Evaluation of the in vitro cytotoxicity and modulation of the inflammatory response by the bioresorbable polymers poly(D,L-lactide-co-glycolide) and poly(L-lactide-co-glycolide).

Bioresorbable polymers composed of poly(D,L-lactide-co-glycolide) (PDLLGA) and poly(L-lactide-co-glycolide) (PLLGA) have become increasingly popular for the preparation of bone substitute constructs. However, there are reports of a delayed inflammatory reaction occurring months or years after implantation. Due to the long polymer degradation times, in vitro tests carried out at physiological temperature, 37°C, tend to assess only the short-term biocompatibility of these materials. The aim of this work is to develop an in vitro protocol that can be used to assess the long-term cytotoxicity of bioresorbable polymers in a time efficient manner. This study used a previously developed and validated accelerated degradation protocol to obtain samples of PDLLGA and PLLGA at increasing levels of degradation. Samples were then applied to standard ISO 10993-5 direct contact cytotoxicity testing and it was found that PDLLGA samples showed increasing levels of cytotoxicity at the later stages of degradation, with PLLGA samples demonstrating significantly less cytotoxic behaviour. Following concern that accumulation of acidic degradation products in a closed multi-well culture environment could overestimate cytotoxicity, we developed and validated a new dynamic flow culture methodology, for testing the cytotoxicity of these degradable materials, by adapting a commercial "organ on a chip" flow culture system, Quasi Vivo®. In addition to cytotoxicity testing, we have carried out profiling of inflammatory cytokines released by cells in response to degraded PDLLGA and PLLGA, and have suggested mechanism by which lactide-based bioresorbable materials could modulate the inflammatory response through the G-protein coupled receptor (GPCR), hydroxycarboxylic acid receptor 1 (HCA1). STATEMENT OF SIGNIFICANCE: Bioresorbable materials naturally disintegrate over time when implanted into the body. They are often used to make screws and clips for repair of broken bones. Unfortunately, some patients can react badly to the material, resulting in painful inflammation. Biomaterials scientists are interested in developing materials that are more compatible with the body. However, it is very difficult to predict the long-term compatibility of bioresorbable materials in the lab. In our study, we have developed a method that will allow us to study the effects of the materials as they continue to break down. This will help us understand why the materials may cause inflammation, and will support research into the development of new and improved materials for bone repair.

Blueprints for the Next Generation of Bioinspired and Biomimetic Mineralised Composites for Bone Regeneration.

Coccolithophores are unicellular marine phytoplankton, which produce intricate, tightly regulated, exoskeleton calcite structures. The formation of biogenic calcite occurs either intracellularly, forming 'wheel-like' calcite plates, or extracellularly, forming 'tiled-like' plates known as coccoliths. Secreted coccoliths then self-assemble into multiple layers to form the coccosphere, creating a protective wall around the organism. The cell wall hosts a variety of unique species-specific inorganic morphologies that cannot be replicated synthetically. Although biomineralisation has been extensively studied, it is still not fully understood. It is becoming more apparent that biologically controlled mineralisation is still an elusive goal. A key question to address is how nature goes from basic building blocks to the ultrafine, highly organised structures found in coccolithophores. A better understanding of coccolithophore biomineralisation will offer new insight into biomimetic and bioinspired synthesis of advanced, functionalised materials for bone tissue regeneration. The purpose of this review is to spark new interest in biomineralisation and gain new insight into coccolithophores from a material science perspective, drawing on existing knowledge from taxonomists, geologists, palaeontologists and phycologists.

Biocompatibility of calcium phosphate bone cement with optimized mechanical properties.

The broad aim of this work was to investigate and optimize the properties of calcium phosphate bone cements (CPCs) for use in vertebroplasty to achieve effective primary fixation of spinal fractures. The incorporation of collagen, both bovine and from a marine sponge (Chondrosia reniformis), into a CPC was investigated. The biological properties of the CPC and collagen-CPC composites were assessed in vitro through the use of human bone marrow stromal cells. Cytotoxicity, proliferation, and osteoblastic differentiation were evaluated using lactate dehydrogenase, PicoGreen, and alkaline phosphatase activity assays, respectively. The addition of both types of collagen resulted in an increase in cytotoxicity, albeit not to a clinically relevant level. Cellular proliferation after 1, 7, and 14 days was unchanged. The osteogenic potential of the CPC was reduced through the addition of bovine collagen but remained unchanged in the case of the marine collagen. These findings, coupled with previous work showing that incorporation of marine collagen in this way can improve the physical properties of CPCs, suggest that such a composite may offer an alternative to CPCs in applications where low setting times and higher mechanical stability are important.

Experimental and computational approach investigating burst fracture augmentation using PMMA and calcium phosphate cements.

The aim of the study was to use a computational and experimental approach to evaluate, compare and predict the ability of calcium phosphate (CaP) and poly (methyl methacrylate) (PMMA) augmentation cements to restore mechanical stability to traumatically fractured vertebrae, following a vertebroplasty procedure. Traumatic fractures (n = 17) were generated in a series of porcine vertebrae using a drop-weight method. The fractured vertebrae were imaged using µCT and tested under axial compression. Twelve of the fractured vertebrae were randomly selected to undergo a vertebroplasty procedure using either a PMMA (n = 6) or a CaP cement variation (n = 6). The specimens were imaged using µCT and re-tested. Finite element models of the fractured and augmented vertebrae were generated from the µCT data and used to compare the effect of fracture void fill with augmented specimen stiffness. Significant increases (p < 0.05) in failure load were found for both of the augmented specimen groups compared to the fractured group. The experimental and computational results indicated that neither the CaP cement nor PMMA cement could completely restore the vertebral mechanical behavior to the intact level. The effectiveness of the procedure appeared to be more influenced by the volume of fracture filled rather than by the mechanical properties of the cement itself.

Development of calcium phosphate cement for the augmentation of traumatically fractured porcine specimens using vertebroplasty.

The study aim was to develop and apply an experimental technique to determine the biomechanical effect of polymethylmethacrylate (PMMA) and calcium phosphate (CaP) cement on the stiffness and strength of augmented vertebrae following traumatic fracture. Twelve burst type fractures were generated in porcine three-vertebra segments. The specimens were randomly split into two groups (n=6), imaged using microCT and tested under axial loading. The two groups of fractured specimens underwent a vertebroplasty procedure, one group was augmented with CaP cement designed and developed at Queen's University Belfast. The other group was augmented with PMMA cement (WHW Plastics, Hull, UK). The specimens were imaged and re-tested . An intact single vertebra specimen group (n=12) was also imaged and tested under axial loading. A significant decrease (p<0.01) was found between the stiffness of the fractured and intact groups, demonstrating that the fractures generated were sufficiently severe, to adversely affect mechanical behaviour. Significant increase (p<0.01) in failure load was found for the specimen group augmented with the PMMA cement compared to the pre-augmentation group, conversely, no significant increase (p<0.01) was found in the failure load of the specimens augmented with CaP cement, this is attributed to the significantly (p<0.05) lower volume of CaP cement that was successfully injected into the fracture, compared to the PMMA cement. The effect of the percentage of cement fracture fill, cement modulus on the specimen stiffness and ultimate failure load could be investigated further by using the methods developed within this study to test a more injectable CaP cement.

The potential of electron beam radiation for simultaneous surface modification and bioresorption control of PLLA.

Bioresorbable polymers have been widely investigated as materials exhibiting significant potential for successful application in the fields of tissue engineering and drug delivery. Further to the ability to control degradation, surface engineering of polymers has been highlighted as a key method central to their development. Previous work has demonstrated the ability of electron beam (e-beam) technology to control the degradation profiles and bioresorption of a number of commercially relevant bioresorbable polymers (poly-l-lactic acid (PLLA), L-lactide/DL-lactide co-polymer (PLDL) and poly(lactic-co-glycolic acid (PLGA)). This work investigates the further potential of e-beam technology to impart added biofunctionality through the manipulation of polymer (PLLA) surface properties. PLLA samples were subjected to e-beam treatments in air, with varying beam energies and doses. Surface characterization was then performed using contact angle analysis, X-ray photoelectron spectroscopy (XPS), Raman spectroscopy, and atomic force microscopy. Results demonstrated a significant increase in surface wettability post e-beam treatment. In correlation with this, XPS data showed the introduction of oxygen-containing functional groups to the surface of PLLA. Raman spectroscopy indicated chain scission in the near surface region of PLLA (as predicted). However, e-beam effects on surface properties were not shown to be dependent on beam energy or dose. E-beam irradiation did not seem to affect the surface roughness of PLLA as a direct consequence of the treatment.

Development of a bovine collagen-apatitic calcium phosphate cement for potential fracture treatment through vertebroplasty.

The aim of this study was to examine the potential of incorporating bovine fibres as a means of reinforcing a typically brittle apatite calcium phosphate cement for vertebroplasty. Type I collagen derived from bovine Achilles tendon was ground cryogenically to produce an average fibre length of 0.96±0.55 mm and manually mixed into the powder phase of an apatite-based cement at 1, 3 or 5 wt.%. Fibre addition of up to 5 wt.% had a significant effect (P ≤ 0.001) on the fracture toughness, which was increased by 172%. Adding ≤ 1 wt.% bovine collagen fibres did not compromise the compressive properties significantly, however, a decrease of 39-53% was demonstrated at ≥ 3wt.% fibre loading. Adding bovine collagen to the calcium phosphate cement reduced the initial and final setting times to satisfy the clinical requirements stated for vertebroplasty. The cement viscosity increased in a linear manner (R²=0.975) with increased loading of collagen fibres, such that the injectability was found to be reduced by 83% at 5 wt.% collagen loading. This study suggests for the first time the potential application of a collagen-reinforced calcium phosphate cement as a viable option in the treatment of vertebral fractures, however, issues surrounding efficacious cement delivery need to be addressed.

Through-thickness control of polymer bioresorption via electron beam irradiation.

Predicable and controlled degradation is not only central to the accurate delivery of bioactive agents and drugs, it also plays a vital role in key aspects of bone tissue engineering. The work addressed in this paper investigates the utilisation of e-beam irradiation in order to achieve a controlled (surface) degradation profile. This study focuses on the modification of commercially and clinically relevant materials, namely poly(L-lactic acid) (PLLA), poly(L-lactide-hydroxyapatite) (PLLA-HA), poly(L-lactide-glycolide) co-polymer (PLG) and poly(L-lactide-DL-lactide) co-polymer (PLDL). Samples were subjected to irradiation treatments using a 0.5MeV electron beam with delivered surface doses of 150 and 500 kGy. In addition, an acrylic attenuation shield was used for selected samples to control the penetration of the e-beam. E-beam irradiation induced chain scission in all polymers, as characterized by reduced molecular weights and glass transition temperatures (T(g)). Irradiation not only produced changes in the physical properties of the polymers but also had associated effects on surface erosion of the materials during hydrolytic degradation. Moreover, the extent to which both mechanical and hydrolytic degradation was observed is synonymous with the estimated penetration of the beam (as controlled by the employment of an attenuation shield).

Special issue: select papers from the 29th Northern Ireland Biomedical Engineering Conference, Belfast, UK, April 2009.

Each year, NIBES hosts a spring conference that is jointly organised by Queen's University of Belfast and University of Ulster. The 29th NIBES Spring meeting took place on 8th April 2009 at Queen's University of Belfast. NIBES 2009 had an impressive scientific program with two international leading plenary speakers and 28 oral presentations.

The modification of PLA and PLGA using electron-beam radiation.

The degradable polymers polylactide (PLA) and polylactide-co-glycolide (PLGA) have found widespread use in modern medical practice. However, their slow degradation rates and tendency to lose strength before mass have caused problems. The aim of this study was to ascertain whether treatment with e-beam radiation could address these problems. Samples of PLA and PLGA were manufactured and placed in layered stacks, 8.1 mm deep, before exposure to 50 kGy of e-beam radiation from a 1.5 MeV accelerator. Gel permeation chromatography testing showed that the molecular weight of both materials was depth-dependent following irradiation, with samples nearest to the treated surface showing a reduced molecular weight. Samples deeper than 5.4 mm were unaffected. Computer modeling of the transmission of a 1.5 MeV e-beam in these materials corresponded well with these findings. An accelerated mass-loss study of the treated materials found that the samples nearest the irradiated surface initiated mass loss earlier, and at later stages showed an increased percentage mass loss. It was concluded that e-beam radiation could modify the degradation of bioabsorbable polymers to potentially improve their performance in medical devices, specifically for improved orthopedic fixation.

The effect of patient gait on the material properties of UHMWPE in hip replacements.

The wear of ultra-high molecular weight polyethylene (UHMWPE) acetabular components in total hip replacements (THRs) has been shown to be highly dependent on the direction of shear. Greatly reduced wear rates have been reported for unidirectional, compared to multidirectional, articulation in vitro. This work for the first time enables investigation of a relationship between clinical wear conditions, as determined by patient gait path, and the mechanical and structural changes that occur within the UHMWPE acetabular component. Individual patients' wear paths were determined prior to revision operation from hip joint kinematics measured by clinical gait analysis. The material properties of the acetabular components removed during the revision operation were subsequently analysed. A technique using Fourier transform infra- red analysis (FTIR) was developed to quantify the orientation of the individual UHMWPE lamellae. This study shows that there is a direct relationship between a patient's clinical gait path and the molecular properties of their UHMWPE acetabular socket. Patient kinematics are an important factor affecting the wear and long-term biocompatibility of UHMWPE used as a bearing surface in THR.