Correlation between biological responses in vitro and in vivo to Ca-doped sol-gel coatings assessed using proteomic analysis.

Poor correlation between the results of in vitro testing and the subsequent in vivo experiments hinders the design of biomaterials. Thus, new characterisation methods are needed. This study used proteomic and histological techniques to analyse the effects of Ca-doped biomaterials in vitro and in vivo and verify the correlation between the two systems. The sol-gel route was employed to synthesise coatings functionalised with 0.5 and 5 wt% of CaCl2. Morphology of the coatings was examined using SEM; the Ca2+ ion release from the materials was analysed by means of ICP-AES spectroscopy. The osteogenic and inflammatory responses were inspected in vitro in human osteoblasts (HOb) and TPH-1 monocytes. The in vivo experiments used a rabbit model. The nLC-MS/MS-based proteomic methods were utilised to analyse the proteins adhering to the material samples incubated with human serum or examine protein expression in the tissues close to the implants. Ca-doped biomaterials caused a remarkable increase in the adsorption of coagulation-related proteins, both in vitro (PLMN, THRB, FIBA and VTNC) and in vivo (FBLN1, G1U978). Enhanced affinity to these materials was also observed for proteins involved in inflammation (CO5, C4BPA, IGHM and KV302 in vitro; CARD6, DDOST and CD14 in vivo) and osteogenic functions (TETN, PEDF in vitro; FBN1, AHSG, MYOC in vivo). The results obtained using different techniques were well matched, with a good correlation between the in vitro and in vivo experiments. Thus, the proteomic analysis of biological responses to biomaterials in vitro is a useful tool for predicting their impact in vivo.

Bioactive zinc-doped sol-gel coating modulates protein adsorption patterns and in vitro cell responses.

Zinc is an essential element with an important role in stimulating the osteogenesis and mineralization and suppressing osteoclast differentiation. In this study, new bioactive ZnCl2-doped sol-gel materials were designed to be applied as coatings onto titanium. The biomaterials were physicochemically characterized and the cellular responses evaluated in vitro using MC3T3-E1 osteoblasts and RAW264.7 macrophages. The effect of Zn on the adsorption of human serum proteins onto the material surface was evaluated through nLC-MS/MS. The incorporation of Zn did not affect the crosslinking of the sol-gel network. A controlled Zn2+ release was obtained, reaching values below 10 ppm after 21 days. The materials were no cytotoxic and lead to increased gene expression of ALP, TGF-ß, and RUNX2 in the osteoblasts. In macrophages, an increase of IL-1ß, TGF-ß, and IL-4 gene expression was accompanied by a reduced TNF-α liberation. Proteomic results showed changes in the adsorption patterns of proteins associated with immunological, coagulative, and regenerative functions, in a Zn dose-dependent manner. The variations in protein adsorption might lead to the downregulation of the NF-κB pathway, thus explain the observed biological effects of Zn incorporation into biomaterials. Overall, these coatings demonstrated their potential to promote bone tissue regeneration.

#EUROmicroMOOC: using Twitter to share trends in Microbiology worldwide.

Twitter is one of the most popular social media networks that, in recent years, has been increasingly used by researchers as a platform to share science and discuss ongoing work. Despite its popularity, Twitter is not commonly used as a medium to teach science. Here, we summarize the results of #EUROmicroMOOC: the first worldwide Microbiology Massive Open Online Course taught in English using Twitter. Content analytics indicated that more than 3 million users saw posts with the hashtag #EUROmicroMOOC, which resulted in over 42 million Twitter impressions worldwide. These analyses demonstrate that free Microbiology MOOCs shared on Twitter are valuable educational tools that reach broad audiences throughout the world. We also describe our experience teaching an entire Microbiology course using Twitter and provide recommendations when using social media to communicate science to a broad audience.

Complement proteins regulating macrophage polarisation on biomaterials.

One of the events occurring when a biomaterial is implanted in an host is the protein deposition onto its surface, which might regulate cell responses. When a biomaterial displays a compromised biocompatibility, distinct complement pathways can be activated to produce a foreign body reaction. In this article, we have designed different types of biomaterial surfaces to study the inflammation process. Here, we used different concentrations of (3-glycidoxypropyl)-trimethoxysilane (GPTMS), an organically-modified alkoxysilane as a precursor for the synthesis of various types of sol-gel materials functionalizing coatings for titanium implants to regulate biological responses. Our results showed that greater GPTMS surface concentrations induced greater secretion of TNF-α and IL-10 on RAW 264.7 macrophages. When implanted into rabbit tibia, osseointegration decreased with higher GPTMS concentrations. Interestingly, higher deposition of complement-related proteins C-reactive protein (CRP) and ficolin-2 (FCN2), two main activators of distinct complement pathways, was observed. Taking all together, inflammatory potential increase seems to be GPTMS concentration-dependent. Our results show that a greater adsorption of complement proteins can condition macrophage polarization.

Proteomic analysis of calcium-enriched sol-gel biomaterials.

Calcium is an element widely used in the development of biomaterials for bone tissue engineering as it plays important roles in bone metabolism and blood coagulation. The Ca ions can condition the microenvironment at the tissue-material interface, affecting the protein deposition process and cell responses. The aim of this study was to analyze the changes in the patterns of protein adsorption on the silica hybrid biomaterials supplemented with different amounts of CaCl2, which can function as release vehicles. This characterization was carried out by incubating the Ca-biomaterials with human serum. LC-MS/MS analysis was used to characterize the adsorbed protein layers and compile a list of proteins whose affinity for the surfaces might depend on the CaCl2 content. The attachment of pro- and anti-clotting proteins, such as THRB, ANT3, and PROC, increased significantly on the Ca-materials. Similarly, VTNC and APOE, proteins directly involved on osteogenic processes, attached preferentially to these surfaces. To assess correlations with the proteomic data, these formulations were tested in vitro regarding their osteogenic and inflammatory potential, employing MC3T3-E1 and RAW 264.7 cell lines, respectively. The results confirmed a Ca dose-dependent osteogenic and inflammatory behavior of the materials employed, in accordance with the protein attachment patterns.

The effect of strontium incorporation into sol-gel biomaterials on their protein adsorption and cell interactions.

It is known strontium can both inhibit the osteoclast formation and stimulate the osteoblast maturation, so biomaterials containing this element can favour bone structure stabilisation. The addition of Sr to biomaterials could affect their interactions with proteins and cells. Here, a silica-hybrid sol-gel network doped with different amounts of SrCl2 and applied as coatings on titanium discs was examined. in vitro analysis was performed to determine the potential effect of Sr in the coatings, showing enhanced gene expression of osteogenic markers (alkaline phosphatase and transforming growth factor-ß) in MC3T3-E1 incubated with Sr-doped biomaterials. The examination of inflammatory markers (tumour necrosis factor-α and interleukin 10) in RAW 264.7 macrophages revealed an anti-inflammatory potential of these materials. Proteins adsorbed onto the coatings incubated with human serum (3 h at 37 °C) were also analysed; mass spectrometry was used to characterise the proteins adhering to materials with different Sr content. Adding Sr to the coatings increased their affinity to APOE and VTNC proteins (associated with anti-inflammatory and osteogenic functions). Moreover, the proteins involved in coagulation processes, such as prothrombin, were more abundant on the coatings containing Sr than on the base sol-gel surfaces. Correlations between gene expression and proteomic results were also examined.

Synthesis and characterization of silica-chitosan hybrid materials as antibacterial coatings for titanium implants.

To avoid dental implant-related infections and to promote the osseointegration of titanium implants, the application of silicon and chitosan containing coatings is proposed. Silicon is a well-known osteogenic element and chitosan was selected to confer the antibacterial properties. The synthesis of hybrid silica-chitosan coatings using the sol-gel process is presented and the characterization using 29Si-NMR to verify the correct formation of the network is discussed. The 13C NMR spectroscopy was used to confirm the covalent union between chitosan and the silicon network. Hydrolytic degradation and silicon release studies showed the effective silicon release from the hybrids and, hence, the possibility to promote bone formation. The introduction of different amounts of chitosan and tetraethyl orthosilicate (TEOS) modulated the Si release. The analysis of cell cultures in vitro demonstrated that the hybrid coatings were not cytotoxic and promoted cell proliferation on their surfaces. The coatings containing 5%-10% chitosan had substantial antibacterial properties.

Osseointegration mechanisms: a proteomic approach.

The prime objectives in the development of biomaterials for dental applications are to improve the quality of osseointegration and to short the time needed to achieve it. Design of implants nowadays involves changes in the surface characteristics to obtain a good cellular response. Incorporating osteoinductive elements is one way to achieve the best regeneration possible post-implantation. This study examined the osteointegrative potential of two distinct biomaterials: sandblasted acid-etched titanium and a silica sol-gel hybrid coating, 70% MTMOS-30% TEOS. In vitro, in vivo, and proteomic characterisations of the two materials were conducted. Enhanced expression levels of ALP and IL-6 in the MC3T3-E1 cells cultured with coated discs, suggest that growing cells on such surfaces may increase mineralisation levels. 70M30T-coated implants showed improved bone growth in vivo compared to uncoated titanium. Complete osseointegration was achieved on both. However, coated implants displayed osteoinductive properties, while uncoated implants demonstrated osteoconductive characteristics. Coagulation-related proteins attached predominantly to SAE-Ti surface. Surface properties of the material might drive the regenerative process of the affected tissue. Analysis of the proteins on the coated dental implant showed that few proteins specifically attached to its surface, possibly indicating that its osteoinductive properties depend on the silicon delivery from the implant.

Bioactive potential of silica coatings and its effect on the adhesion of proteins to titanium implants.

There is an ever-increasing need to develop dental implants with ideal characteristics to achieve specific and desired biological response in the scope of improve the healing process post-implantation. Following that premise, enhancing and optimizing titanium implants through superficial treatments, like silica sol-gel hybrid coatings, are regarded as a route of future research in this area. These coatings change the physicochemical properties of the implant, ultimately affecting its biological characteristics. Sandblasted acid-etched titanium (SAE-Ti) and a silica hybrid sol-gel coating (35M35G30T) applied onto the Ti substrate were examined. The results of in vitro and in vivo tests and the analysis of the protein layer adsorbed to each surface were compared and discussed. In vitro analysis with MC3T3-E1 osteoblastic cells, showed that the sol-gel coating raised the osteogenic activity potential of the implants (the expression of osteogenic markers, the alkaline phosphatase (ALP) and IL-6 mRNAs, increased). In the in vivo experiments using as model rabbit tibiae, both types of surfaces promoted osseointegration. However, the coated implants demonstrated a clear increase in the inflammatory activity in comparison with SAE-Ti. Mass spectrometry (LC-MS/MS) analysis showed differences in the composition of protein layers formed on the two tested surfaces. Large quantities of apolipoproteins were found attached predominantly to SAE-Ti. The 35M35G30T coating adsorbed a significant quantity of complement proteins, which might be related to the material intrinsic bioactivity, following an associated, natural and controlled immune response. The correlation between the proteomic data and the in vitro and in vivo outcomes is discussed on this experimental work.

Preparation and characterization of injectable PMMA-strontium-substituted bioactive glass bone cement composites.

In most minimally-invasive procedures used to address severe pain arising from compression fractures of the vertebral bodies, such as percutaneous vertebroplasty (PVP), a poly(methyl methacrylate) (PMMA) bone cement is used. Shortcomings of this type of cement, such as high exotherm temperature and lack of bioactivity, are well known. We prepared different formulations of a composite bone cement, whose solid constituents consisted of PMMA beads and particles of a bioactive glass (BG), where 0-20%(w/w) of the calcium component was substituted by strontium. The difference between the formulations was in the relative amounts of the solid phase constituents and in the Sr-content of BG. We determined the influence of the mixture of solid phase constituents of the cement formulation on a collection of properties, such as maximum exotherm temperature (Tmax ), setting time (tset ), and injectability (I). The selection of the PMMA beads was crucial to obtain cement composite formulations capable to be efficiently injected. Results allowed to select nine solid phase mixtures to be further tested. Then, we determined the influence of the composition of these composite bone cements on Tmax , tset , I, and cell proliferation. The results showed that the performance of various of the selected composite cements was better than that of PMMA cement reference, with lower Tmax , lower tset , and higher I. We found that incorporation of Sr-substituted BGs into these materials bestows bioactivity properties associated with the role of Sr in bone formation, leading to some composite cement formulations that may be suitable for use in PVP. © 2017 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater, 106B: 1245-1257, 2018.

Proteomic analysis of silica hybrid sol-gel coatings: a potential tool for predicting the biocompatibility of implants in vivo.

The interactions of implanted biomaterials with the host organism determine the success or failure of an implantation. Normally, their biocompatibility is assessed using in vitro tests. Unfortunately, in vitro and in vivo results are not always concordant; new, effective methods of biomaterial characterisation are urgently needed to predict the in vivo outcome. As the first layer of proteins adsorbed onto the biomaterial surfaces might condition the host response, mass spectrometry analysis was performed to characterise these proteins. Four distinct hybrid sol-gel biomaterials were tested. The in vitro results were similar for all the materials examined here. However, in vivo, the materials behaved differently. Six of the 171 adsorbed proteins were significantly more abundant on the materials with weak biocompatibility; these proteins are associated with the complement pathway. Thus, protein analysis might be a suitable tool to predict the in vivo outcomes of implantations using newly formulated biomaterials.

Synthesis of hybrid sol-gel materials and their biological evaluation with human mesenchymal stem cells.

Surface engineering of biomaterials could promote the osseointegration of implants. In this work, two types of hybrid sol-gel materials were developed to stimulate cell attachment, proliferation and differentiation of osteogenic cells. One type was synthesised from vinyl triethoxysilane (VTES) and tetraethyl-orthosilicate (TEOS) at different molar ratios, while the other from VTES and hydroxyapatite particles (HAp). Hybrid materials were systematically investigated using nuclear magnetic resonance, Fourier transform infrared spectroscopy and contact angle metrology. The biocompatibility and osseoinduction of the coatings were evaluated by measuring mesenchymal stem cell proliferation using MTT assays and analysing the mineralised extracellular matrix production by quantifying calcium-rich deposits. The results highlighted the versatility of these coatings in obtaining different properties by changing the molar ratio of the VTES:TEOS precursors. Thus, mineralisation was stimulated by increasing TEOS content, while the addition of HAp improved cell proliferation but worsened mineralisation.

Prevalence and significance of parasites of horses in some States of northern Nigeria.

This study was conducted to determine the prevalence and significance of parasites of horses in northern Nigeria. Blood and faecal samples were randomly collected from 243 horses from different stables in some states of northern Nigeria for laboratory analyses. Fifty-seven horses (23.5%) were found infected with parasites. The hemoparasites detected, 21 (8.6%), include Theileria equi, Babesia caballi, Trypanosoma vivax and Trypanosoma evansi. The endoparasites encountered, 29 (11.9%) were Strongylus spp., Strongyloides spp., Oxyuris equi, Parascaris equorum, Paragonimus spp. and Dicrocoelium spp., 3 (1.2%) was Eimeria spp. Four horses (1.6%) had mixed infection of hemo- and endoparasites. This preliminary finding shows that parasitism is a problem in the horse stables examined, and calls for proper stable hygiene, routine tick control and regular deworming programme.

Intestinal bioaccessibility of polyphenols and antioxidant capacity of pulp and seeds of cactus pear.

The aim of this work was to estimate the intestinal bioaccessibility of polyphenols and antioxidant capacity of cactus pear using an in vitro gastrointestinal model. The amount of polyphenols released from the food matrix by the action of digestive enzymes was about 69-83% in pulp and 0-14% in seeds. The rest of polyphenols was associated with indigestible compounds conferring antioxidant capacity. The results suggest that these compounds could reach the colon and induce healthy effects.

Construction and evaluation of an ORFeome-based Brucella whole-genome DNA microarray.

The genus Brucella contains bacteria producing a zoonosis of large sanitary and economical impact. The complete nucleotide sequence of eight Brucella isolates is currently available. This information can be used for high throughput approaches to the biology of this genus such as the construction of comprehensive collections of ORF clones or ORFeomes. The ORFeome of Brucella melitensis was a first contribution to this goal. Using the Brucella ORFeome as starting material we have amplified each ORF and printed them in duplicate onto coated glass slides along with the appropriate positive and negative controls. Quality control of the microarray was performed by image analysis after ethidium bromide staining. This Brucella DNA microarray was used to determine the global transcriptional profile of Brucella abortus grown under laboratory conditions. Two sets of genes representing strongly and poorly expressed genes have been defined. The occurrence of several genes of the same operon in the same data set has been taken as additional proof of the significance of the results. The two sets have been validated by RT-PCR of retrotranscribed RNA. Among the more abundant transcripts we found ribosomal proteins, Krebs cycle and oxidative phosphorylation enzymes. virB, flagellar components and other genes related with virulence and intracellular growth were in the poorly transcribed set. This report demonstrated the usefulness of the ORFeome for the construction of a PCR product microarray for the analysis of global gene expression in Brucella and also applicable to other microorganisms. The results provided here represent a comprehensive description of the global transcriptional profile of B. abortus grown under laboratory conditions and, at the same time, validate the use of this Brucella microarray for the study of the biology and pathogenesis of Brucella through the analysis of gene expression under any experimental conditions.

Antioxidative effect of dietary grape pomace concentrate on lipid oxidation of chilled and long-term frozen stored chicken patties.

Grape pomace concentrate (GPC) is a natural source of phenolic compounds with high antioxidant capacity. The effect of a diet containing GPC on lipid peroxidation levels (TBARS) and antioxidant capacity (ABTS method) of raw and cooked chicken breast meat patties stored in chilled conditions (4°C) for 0, 3, 6, 13 and 20days, and long-term frozen storage (6months) was investigated. Chickens were fed GPC at levels of 0, 30 and 60mg/kg from 3 to 6weeks of age. Dietary GPC did not affect chicken performance. Lipid oxidation (TBARS value) was significantly increased by the storage time (0-20days and 6months of storage, respectively) in raw and cooked samples. Dietary GPC significantly caused an inhibitory effect on lipid oxidation of raw and cooked breast chicken patties compared with samples obtained from birds fed the control diet at 20days and long-term frozen storage (6months). Radical scavenging capacity was significantly increased at 20days in cooked samples and significantly reduced at 6months of storage in raw and cooked samples. The higher concentration of dietary GPC increased the ABTS values only in the raw samples. These results indicated that dietary grape pomace concentrate could be effective in inhibiting lipid oxidation of chilled and long-term frozen stored chicken patties.

Serosurvey for Toxoplasma gondii in dogs in Maiduguri, Borno State, Nigeria.

The seroprevalence of Toxoplasma gondii (T. gondii) antibodies in dogs in Maiduguri, the capital of Borno State, Nigeria, was determined using the Latex Aagglutination Test (LAT). Antibodies (LAT titer > 1:64) to T. gondii were found in 42 (25%) of the animals examined. Antibody titers in positive dogs ranged from 1:64 (15 dogs) to 1:2048 (3 dogs). There was a significant statistical difference (P < 0.05) between age groups of dogs, the prevalence being higher in dogs > or = 3 years (chi2 = 13.73 P = 0.0002, OR 2.80 CI95% 1.28-6.13 P = 0.008). Pure Alsatians and their crosses were less likely to be seropositive (OR cross breed 0.28 CI95% 0.13-0.61 P = 0.001, OR Alsatian 0.16 CI95% 0.04-0.58 P = 0.002). The high prevalence of T. gondii infection that we found in dogs suggests a need for a larger survey to determine the national prevalence and identify possible risk factors in different agro-climatic zones. Such a study will help in formulating nation-wide control measures for toxoplasmosis.

Injectable acrylic bone cements for vertebroplasty based on a radiopaque hydroxyapatite. Formulation and rheological behaviour.

The utilization of injectable acrylic bone cement is crucial to the outcome of vertebroplasty and kyphoplasty. However, only a few cements that are in clinical use today are formulated specifically for use in these procedures and even these formulations are not regarded as "ideal" injectable bone cements. The aim of this work is to prepare bioactive bone cements by adding strontium hydroxyapatite (SrHA) to a cement formulation based on polymethylmethacrylate. Thus, the cement combines the immediate mechanical support given by the setting of the acrylic matrix with optimum radiopacity and bioactivity due to the incorporation of the SrHA. Formulations of bioactive cement were prepared with 10 and 20 wt% of SrHA as synthesised and after a surface treatment with the monomer. Cements loaded with treated particles showed an enhancement of their handling properties, and hence, an improvement on their rheological behaviour, injectabilities and compressive parameters. Further experiments were also carried out to determine their bioactivity and biocompatibility and results appear in other publication.

Evaluation of a multiplex PCR assay (Bruce-ladder) for molecular typing of all Brucella species, including the vaccine strains.

An evaluation of a multiplex PCR assay (Bruce-ladder) was performed in seven laboratories using 625 Brucella strains from different animal and geographical origins. This robust test can differentiate in a single step all of the classical Brucella species, including those found in marine mammals and the S19, RB51, and Rev.1 vaccine strains.

Effect of grape pomace concentrate and vitamin E on digestibility of polyphenols and antioxidant activity in chickens.

Grape pomace provides a rich source of polyphenols that have the capacity to act as powerful antioxidants. An experiment was conducted to study the effect of inclusion of grape pomace concentrate (GPC) at levels of 15, 30, and 60 g/kg and alpha-tocopheryl acetate (200 mg/kg) in broiler chicks (21 to 42 d of age) on performance; digestive organ sizes; protein; fat; hydrolyzable polyphenol and condensed tannin digestibilities; the anti-oxidant activity of diet, serum, ileal content, and excreta; and the susceptibility to oxidation of breast meat during refrigerated storage. The inclusion of GPC did not affect the performance; the apparent ileal digestibility of CP; the relative abdominal fat, liver, pancreas, and spleen weight; and the relative intestinal length. Fat digestibility was reduced in birds fed control and GPC diets compared with birds fed vitamin E. Ileal and fecal digestibility of hydrolyzable polyphenols and condensed tannins reached values in a range of 56 to 73% and 14 to 47%, respectively. The GPC diets reduced ileal and fecal digestibility of hydrolyzable polyphenols. Antioxidant activity in GPC diet, ileal content, and excreta [2, 2-azinobis (3-ethilenzotiazolin)-6-sulfonate method] and GPC diet (ferric antioxidant power method) exhibited higher scavenging free radical capacity than control and vitamin E diets. The lipid oxidation in breast meat was lower in the birds fed the supplemented vitamin E diet than the control diet after 1, 4, and 7 d of refrigerated storage. Oxidative stability in breast meat at 1, 4, and 7 d of storage was equivalent in GPC diets compared with the vitamin E diet. In conclusion, the inclusion of GPC (up to 60 g/kg) did not impair chicken growth performance, digestive organ sizes, and protein digestibility. Hydrolyzable polyphenols were more bioavailable than condensed tannins. Antioxidant activity in diet, excreta, ileal content, and breast muscle were increased in GPC diets. The GPC supplementation was equally as effective in antioxidant potential as vitamin E. On the basis of these observations, we concluded that GPC could be a new source of antioxidant in animal nutrition.