Correction to: Process-induced degradation of bioresorbable PDLGA in bone tissue scaffold production.

The original version of this article unfortunately contained a mistake. E. Themistou was missing from the author group and so is now included with this erratum.

Methylpyrrole inhibitors of BET bromodomains.

An NMR fragment screen for binders to the bromodomains of BRD4 identified 2-methyl-3-ketopyrroles 1 and 2. Elaboration of these fragments guided by structure-based design provided lead molecules with significant activity in a mouse tumor model. Further modifications to the methylpyrrole core provided compounds with improved properties and enhanced activity in a mouse model of multiple myeloma.

SAR and characterization of non-substrate isoindoline urea inhibitors of nicotinamide phosphoribosyltransferase (NAMPT).

Herein we disclose SAR studies that led to a series of isoindoline ureas which we recently reported were first-in-class, non-substrate nicotinamide phosphoribosyltransferase (NAMPT) inhibitors. Modification of the isoindoline and/or the terminal functionality of screening hit 5 provided inhibitors such as 52 and 58 with nanomolar antiproliferative activity and preclinical pharmacokinetics properties which enabled potent antitumor activity when dosed orally in mouse xenograft models. X-ray crystal structures of two inhibitors bound in the NAMPT active-site are discussed.

Process-induced degradation of bioresorbable PDLGA in bone tissue scaffold production.

Process-induced degradation of clinically relevant resorbable polymers was investigated for two thermal techniques, filament extrusion followed by fused deposition modelling (FDM). The aim was to develop a clear understanding of the relationship between temperature, processing time and resultant process-induced degradation. This acts to address the current knowledge gap in studies involving thermal processing of resorbable polymers. Poly(DL-lactide-co-glycolide) (PDLGA) was chosen for its clinically relevant resorption properties. Furthermore, a comparative study of controlled thermal exposure was conducted through compression moulding PDLGA at a selected range of temperatures (150-225 °C) and times (0.5-20 min). Differential scanning calorimetry (DSC) and gel permeation chromatography (GPC) were used to characterise thermally induced degradation behaviour. DSC proved insensitive to degradation effects, whereas GPC demonstrated distinct reductions in molecular weight allowing for the quantification of degradation. A near-exponential pattern of degradation was identified. Through the application of statistical chain scission equations, a predictive plot of theoretical degradation was created. Thermal degradation was found to have a significant effect on the molecular weight with a reduction of up to 96% experienced in the controlled processing study. The proposed empirical model may assist prediction of changes in molecular weight, however, accuracy limitations are highlighted for twin-screw extrusion, accredited to high-shear mixing. The results from this study highlight the process sensitivity of PDLGA and proposes a methodology for quantification and prediction, which contributes to efforts in understanding the influence of manufacture on performance of degradable medical implants.

Osteogenic cell response to 3-D hydroxyapatite scaffolds developed via replication of natural marine sponges.

Bone tissue engineering may provide an alternative to autograft, however scaffold optimisation is required to maximize bone ingrowth. In designing scaffolds, pore architecture is important and there is evidence that cells prefer a degree of non-uniformity. The aim of this study was to compare scaffolds derived from a natural porous marine sponge (Spongia agaricina) with unique architecture to those derived from a synthetic polyurethane foam. Hydroxyapatite scaffolds of 1 cm(3) were prepared via ceramic infiltration of a marine sponge and a polyurethane (PU) foam. Human foetal osteoblasts (hFOB) were seeded at 1 × 10(5) cells/scaffold for up to 14 days. Cytotoxicity, cell number, morphology and differentiation were investigated. PU-derived scaffolds had 84-91% porosity and 99.99% pore interconnectivity. In comparison marine sponge-derived scaffolds had 56-61% porosity and 99.9% pore interconnectivity. hFOB studies showed that a greater number of cells were found on marine sponge-derived scaffolds at than on the PU scaffold but there was no significant difference in cell differentiation. X-ray diffraction and inductively coupled plasma mass spectrometry showed that Si ions were released from the marine-derived scaffold. In summary, three dimensional porous constructs have been manufactured that support cell attachment, proliferation and differentiation but significantly more cells were seen on marine-derived scaffolds. This could be due both to the chemistry and pore architecture of the scaffolds with an additional biological stimulus from presence of Si ions. Further in vivo tests in orthotopic models are required but this marine-derived scaffold shows promise for applications in bone tissue engineering.

Connecting COX-2 and Wnt in cancer.

Both the cyclooxygenase-2 (COX-2) and Wnt signaling cascades are active in the majority of colorectal cancers. Nevertheless, a direct link between these two key pathways has remained elusive. Recent reports show that one of the bioactive products of COX-2, prostaglandin E2, activates components of the canonical Wnt signaling system. The findings reviewed below reveal important crosstalk between these pathways, which may provide opportunities for the development of new drugs for treatment and/or prevention of colorectal cancer.

Effect of patient sex on general anaesthesia and recovery.

BACKGROUND: Numerous studies have shown that women emerge faster from general anaesthesia than men, and differ in their postoperative recovery profile. The extent and underlying mechanisms for these sex-related differences in general anaesthesia are unclear. METHODS: In a multicentre, prospective, matched cohort study, 500 ASA physical status I or II patients of either sex undergoing general anaesthesia for elective surgery were recruited. All subjects received a general anaesthetic through inhalation. Anaesthetic drugs and doses used, bispectral index (BIS) scores, recovery times, pain scores, and 40-item quality of recovery (QoR-40) scores for 3 days after general anaesthesia were recorded. RESULTS: Women had higher BIS scores at similar concentrations of anaesthesia (P<0.05). Time to eye-opening (P<0.01) and time to obeying commands (P<0.01) were shorter in women. Duration of recovery room stay was longer in women, who also had higher pain scores and need for treatment of nausea and vomiting (all P<0.001). QoR-40 scores for the first 3 days after general anaesthesia were lower in women (P<0.001). Plasma progesterone concentrations in women negatively correlated with the time to eye-opening (ρ=-0.53, P=0.01). CONCLUSIONS: Patient sex is an independent factor influencing the response to anaesthesia and recovery after surgery. Women emerged faster from general anaesthesia but their overall quality of recovery was poorer. Female sex hormones, particularly progesterone, might be involved, with premenopausal women having faster recovery time but poor overall recovery.

A study of the relationship between process conditions and mechanical strength of mineralized red algae in the preparation of a marine-derived bone void filler.

Bone void fillers that can enhance biological function to augment skeletal repair have significant therapeutic potential in bone replacement surgery. This work focuses on the development of a unique microporous (0.5-10 microm) marine-derived calcium phosphate bioceramic granule. It was prepared from Corallina officinalis, a mineralized red alga, using a novel manufacturing process. This involved thermal processing, followed by a low pressure-temperature chemical synthesis reaction. The study found that the ability to maintain the unique algal morphology was dependent on the thermal processing conditions. This study investigates the effect of thermal heat treatment on the physiochemical properties of the alga. Thermogravimetric analysis was used to monitor its thermal decomposition. The resultant thermograms indicated the presence of a residual organic phase at temperatures below 500 degrees C and an irreversible solid-state phase transition from mg-rich-calcite to calcium oxide at temperatures over 850 degrees C. Algae and synthetic calcite were evaluated following heat treatment in an air-circulating furnace at temperatures ranging from 400 to 800 degrees C. The highest levels of mass loss occurred between 400-500 degrees C and 700-800 degrees C, which were attributed to the organic and carbonate decomposition respectively. The changes in mechanical strength were quantified using a simple mechanical test, which measured the bulk compressive strength of the algae. The mechanical test used may provide a useful evaluation of the compressive properties of similar bone void fillers that are in granular form. The study concluded that soak temperatures in the range of 600 to 700 degrees C provided the optimum physiochemical properties as a precursor to conversion to hydroxyapatite (HA). At these temperatures, a partial phase transition to calcium oxide occurred and the original skeletal morphology of the alga remained intact.

A review on diatom biosilicification and their adaptive ability to uptake other metals into their frustules for potential application in bone repair.

Diatoms are unicellular eukaryotic algae that have a distinctive siliceous cell wall (frustule) with unique architectures. The nanotopography of the frustule is perfectly replicated between generations, offering a source of highly intricate and identical silica microparticles. In recent years, the ability to alter their cell wall chemistry both in terms of functionalisation with organic moieties or by incorporation of the metal ions in their frustules has increased interest in their utility for catalysis technologies, and semiconductor and biomedical applications. Herein we review the fundamental biological mechanisms in which diatoms produce their frustule and their ability to substitute different metal ions in their frustule fabrication process. The review focuses on the potential of diatom frustules as a naturally derived biomaterial in bone tissue engineering applications and how their cell walls, comprising biogenic silica, could either partially or fully incorporate other bone therapeutic metal ions, e.g., titanium or calcium, into their frustule. The use of diatom frustules in bone repair also potentially offers a 'greener', more environmentally friendly, biomaterial as they can naturally synthesise oxides of silicon and other metals into their frustules under ambient conditions at a relatively neutral pH. This process would negate the use of harsh organic chemicals and high-temperature processing conditions, often used in the fabrication of silica based biomaterials, e.g., bioactive glass.

Filament extrusion of bioresorbable PDLGA for additive manufacturing utilising diatom biosilica to inhibit process-induced thermal degradation.

Bone scaffolds are often fabricated by initially producing custom-made filaments by twin-screw extruder and subsequently fabricating into 3D scaffolds using fused deposition modelling. This study aims to directly compare the effect of two alternative silica-rich filler materials on the thermo-mechanical properties of such scaffolds after extrusion and printing. Poly (DL-lactide-co-glycolide) (PDLGA) was blended with either 45S5 Bioglass (5 wt %) or Biosilica (1 and 5 wt%) isolated from Cyclotella meneghiniana a freshwater diatom were tested. Diatom-PDLGA was found to have similar mechanical strength and ductility to pure-PDLGA, whereas Bioglass-PDLGA was found induce a more brittle behaviour. Bioglass-PDLGA was also found to have the lowest toughness in terms of energy absorption to failure. The TGA results suggested that significant thermal degradation in both the Bioglass filaments and scaffolds had occurred as a result of processing. However, diatom biosilica was found to inhibit thermal degradation of the PDLGA. Furthermore, evidence suggested the agglomeration of Bioglass particles occurred during processing the Bioglass-PDLGA filaments. Overall, diatom biosilica was found to be a promising candidate as a bone filler additive in 3D printed PDLGA scaffolds, whereas Bioglass caused some potentially detrimental effects on performance.

A comparison of the degradation behaviour of 3D printed PDLGA scaffolds incorporating bioglass or biosilica.

Silica incorporation into biomaterials, such as Bioglass and Si-substituted calcium phosphate ceramics has received significant attention in bone tissue engineering over the last few decades. This study aims to explore the dissolution behaviour of natural biosilica isolated from a freshwater diatom, Cyclotella meneghiniana, that has been incorporated into 3D printed poly (DL-lactide -co - glycolide) (PDLGA) scaffolds using extrusion and additive manufacturing. In the study, two different dry weight percentage (1 wt% & 5 wt%) of diatom-silica were incorporated into PDLGA scaffolds that were then degraded in phosphate buffered saline (PBS) cell free media. In addition, pure PDLGA scaffolds and 5 wt% Bioglass scaffolds were used as control groups. The degradation study was performed over 26-weeks. The release rate of Si4+ ions from diatom-PDLGA scaffolds was found to increase exponentially with respect to time. The compressive strength of scaffolds was also measured with the Diatom-PDLGA scaffolds found to maintain their strength for longer than either pure PDLGA scaffolds or 5 wt% Bioglass scaffolds. 13C NMR data showed that diatom biosilica containing scaffolds had less degradation than pure or bioglass-containing scaffolds at comparable time-points. Overall, the Diatom-PDLGA scaffolds were found to have more desirable physiochemical properties for bone repair compared to Bioglass.

Sintering of biphasic calcium phosphates.

Biphasic calcium phosphate (BCP) discs were fabricated and then sintered using two different sintering programs to establish whether the phases present could be controlled at low and high sintering temperatures. X-ray diffraction (XRD) was used to establish the phases present after sintering and scanning electron microscopy (SEM) determined the microstructure. Sintering program 1 involved a simple heating and cooling schedule and temperatures of 1100, 1250, 1275 and 1300 degrees C. It produced samples containing an additional alpha-tricalcium phosphate (alpha-TCP) phase at temperatures above 1100 degrees C. The original ratio of hydroxyapatite/beta-tricalcium phosphate (HA/beta-TCP) could not be maintained above this temperature. Sintering program 2 combined the heating and cooling schedules of the first program with a 900 degrees C hold stage to allow alpha-TCP to beta-TCP conversion to take place. At temperatures of 1250 and 1275 degrees C, this program was successful in completely removing the alpha-TCP phase and preserving the HA:beta-TCP ratio. The SEM results show that the surface morphology of the discs was not greatly affected by choice of sintering program.

Thermal preparation of highly porous calcium phosphate bone filler derived from marine algae.

A sustainable marine-derived bioceramic with a unique porous structure has been developed for hard tissue repair. The conversion of alga was achieved through a novel technique, involving well controlled thermal processing followed by low pressure-temperature hydrothermal synthesis. In its preparation, a heat treatment step was required to remove the organic compounds from the algae, which reinforces the mineralised matrices. Its removal is necessary to prevent issue such as immune biocompatibility and ensure phase purity of the resultant biomaterial. This paper investigates the hydrothermal technique used for the transformation of mineralised red algae to hydroxyapatite that preserves the algae's unique structure. It specifically focuses on the effects of heat treatment on the morphology of the algae, TGA, SEM and hot stage XRD to quantity the changes.

Optimisation of the mechanical and handling properties of an injectable calcium phosphate cement.

Calcium phosphate cements have the potential to be successful in minimally invasive surgical techniques, like that of vertebroplasty, due to their ability to be injected into a specific bone cavity. These bone cements set to produce a material similar to that of the natural mineral component in bone. Due to the ceramic nature of these materials they are highly brittle and it has been found that they are difficult to inject. This study was carried out to determine the factors that have the greatest effect on the mechanical and handling properties of an apatitic calcium phosphate cement with the use of a Design of Experiments (DoE) approach. The properties of the cement were predominantly influenced by the liquid:powder ratio and weight percent of di-sodium hydrogen phosphate within the liquid phase. An optimum cement composition was hypothesised and tested. The mechanical properties of the optimised cement were within the clinical range for vertebroplasty, however, the handling properties still require improvement.

Effects of leptin Arg25Cys on peripheral mononuclear cell counts and antibody response to vaccination in beef cattle.

A single nucleotide polymorphism (SNP) in the leptin gene that results in Arg25Cys has been associated with beef carcass quality and milk composition in dairy cattle. However, leptin (LEP) also plays a role in immune performance and hence it was important to determine whether selection based on this SNP would negatively affect immune cell numbers or antibody production. LEP c.73C>T was assessed for effects on immune cell counts and antibody titres in 27 beef cattle herds (n = 556). A commercial rabies vaccine had been administered to these animals. Prior to being vaccinated, counts of several important mononuclear cells (total and activated B lymphocytes, total and activated T helper and T cytotoxic, WC1 T lymphocytes and monocytes) as well as baseline serum antibody titres were determined for each animal. On day 21, antibody titres were measured and a booster vaccine was administered. Finally on day 42, antibody titres and mononuclear cell types were again counted. Counts of six different cell types were significantly associated with the LEP genotype; however, no consistent patterns were observed between LEP genotype (TT, CT or CC) and peripheral blood mononuclear cell populations. Significant differences in the production of rabies antibodies in response to vaccination were observed relative to LEP genotype. Our results suggest that selection for either the C or T allele would not detrimentally impact on the measured indicators of immune function in beef cattle.

Fishy-egg tainting is recessively inherited when brown-shelled layers are fed canola meal.

Feeding canola meal to brown-shelled laying hens can result in the production of eggs with a fishy odor. This fishy taint is caused by the accumulation of trimethylamine (TMA) in the yolk. Trimethylamine is produced by the bacterial fermentation of choline in the lower gut. Fishy-egg tainting is caused by a SNP in flavin-containing monooxygenase 3 (FMO3 c.984A > T), rendering the hen unable to metabolize TMA into the nonodorous TMA N-oxide. The purpose of this study was to characterize the inheritance pattern of fishy-egg tainting when hens are fed canola meal at levels reflecting maximum use based on conventional formulation of laying hen diets. Additionally, we wished to examine the effect of choline source (choline chloride vs. canola meal) on egg tainting. In the first of 2 experiments, 6 hens per genotype (AA, AT, and TT) were allocated to each of 5 dietary treatments (0, 6, 12, 18, or 24% canola meal) for 4 wk. Three yolks per hen collected in the last week of the trial were analyzed for TMA concentration. There was a significant linear regression (P < 0.05) between yolk TMA concentration and dietary canola meal level for hens of the TT but not the AA or AT genotypes. In the second experiment, 6 hens of the TT (homozygous tainting) genotype were each assigned to 1 of 9 dietary treatments: the 5 diets used in the first experiment plus 4 diets that used choline chloride to match the total choline concentration of the 6, 12, 18, and 24% canola meal diets, respectively. Three yolks per hen were analyzed for TMA concentration. A significant response in yolk TMA concentration was seen with the canola meal diets but not the choline chloride diets. We conclude that fishy-egg tainting is recessively expressed when hens are fed canola meal at levels from 12 up to 24% inclusion. We also conclude that choline chloride, at levels typical of commercial egg production, will not lead to egg tainting.

High-solid-content hydroxyapatite slurry for the production of bone substitute scaffolds.

Key to various bone substitute scaffold production techniques is the development of free-flowing ceramic slurry with optimum theological properties. The aim is to achieve a colloidal suspension with as high a solid content as possible while maintaining a low viscosity which easily penetrates the pores of relevant sacrificial templates. The following investigation describes the optimization of a hydroxyapatite slip and demonstrates its potential application in scaffold production. Using predominantly spherical particles of hydroxyapatite of between 0.82 microm and 16.2 microm, coupled with a 2 wt % addition of the anionic polyelectrolyte, ammonium polyacrylate, an 80 wt % (55.9 vol%) hydroxyapatite solid loaded slip with a viscosity of approximately 126mPas has been developed. Its ability to infiltrate and replicate porous preforms has been shown using polyurethane foam. The enhanced particle packing achieved has allowed for the production of scaffolds with highly dense and uniform grain structures. The results represent a significant improvement in current slurry production techniques and can be utilized to develop high-density ceramic bone substitute scaffolds.

Targeting cyclooxygenase-2 and the epidermal growth factor receptor for the prevention and treatment of intestinal cancer.

Clinical and animal studies indicate a role for cyclooxygenase-2 (COX-2) and the epidermal growth factor receptor (EGFR) in the development and progression of intestinal polyps and cancers. Although this combination of enzyme inhibition has shown synergy in intestinal polyp and tumor models, the exact mechanism for these effects remains undefined. Therefore, we sought to define the molecular mechanisms through which this process occurs. We observed a significant reduction in the number and size of small intestinal polyps in APC(min+/-) mice treated with either celecoxib (a selective COX-2 inhibitor) or erlotinib (Tarceva, an EGFR inhibitor). However, in combination, there was an overall prevention in the formation of polyps by over 96%. Furthermore, we observed a 70% reduction of colorectal xenograft tumors in mice treated with the combination and microarray analysis revealed genes involved in cell cycle progression were negatively regulated. Although we did not observe significant changes in mRNAs of genes with known apoptotic function, there was a significant increase of apoptosis in tumors from animals treated with the combination. The inhibition of EGFR also induced the down-regulation of COX-2 and further inhibited prostaglandin E2 formation. We observed similar effects on the prevention of intestinal adenomas and reduction of xenograft tumor volume when nonselective COX inhibitors were used in combination with erlotinib. Together, these findings suggest that the inhibition of both COX-2 and EGFR may provide a better therapeutic strategy than either single agent through a combination of decreased cellular proliferation and prostaglandin signaling as well as increased apoptosis.

Enhanced release of calcium phosphate additives from bioresorbable orthopaedic devices using irradiation technology is non-beneficial in a rabbit model: An animal study.

OBJECTIVES: Bioresorbable orthopaedic devices with calcium phosphate (CaP) fillers are commercially available on the assumption that increased calcium (Ca) locally drives new bone formation, but the clinical benefits are unknown. Electron beam (EB) irradiation of polymer devices has been shown to enhance the release of Ca. The aims of this study were to: 1) establish the biological safety of EB surface-modified bioresorbable devices; 2) test the release kinetics of CaP from a polymer device; and 3) establish any subsequent beneficial effects on bone repair in vivo. METHODS: ActivaScrew Interference (Bioretec Ltd, Tampere, Finland) and poly(L-lactide-co-glycolide) (PLGA) orthopaedic screws containing 10 wt% ß-tricalcium phosphate (ß-TCP) underwent EB treatment. In vitro degradation over 36 weeks was investigated by recording mass loss, pH change, and Ca release. Implant performance was investigated in vivo over 36 weeks using a lapine femoral condyle model. Bone growth and osteoclast activity were assessed by histology and enzyme histochemistry. RESULTS: Calcium release doubled in the EB-treated group before returning to a level seen in untreated samples at 28 weeks. Extensive bone growth was observed around the perimeter of all implant types, along with limited osteoclastic activity. No statistically significant differences between comparative groups was identified. CONCLUSION: The higher than normal dose of EB used for surface modification did not adversely affect tissue response around implants in vivo. Surprisingly, incorporation of ß-TCP and the subsequent accelerated release of Ca had no significant effect on in vivo implant performance, calling into question the clinical evidence base for these commercially available devices.Cite this article: I. Palmer, S. A. Clarke, F. J Buchanan. Enhanced release of calcium phosphate additives from bioresorbable orthopaedic devices using irradiation technology is non-beneficial in a rabbit model: An animal study. Bone Joint Res 2019;8:266-274. DOI: 10.1302/2046-3758.86.BJR-2018-0224.R2.

Feedlot performance and immune function analysis of implanted and non-implanted steers selected for alcohol dehydrogenase 1 C (ADH1C) genotype and fed a low vitamin A diet.

Previous studies have shown that the interaction between limiting vitamin A (VA) and an alcohol dehydrogenase 1 C (ADH1C) variant in beef cattle results in increased intramuscular fat in the longissimus thoracis muscle in one genotype when fed low dietary VA. Although quality grade is important for increased profitability and improving taste characteristics of beef products, limiting VA too drastically can impair animal welfare. The objectives of this study were to determine if this marker-assisted management strategy would be effective, and whether any impairment in immune function would occur in a feedlot setting. Mixed breed beef steers (n=2000) were sorted into 40 feedlot pens so that all combinations of ADH1C genotype (TT or CT), VA level (50% or 100% of recommended) and hormonal implant status (implanted (IMP) or non-implanted (NI)) were equally represented within the population. The VA×ADH1C interaction was not observed. An implant status × ADH1C interaction was observed with average daily gain (ADG; P=0.03). Steers that were IMP and CT had higher ADG than IMP TT (CT=1.69 and TT=1.62 kg/day), whereas both genotypes in the NI steers were lower (CT=1.29 and TT=1.32 kg/day). Implant status was shown to affect dry matter intake (DMI; IMP=8.55 and NI=7.87 kg; P<0.01), total days-on-feed (IMP=164.4 and NI 210.5 days; P<0.01), USDA yield grade (YIELD; IMP=2.40 and NI=2.77; P<0.01), marbling score (MARB; IMP=392 and NI=455; P<0.01), longissimus thoracis area (LTA; IMP=85.0 and NI=80.7 cm2; P=0.01) and backfat thickness (FAT; IMP=8.0 and NI 10.0 mm; P<0.01). Overall, IMP animals finished on fewer total days-on-feed with higher ADG, DMI, larger LTA, and lower YIELD, MARB and FAT. To investigate immune function parameters, crossbred steers (n=18) were selected from a prior feeding trial so that all combinations of ADH1C (TT, CT and CC) and VA (25% or 75%) were equally represented. Blood cell count analysis and peripheral blood mononuclear cell proliferation and stimulation assays were conducted. None of these immune parameters were affected by VA level. Treatment and mortality records were examined in the 2000 steer population, where no correlations with ADH1C, implant status or VA level were observed. Due to no VA × ADH1C interaction, this nutrigenetic marker-assisted management strategy is not effective at this time in commercial beef cattle feedlots, however, supplementing VA at a level as low as 25% of recommended in finishing rations would likely not result in signs of immune dysfunction.