash-out results. The strength (p<0.05-0.01) and stiffness (p<0.01) of samples significantly increased by cement injection in comparison to control, the extent of this increase was greater with increasing cement viscosity.

Linear correlation analysis showed a definite association between the mechanical properties and viscosity of injected cement and was dependent on the amount of cement retained within the synthetic bone post-setting.

Introduction: Computational modelling of the spine offers a particularly difficult challenge to analysts due to its complex structure and high level of functionality. Previous studies [Wijayathunga, 2008; Jones, 2007] have shown that finite element (FE) predictions of vertebral stiffness are highly sensitive to the applied boundary conditions and therefore validation requires careful matching between the experimental and simulated situation. The aim of this study was to develop and experimentally validate specimen specific FE models of porcine vertebrae in order to accurately predict the stiffness of single vertebra specimens.

METHOD: Nine single vertebra specimens were excised from the thoracolumbar region of two porcine spines. The specimens were mounted between two parallel PMMA housings and each specimen was imaged using a micro computed tomography (μCT) system (μCT80; Scanco Medical, Switzerland). In order to accurately match the experimental conditions, a radio-opaque marker was positioned on the specimen housing at the point of load application. The vertebrae were separated into two groups: a development set (set 1) consisting of three specimens and a validation set (set 2) of six specimens. Specimens from set 1 were used to establish the optimum method of conversion from image greyscale, to element material properties. The models in set 2 were used to assess the accuracy of the stiffness predictions for each model. The vertebrae were tested in a materials testing machine (AGS-10kNG; Shimadzu Corp., Japan) under axial compression and the stiffness for each specimen was calculated. The μCT data was imported into an image processing package (Scan IP, Simpleware, UK). The software enabled the images to be segmented and the vertebra, cement housings and position of load application to be identified. The segmented images were down-sampled to 1mm voxels, enabling a FE mesh to be generated (Scan FE; Simpleware, UK) based on direct voxel to element conversion. The Young’s modulus of each bone element was assigned, based on the greyscale of the corresponding image voxel. The PMMA housing plates were assigned homogeneous material properties (E = 2.45 GPa). Abaqus CAE 6.8 (Simula, Providence, Rhode Island, USA) was used for the processing and post-processing of all the models.

Results: The mean experimental stiffness was 4321 N/ mm (standard deviation = 415 N/mm). The optimum conversion factor was calculated for set 1, which yielded a root mean squared (RMS) percentage error of 7.5% when compared with the experimental stiffness. Using this optimised scale factor, FE models of specimens from set 2 were created. The predicted stiffnesses for set 2 were compared to the corresponding experimental values and yielded an RMS error of 10.1%.

Conclusion: The results indicate that specimen specific models can provide good agreement with the corresponding experimental specimen stiffness. In addition, the method employed in this study proved robust enough to be applied to vertebral tissue obtained from different animals of the same species. This method will now be developed to assess treatments for traumatic spinal injuries.