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.

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.

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.

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.

Exploring the Interplay of Antimicrobial Properties and Cellular Response in Physically Crosslinked Hyaluronic Acid/ε-Polylysine Hydrogels.

The reduction of tissue cytotoxicity and the improvement of cell viability are of utmost significance, particularly in the realm of green chemistry. Despite substantial progress, the threat of local infections remains a concern. Therefore, hydrogel systems that provide mechanical support and a harmonious balance between antimicrobial efficacy and cell viability are greatly needed. Our study explores the preparation of physically crosslinked, injectable, and antimicrobial hydrogels using biocompatible hyaluronic acid (HA) and antimicrobial ε-polylysine (ε-PL) in different weight ratios (10 wt% to 90 wt%). The crosslinking was achieved by forming a polyelectrolyte complex between HA and ε-PL. The influence of HA content on the resulting HA/ε-PL hydrogel physicochemical, mechanical, morphological, rheological, and antimicrobial properties was evaluated, followed by an inspection of their in vitro cytotoxicity and hemocompatibility. Within the study, injectable, self-healing HA/ε-PL hydrogels were developed. All hydrogels showed antimicrobial properties against S. aureus, P. aeruginosa, E. coli, and C. albicans, where HA/ε-PL 30:70 (wt%) composition reached nearly 100% killing efficiency. The antimicrobial activity was directly proportional to ε-PL content in the HA/ε-PL hydrogels. A decrease in ε-PL content led to a reduction of antimicrobial efficacy against S. aureus and C. albicans. Conversely, this decrease in ε-PL content in HA/ε-PL hydrogels was favourable for Balb/c 3T3 cells, leading to the cell viability of 152.57% for HA/ε-PL 70:30 and 142.67% for HA/ε-PL 80:20. The obtained results provide essential insights into the composition of the appropriate hydrogel systems able to provide not only mechanical support but also the antibacterial effect, which can offer opportunities for developing new, patient-safe, and environmentally friendly biomaterials.

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 containing calcium phosphates: Potential antibacterial agents or fictitious truth.

Amidst an ever-increasing demand for the enhancement of the lifestyle and the modulation of modern diseases, the functionalization of biomaterials is of utmost importance. One of the leading materials for the aforementioned purpose have been calcium phosphates (CaPs). They have been widely used in bone regeneration displaying favourable regenerative potential and biological properties. Many studies have placed their entire focus on facilitating the osteogenic differentiation of stem cells and bone progenitor cells, while the aspect of antibacterial properties has been surmounted. Nevertheless, increasing antibiotic resistance of bacteria requires the development of new materials and the usage of alternative approaches such as ion doping. Gallium (Ga) has been the potential star on the rise among the ions. However, the obstacle that accompanies gallium is the scarcity of research performed and the variety of amalgamations. The question that imposes itself is how a growing field of therapeutics can be further entwined with advances in material science, and how will the incorporation of gallium bring a new outlook. The present study offers a comprehensive overview of state-of-the-art gallium containing calcium phosphates (GaCaPs), their synthesis methods, antibacterial properties, and biocompatibility. Considering their vast potential as antibacterial agents, the need for a methodical perspective is highly necessary to determine if it is a direction on the brink of recognition or a fruitless endeavour. STATEMENT OF SIGNIFICANCE: Although several studies have been published on various metal ions-containing calcium phosphates, to this date there is no systematic overview pointing out the properties and benefits of gallium containing calcium phosphates. Here we offer a critical overview, including synthesis, structure and biological properties of gallium containing calcium phosphates.

Incorporation of Barium Ions into Biomaterials: Dangerous Liaison or Potential Revolution?

In the present manuscript, a brief overview on barium, its possible utilization, and the aftermath of its behavior in organisms has been presented. As a bivalent cation, barium has the potential to be used in a myriad of biochemical reactions. A number of studies have exhibited both the unwanted outcome barium displayed and the advantages of barium laden compounds, tested in in vitro and in vivo settings. The plethora of prospective manipulations covered the area of hydrogels and calcium phosphates, with an end goal of examining barium's future in the tissue engineering. However, majority of data revert to the research conducted in the 20th century, without investigating the mechanisms of action using current state-of-the-art technology. Having this in mind, set of questions that are needed for possible future research arose. Can barium be used as a substitute for other biologically relevant divalent cations? Will the incorporation of barium ions hamper the execution of the essential processes in the organism? Most importantly, can the benefits outweigh the harm?

Octacalcium phosphate: Innovative vehicle for the local biologically active substance delivery in bone regeneration.

Disadvantages of conventional drug delivery systems (DDS), such as systemic circulation, interaction with physiochemical factors, reduced bioavailability, and insufficient drug concentration at bone defect site, have underlined the importance of developing efficacious local drug delivery systems. Octacalcium phosphate (OCP) is presumed to be the precursor of biologically formed apatite, owing to its similarity to hydroxyapatite (HAp) and readiness to convert to it. Specific crystal structure of OCP is constructed of compiled apatite layers and water layers, which make possible the incorporation of various ions in its structure, making it feasible to alter the overall effect OCP has in the system. Next to that intrinsic property, characteristics as high solubility, biodegradability and osteoconductivity have made it indispensable to tailor OCP as a carrier material. In this review, we present the main characteristics and progress done on utilizing OCP as an innovative vehicle and provide suggestions for possible research pathways and advantages for local drug delivery in bone tissue engineering. STATEMENT OF SIGNIFICANCE: Octacalcium phosphate (OCP), being a precursor to biologically formed apatite, has many assets when compared to other calcium phosphates. Owing to its highly pertinent structure, it is being used as a vehicle for biologically active substances or ions for bone regeneration. However, orchestrating drug delivery systems with OCP, in order to achieve the best possible outcome, is still a pioneering concept, and the all-encompassing data is still scarce. Although several articles have been published on this matter, to this date there is no systematic overview pointing out the benefits that OCP can bring in the field of drug delivery. Here we offer a comprehensive overview, starting from the OCP synthesis to its structure, morphology, and the biological significance OCP has.