Chemically Pretreated Densification of Juniper Wood for Potential Use in Osteosynthesis Bone Implants.

The aim of the study was to perform treatment of juniper wood to obtain wood material with a density and mechanical properties comparable to bone, thus producing a potential material for use in osteosynthesis bone implants. In the first step, partial delignification of wood sample was obtained by Kraft cooking. The second step was extraction with ethanol, ethanol-water mixture, saline, and water to prevent the release of soluble compounds and increase biocompatibility. In the last step, the thermal densification at 100 °C for 24 h was implemented. The results obtained in the dry state are equivalent to the properties of bone. The swelling of chemically pre-treated densified wood was reduced compared to chemically untreated densified wood. Samples showed no cytotoxicity by in vitro cell assays. The results of the study showed that it is possible to obtain noncytotoxic wood samples with mechanical properties equivalent to bones by partial delignification, extraction, and densification. However, further research is needed to ensure the material's shape stability, water resistance, and reduced swelling.

Improved visualisation of ACP-engineered osteoblastic spheroids: a comparative study of contrast-enhanced micro-CT and traditional imaging techniques.

This study investigates osteoblastic cell spheroid cultivation methods, exploring flat-bottom, U-bottom, and rotary flask techniques with and without amorphous calcium phosphate (ACP) supplementation to replicate the 3D bone tissue microenvironment. ACP particles derived from eggshell waste exhibit enhanced osteogenic activity in 3D models. However, representative imaging of intricate 3D tissue-engineered constructs poses challenges in conventional imaging techniques due to notable scattering and absorption effects in light microscopy, and hence limited penetration depth. We investigated contrast-enhanced micro-CT as a methodological approach for comprehensive morphological 3D-analysis of thein-vitromodel and compared the technique with confocal laser scanning microscopy, scanning electron microscopy and classical histology. Phosphotungstic acid and iodine-based contrast agents were employed for micro-CT imaging in laboratory and synchrotron micro-CT imaging. Results revealed spheroid shape variations and structural integrity influenced by cultivation methods and ACP particles. The study underscores the advantage of 3D spheroid models over traditional 2D cultures in mimicking bone tissue architecture and cellular interactions, emphasising the growing demand for novel imaging techniques to visualise 3D tissue-engineered models. Contrast-enhanced micro-CT emerges as a promising non-invasive imaging method for tissue-engineered constructs containing ACP particles, offering insights into sample morphology, enabling virtual histology before further analysis.

Development of nanocomposite hydrogel using citrate-containing amorphous calcium phosphate and gelatin methacrylate.

Nanocomposite hydrogels are suitable in bone tissue engineering due to their resemblance with the extracellular matrix, ability to match complex geometries, and ability to provide a framework for cell attachment and proliferation. The nanocomposite hydrogel comprises organic and inorganic counterparts. Gelatin methacrylate (GELMA) is an extensively used organic biomaterial in tissue engineering due to its excellent biocompatibility, biodegradability, and bioactivity. The photo-crosslinking of GELMA presents a challenge when aiming to create thicker nanocomposite hydrogels due to opacity induced by fillers, which obstructs the penetration of ultraviolet (UV) light. Therefore, using a chemical crosslinking approach, we have developed nanocomposite GELMA hydrogel in this study by incorporating citrate-containing amorphous calcium phosphate (ACP_CIT). Ammonium persulfate (APS) and Tetramethylethylenediamine (TEMED) were deployed to crosslink the methacrylate group of GELMA. The oscillatory shear tests have confirmed that crosslinking enhances both storage (G') and loss modulus (G″) of GELMA. Subsequently, incorporation of ACP_CIT in GELMA hydrogel shows further enhancement in G' and G″ values. In vitro analysis of the developed hydrogels revealed that chemical crosslinking and incorporation of ACP_CIT do not compromise the cytocompatibility of the GELMA. Hence, for developing nanocomposite GELMA hydrogels employing APS/TEMED crosslinking emerges as a promising alternative to photo-crosslinking.

Ultrafast and Reproducible Synthesis of Tailor-Made Octacalcium Phosphate.

Octacalcium phosphate (OCP) has excellent bone formation ability and a good resorption rate as compared to the commercial bone substitutes [e.g., Bio-Oss (Geistlich Pharma AG) and MBCP+ (Biomatlante)], as well as synthesized biomaterials (hydroxyapatite and tricalcium phosphate). The synthesis approach to obtain phase-pure OCP possesses a great challenge due to its complex reaction mechanism and narrow synthesis window. Thus, the current study aimed to overcome the synthesis challenges and to define the precise reaction conditions required for controllable and reproducible synthesis of OCP. Using the principles of the coprecipitation method, novel synthesis protocols were developed ensuring ultrafast synthesis of OCP within minutes. XRD analysis confirmed that phase-pure OCP was obtained. These processing schemes enabled the synthesis of tailor-made OCP with specific surface areas ranging from 16 to 91 m2/g. The synthesized OCPs exhibited plate-like morphology. The interaction of the synthesized OCPs with MC3T3-E1 cells was found to be nontoxic, confirming their cytocompatibility. The synthesis approach developed in this study indicates that challenges such as the reaction volume, stirring rate, and flow rate need to be addressed in the future to upscale the technology.

Calcium Phosphates: A Key to Next-Generation In Vitro Bone Modeling.

The replication of bone physiology under laboratory conditions is a prime target behind the development of in vitro bone models. The model should be robust enough to elicit an unbiased response when stimulated experimentally, giving reproducible outcomes. In vitro bone tissue generation majorly requires the availability of cellular components, the presence of factors promoting cellular proliferation and differentiation, efficient nutrient supply, and a supporting matrix for the cells to anchor - gaining predefined topology. Calcium phosphates (CaP) are difficult to ignore while considering the above requirements of a bone model. Therefore, the current review focuses on the role of CaP in developing an in vitro bone model addressing the prerequisites of bone tissue generation. Special emphasis is given to the physico-chemical properties of CaP that promote osteogenesis, angiogenesis and provide sufficient mechanical strength for load-bearing applications. Finally, the future course of action is discussed to ensure efficient utilization of CaP in the in vitro bone model development field.

Challenging applicability of ISO 10993-5 for calcium phosphate biomaterials evaluation: Towards more accurate in vitro cytotoxicity assessment.

Research on biomaterials typically starts with cytocompatibility evaluation, using the ISO 10993-5 standard as a reference that relies on extract tests to determine whether the material is safe (cell metabolic activity should exceed 70 %). However, the generalized approach within the standard may not accurately reflect the material's behavior in direct contact with cells, raising concerns about its effectiveness. Calcium phosphates (CaPs) are a group of materials that, despite being highly biocompatible and promoting bone formation, still exhibit inconsistencies in basic cytotoxicity evaluations. Hence, in order to test the cytocompatibility dependence on different experimental setups and material-cell interactions, we used amorphous calcium phosphate, α-tricalcium phosphate, hydroxyapatite, and octacalcium phosphate (0.1 mg/mL to 5 mg/mL) with core cell lines of bone microenvironment: mesenchymal stem cells, osteoblast-like and endothelial cells. All materials have been characterized for their physicochemical properties before and after cellular contact and once in vitro assays were finalized, groups identified as 'cytotoxic' were further analyzed using a modified Annexin V apoptosis assay to accurately determine cell death. The obtained results showed that indirect contact following ISO standards had no sensitivity of tested cells to the materials, but direct contact tests at physiological concentrations revealed decreased metabolic activity and viability. In summary, our findings offer valuable guidelines for handling biomaterials, especially in powder form, to better evaluate their biological properties and avoid false negatives commonly associated with the traditional standard approach.

Apatite/Chitosan Composites Formed by Cold Sintering for Drug Delivery and Bone Tissue Engineering Applications.

In the biomedical field, nanocrystalline hydroxyapatite is still one of the most attractive candidates as a bone substitute material due to its analogies with native bone mineral features regarding chemical composition, bioactivity and osteoconductivity. Ion substitution and low crystallinity are also fundamental characteristics of bone apatite, making it metastable, bioresorbable and reactive. In the present work, biomimetic apatite and apatite/chitosan composites were produced by dissolution-precipitation synthesis, using mussel shells as a calcium biogenic source. With an eye on possible bone reconstruction and drug delivery applications, apatite/chitosan composites were loaded with strontium ranelate, an antiosteoporotic drug. Due to the metastability and temperature sensitivity of the produced composites, sintering could be carried out by conventional methods, and therefore, cold sintering was selected for the densification of the materials. The composites were consolidated up to ~90% relative density by applying a uniaxial pressure up to 1.5 GPa at room temperature for 10 min. Both the synthesised powders and cold-sintered samples were characterised from a physical and chemical point of view to demonstrate the effective production of biomimetic apatite/chitosan composites from mussel shells and exclude possible structural changes after sintering. Preliminary in vitro tests were also performed, which revealed a sustained release of strontium ranelate for about 19 days and no cytotoxicity towards human osteoblastic-like cells (MG63) exposed up to 72 h to the drug-containing composite extract.

Doxorubicin loaded octacalcium phosphate particles as controlled release drug delivery systems: Physico-chemical characterization, in vitro drug release and evaluation of cell death pathway.

Mastering new and efficient ways to obtain successful drug delivery systems (DDS) with controlled release became a paramount quest in the scientific community. Increase of malignant bone tumors and the necessity to optimize an approach of localized drug delivery require research to be even more intensified. Octacalcium phosphate (OCP), with a number of advantages over current counterparts is extensively used in bone engineering. The aim of the present research was to synthesize bioactive and biocompatible doxorubicin (DOX) containing OCP particles. DOX-OCP was successfully obtained in situ in an exhaustive range of added drug (1-20 wt%, theoretical loading). Based on XRD, above 10 wt% of DOX, OCP formation was inhibited and the obtained product was low crystalline α-TCP. In-vitro drug release was performed in pH 7.4 and 6.0. In both pH environments DOX had a continuous release over six weeks. However, the initial drug burst for pH 7.4, in the first 24 h, ranged from 15.9 ± 1.3 % to 33.5 ± 12 % and for pH 6.0 23.7 ± 1.5 % to 36.2 ± 12 %.The DOX-OCP exhibited an inhibitory effect on viability of osteosarcoma cell lines MG63, U2OS and HOS. In contrast, MC3T3-E1 cells (IC50 > 0.062 µM) displayed increased viability and proliferation from 3rd to 7th day. Testing of the DDS on ferroptotic markers (CHAC1, ACSL4 and PTGS2) showed that OCP-DOX does not induce ferroptotic cell death. Moreover, the evaluation of protein levels of cleaved PARP, by western blotting analysis, corroborated that apoptosis is the main pathway of programmed cell death in osteosarcoma cells induced by DOX-OCP.

Incorporating strontium enriched amorphous calcium phosphate granules in collagen/collagen-magnesium-hydroxyapatite osteochondral scaffolds improves subchondral bone repair.

Osteochondral defect repair with a collagen/collagen-magnesium-hydroxyapatite (Col/Col-Mg-HAp) scaffold has demonstrated good clinical results. However, subchondral bone repair remained suboptimal, potentially leading to damage to the regenerated overlying neocartilage. This study aimed to improve the bone repair potential of this scaffold by incorporating newly developed strontium (Sr) ion enriched amorphous calcium phosphate (Sr-ACP) granules (100-150 µm). Sr concentration of Sr-ACP was determined with ICP-MS at 2.49 ± 0.04 wt%. Then 30 wt% ACP or Sr-ACP granules were integrated into the scaffold prototypes. The ACP or Sr-ACP granules were well embedded and distributed in the collagen matrix demonstrated by micro-CT and scanning electron microscopy/energy dispersive x-ray spectrometry. Good cytocompatibility of ACP/Sr-ACP granules and ACP/Sr-ACP enriched scaffolds was confirmed with in vitro cytotoxicity assays. An overall promising early tissue response and good biocompatibility of ACP and Sr-ACP enriched scaffolds were demonstrated in a subcutaneous mouse model. In a goat osteochondral defect model, significantly more bone was observed at 6 months with the treatment of Sr-ACP enriched scaffolds compared to scaffold-only, in particular in the weight-bearing femoral condyle subchondral bone defect. Overall, the incorporation of osteogenic Sr-ACP granules in Col/Col-Mg-HAp scaffolds showed to be a feasible and promising strategy to improve subchondral bone repair.

Fusion and classification algorithm of octacalcium phosphate production based on XRD and FTIR data.

The present manuscript tested an automated analysis sequence to provide a decision support system to track the OCP synthesis from [Formula: see text]-TCP over time. Initially, the XRD and FTIR signals from a hundredfold scaled-up hydrolysis of OCP from [Formula: see text]-TCP were fused and modeled by the curve fitting based on the significantly established maxima from the literature and nine features extracted from the fitted shapes. Afterward, the analysis sequence enclosed the machine learning techniques for feature ranking, spatial filtering, and dimensionality reduction to support the automatic recognition of the synthesis stages. The proposed analysis pipeline for OCP identification might be the foundation for a decision support system explicitly targeting OCP synthesis. Future projects will exploit the suggested methodology for pinpointing the OCP production over time (including the intermediary phases present in the OCP formation) and for evaluating whether biological variables might be merged with biomaterial properties to build a unified model of tissue response to the implant.

Cell-Laden 3D Printed GelMA/HAp and THA Hydrogel Bioinks: Development of Osteochondral Tissue-like Bioinks.

Osteochondral (OC) disorders such as osteoarthritis (OA) damage joint cartilage and subchondral bone tissue. To understand the disease, facilitate drug screening, and advance therapeutic development, in vitro models of OC tissue are essential. This study aims to create a bioprinted OC miniature construct that replicates the cartilage and bone compartments. For this purpose, two hydrogels were selected: one composed of gelatin methacrylate (GelMA) blended with nanosized hydroxyapatite (nHAp) and the other consisting of tyramine-modified hyaluronic acid (THA) to mimic bone and cartilage tissue, respectively. We characterized these hydrogels using rheological testing and assessed their cytotoxicity with live-dead assays. Subsequently, human osteoblasts (hOBs) were encapsulated in GelMA-nHAp, while micropellet chondrocytes were incorporated into THA hydrogels for bioprinting the osteochondral construct. After one week of culture, successful OC tissue generation was confirmed through RT-PCR and histology. Notably, GelMA/nHAp hydrogels exhibited a significantly higher storage modulus (G') compared to GelMA alone. Rheological temperature sweeps and printing tests determined an optimal printing temperature of 20 °C, which remained unaffected by the addition of nHAp. Cell encapsulation did not alter the storage modulus, as demonstrated by amplitude sweep tests, in either GelMA/nHAp or THA hydrogels. Cell viability assays using Ca-AM and EthD-1 staining revealed high cell viability in both GelMA/nHAp and THA hydrogels. Furthermore, RT-PCR and histological analysis confirmed the maintenance of osteogenic and chondrogenic properties in GelMA/nHAp and THA hydrogels, respectively. In conclusion, we have developed GelMA-nHAp and THA hydrogels to simulate bone and cartilage components, optimized 3D printing parameters, and ensured cell viability for bioprinting OC constructs.

Calcium phosphates enhanced with liposomes - the future of bone regeneration and drug delivery.

Effective healing and regeneration of various bone defects is still a major challenge and concern in modern medicine. Calcium phosphates have emerged as extensively studied bone substitute materials due to their structural and chemical resemblance to the mineral phase of bone, along with their versatile properties. Calcium phosphates present promising biological characteristics that make them suitable for bone substitution, but a critical limitation lies in their low osteoinductivity. To supplement these materials with properties that promote bone regeneration, prevent infections, and cure bone diseases locally, calcium phosphates can be biologically and therapeutically modified. A promising approach involves combining calcium phosphates with drug-containing liposomes, renowned for their high biocompatibility and ability to provide controlled and sustained drug delivery. Surprisingly, there is a lack of research focused on liposome-calcium phosphate composites, where liposomes are dispersed within a calcium phosphate matrix. This raises the question of why such studies are limited. In order to provide a comprehensive overview of existing liposome and calcium phosphate composites as bioactive substance delivery systems, the authors review the literature exploring the interactions between calcium phosphates and liposomes. Additionally, it seeks to identify potential interactions between calcium ions and liposomes, which may impact the feasibility of developing liposome-containing calcium phosphate composite materials. Liposome capacity to protect bioactive compounds and facilitate localized treatment can be particularly valuable in scenarios involving bone regeneration, infection prevention, and the management of bone diseases. This review explores the implications of liposomes and calcium phosphate material containing liposomes on drug delivery, bioavailability, and stability, offering insights into their advantages.

Human Osteoblasts' Response to Biomaterials for Subchondral Bone Regeneration in Standard and Aggressive Environments.

Osteochondral lesions, when not properly treated, may evolve into osteoarthritis (OA), especially in the elderly population, where altered joint function and quality are usual. To date, a collagen/collagen-magnesium-hydroxyapatite (Col/Col-Mg-HAp) scaffold (OC) has demonstrated good clinical results, although suboptimal subchondral bone regeneration still limits its efficacy. This study was aimed at evaluating the in vitro osteogenic potential of this scaffold, functionalized with two different strategies: the addition of Bone Morphogenetic Protein-2 (BMP-2) and the incorporation of strontium (Sr)-ion-enriched amorphous calcium phosphate (Sr-ACP) granules. Human osteoblasts were seeded on the functionalized scaffolds (OC+BMP-2 and OC+Sr-ACP, compared to OC) under stress conditions reproduced with the addition of H2O2 to the culture system, as well as in normal conditions, and evaluated in terms of morphology, metabolic activity, gene expression, and matrix synthesis. The OC+BMP-2 scaffold supported a better osteoblast morphology and stimulated scaffold colonization, cell activity, and extracellular matrix secretion, especially in the stressed culture environment but also in normal culture conditions, with increased expression of genes related to osteoblast differentiation. In conclusion, the incorporation of BMP-2 into the Col/Col-Mg-HAp scaffold also represents an improvement of the osteochondral scaffold in more challenging conditions, supporting further preclinical studies to optimize it for use in clinical practice.

Octacalcium Phosphate-Laden Hydrogels on 3D-Printed Titanium Biomaterials Improve Corrosion Resistance in Simulated Biological Media.

The inflammatory-associated corrosion of metallic dental and orthopedic implants causes significant complications, which may result in the implant's failure. The corrosion resistance can be improved with coatings and surface treatments, but at the same time, it might affect the ability of metallic implants to undergo proper osteointegration. In this work, alginate hydrogels with and without octacalcium phosphate (OCP) were made on 3D-printed (patterned) titanium alloys (Ti Group 2 and Ti-Al-V Group 23) to enhance their anticorrosion properties in simulated normal, inflammatory, and severe inflammatory conditions in vitro. Alginate (Alg) and OCP-laden alginate (Alg/OCP) hydrogels were manufactured on the surface of 3D-printed Ti substrates and were characterized with wettability analysis, XRD, and FTIR. The electrochemical characterization of the samples was carried out with open circuit potential, potentiodynamic polarization, and electrochemical impedance spectroscopy (EIS). It was observed that the hydrophilicity of Alg/OCP coatings was higher than that of pure Alg and that OCP phase crystallinity was increased when samples were subjected to simulated biological media. The corrosion resistance of uncoated and coated samples was lower in inflammatory and severe inflammatory environments vs. normal media, but the hydrogel coatings on 3D-printed Ti layers moved the corrosion potential towards more nobler values, reducing the corrosion current density in all simulated solutions. These measurements revealed that OCP particles in the Alg hydrogel matrix noticeably increased the electrical charge transfer resistance at the substrate and coating interface more than with Alg hydrogel alone.

Amorphous Calcium Phosphate and Amorphous Calcium Phosphate Carboxylate: Synthesis and Characterization.

Amorphous calcium phosphate (ACP) is the first solid phase precipitated from a supersaturated calcium phosphate solution. Naturally, ACP is formed during the initial stages of biomineralization and stabilized by an organic compound. Carboxylic groups containing organic compounds are known to regulate the nucleation and crystallization of hydroxyapatite. Therefore, from a biomimetic point of view, the synthesis of carboxylate ions containing ACP (ACPC) is valuable. Usually, ACP is synthesized with fewer steps than ACPC. The precipitation reaction of ACP is rapid and influenced by pH, temperature, precursor concentration, stirring conditions, and reaction time. Due to phosphates triprotic nature, controlling pH in a multistep approach becomes tedious. Here, we developed a new ACP and ACPC synthesis approach and thoroughly characterized the obtained materials. Results from vibration spectroscopy, nuclear magnetic resonance (NMR), X-ray photoelectron spectroscopy (XPS), true density, specific surface area, and ion release studies have shown a difference in the physiochemical properties of the ACP and ACPC. Additionally, the effect of a carboxylic ion type on the physiochemical properties of ACPC was characterized. All of the ACPs and ACPCs were synthesized in sterile conditions, and in vitro analysis was performed using MC-3T3E1 cells, revealing the cytocompatibility of the synthesized ACPs and ACPCs, of which the ACPC synthesized with citrate showed the highest cell viability.

Activated Carbon and Clay Pellets Coated with Hydroxyapatite for Heavy Metal Removal: Characterization, Adsorption, and Regeneration.

In the present work, activated-carbon-containing pellets were preparedby direct chemical activation of sawdust, using clays as a binder. The obtained pellets (ACC) were coated with hydroxyapatite (HAp) nanoparticles (ACC-HAp) to improve adsorption towards Pb(II), Cu(II), Zn(II), and Ni(II). The pellets were characterized by scanning electron microscopy (SEM), by Fourier transform infrared spectroscopy (FTIR), and with a gas sorptometer. The effect of pH, contact time, and initial concentration on adsorption performance was investigated. Additionally, desorption studies were performed, and the regeneration influence on compressive strength and repeated Pb(II) adsorption was investigated. The results showed that, after coating ACC pellets with HAp nanoparticles, the adsorption capacity increased for all applied heavy metal ions. Pb(II) was adsorbed the most, and the best results were achieved at pH 6. The adsorption process followed the pseudo-second-order kinetic model. The adsorption isotherm of Pb(II) is better fitted to the Langmuir model, showing the maximum adsorption capacity of 56 and 47 mg/g by ACC-HAp and ACC pellets, respectively. The desorption efficiency of Pb(II)-loaded ACC-HAp pellets increased by lowering the pH of the acid, resulting in the dissolution of the HAp coating. The best desorption results were achieved with HCl at pH 1 and 1.5. Therefore, the regeneration procedure consisted of desorption, rinsing with distilled water, and re-coating with HAp nanoparticles. After the regeneration process, the Pb(II) adsorption was not affected. However, the desorption stage within the regeneration process decreased the compressive strength of the pellets.

Role of carboxylic organic molecules in interfibrillar collagen mineralization.

Bone is a composite material made up of inorganic and organic counterparts. Most of the inorganic counterpart accounts for calcium phosphate (CaP) whereas the major organic part is composed of collagen. The interfibrillar mineralization of collagen is an important step in the biomineralization of bone and tooth. Studies have shown that synthetic CaP undergoes auto-transformation to apatite nanocrystals before entering the gap zone of collagen. Also, the synthetic amorphous calcium phosphate/collagen combination alone is not capable of initiating apatite nucleation rapidly. Therefore, it was understood that there is the presence of a nucleation catalyst obstructing the auto-transformation of CaP before entering the collagen gap zone and initiating rapid nucleation after entering the collagen gap zone. Therefore, studies were focused on finding the nucleation catalyst responsible for the regulation of interfibrillar collagen mineralization. Organic macromolecules and low-molecular-weight carboxylic compounds are predominantly present in the bone and tooth. These organic compounds can interact with both apatite and collagen. Adsorption of the organic compounds on the apatite nanocrystal governs the nucleation, crystal growth, lattice orientation, particle size, and distribution. Additionally, they prevent the auto-transformation of CaP into apatite before entering the interfibrillar compartment of the collagen fibril. Therefore, many carboxylic organic compounds have been utilized in developing CaP. In this review, we have covered different carboxylate organic compounds governing collagen interfibrillar mineralization.

Exploring the Formation Kinetics of Octacalcium Phosphate from Alpha-Tricalcium Phosphate: Synthesis Scale-Up, Determination of Transient Phases, Their Morphology and Biocompatibility.

Even with decades of research studies behind octacalcium phosphate (OCP), determination of OCP phase formation has proved to be a cumbersome challenge. Even though obtaining a large quantity of OCP is important for potential clinical uses, it still remains a hindrance to obtain high yields of pure OCP. Taking that into consideration, the purpose of this study was to scale-up OCP synthesis for the first time and to use a multi-technique approach to follow the phase transformation pathway at multiple time points. In the present study, OCP has been synthesized from α-tricalcium phosphate (α-TCP), and subsequently scaled-up tenfold and hundredfold (100 mg → 10 g). The hydrolysis mechanism has been followed and described by using XRD and FTIR spectroscopy, as well as Raman and SEM. Gradual transformation into the OCP phase transpired through dicalcium phosphate dihydrate (brushite, DCPD, up to ~36%) as an intermediary phase. Furthermore, the obtained transitional phases and final OCP phases (across all scale-up levels) were tested with human bone marrow-derived mesenchymal stem cells (hBMSCs), in order to see how different phase mixtures affect the cell viability, and also to corroborate the safety of the scaled-up product. Twelve out of seventeen specimens showed satisfactory percentages of cell viability and confirmed the prospective use of scaled-up OCP in further in vitro studies. The present study, therefore, provides the first scale-up process of OCP synthesis, an in depth understanding of the formation pathway, and investigation of the parameters able to contribute in the OCP phase formation.

Gallium-Doped Hydroxyapatite Shows Antibacterial Activity against Pseudomonas aeruginosa without Affecting Cell Metabolic Activity.

Calcium phosphates (CaPs) have been used in bone regeneration for decades. Among the described CaPs, synthetic hydroxyapatite (HAp) has a chemical composition similar to that of natural bone. Gallium-containing compounds have been studied since the 1970s for the treatment of autoimmune diseases and have shown beneficial properties, such as antibacterial activity and inhibition of osteoclast activity. In this study, we synthesized hydroxyapatite (HAp) powder with Ga doping ratios up to 6.9 ± 0.5 wt% using the wet chemical precipitation method. The obtained products were characterized using XRD, BET, FTIR, and ICP-MS. Ga3+ ion release was determined in the cell culture media for up to 30 days. Antibacterial activity was assessed against five bacterial species: Pseudomonas aeruginosa, Escherichia coli, Staphylococcus aureus, Staphylococcus epidermidis, and Streptococcus pyogenes. The biocompatibility of the GaHAp samples was determined in human fibroblasts (hTERT-BJ1) through direct and indirect tests. The structure of the synthesized products was characteristic of HAp, as revealed with XRD and FTIR, although the addition of Ga caused a decrease in the crystallite size. Ga3+ was released from GaHAp paste in a steady manner, with approximately 40% being released within 21 days. GaHAp with the highest gallium contents, 5.5 ± 0.1 wt% and 6.9 ± 0.5 wt%, inhibited the growth of all five bacterial species, with the greatest activity being against Pseudomonas aeruginosa. Biocompatibility assays showed maintained cell viability (~80%) after seven days of indirect exposure to GaHAp. However, when GaHAp with Ga content above 3.3 ± 0.4 wt% was directly applied on the cells, a decrease in metabolic activity was observed on the seventh day. Overall, these results show that GaHAp with Ga content below 3.3 ± 0.4 wt% has attractive antimicrobial properties, without affecting the cell metabolic activity, creating a material that could be used for bone regeneration and prevention of infection.

Effect of Temporary Cement, Surface Pretreatment and Tooth Area on the Bond Strength of Adhesively Cemented Ceramic Overlays-An In Vitro Study.

Several viewpoints have been reported regarding the effect of temporary cements, different surface pretreatment protocols before adhesive cementation, and predictive factors. This in vitro study tested if temporary cement, pretreatment of the tooth surface, the size of enamel or dentine influence adhesive cementation to zirconia ceramics. Twenty premolars were prepared for determination of enamel and dentin area, bond strength test and failure analysis. The samples were divided into two groups: untreated prior adhesive cementation (n = 10) and with temporary cementation done, pretreated prior adhesive cementation (n = 10). Zirconia overlays (Katana Zirconia STML) were cemented on the grounded flat teeth surfaces using Panavia V5. An additional six premolars underwent dentine tubule analysis with SEM to detect temporary cement residues after temporary cementation on an untreated tooth surface (n = 3) and on a pretreated surface (n = 3). The independent sample t-test was used to compare the two groups and the means of the total tooth, dentin or enamel areas did not differ significantly between the untreated and pretreated specimens. The mean tensile bond strength was significantly (p = 0.005) higher in the pretreated specimens (337N) than in the untreated ones (204N). The overall multivariable linear regression model with three predictors (surface pre-treatment, enamel area and dentine area) was significant (p = 0.003), among which the size of enamel was the strongest predictor (ß = 0.506; p = 0.049), followed by the pretreatment effect (ß = 0.478; p = 0.001) and the size of dentin area (ß = -0.105; p = 0.022).