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Scarring/Opacity on the surface of the eye and vascularisation following infectious diseases, inflammation and corneal trauma are often a leading cause of blindness. The 'gold standard' treatment to prevent corneal scarring is the application of amniotic membrane (AM) to the ocular surface in the acute stage of injury. Although clinically effective, the use of the AM is associated with biological variability and unpredictable responses. Potential health risks including disease transmission, significant ethical issues surrounding the tissue donation process and stringent regulations/storage conditions, preclude widespread use. Consequently, there is a demand for the development of a new, synthetic alternative, that is stable at room temperature, capable of protecting the wound and has the capacity to deliver anti-scarring and anti-inflammatory mediators. Here we have developed a micro-structured fluid gel eye drop, to deliver a potent anti-scarring molecule, decorin. We have compared the release of decorin from the formulated dressing to a typical gel film, demonstrating enhanced release for the fluid gel eye-drops. Therefore, we have investigated the effect of the fluid gel system in 2D human corneal fibroblast culture models, as well as shown the retention of the gellan fluid gel in an in vivo rat model. At the same time the efficacy of the fluid gel eye drop was studied in an organ culture model, whereby the fluid gel containing decorin, significantly (P 0.05) increased re-epithelialisation within 4 days of treatment.

Pyrophosphate ions are both inhibitors of HA formation and substrates for phosphatase enzymes. Unlike polyphosphates their hydrolysis results simultaneously in the complete loss of mineral formation inhibition and a localised elevation in orthophosphate ion concentration. Despite recent advances in our knowledge of the role of the pyrophosphate ion, very little is known about the effects of pyrophosphate on bone formation and even less is known about its local delivery. In this work we first developed a self setting pyrophosphate based calcium cement system with appropriate handling properties and then compared its in vivo degradation properties with those of a non-pyrophosphate containing control. Contrary to expectation, the presence of the pyrophosphate phase in the cement matrix did not inhibit mineralisation of the healing bone around the implant, but actually appeared to stimulate it. In vitro evidence suggested that enzymatic action accelerated dissolution of the inorganic pyrophosphate ions, causing a simultaneous loss of their mineralisation inhibition and a localised rise in supersaturation with respect to HA. This is thought to be a rare example of a biologically responsive inorganic material and these materials seem to be worthy of further investigation. Bioceramics to date have mainly been limited to orthophosphate, silicate and carbonate salts of calcium, here we report the successful application of a pyrophosphate material as a degradable osteoconductive bone repair cement.

There is a need for particles which exhibit controlled release of therapeutic agents delivered via the inhalational route, for tissue specific applications such as anti-cancer, bronchodilators and antiviral agents as well as drugs for systemic action. The aim of this study was to assess the acute toxicity, distribution and capacity of the microspheres to exhibit controlled release properties in an in vivo model of airway inflammation. Calcium pyrophosphate nanofibrous microspheres were loaded with dexamethasone phosphate (Dex-P); the profile of drug release was studied in vitro and validated in vivo. Unloaded microspheres were administered intra-tracheally (i.t.) to rats to assess the tissue reaction. The anti-inflammatory properties of the Dex-P loaded microspheres against an inflammatory agent (compound 48/80), were evaluated in vivo. Unloaded microspheres did not cause an inflammatory response when given at doses below 3 mg, and appeared to be eliminated through mucus clearance mechanisms. Microspheres loaded with Dex-P but not Dex-P alone, were capable of inhibiting eosinophil and total inflammatory cell increases in bronchoalveolar lavage fluid for 42 h following a single application. These observations demonstrated that calcium pyrophosphate nanofibrous microspheres displayed in vivo controlled release properties, were well tolerated and did not accumulate in the lung.

Limiting cell proliferation without reducing cell viability for in vivo tissue engineering applications is important in co-culture applications where the growth of one cell type must be inhibited to prevent overgrowth of the scaffold at the expense of another cell type. Also, it is vital for maintaining viability of cells in large constructs before vascularisation occurs. In this study we have shown by means of the Thiazolyl blue (MTT) assay and immuno-staining for proliferating cell nuclear antigen (PCNA) that encapsulating fibroblasts in 2% and 5%w/v calcium-alginate at a density of 7.5 x 10(5)cells/ml as uniformly dispersed entities, enabled cells to maintain viability and caused a reversible mitotic inhibition. Alginate encapsulation also caused reversible metabolic inhibition as demonstrated by the MTT assay and fluorescent staining for mitochondrial membrane potential. Histological evaluation of the alginate constructs containing fibroblasts showed that mitotic and metabolic inhibition was possibly due to cell isolation during the first five weeks of culture. The alginate scaffold degraded with time releasing encapsulated fibroblasts. Upon implantation to a wound site this should ensure that encapsulated cells are able to replace the damaged tissue after sufficient proliferation of the co-cultured cell type or sufficient vascularisation of the construct.

Cobalt-chromium-molybdenum (CoCrMo) alloy-based metal-on-metal prostheses have been the implant of choice for total hip replacement in younger patients. However 6.2% of patients require revision of their CoCrMo total hip replacement (THR) implant within five years of surgery and their use was restricted in 2013. We aimed to determine if there were individual differences in the immune response to wear debris that might indicate a poor outcome with a CoCrMo prosthesis. Blood from 22 donors was incubated with CoCrMo particles (>99.9% less than 10 μm diameter) generated by a wear simulator for 24 h. T cell phenotype was assessed by immunostaining and secretion of 8 different pro- and anti-inflammatory cytokines was measured using multiplex technology. Clear differences were seen between individuals in the induction of Th17 and Th1 responses, with some donors showing pro-inflammatory responses (increased IL17 or IFNγ) and others showing anti-inflammatory responses (decreased IL17 or IFNγ). The only differences seen for gender and age related to increased IL-10 expression from T cells in females (p = 0.008) and a trend towards decreased IL-6 expression systemically for older donors (p = 0.058). We conclude that individuals show differential responses to CoCrMo wear debris and that these responses could give early indications of the suitability of the patient for a metal-on-metal prosthesis.

The reactivity of acid base cements forming hydroxyapatite (HA) such as, tetracalcium phosphate, and dicalcium phosphate anhydride or dicalcium phosphate dihydrate, is normally adjusted by altering the particle size and hence the specific surface area of the compounds. Amorphous calcium phosphates, prepared by precipitation from supersaturated solutions, can also react to form apatitic cements since they are thermodynamic unstable with respect to HA and have a setting reaction more independent of particle size. In this report we show for the first time that prolonged high-energy ball milling of β -tricalcium phosphate ( β -TCP), led to mechanically induced phase transformation from the crystalline to the amorphous state. The process increased the thermodynamic solubility of the β -TCP compared to the unmilled material by up to nine times and accelerated the normally slow reaction with water. By using a 2.5% Na 2 HPO 4 solution setting times were reduced to 5–16 min rather than hours. X-ray diffraction analyses indicated that the amorphous fraction within the materials was responsible for the primary setting reaction and hardening of the cements, while the crystalline fraction remained unreacted and converted only slowly to HA. Mechanically activated β -TCP cements were produced with compressive and diametral tensile strengths of up to 50 and 7 MPa respectively. The effect of preparation and setting parameters on the physical and chemical properties of mechanically activated β -TCP cement was investigated.

Depending upon local conditions, brushite (CaHPO4 � 2H2O) cements may be largely resorbed or (following hydrolysis to hydroxyapatite) remain stable in vivo. To determine which factors influence cement resorption, previous studies have investigated the solution-driven degradation of brushite cements in vitro in the absence of any cells. However, the mechanism of cell-mediated biodegradation of the brushite cement is still unknown. The aim of the current study was to observe the cell-mediated biodegradation of brushite cement formulations in vitro. The cements were aged in the presence of a murine cell line (RAW264.7), which had the potential to form osteoclasts in the presence of the receptor for nuclear factor kappa B ligand (RANKL) in vitro, independently of macrophage colony stimulating factor (M-CSF). The cytotoxicity of the cements on RAW264.7 cells and the calcium and phosphate released from materials to the culture media were analysed. Scanning electron microscopy (SEM) and focused ion beam (FIB) microscopy were used to characterise the ultrastructure of the cells. The results showed that the RAW264.7 cell line formed multinucleated TRAP positive osteoclast-like cells, capable of ruffled border formation and lacunar resorption on the brushite calcium phosphate cement in vitro. In the osteoclast-like cell cultures, ultrastuctural analysis by SEM revealed phenotypic characteristics of osteoclasts including formation of a sealing zone and ruffled border. Penetration of the surface of the cement, was demonstrated using FIB, and this showed the potential demineralising effect of the cells on the cements. This study has set up a useful model to investigate the cell-mediated cement degradation in vitro. r 2006 Elsevier Ltd. All rights reserved.

A new calcium phosphate cement is reported, which sets to form a matrix consisting of brushite, dicalcium pyrophosphate dihydrate and an amorphous phase following the mixture of β-tricalcium phosphate with an aqueous pyrophosphoric acid solution. This reactant combination set within a clinically relevant time-frame (approximately 10 min) and exhibited a higher compressive strength (25 MPa) than previously reported brushite cements. The in vitro degradation of the β-tricalcium phosphate–pyrophosphoric acid cement was tested in both phosphate buffered saline and bovine serum. The pyrophosphate ion containing cement reported here was found not to be hydrolysed to form hydroxyapatite in vitro like β-tricalcium phosphate–orthophosphoric acid solution cements. This finding is significant since the formation of hydroxyapatite by hydrolysis is thought to retard in vivo degradation of brushite cements. When aged in bovine serum, the cement lost considerably more mass than when aged in phosphate buffered saline, indicating that proteins, most likely phosphatase enzymes played an important role in the degradation. As pyrophosphate ions are thought to be the source of orthophosphate ions during bone mineralisation, this new class of bone cement offers a route to new degradable synthetic bone grafting materials.

Potassium and sodium containing nanoapatite cements were produced by the reaction of mechanically activated CaNaPO 4 (CSP), CaKPO 4 (CPP) and Ca 2 KNa(PO 4 ) 2 (CPCP) with a 2.5% Na 2 HPO 4 solution. The cements exhibited clinically acceptable setting times of approximately 5 min and compressive strengths of 5–10 MPa. The antimicrobial properties of the cements were tested with the agar diffusion test using Streptococcus salvarius , Staphylococcus epidermis and Candida albicans . All types of alkali ion containing cements showed a significantly higher antimicrobial potency with inhibition zones of approx. 4–11 mm than a commercial calcium hydroxide cement which resulted in small inhibition zones around the cement samples of a maximum of 1.5 mm. The antimicrobial properties of all the cements were not found to diminish even after longer incubation times. This behaviour was attributed to the formation of soluble alkaline metal phosphates during setting which increased the pH value in the agar gel around the alkali containing calcium phosphate cement to 8.5–10.7 compared to 6.5–8.0 for the Ca(OH) 2 product. The high antimicrobial potency of alkali–calcium phosphate cements may find an application in dentistry as pulp capping agents, root fillers or cavity liners.

A broadening of the indications for which calcium phosphate cements (CPC) can be used, for example, in the field of vertebroplasty, would require injectable and higher strength materials. Unmodified CPC are not injectable due to a filter-pressing effect during injection. In this work we demonstrated that an effective method for improving the injection properties of CPC was by the use of sodium citrate solution as a liquid component. Cement consisting of tetracalcium phosphate (TTCP) and monetite (DCPA) mixed with water up to a powder:liquid ratio (P:L) of 3.3 g/ml had an injectability of approximately 60%. The use of 500 mM trisodium citrate solution instead of water decreased the viscosity of the cement paste to a point, where complete injectability (>95%) through an 800 microm diameter hypodermic needle could be achieved at low loads. The reduction in water demand of the cement effected by the use of sodium citrate enabled high P:L mixes to be formed which were 400% stronger than cements made with water. The effect was less pronounced with compacted cements such that at 9 MPa applied pressure, 58% improvement was obtained and at 50 MPa 36% improvement was measured yielding a cement with a compressive strength of 154 MPa. The liquefying effect of sodium citrate was thought to derive from a strong increase in the surface charge of both the reactants and the product as determined by zeta-potential measurement.

In vivo studies investigating the use of brushite cements have demonstrated mixed results with one or more of dissolution, hydrolysis, fragmentation and long term stability being demonstrated. It has been suggested that sample volume, implant location, and species can affect in vivo behaviour. As few in vitro studies on this cement system have been performed, this study aimed to compare the effects of static and dynamic in vitro ageing protocols on the phase composition, weight loss and mechanical properties of brushite cement. The effects of immersion liquid to cement volume ratio (LCVR) and sample volume on phase composition were investigated and comparative in vitro experiments were also performed in foetal bovine serum. It was determined that the weight loss after 28 days was up to seven times higher in serum than in phosphate buffered saline (PBS) and that fragmentation accounted for most of the weight loss observed. Hydroxyapatite was formed in PBS but not in serum when aged in refreshed media at all LCVRs investigated. This study has highlighted that LCVR, media refresh rate and media composition are critical to brushite cement performance. It appears that brushite cement removal from an implant site may be complex and dependent on physiological processes other than simple dissolution. A better understanding of these processes could provide the means to engineer more precise calcium phosphate cement degradation profiles.

Unlike sintered hydroxyapatite there is evidence to suggest that calcium phosphate cement (CPC) is actively remodelled in vivo and because CPC is formed by a low-temperature process, thermally unstable compounds such as proteins may be incorporated into the matrix of the cement which can then be released after implantation. The efficacy of a macroporous CPC as a bone tissue engineering scaffold has been reported; however, there have been few previous studies on the effect of macroporosity on the mechanical properties of the CPC. This study reports a novel method for the formation of macroporous CPC scaffolds, which has two main advantages over the previously reported manufacturing route: the cement matrix is considerably denser than CPC formed from slurry systems and the scaffold is formed at temperatures below room temperature. A mixture of frozen sodium phosphate solution particles and CPC powder were compacted at 106 MPa and the sodium phosphate was allowed to melt and simultaneously set the cement. The effect of the amount of porogen used during processing on the porosity, pore size distribution and compressive strength of the scaffold was investigated. It was found that macroporous CPC could reliably be fabricated using cement:ice ratios as low as 5:2.