A Comparative Study on Physicochemical Properties and In Vitro Biocompatibility of Sr-Substituted and Sr Ranelate-Loaded Hydroxyapatite Nanoparticles.

Synthetic hydroxyapatite nanoparticles (nHAp) possess compositional and structural similarities to those of bone minerals and play a key role in bone regenerative medicine. Functionalization of calcium phosphate biomaterials with Sr, i.e., bone extracellular matrix trace element, has been proven to be an effective biomaterial-based strategy for promoting osteogenesis in vitro and in vivo. Functionalizing nHAp with Sr2+ ions or strontium ranelate (SrRAN) can provide favorable bone tissue regeneration by locally delivering bioactive molecules to the bone defect microenvironment. Moreover, administering an antiosteoporotic drug, SrRAN, directly into site-specific bone defects could significantly reduce the necessary drug dosage and the risk of possible side effects. Our study evaluated the impact of the Sr source (Sr2+ ions and SrRAN) used to functionalize nHAp by wet precipitation on its in vitro cellular activities. The systematic comparison of physicochemical properties, in vitro Sr2+ and Ca2+ ion release, and their effect on in vitro cellular activities of the developed Sr-functionalized nHAp was performed. The ion release tests in TRIS-HCl demonstrated a 21-day slow and continuous release of the Sr2+ and Ca2+ ions from both Sr-substituted nHAp and SrRAN-loaded HAp. Also, SrRAN and Sr2+ ion release kinetics were evaluated in DMEM to understand their correlation with in vitro cellular effects in the same time frame. Relatively low concentration (up to 2 wt %) of Sr in the nHAp led to an increase in the alkaline phosphatase activity in preosteoblasts and expression of collagen I and osteocalcin in osteoblasts, demonstrating their ability to boost bone formation.

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.

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 the Biopolymer Carrier on Staphylococcus aureus Bacteriophage Lytic Activity.

The use of implant materials is always associated with the risk of infection. Moreover, the effectiveness of antibiotics is reduced due to antibiotic-resistant pathogens. Thus, selecting the appropriate alternative antimicrobials for local delivery systems is correlated with successful infection management. We evaluated immobilization of the S. aureus specific bacteriophages in clinically recognized biopolymers, i.e., chitosan and alginate, to control the release profile of the antimicrobials. The high-titre S. aureus specific bacteriophages were prepared from commercial bacteriophage cocktails. The polymer mixtures with the propagated bacteriophages were then prepared. The stability of the S. aureus bacteriophages in the biopolymer solutions was assessed. In the case of chitosan, no plaques indicating the presence of the lytic bacteriophages were observed. The titre reduction of the S. aureus bacteriophages in the Na-alginate was below 1 log unit. Furthermore, the bacteriophages retained their lytic activity in the alginate after crosslinking with Ca2+ ions. The release of the lytic S. aureus bacteriophages from the Ca-alginate matrices in the TRIS-HCl buffer solution (pH 7.4 ± 0.2) was determined. After 72 h-0.292 ± 0.021% of bacteriophages from the Ca-alginate matrices were released. Thus, sustained release of the lytic S. aureus bacteriophages can be ensured.

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.

Nanosized calcium deficient hydroxyapatites for tooth enamel protection.

Calcium phosphates (CaP) are extensively studied as additives to dental care products for tooth enamel protection against caries. However, it is not clear yet whether substituted CaP could provide better enamel protection. In this study we produced, characterized and tested in vitro substituted and co-substituted calcium deficient hydroxyapatite (CDHAp) with Sr2+ and F- ions. X-ray powder diffractometry, Fourier transformation infrared spectroscopy, scanning electron microscopy, energy-dispersive X-ray analysis, Brunauer-Emmett-Teller were used to characterize synthesized powders and also cytotoxicity was evaluated. pH = f(t) test was performed to estimate, weather synthesized CDHAp suspensions are able to increase pH of experimental media after acid addition. Synthesis products were incorporated into paste to perform in vitro remineralization on the bovine enamel. In addition to mentioned instrumental methods, profilometry was used for evaluation of remineralised enamel samples. The obtained results confirmed formation of CDHAp substituted with 1.5-1.6 wt% of fluoride and 7.4-7.8 wt% of strontium. pH = f(t) experiment pointed out that pH increased by approximately 0.3 within 10 min after acid addition for all CDHAp suspensions. A new layer of the corresponding CDHAp was formed on the enamel. Its thickness increased by 0.8 ± 0.1 µm per day and reached up to 5.8 µm after 7 days. Additionally, octa calcium phosphates were detected on the surface of control samples. In conclusion, we can assume that CDHAp substituted with Sr2+ and/or F- could be used as an effective additive to dental care products promoting formation of protecting layer on the enamel, but there was no significant difference among sample groups.

Development of local strontium ranelate delivery systems and long term in vitro drug release studies in osteogenic medium.

It has been recognized that the operative stabilization of osteoporotic fractures should be followed up with an appropriate osteoporosis treatment in order to decrease the risk of repeated fractures. Despite the good clinical results of strontium ranelate (SrRan) towards the osteoporosis treatment, high drug doses and long treatment period cause an increased risk of serious side effects. Novel local SrRan/poly(lactic acid) (SrRan/PLA) delivery systems containing from 3.57 ± 0.28 wt% to 24.39 ± 0.91 wt% of active substance were developed. In order to resemble the naturally occurring processes, osteogenic media (OM) was used as a release medium for long term (121 days) in vitro drug release studies and UV/VIS method for the determination of SrRan content in OM was developed and validated. Biomimetic calcium phosphate precipitates were found on the surface and in the pores of prepared delivery system after microcapsule exposure to OM for 121 days as well as SrRan particles, indicating that the release of the drug have not been completed within 121 days. In vitro cell viability evaluation approved no cytotoxic effects of microcapsule suspensions and extracts.