Bioactive Glass Modified with Zirconium Incorporation for Dental Implant Applications: Fabrication, Structural, Electrical, and Biological Analysis.

Implantology is crucial for restoring aesthetics and masticatory function in oral rehabilitation. Despite its advantages, certain issues, such as bacterial infection, may still arise that hinder osseointegration and result in implant rejection. This work aims to address these challenges by developing a biomaterial for dental implant coating based on 45S5 Bioglass® modified by zirconium insertion. The structural characterization of the glasses, by XRD, showed that the introduction of zirconium in the Bioglass network at a concentration higher than 2 mol% promotes phase separation, with crystal phase formation. Impedance spectroscopy was used, in the frequency range of 102-106 Hz and the temperature range of 200-400 K, to investigate the electrical properties of these Bioglasses, due to their ability to store electrical charges and therefore enhance the osseointegration capacity. The electrical study showed that the presence of crystal phases, in the glass ceramic with 8 mol% of zirconium, led to a significant increase in conductivity. In terms of biological properties, the Bioglasses exhibited an antibacterial effect against Gram-positive and Gram-negative bacteria and did not show cytotoxicity for the Saos-2 cell line at extract concentrations up to 25 mg/mL. Furthermore, the results of the bioactivity test revealed that within 24 h, a CaP-rich layer began to form on the surface of all the samples. According to our results, the incorporation of 2 mol% of ZrO2 into the Bioglass significantly improves its potential as a coating material for dental implants, enhancing both its antibacterial and osteointegration properties.

Extensive Investigation on the Effect of Niobium Insertion on the Physical and Biological Properties of 45S5 Bioactive Glass for Dental Implant.

Dental implants have emerged as one of the most consistent and predictable treatments in the oral surgery field. However, the placement of the implant is sometimes associated with bacterial infection leading to its loss. In this work, we intend to solve this problem through the development of a biomaterial for implant coatings based on 45S5 Bioglass® modified with different amounts of niobium pentoxide (Nb2O5). The structural feature of the glasses, assessed by XRD and FTIR, did not change in spite of Nb2O5 incorporation. The Raman spectra reveal the Nb2O5 incorporation related to the appearance of NbO4 and NbO6 structural units. Since the electrical characteristics of these biomaterials influence their osseointegration ability, AC and DC electrical conductivity were studied by impedance spectroscopy, in the frequency range of 102-106 Hz and temperature range of 200-400 K. The cytotoxicity of glasses was evaluated using the osteosarcoma Saos-2 cells line. The in vitro bioactivity studies and the antibacterial tests against Gram-positive and Gram-negative bacteria revealed that the samples loaded with 2 mol% Nb2O5 had the highest bioactivity and greatest antibacterial effect. Overall, the results showed that the modified 45S5 bioactive glasses can be used as an antibacterial coating material for implants, with high bioactivity, being also non-cytotoxic to mammalian cells.

Production of Electrospun Fast-Dissolving Drug Delivery Systems with Therapeutic Eutectic Systems Encapsulated in Gelatin.

Fast-dissolving delivery systems (FDDS) have received increasing attention in the last years. Oral drug delivery is still the preferred route for the administration of pharmaceutical ingredients. Nevertheless, some patients, e.g. children or elderly people, have difficulties in swallowing solid tablets. In this work, gelatin membranes were produced by electrospinning, containing an encapsulated therapeutic deep-eutectic solvent (THEDES) composed by choline chloride/mandelic acid, in a 1:2 molar ratio. A gelatin solution (30% w/v) with 2% (v/v) of THEDES was used to produce electrospun fibers and the experimental parameters were optimized. Due to the high surface area of polymer fibers, this type of construct has wide applicability. With no cytotoxicity effect, and showing a fast-dissolving release profile in PBS, the gelatin fibers with encapsulated THEDES seem to have promising applications in the development of new drug delivery systems.

The importance of the Abn2 calcium cluster in the endo-1,5-arabinanase activity from Bacillus subtilis.

Arabinanase is a glycosyl hydrolase that is able to cleave the glycosidic bonds of α-1,5-L-arabinan, releasing arabino-oligosaccharides and L-arabinose. The enzyme has two domains, an N-terminal catalytic domain with a characteristic ß-propeller fold and a C-terminal domain whose function is unknown. A calcium ion, located near the catalytic site, serves to stabilize the N-terminal domain, but it has also been proposed to play a key role in the enzyme mechanism. The present work describes the structure of an inactive mutant of the wild-type enzyme (H318Q) and in which the calcium ion has been adventitiously replaced by nickel. These structural studies, together with functional and modelling studies, clearly support the role of the calcium ion in the overall reaction mechanism.

The Effect of Iron Oxide Insertion on the In Vitro Bioactivity, and Antibacterial Properties of the 45S5 Bioactive Glass.

The aging population and increasing incidence of trauma among younger age groups have heightened the increasing demand for reliable implant materials. Effective implant materials must demonstrate rapid osseointegration and strong antibacterial properties to ensure optimal patient outcomes and decrease the chance of implant rejection. This study aims to enhance the bone-implant interface by utilizing 45S5 bioglass modified with various concentrations of Fe3O4 as a coating material. The effect of the insertion of Fe3O4 into the bioglass structure was studied using Raman spectroscopy which shows that with the increase in Fe3O4 concentration, new vibration bands associated with Fe-related structural units appeared within the sample. The bioactivity of the prepared glasses was evaluated using immersion tests in simulated body fluid, revealing the formation of a calcium phosphate-rich layer within 24 h on the samples, indicating their potential for enhanced tissue integration. However, the sample modified with 8 mol% of Fe3O4 showed low reactivity, developing a calcium phosphate-rich layer within 96 h. All the bioglasses showed antibacterial activity against the Gram-positive and Gram-negative bacteria. The modified bioglass did not present significant antibacterial properties compared to the bioglass base.

Fabrication, Structural and Biological Characterization of Zinc-Containing Bioactive Glasses and Their Use in Membranes for Guided Bone Regeneration.

Polymeric membranes are widely used in guided bone regeneration (GBR), particularly in dentistry. In addition, bioactive glasses can be added to the polymers in order to develop a matrix that is osteoconductive and osteoinductive, increasing cell adhesion and proliferation. The bioactive glasses allow the insertion into its network of therapeutic ions in order to add specific biological properties. The addition of zinc into bioactive glasses can promote antibacterial activity and induce the differentiation and proliferation of the bone cells. In this study, bioactive glasses containing zinc (0.25, 0.5, 1 and 2 mol%) were developed and structurally and biologically characterized. The biological results show that the Zn-containing bioactive glasses do not present significant antibacterial activity, but the addition of zinc at the highest concentration does not compromise the bioactivity and promotes the viability of Saos-2 cells. The cell culture assays in the membranes (PCL, PCL:BG and PCL:BGZn2) showed that zinc addition promotes cell viability and an increase in alkaline phosphatase (ALP) production.

Antibacterial Biomaterial Based on Bioglass Modified with Copper for Implants Coating.

Biofilm-related implant infections pose a substantial threat to patients, leading to inflammation in the surrounding tissue, and often resulting in implant loss and the necessity for additional surgeries. Overcoming this implantology challenge is crucial to ensure the success and durability of implants. This study shows the development of antibacterial materials for implant coatings by incorporating copper into 45S5 Bioglass®. By combining the regenerative properties of Bioglass® with the antimicrobial effects of copper, this material has the potential to prevent infections, enhance osseointegration and improve the long-term success of implants. Bioglasses modified with various concentrations of CuO (from 0 to 8 mol%) were prepared with the melt-quenching technique. Structural analysis using Raman and FTIR spectroscopies did not reveal significant alterations in the bioglasses structure with the addition of Cu. The antibacterial activity of the samples was assessed against Gram-positive and Gram-negative bacteria, and the results demonstrated significant inhibition of bacterial growth for the bioglass with 0.5 mol% of CuO. Cell viability studies indicated that the samples modified with up to 4 mol% of CuO maintained good cytocompatibility with the Saos-2 cell line at extract concentrations up to 25 mg/mL. Furthermore, the bioactivity assessment demonstrated the formation of a calcium phosphate (CaP)-rich layer on the surfaces of all bioglasses within 24 h. Our findings show that the inclusion of copper in the bioglass offers a significant enhancement in its potential as a coating material for implants, resulting in notable advancements in both antibacterial efficacy and osteointegration properties.

Bioactive Glasses Containing Strontium or Magnesium Ions to Enhance the Biological Response in Bone Regeneration.

The non-surgical treatments are being required to reconstruct damaged tissue, prioritizing our body's natural healing process. Thus, the use of bioactive materials such as bioactive glass has been studied to support the repair and restoration of hard and soft tissue. Thus, in this work Bioglass 45S5 was developed, adding 1 and 2%mol of SrO or MgO and the physical and biological properties were evaluated. The addition of MgO and SrO at the studied concentrations promoted the slight increase in non-bridging oxygens number, observed through the temperature shift in phase transitions to lower values compared to Bioglass 45S5. The insertion of the ions also showed a positive effect on Saos-2 cell viability, decreasing the cytotoxic of Bioglass 45S5. Besides the Ca/P ratio on the pellets surface demonstrating no evidence of higher reactivity between Bioglass 45S5 and Bioglass with Sr and Mg, micrographs show that at 24 h the Ca/P rich layer is denser than in Bioglass 45S5 after the contact with simulated body fluid. The samples with Sr and Mg show a higher antibacterial effect compared to Bioglass 45S5. The addition of the studied ions may benefit the biological response of Bioglass 45S5 in dental applications as scaffolds or coatings.

Biocompatibility, Bioactivity, and Antibacterial Behaviour of Cerium-Containing Bioglass®.

The main reason for the increased use of dental implants in clinical practice is associated with aesthetic parameters. Implants are also presented as the only technique that conserves and stimulates natural bone. However, there are several problems associated with infections, such as peri-implantitis. This disease reveals a progressive inflammatory action that affects the hard and soft tissues surrounding the implant, leading to implant loss. To prevent the onset of this disease, coating the implant with bioactive glasses has been suggested. In addition to its intrinsic function of promoting bone regeneration, it is also possible to insert therapeutic ions, such as cerium. Cerium has several advantages when the aim is to improve osseointegration and prevent infectious problems with dental implant placement. It promotes increased growth and the differentiation of osteoblasts, improves the mechanical properties of bone, and prevents bacterial adhesion and proliferation that may occur on the implant surface. This antibacterial effect is due to its ability to disrupt the cell wall and membrane of bacteria, thus interfering with vital metabolic functions such as respiration. In addition, its antioxidant effect reverses oxidative stress after implantation in bone. In this work, Bioglass 45S5 with CeO2 with different percentages (0.25, 0.5, 1, and 2 mol%) was developed by the melt-quenching method. The materials were analyzed in terms of morphological, structural, and biological (cytotoxicity, bioactivity, and antibacterial activity) properties. The addition of cerium did not promote structural changes to the bioactive glass, which shows no cytotoxicity for the Saos-2 cell line up to 25 mg/mL of extract concentration for all cerium contents. For the maximum cerium concentration (2 mol%) the bioactive glass shows an evident inhibitory effect for Escherichia coli and Streptococcus mutans bacteria. Furthermore, all samples showed the beginning of the deposition of a CaP-rich layer on the surface of the material after 24 h.

Subcritical Water Extraction and Hydrolysis of Cod (Gadus morhua) Frames to Produce Bioactive Protein Extracts.

The valorization of Atlantic cod (Gadus morhua) frames from a filleting industry was investigated using subcritical water extraction and hydrolysis (SBW) at different temperatures (90, 140, 190 and 250 °C) and 100 bar to obtain extracts rich in proteins, peptides and amino acids. Up to 57.7 g of extract per 100 g of codfish frames were obtained, with nearly total recovery of the protein fraction. At each temperature, protein extracts of decreasing molecular weight were obtained, according to SEC-GPC results. Most of the protein present in the raw material and extracts was collagen and collagen fragments, as suggested by the amino acid profile. Codfish SBW extracts did not show cytotoxicity in the range of concentrations tested and the protein extract obtained at the lowest temperature (90 °C) showed the highest anti-inflammatory potential in human intestinal epithelium cell model. The mineralized residue left after SBW treatment of cod frames was identified as practically pure, crystalline, hydroxyapatite, that may find applications in biomedical field and hard-tissue engineering. This study shows the possible valorization of cod frames using green extraction methods such as SBW process to obtain protein extracts for food and nutraceutical applications.

A multitask ATPase serving different ABC-type sugar importers in Bacillus subtilis.

Bacillus subtilis is able to utilize arabinopolysaccharides derived from plant biomass. Here, by combining genetic and physiological analyses we characterize the AraNPQ importer and identify primary and secondary transporters of B. subtilis involved in the uptake of arabinosaccharides. We show that the ABC-type importer AraNPQ is involved in the uptake of α-1,5-arabinooligosaccharides, at least up to four L-arabinosyl units. Although this system is the key transporter for α-1,5-arabinotriose and α-1,5-arabinotetraose, the results indicate that α-1,5-arabinobiose also is translocated by the secondary transporter AraE. This broad-specificity proton symporter is the major transporter for arabinose and also is accountable for the uptake of xylose and galactose. In addition, MsmX is shown to be the ATPase that energizes the incomplete AraNPQ importer. Furthermore, the results suggest the existence of at least one more unidentified MsmX-dependent ABC importer responsible for the uptake of nonlinear α-1,2- and α-1,3-arabinooligosaccharides. This study assigns MsmX as a multipurpose B. subtilis ATPase required to energize different saccharide transporters, the arabinooligosaccharide-specific AraNPQ-MsmX system, a putative MsmX-dependent ABC transporter specific for nonlinear arabinooligosaccharides, and the previously characterized maltodextrin-specific MdxEFG-MsmX system.

Multitask ATPases (NBDs) of bacterial ABC importers type I and their interspecies exchangeability.

ATP-binding cassette (ABC) type I importers are widespread in bacteria and play a crucial role in its survival and pathogenesis. They share the same modular architecture comprising two intracellular nucleotide-binding domains (NBDs), two transmembrane domains (TMDs) and a substrate-binding protein. The NBDs bind and hydrolyze ATP, thereby generating conformational changes that are coupled to the TMDs and lead to substrate translocation. A group of multitask NBDs that are able to serve as the cellular motor for multiple sugar importers was recently discovered. To understand why some ABC importers share energy-coupling components, we used the MsmX ATPase from Bacillus subtilis as a model for biological and structural studies. Here we report the first examples of functional hybrid interspecies ABC type I importers in which the NBDs could be exchanged. Furthermore, the first crystal structure of an assigned multitask NBD provides a framework to understand the molecular basis of the broader specificity of interaction with the TMDs.

Probing key DNA contacts in AraR-mediated transcriptional repression of the Bacillus subtilis arabinose regulon.

In the absence of arabinose, the AraR transcription factor represses the expression of genes involved in the utilization of arabinose, xylose and galactose in Bacillus subtilis. AraR exhibits a chimeric organization: the N-terminal DNA-binding region belongs to the GntR family and the C-terminal effector-binding domain is homologous to the GalR/LacI family. Here, the AraR-DNA-binding interactions were characterized in vivo and in vitro. The effect of residue substitutions in the AraR N-terminal domain and of base-pair exchanges into an AraR-DNA-binding operator site were examined by assaying for AraR-mediated regulatory activity in vivo and DNA-binding activity in vitro. The results showed that residues K4, R45 and Q61, located in or near the winged-helix DNA-binding motif, were the most critical amino acids required for AraR function. In addition, the analysis of the various mutations in an AraR palindromic operator sequence indicated that bases G9, A11 and T16 are crucial for AraR binding. Moreover, an AraR mutant M34T was isolated that partially suppressed the effect of mutations in the regulatory cis-elements. Together, these findings extend the knowledge on the nature of AraR nucleoprotein complexes and provide insight into the mechanism that underlies the mode of action of AraR and its orthologues.

Therapeutic Role of Deep Eutectic Solvents Based on Menthol and Saturated Fatty Acids on Wound Healing.

The breakthroughs achieved in green solvents promote the emergence of therapeutic deep eutectic solvents (THEDES), which possess intriguing possible applications in the biomedical field. Herein, the main aim was to unravel the biomedical potential of hydrophobic THEDES based in menthol and saturated fatty acids with different chain lengths (e.g., stearic acid (SA), myristic acid (MA), and lauric acid (LA)). Our comprehensive strategy resulted in the thermophysical characterization of different formulations, which allow one to identify the most suitable molar ratio, as well as the intermolecular interactions behind the successful formation of THEDES. The evaluation of their biological performance was also performed toward bacteria and HaCaT cells. Among the different formulations of THEDES, the one based on menthol and SA establishes stronger hydrogen bonding interactions, being also the most promising formulation because it did not elicit any relevant cytotoxicity, and potentiated wound healing, while presenting antibacterial properties against Staphylococcus epidermis and Staphylococcus aureus strains, some of which were methicillin resistant. This work provides clues on the future use of THEDES based on menthol:SA in wound dressings.

Characterization of abn2 (yxiA), encoding a Bacillus subtilis GH43 arabinanase, Abn2, and its role in arabino-polysaccharide degradation.

The extracellular depolymerization of arabinopolysaccharides by microorganisms is accomplished by arabinanases, xylanases, and galactanases. Here, we characterize a novel endo-alpha-1,5-l-arabinanase (EC 3.2.1.99) from Bacillus subtilis, encoded by the yxiA gene (herein renamed abn2) that contributes to arabinan degradation. Functional studies by mutational analysis showed that Abn2, together with previously characterized AbnA, is responsible for the majority of the extracellular arabinan activity in B. subtilis. Abn2 was overproduced in Escherichia coli, purified from the periplasmic fraction, and characterized with respect to substrate specificity and biochemical and physical properties. With linear-alpha-1,5-l-arabinan as the preferred substrate, the enzyme exhibited an apparent K(m) of 2.0 mg ml(-1) and V(max) of 0.25 mmol min(-1) mg(-1) at pH 7.0 and 50 degrees C. RNA studies revealed the monocistronic nature of abn2. Two potential transcriptional start sites were identified by primer extension analysis, and both a sigma(A)-dependent and a sigma(H)-dependent promoter were located. Transcriptional fusion studies revealed that the expression of abn2 is stimulated by arabinan and pectin and repressed by glucose; however, arabinose is not the natural inducer. Additionally, trans-acting factors and cis elements involved in transcription were investigated. Abn2 displayed a control mechanism at a level of gene expression different from that observed with AbnA. These distinct regulatory mechanisms exhibited by two members of extracellular glycoside hydrolase family 43 (GH43) suggest an adaptative strategy of B. subtilis for optimal degradation of arabinopolysaccharides.

Overproduction, crystallization and preliminary X-ray characterization of Abn2, an endo-1,5-alpha-arabinanase from Bacillus subtilis.

Two Bacillus subtilis extracellular endo-1,5-alpha-L-arabinanases, AbnA and Abn2, belonging to glycoside hydrolase family 43 have been identified. The recently characterized Abn2 protein hydrolyzes arabinan and has low identity to other reported 1,5-alpha-L-arabinanases. Abn2 and its selenomethionine (SeMet) derivative have been purified and crystallized. Crystals appeared in two different space groups: P1, with unit-cell parameters a = 51.9, b = 57.6, c = 86.2 A, alpha = 82.3, beta = 87.9, gamma = 63.6 degrees , and P2(1)2(1)2(1), with unit-cell parameters a = 57.9, b = 163.3, c = 202.0 A. X-ray data have been collected for the native and the SeMet derivative to 1.9 and 2.7 A resolution, respectively. An initial model of Abn2 is being built in the SeMet-phased map.

trans-Acting factors and cis elements involved in glucose repression of arabinan degradation in Bacillus subtilis.

In Bacillus subtilis, the synthesis of enzymes involved in the degradation of arabinose-containing polysaccharides is subject to carbon catabolite repression (CCR). Here we show that CcpA is the major regulator of repression of the arabinases genes in the presence of glucose. CcpA acts via binding to one cre each in the promoter regions of the abnA and xsa genes and to two cres in the araABDLMNPQ-abfA operon. The contributions of the coeffectors HPr and Crh to CCR differ according to growth phase. HPr dependency occurs during both exponential growth and the transitional phase, while Crh dependency is detected mainly at the transitional phase. Our results suggest that Crh synthesis may increase at the end of exponential growth and consequently contribute to this effect, together with other factors.

The MsmX ATPase plays a crucial role in pectin mobilization by Bacillus subtilis.

Carbohydrates from plant cell walls are often found as heteropolysaccharides intertwined with each other. For competitive advantage against other microorganisms, and ability to fully exploit available carbon and energy sources, Bacillus subtilis possesses a high number of proteins dedicated to the uptake of mono- and oligosaccharides. Here, we characterize transporter complexes, belonging to the ATP-binding cassette (ABC) superfamily, involved in the uptake of oligosaccharides commonly found in pectin. The uptake of these carbohydrates is shown to be MsmX-dependent, assigning a key role in pectin mobilization for MsmX, a multipurpose ATPase serving several distinct ABC-type I sugar importers. Mutagenesis analysis of the transmembrane domains of the AraNPQ MsmX-dependent importer revealed putative residues for MsmX interaction. Interestingly however, although MsmX is shown to be essential for energizing various ABC transporters we found that a second B. subtilis ATPase, YurJ, is able to complement its function when placed in trans at a different locus of the chromosome.

Dissolution enhancement of active pharmaceutical ingredients by therapeutic deep eutectic systems.

A therapeutic deep eutectic system (THEDES) is here defined as a deep eutectic solvent (DES) having an active pharmaceutical ingredient (API) as one of the components. In this work, THEDESs are proposed as enhanced transporters and delivery vehicles for bioactive molecules. THEDESs based on choline chloride (ChCl) or menthol conjugated with three different APIs, namely acetylsalicylic acid (AA), benzoic acid (BA) and phenylacetic acid (PA), were synthesized and characterized for thermal behaviour, structural features, dissolution rate and antibacterial activity. Differential scanning calorimetry and polarized optical microscopy showed that ChCl:PA (1:1), ChCl:AA (1:1), menthol:AA (3:1), menthol:BA (3:1), menthol:PA (2:1) and menthol:PA (3:1) were liquid at room temperature. Dissolution studies in PBS led to increased dissolution rates for the APIs when in the form of THEDES, compared to the API alone. The increase in dissolution rate was particularly noticeable for menthol-based THEDES. Antibacterial activity was assessed using both Gram-positive and Gram-negative model organisms. The results show that all the THEDESs retain the antibacterial activity of the API. Overall, our results highlight the great potential of THEDES as dissolution enhancers in the development of novel and more effective drug delivery systems.

Purification, characterization and functional analysis of an endo-arabinanase (AbnA) from Bacillus subtilis.

Bacillus subtilis synthesizes at least one arabinanase encoded by the abnA gene that is able to degrade the polysaccharide arabinan. Here, we report the expression in Escherichia coli of the full-length abnA coding region with a His6-tag fused to the C-terminus. The recombinant protein was secreted to the periplasmic space and correctly processed by the E. coli signal peptidase. The substrate specificity of purified AbnA, the physico-chemical properties and kinetic parameters were determined. Functional analysis studies revealed Glu 215 as a key residue for AbnA hydrolytic activity and indicated that in addition to AbnA B. subtilis secretes other enzyme(s) able to degrade linear 1,5-alpha-l-arabinan.