Structural Diversity in Antiosteolytic Bisphosphonates: Deciphering Structure-Activity Trends in Ultra Long Controlled Release Phenomena.

Bisphosphonate (BP)-based treatments have been extensively prescribed for bone-related conditions, particularly for osteoporosis. Their low bioavailability creates the need for prescribed dosage increase to reach therapeutic levels but generates a plethora of undesirable side effects. A viable approach to alleviating these issues is to design and exploit controlled release strategies. Herein, the controlled release profiles of 15 structurally characterized BPs (actual drugs and structural analogs) were thoroughly studied from tablets containing three (cellulose, lactose, and silica) or two (cellulose, and silica) excipients in human stomach-simulated pH conditions. The BPs were of two types, alkyl-BPs and amino-BPs. Alkyl-BPs included four derivatives of etidronate (acid, disodium, tetra-sodium, and monopotassium forms), medronic acid, and three analogs of etidronate, in which the -CH3 group was replaced by the moieties -H, -CH2CH2CH3, and -CH2CH2CH2CH2CH3. Amino-BPs included the commercial drugs pamidronate, alendronate, neridronate, and ibandronate, as well as three analog compounds. Release curves were constructed based on data taken from 1H NMR peak integration and were expressed as "% BP release" vs time. The controlled release profiles (initial release rate, plateau value, etc.) were correlated with certain structural features (number of hydrogen and metal-oxygen bonds), showing that the molecular and crystal lattice features of each BP profoundly influence its release characteristics. It was concluded that for all BPs, in general, the initial rate became lower as the total number of lattice interactions increased. For the alkyl-BPs elongation of the alkyl side chain seems to decelerate the release. Amino-BPs, in general, show slower release than the alkyl-BPs. No adverse effects of alkyl- and amino-BP drugs on NIH3T3 cell viability were noted.

A quantitative risk assessment of Listeria monocytogenes from prevalence and concentration data: Application to a traditional ready to eat (RTE) meat product.

Ready to Eat (RTE) cooked meat products are among the most consumed RTE food subcategories in the EU/EEA. They are also associated with the highest number of identified listeriosis cases per year (>850), thus posing a public health risk especially among the susceptible population. This study estimated the risk of listeriosis from Italian head cheese (Coppa di Testa) consumption using a Quantitative Microbiological Risk Assessment (QMRA) based on data of prevalence and starting concentrations of Listeria monocytogenes in the product during a 3-year period (n = 1568). A consumer survey (n = 162) was conducted to provide information on domestic storage time and consumption habits, and storage conditions were determined from recordings of temperatures of domestic refrigerators (n = 57). A probabilistic model was designed for the evaluation of the growth of L. monocytogenes at each stage of the product pathway from production to consumption, using Monte Carlo simulations and employing the @Risk software. Risks associated to consumption of vacuum-packed and sliced-at-retail head cheese were assessed: The model predicted that the risk of listeriosis per serving of vacuum-packed product was in the 10-4 and 10-6 range (mean) for the high-risk and general populations respectively, and listeriosis cases were estimated to be greater than those due to consumption of sliced product (with risks in the range of 10-7 and 10-8). Overall, the model predicted that the mean number of listeriosis cases ranged from 0.001 to 0.24 and from 0.06 to 10 per one hundred thousand people, for the healthy and the high-risk population, respectively. Scenario analyses indicated that better control of the temperature of domestic refrigerators is effective in reducing the predicted risk of listeriosis for the longer stored vacuum-packed product by ~80 % for both the healthy and high-risk populations, whereas a shorter use-by-date of 30 days is an effective risk mitigation measure for both types of packed product. Model assumptions, as well as data gaps are discussed.

Effects of NSAIDs on pre-osteoblast viability and osteogenic differentiation.

Non-steroidal anti-inflammatory drugs (NSAIDs) are widely used in the treatment of a variety of musculoskeletal conditions, injuries and after surgery for postoperative pain management. Their use has been associated with impaired bone healing, possibly due to a multifactorial function, which may include inhibition of osteoblast recruitment and differentiation. However, up to date, there is no consensus regarding the impact of NSAIDs on bone-healing. The aim of the current study was to investigate the effects of five NSAIDs on the cellular functions of mouse MC3T3-E1 pre-osteoblasts. Cells were treated with the non-selective COX inhibitors lornoxicam and diclofenac, the COX-2 selective inhibitors parecoxib, meloxicam and paracetamol, as well as steroidal prednisolone at different doses and exposure times. The PrestoBlue™ technique was used to measure cell viability, an enzymatic assay was employed for alkaline phosphatase (ALP) activity and alizarin red S mineral staining was used to determine osteogenic differentiation. All drugs had a negative impact on pre-osteoblast cell growth, with the exception of paracetamol. Lornoxicam, diclofenac and meloxicam reduced ALP activity, while the other NSAIDs had no effect and prednisolone strongly increased ALP activity. In contrast, calcium deposits were either unaffected or increased by NSAID treatments but were significantly decreased by prednisolone. These results provide evidence that NSAIDs may adversely affect the viability of mouse pre-osteoblast cells but their actions on the osteogenic differentiation are drug-specific. The direct comparison of the effects of different NSAIDs and prednisolone on pre-osteoblasts may serve to place some NSAIDs in a preferential position for analgesic and anti-inflammatory therapy during bone repair.

Bovine lymph nodes as a source of Escherichia coli contamination of the meat.

Ground beef contamination with Escherichia coli is usually a result of carcass faecal contamination during the slaughter process. Carcasses are contaminated when they come into contact with soiled hides or intestinal leakage content during dressing and the evisceration processes. A more recent and compelling hypothesis is that, when lymph nodes are present in manufacturing beef trimmings, they can be a potential source of Enterobacteriaceae contamination of ground beef. The aim of this study was to investigate the occurrence of E. coli in lymph nodes from beef carcasses used for ground meat production, in six slaughter plants situated in central Italy A total of 597 subiliac (precrural) lymph nodes were obtained from 597 cattle carcasses and screened for E. coli by culture. Furthermore, E. coli isolates (one per positive carcass) were tested for stx1, stx2 eaeA and hlyA genes that are commonly used to identify and characterise shiga toxin-producing E. coli (STEC). In addition, the E. coli isolates were profiled for antimicrobial susceptibility. A proportion of 34.2% (204/597) carcasses were positive for E. coli. PCR revealed that 29% (59/204) of E. coli possessed stx1 or stx2 which corresponded to 9.9% of the cattle sampled. Moreover, a combination of stx1 or stx2 and eaeA was found in in 4 isolates (2% among E. coli positive samples and 1% among cattle sampled) and a combination of stx1 or stx2 and eaeA and hly in 1 isolate (0.5% and 0.2%). More than 95% of isolates were susceptible to gentamicin, ceftriaxone, cyprofloxacin and cefotaxime while high rates of resistance were recorded for cephalotin, ampicillin, tetracycline, tripe sulfa and streptomycin. The multivariate analysis identified "age" as the factor most closely related to E. coli positivity (either generic E. coli or STEC) in bovine lymph nodes. In conclusion, subiliac lymph nodes represent a source of E. coli for ground beef. These results are of major importance for risk assessment and improving good manufacturing practices during animal slaughter and ground meat production.

Self-sacrificial MOFs for ultra-long controlled release of bisphosphonate anti-osteoporotic drugs.

In this paper we report drug delivery systems that are based on phosphonate MOFs. These employ biologically-acceptable metal ions (e.g. Ca2+ and Mg2+) and several anti-osteoporosis bisphosphonate drugs (etidronate, pamidronate, alendronate and neridronate), as the organic linkers. These materials have been synthesized, structurally characterized, and studied for the self-sacrificial release (by pH-driven dissolution) of the bisphosphonate active ingredient. They exhibit variable release rates and final % release, depending on the actual structure of the metal-bisphosphonate material. Their cytotoxicity profiles match those of the active ingredients.

Quantitative risk assessment of Listeria monocytogenes in a traditional RTE product.

Ready to Eat (RTE) cooked meat products are among the most consumed RTE food subcategories in the EU. They are also associated with the highest number of listeriosis cases per year. Contamination with Listeria monocytogenes may arise from post-processing and its growth is often supported by the pH and water activity of the product. L. monocytogenes may grow during refrigeration and reach unacceptable levels at the time of consumption, posing a public health risk. The aim of this study was to conduct a Quantitative Microbiological Risk Assessment (QMRA) of L. monocytogenes in a traditional Italian RTE cooked meat product. Data for the risk assessment included prevalence and concentration of the microorganism, temperature-time conditions during transport and storage, information on the growth of the microorganism and its potential for disease (dose-response). These data were obtained from laboratory analysis of product samples (n = 50), a consumer survey (n = 160), recordings of temperatures of domestic refrigerators (n = 60) and were complemented with information from the literature. The data were described with appropriate probability distributions and introduced into a previously described growth model of L. monocytogenes. Based on the above components, a probabilistic model was created to evaluate the growth of L. monocytogenes at each stage of the product pathway (retail storage, transportation and domestic storage) using Monte Carlo simulations. The model design for this pathogen/food product combination, alongside with the findings of the study are included in a separate publication (manuscript under preparation). The results may help risk managers to apply appropriate control measures to minimise the public health risk. The project contributed to further education of the fellow, especially in the use of QMRA risk analysis tools and laid the foundations for future collaborations between the fellow's home institution, the University of Crete, Greece and the University of Perugia, Italy.

Controlling pre-osteoblastic cell adhesion and spreading on glycopolymer brushes of variable film thickness.

Controlling the cell behavior on biocompatible polymer surfaces is critical for the development of suitable medical implant coatings as well as in anti-adhesive applications. Synthetic glycopolymer brushes, based on sugar methacrylate monomers have been reported as robust surfaces to resist protein adsorption and cell adhesion. In this study, poly(D-gluconamidoethyl methacrylate) (PGAMA) brushes of various chain lengths were synthesized directly from initiator functionalized glass substrates using surface-initiated atom transfer radical polymerization. The glycopolymer film thicknesses were determined by ellipsometry, whereas the wettability and the morphology of the surfaces were characterized by static water contact angle measurements and atomic force microscopy, respectively. Stable, grafted films with thicknesses in the dry state between 4 and 20 nm and of low roughness (~1 nm) were obtained by varying the polymerization time. Cell experiments with MC3T3-E1 pre-osteoblasts cultured on the PGAMA brushes were performed to examine the effect of film thickness on the cell morphology, cytoskeleton organization and growth. The results revealed good cell spreading and proliferation on PGAMA layers of low film thickness, whereas cell adhesion was prevented on polymer films with thickness higher than ~10 nm, indicating their potential use in medical implants and anti-adhesive surfaces, respectively.

Effects of NSAIDs on the osteogenic differentiation of human adipose tissue-derived stromal cells.

OBJECTIVES: Non-steroidal anti-inflammatory drugs (NSAIDs), used in the treatment of musculoskeletal pathologies, have been associated with impaired bone healing, possibly through inhibition of osteogenic differentiation. The adipose tissue (AT) is regarded as an attractive source of stromal cells for autologous cell transplantation in the bone. The effects of NSAIDs on human AT-derived stromal cells (hADSCs) are unknown. METHODS: We examined the effect of several NSAIDs including meloxicam, parecoxib, lornoxicam, diclofenac and paracetamol on the proliferation of hADSCs by means of the PrestoBlue® viability assay, and the osteogenic differentiation capacity of hADSCs by means of the alkaline phosphatase (ALP) activity, calcium deposition by alizarin red staining and osteogenic gene expression by semi-quantitative PCR. KEY FINDINGS: Most of the drugs enhanced hADSC cell growth, while either positively affecting or not influencing alkaline phosphatase (ALP) activity, calcium deposition and osteogenic gene expression. Moreover, selective COX-2 inhibitor NSAIDs, such as meloxicam or parecoxib, were advantageous over the non-selective COX-1 and COX-2 inhibitor NSAIDs lornoxicam and diclofenac. CONCLUSIONS: Altogether through this study, we show that NSAIDs, possibly depending on their selectivity for COX inhibition, leave the osteogenic differentiation capacity of hADSCs unaltered or might even enhance it.

Effect of Porosity of Alumina and Zirconia Ceramics toward Pre-Osteoblast Response.

It is acknowledged that cellular responses are highly affected by biomaterial porosity. The investigation of this effect is important for the development of implanted biomaterials that integrate with bone tissue. Zirconia and alumina ceramics exhibit outstanding mechanical properties and are among the most popular implant materials used in orthopedics, but few data exist regarding the effect of porosity on cellular responses to these materials. The present study investigates the effect of porosity on the attachment and proliferation of pre-osteoblastic cells on zirconia and alumina. For each composition, ceramics of three different porosities are fabricated by sintering, and characterized using scanning electron microscopy, energy dispersive X-ray spectroscopy and X-ray powder diffraction. Cell proliferation is quantified, and microscopy is employed to qualitatively support the proliferation results and evaluate cell morphology. Cell adhesion and metabolic activity are found comparable among low porosity zirconia and alumina. In contrast, higher porosity favors better cell spreading on zirconia and improves growth, but does not significantly affect cell response on alumina. Between the highest porosity materials, cell response on zirconia is found superior to alumina. Results show that an average pore size of ~150 µm and ~50% porosity can be considered beneficial to cellular growth on zirconia ceramics.

Calcium phosphate nanoparticles carrying BMP-7 plasmid DNA induce an osteogenic response in MC3T3-E1 pre-osteoblasts.

Functionalized calcium phosphate nanoparticles with osteogenic activity were prepared. Polyethyleneimine-stabilized calcium phosphate nanoparticles were coated with a shell of silica and covalently functionalized by silanization with thiol groups. Between the calcium phosphate surface and the outer silica shell, plasmid DNA which encoded either for bone morphogenetic protein 7 (BMP-7) or for enhanced green fluorescent protein was incorporated as cargo. The plasmid DNA-loaded calcium phosphate nanoparticles were used for the transfection of the pre-osteoblastic MC3T3-E1 cells. The cationic nanoparticles showed high transfection efficiency together with a low cytotoxicity. Their potential to induce an osteogenic response by transfection was demonstrated by measuring the alkaline phosphatase (ALP) activity and calcium deposition with alizarin red staining. The expression of the osteogenic markers Alp, Runx2, ColIa1 and Bsp was investigated by means of real-time quantitative polymerase chain reaction. It was shown that phBMP-7-loaded nanoparticles can provide a means of transient transfection and localized production of BMP-7 in MC3T3-E1 cells, with a subsequent increase of two osteogenic markers, specifically ALP activity and calcium accumulation in the extracellular matrix. Future strategies to stimulate bone regeneration focus into enhancing transfection efficiency and achieving higher levels of BMP-7 produced by the transfected cells.

Proliferation and osteogenic response of MC3T3-E1 pre-osteoblastic cells on porous zirconia ceramics stabilized with magnesia or yttria.

Dense zirconia ceramics are used in bone applications due to their mechanical strength and biocompatibility, but lack osseointegration. A porous interface in contact with bone tissue may lead to better bone bonding but the biological properties of porous zirconia are not widely explored. The present study focuses on the manufacturing of an yttria- (YSZ) and a magnesia-stabilized (MgSZ) porous zirconia, and on their in vitro biological investigation. The sintered ceramics had similar characteristics of porosity, pore size and interconnectivity. Their elastic moduli and compressive strength values were within the range of the values of human cortical bone. MC3T3-E1 pre-osteoblasts were used to investigate the proliferation, alkaline phosphatase (ALP) activity, collagen deposition and expression profile of four genes involved in bone metabolism of cells on porous ceramics. Scanning electron and fluorescence microscopy were employed to visualize cell morphology and growth. Pre-osteoblasts adhered well on both ceramics but cell numbers on YSZ were higher. Cells exhibited an increase in ALP activity and collagen deposition after 14 days on both MgSZ and YSZ, with higher levels on YSZ. Real-time quantitative polymerase chain reaction (qPCR) showed that the expression of bone sialoprotein (Bsp) and collagen type I (col1aI) were significantly higher on YSZ. No significant differences were found in their ability to regulate the early gene expression of Runx2 and Alp. Nevertheless, the biomineralized calcium content was similar on both ceramics after 21 days, indicating that despite chemical differences, both scaffolds direct the pre-osteoblasts toward a mature state capable of mineralizing the extracellular matrix.

Porous alumina, zirconia and alumina/zirconia for bone repair: fabrication, mechanical and in vitro biological response.

Zirconia (ZrO2) and alumina (Al2O3) based ceramics are widely used for load-bearing applications in bone repair due to their excellent mechanical properties and biocompatibility. They are often regarded as bioinert since no direct bone-material interface is created unless a porous structure intercedes, leading to better bone bonding. In this regard, investigating interactions between cells and porous ceramics is of great interest. In the present study, we report on the successful fabrication of sintered alumina A-61, zirconia Z-50 and zirconia/alumina composite ZA-60 ceramics with medium porosities of 61, 50 and 60%, respectively, indicating a bimodal pore size distribution and good interconnectivity. They exhibit elastic moduli of 3-10 GPa and compressive strength values of 60-240 MPa, similar to those of human cortical bone.We performed in vitro cell-material investigations comparing the adhesion, proliferation and differentiation of mouse pre-osteoblasts MC3T3-E1 on the three porous materials. While all three ceramics demonstrate a strong cell attachment, better cell spreading is observed on zirconia-containing substrates. Significantly higher cell growth was quantified on the latter ceramics, revealing an increased alkaline phosphatase activity, higher collagen production and increased calcium biomineralization compared to A-61. Hence, these porous zirconia-containing ceramics elicit superior biological responses over porous alumina of similar porosity, promoting enhanced biological interaction, with potential use as non-degradable bone grafts or as implant coatings.

Mechanisms of alpha-defensin bactericidal action: comparative membrane disruption by Cryptdin-4 and its disulfide-null analogue.

Mammalian alpha-defensins all have a conserved triple-stranded beta-sheet structure that is constrained by an invariant tridisulfide array, and the peptides exert bactericidal effects by permeabilizing the target cell envelope. Curiously, the disordered, disulfide-null variant of mouse alpha-defensin cryptdin-4 (Crp4), termed (6C/A)-Crp4, has bactericidal activity equal to or greater than that of the native peptide, providing a rationale for comparing the mechanisms by which the peptides interact with and disrupt phospholipid vesicles of defined composition. For both live Escherichia coli ML35 cells and model membranes, disordered (6C/A)-Crp4 induced leakage in a manner similar to that of Crp4 but had less overall membrane permeabilizing activity. Crp4 induction of the leakage of the fluorophore from electronegative liposomes was strongly dependent on vesicle lipid charge and composition, and the incorporation of cardiolipin into liposomes of low electronegative charge to mimic bacterial membrane composition conferred sensitivity to Crp4- and (6C/A)-Crp4-mediated vesicle lysis. Membrane perturbation studies using biomimetic lipid/polydiacetylene vesicles showed that Crp4 inserts more pronouncedly into membranes containing a high fraction of electronegative lipids or cardiolipin than (6C/A)-Crp4 does, correlating directly with measurements of induced leakage. Fluorescence resonance energy transfer experiments provided evidence that Crp4 translocates across highly charged or cardiolipin-containing membranes, in a process coupled with membrane permeabilization, but (6C/A)-Crp4 did not translocate across lipid bilayers and consistently displayed membrane surface association. Thus, despite the greater in vitro bactericidal activity of (6C/A)-Crp4, native, beta-sheet-containing Crp4 induces membrane permeabilization more effectively than disulfide-null Crp4 by translocating and forming transient membrane defects. (6C/A)-Crp4, on the other hand, appears to induce greater membrane disintegration.

Measurement of eotaxin (CCL11) in induced sputum supernatants: validation and detection in asthma.

BACKGROUND: Induced sputum is widely used in asthma research; however, for many mediators, the detection methods have not been validated. OBJECTIVE: We sought to optimize the method of detection of eotaxin, an important chemokine acting through the CCR3 receptor on eosinophils, basophils, and T(H)2 cells. METHODS: Induced sputum from normal and asthmatic subjects was processed with dithioerythritol (DTE) or PBS; recovery of eotaxin was assessed by means of ELISA before and after spiking with recombinant eotaxin. Furthermore, the effects of removing DTE by means of ultrafiltration or the addition of protease inhibitors and high-speed centrifugation on endogenous levels and spiking recovery of eotaxin were assessed. RESULTS: Endogenous eotaxin was undetectable in DTE-processed samples, with a mean of only 30% (SD, 13%) spike recovery. DTE had no effect on the immunoassay capture antibody but dramatically reduced the detection of recombinant eotaxin. Removal of DTE from sputum before immunoassay did not improve detection, although it restored the recovery of a subsequent eotaxin spike. In contrast, PBS-processed sputum resulted in an eotaxin spike recovery of 101% (SD, 20%). Addition of protease inhibitors or high-speed centrifugation had no effect on eotaxin detection. By using this optimized protocol, eotaxin levels in PBS-processed sputum samples were found to be significantly increased in asthmatic sputum (P<.05). CONCLUSION: Measurement of eotaxin by means of immunoassay is adversely affected by DTE, possibly through irreversible denaturation of epitopes, which makes eotaxin undetectable by using the immunoassay antibody. Sputum samples should be processed into PBS for assessment of eotaxin, which is present at increased levels in asthmatic sputum.

Contribution of eotaxin-1 to eosinophil chemotactic activity of moderate and severe asthmatic sputum.

The CC chemokine eotaxin-1 (CCL11) is chemotactic for eosinophils, basophils, and type 2 helper T cells and may play a role in allergic inflammation. We investigated its contribution as an eosinophil chemoattractant in asthmatic airway secretions (sampled as induced sputum), which possess chemotactic activity for eosinophils and T cells. Sputum samples collected from healthy subjects and subjects with mild, stable-moderate, unstable-moderate, and severe asthma were processed with phosphate-buffered saline and assayed for eotaxin by ELISA and for eosinophil chemotactic activity by fluorescence-based chemotaxis assay. The contribution of eotaxin to chemotactic activity was studied by using a high-affinity neutralizing human anti-eotaxin antibody, CAT-213. Sputum eotaxin concentration was significantly raised in moderate and severe asthma (p < 0.05 versus healthy control subjects) but not in mild asthma. Chemotactic activity was significantly increased in all asthmatic groups relative to healthy subjects (p < 0.05) and was significantly inhibited by CAT-213 (100 nM) in subjects with moderate and severe asthma, with median inhibition of 52% (p < 0.05), 78% (p < 0.0001), and 86% (p < 0.0001), respectively, in samples representing stable-moderate, unstable-moderate, and severe asthma. Eotaxin contributed to the eosinophil chemotactic activity of sputum from subjects with more severe forms of asthma but not mild asthma, suggesting that its contribution is more important in more severe disease. This activity is inhibited significantly by CAT-213.