Crystallographic plane-induced selective mineralization of nanohydroxyapatite on fibrous-grained titanium promotes osteointegration and biocorrosion resistance.

The (002) crystallographic plane-oriented hydroxyapatite (HA) and anatase TiO2 enable favorable hydrophilicity, osteogenesis, and biocorrosion resistance. Thus, the crystallographic plane control in HA coating and crystalline phase control in TiO2 is vital to affect the surface and interface bioactivity and biocorrosion resistance of titanium (Ti) implants. However, a corresponding facile and efficient fabrication method is absent to realize the HA(002) mineralization and anatase TiO2 formation on Ti. Herein, we utilized the predominant Ti(0002) plane of the fibrous-grained titanium (FG Ti) to naturally form anatase TiO2 and further achieve a (002) basal plane oriented nanoHA (nHA) film through an in situ mild hydrothermal growth strategy. The formed FG Ti-nHA(002) remarkably improved hydrophilicity, mineralization, and biocorrosion resistance. Moreover, the nHA(002) film reserved the microgroove-like topological structure on FG Ti. It could enhance osteogenic differentiation through promoted contact guidance, showing one order of magnitude higher expression of osteogenic-related genes. On the other hand, the nHA(002) film restrained the osteoclast activity by blocking actin ring formation. Based on these capacities, FG Ti-nHA(002) improved new bone growth and binding strength in rabbit femur implantation, achieving satisfactory osseointegration within 2 weeks.

Antibiotic-eluting scaffolds with responsive dual-release kinetics facilitate bone healing and eliminate S. aureus infection.

Osteomyelitis (OM) is a progressive, inflammatory infection of bone caused predominately by Staphylococcus aureus. Herein, we engineered an antibiotic-eluting collagen-hydroxyapatite scaffold capable of eliminating infection and facilitating bone healing. An iterative freeze-drying and chemical crosslinking approach was leveraged to modify antibiotic release kinetics, resulting in a layered dual-release system whereby an initial rapid release of antibiotic to clear infection was followed by a sustained controlled release to prevent reoccurrence of infection. We observed that the presence of microbial collagenase accelerated antibiotic release from the crosslinked layer of the scaffold, indicating that the material is responsive to microbial activity. As exemplar drugs, vancomycin and gentamicin-eluting scaffolds were demonstrated to be bactericidal, and supported osteogenesis in vitro. In a pilot murine model of OM, vancomycin-eluting scaffolds were observed to reduce S. aureus infection within the tibia. Finally, in a rabbit model of chronic OM, gentamicin-eluting scaffolds both facilitated radial bone defect healing and eliminated S. aureus infection. These results show that antibiotic-eluting collagen-hydroxyapatite scaffolds are a one-stage therapy for OM, which when implanted into infected bone defects simultaneously eradicate infection and facilitate bone tissue healing.

Preparation and characterization of mesoporous HA coating with paclitaxel loaded lignin nanospheres on titanium surface.

Background: Primary malignant bone tumor is a disease that can lead to death. The usually applied clinical treatment strategy is surgical resection of the primary tumor. However, tumor cells are difficult to clean up, easy to make the tumor recurrence, and the bone defect caused by surgical resection also hindered the postoperative recovery. Materials and methods: Herein, in this work, mesoporous hydroxyapatite (HA) coating with petal-structure was prepared on titanium (Ti) implant surfaces by micro-arc oxidation (MAO) to accelerate the bone growth, and then paclitaxel (PTX) loaded lignin nanospheres were deposited into the HA coatings to get a sustained release for killing residual tumor cells. Results: The results showed that many gaps and holes of micro-scale were formed in the petal-structured HA coatings, they worked as traps for the PTX loaded nanospheres to enhance the deposited amount and immobilization stability, playing good role of drug loading platform. The encapsulation of PTX by lignin ensured a lower release rate and a higher sustaining release time when compared with the PTX without encapsulation. In addition, the HA coating with PTX loaded lignin nanospheres showed higher killing effect to tumor cells than to osteoblast. Conclusion: The mesoporous HA coating with paclitaxel loaded lignin nanospheres endowed the titanium surface with good biological property and tumor cell-killing effect, so the obtained Ti-based material had a highly hopeful application as the localized implant for therapy of primary malignant bone tumor.

Analyzing the Bioactivity of a Novel Bone Cement: An In Vitro Study.

AIM: To analyze the effect of bioactive bone cement (BBC) placed in a phosphate buffer saline solution in comparison to mineral trioxide aggregate (MTA). METHODOLOGY: Ten samples each of BBC (group 1) and MTA (group 2) were prepared and stored in a phosphate buffer saline solution. After three days of storage, white precipitates were formed on the surface of the samples. The solution with precipitates from each sample was analyzed for the presence of calcium and phosphate ions with coupled plasma atomic spectroscopy. RESULTS: BBC showed a significant amount of calcium and phosphate release after a seven-day storage period in phosphate buffer saline solution. Calcium release was significantly higher in group 1 (MTA) (p < 0.001) compared to that in group 2 (BBC), while group 2 (BBC) (p < 0.001) exhibited greater phosphate release compared to group 1 (MTA). CONCLUSION: BBC (group 2) retains its bioactivity when it comes into contact with a stimulated oral environment (STF). This demonstrates that BBC is bioactive in a simulated oral environment. Moreover, it retained good handling properties and could be easily manipulated into a dough form. Clinically, in cases of apical surgery, internal resorption or perforation repair where material placement poses difficulty, BBC will prove to be beneficial.

Abalone shell-derived Mg-doped mesoporous hydroxyapatite microsphere drug delivery system loaded with icariin for inducing apoptosis of osteosarcoma cells.

Hydroxyapatite (HAP) porous microspheres with very high specific surface area and drug loading capacity, as well as excellent biocompatibility, have been widely used in tumour therapy. Mg2+ is considered to be a key factor in bone regeneration, acting as an active agent to stimulate bone and cartilage formation, and is effective in accelerating cell migration and promoting angiogenesis, which is essential for bone tissue repair, anti-cancer, and anti-infection. In this study, abalone shells from a variety of sources were used as raw materials, and Mg2+-doped abalone shell-derived mesoporous HAP microspheres (Mg-HAP) were prepared by hydrothermal synthesis as Mg2+/ icariin smart dual delivery system (ICA-Mg-HAP, IMHA). With increasing of Mg2+ doping, the surface morphology of HAP microspheres varied from collapsed macroporous to mesoporous to smooth and non-porous, which may be due to Mg2+ substitution or coordination in the HAP lattice. At 30% Mg2+ doping, the Mg-HAP microspheres showed a more homogeneous mesoporous morphology with a high specific surface area (186.06 m2/g). The IMHA microspheres showed high drug loading (7.69%) and encapsulation rate (83.29%), sustained Mg2+ release for more than 27 days, sustained and stable release of icariin for 60 hours, and good responsiveness to pH (pH 6.4 > pH 5.6). In addition, the IMHA delivery system stimulated the rapid proliferation of bone marrow mesenchymal stem cells and induced apoptosis in MG63 cells by blocking the G2 phase cycle of osteosarcoma cells and stimulating the high expression of apoptotic genes (Bcl-2, caspase-3, -8, -9). This suggests that the abalone shell-based IMHA may have potential applications in drug delivery and tumour therapy.

Energy-dispersive Laue diffraction analysis of the influence of statherin and histatin on the crystallographic texture during human dental enamel demineralization.

Energy-dispersive Laue diffraction (EDLD) is a powerful method to obtain position-resolved texture information in inhomogeneous biological samples without the need for sample rotation. This study employs EDLD texture scanning to investigate the impact of two salivary peptides, statherin (STN) and histatin-1 (HTN) 21 N-terminal peptides (STN21 and HTN21), on the crystallographic structure of dental enamel. These proteins are known to play crucial roles in dental caries progression. Three healthy incisors were randomly assigned to three groups: artificially demineralized, demineralized after HTN21 peptide pre-treatment and demineralized after STN21 peptide pre-treatment. To understand the micro-scale structure of the enamel, each specimen was scanned from the enamel surface to a depth of 250 µm using microbeam EDLD. Via the use of a white beam and a pixelated detector, where each pixel functions as a spectrometer, pole figures were obtained in a single exposure at each measurement point. The results revealed distinct orientations of hydroxyapatite crystallites and notable texture variation in the peptide-treated demineralized samples compared with the demineralized control. Specifically, the peptide-treated demineralized samples exhibited up to three orientation populations, in contrast to the demineralized control which displayed only a single orientation population. The texture index of the demineralized control (2.00 ± 0.21) was found to be lower than that of either the STN21 (2.32 ± 0.20) or the HTN21 (2.90 ± 0.46) treated samples. Hence, texture scanning with EDLD gives new insights into dental enamel crystallite orientation and links the present understanding of enamel demineralization to the underlying crystalline texture. For the first time, the feasibility of EDLD texture measurements for quantitative texture evaluation in demineralized dental enamel samples is demonstrated.

Fabrication of 3D bioactive melt electrowriting composite scaffold with high osteogenic potential.

A key challenge in using melt electrowriting (MEW) technology is incorporating large amounts of bioactive inorganic materials, such as hydroxyapatite (HA). In the present study, following optimization of the fabrication parameters, 40 %-HA (HA40) nanoparticles were pre-mixed into medical-grade polycaprolactone (PCL) and processed using the MEW (MEW) technique to mimic the structure and function of the natural extracellular matrix (ECM) for bone regeneration. The HA40 fibrous composite scaffolds showed continuous writing and obtained a well-connected and orderly stacked fibre with a small diameter size (67 ± 8.5 µm). A major result of the present study was the successful enrichment and accumulation of the HA particles, which mostly occurred on the MEW fibre external surfaces. This design allows for direct interfacial interaction with human periodontal ligament cells (hPDLCs). We systematically investigated the behaviour and function of hPDLCs on the HA40 composite scaffold, alongside parameters related to mineralization. The HA40 scaffold demonstrated significantly higher metabolic activity and enhanced expression of osteopontin compared to PCL-only scaffolds, as well as increased levels of ALP and COL1. The study's findings demonstrate that bioactive composite scaffolds, incorporating 40 % HA into m-PCL via MEW, effectively enhance the biological response of the ECM and are promising for potential applications in bone regeneration.

Strontium-containing mineralized phospholipid coatings improve osseointegration in osteoporotic rats.

Surface treatments play an important role in enhancing the osseointegration of Titanium (Ti) and its alloys. This study introduces a method employing biomimetic hydroxyapatite (Hap) deposition guided by molecularly organized phospholipids, affixed to the metal implant surface. Using the Langmuir-Blodgett technique, phospholipids were deposited onto Ti-screws by using CaCl2 or CaCl2/SrCl2 aqueous solution in the subphase of a Langmuir trough in the target proportion (i.e. 10 and 90 mol% of Sr2+ in relation of Ca2+) followed by immersion in phosphate buffer and in supersaturated simulated body fluid. Coating composition and morphology were evaluated using infrared spectroscopy and scanning electron microscopy, respectively, while contact angle measurements assessed coating wettability and surface energy. Randomized screws were then implanted into the tibias of healthy and osteoporotic female rats (G1: Control-Machined, G2: Hap, G3: HapSr10, G4: HapSr90). Osseointegration, assessed 60 days post-implantation, included reverse torque, fluorochrome area, bone tissue-screw contact area, and linear extent of bone-screw contact. Results, grouped by surface treatment (Machined, Hap, HapSr10, HapSr90), revealed that the deposition of Hap, HapSr10, and HapSr90 resulted in thin and rough coatings composed of hydroxyapatite (Hap) on the screw surface with nanoscale pores. The coatings resulted in increased wettability and surface energy of Ti surfaces. The minerals are chemically similar to natural bone apatite as revealed by FTIR analysis. In vivo analyses indicated higher torque values for strontium-containing surfaces in the osteoporotic group (p = 0.02) and, in the control group superior torque for screw removal on the Hap surface (p = 0.023). Hydroxyapatite-treated surfaces enhance morphology, composition, and reactivity, promoting screw osseointegration in healthy and osteoporotic female rats. The incorporation of strontium into the mineral phase has been proposed to not only stimulate osteoblast activity but also reduce osteoclastic resorption, which may explain the improved outcomes observed here in experimental osteoporotic conditions.

The impact of the physicochemical properties of calcium phosphate ceramics on biocompatibility and osteogenic differentiation of mesenchymal stem cells.

OBJECTIVE: In this study we have focused on biocompatibility and osteoinductive capacity analysis of self-manufactured single-phase (HAP) and two-phase (HAP and ß-ТСР) bioactive ceramics with various chemical modifications (Fig. 1). RESULTS: We demonstrate a reduction in solubility for all analyzed composite after the treatment with H2O and H2O2, accompanied by an enhancement in adsorption activity. This modification also resulted in an increase in micro- and macroporosity, along with a rise in the open porosity. Adipose-derived mesenchymal stromal cells demonstrated excellent cell adhesion and survival when cultured with these ceramics. Calcium phosphate ceramics (H-500, HT-500, and HT-1 series) stimulated alkaline phosphatase expression, promoted calcium deposition, and enhanced osteopontin expression in ADSCs, independently inducing osteogenesis without additional osteogenic stimuli. These findings underscore the promising potential of HAP-based bioceramics for bone regeneration/reconstruction.

Green synthesis of novel Z-scheme SnS2/HAp nanocomposite using Ocimum tenuiflorum leaf extract and investigation of its photocatalytic activity.

The present study focuses on the green synthesis of a novel Z-scheme SnS2/HAp photocatalyst using Ocimum tenuiflorum (tulsi) leaf extract as a stabilizing agent. This approach not only emphasizes sustainability but also adds value to waste by extracting hydroxyapatite (HAp) from Labeo rohita fish scales, addressing the challenge of their disposal. The synthesized photocatalyst was thoroughly characterized using a range of analytical techniques to evaluate its crystal structure, optical properties, morphology, and elemental composition. The photocatalytic activity of the SnS2/HAp composite was assessed through the degradation of gentian violet (GV) dye, a representative organic pollutant. Various reaction parameters were optimized to enhance the degradation efficiency, and the photocatalyst's performance was further tested across different water matrices. Under optimal conditions, the SnS2/HAp photocatalyst achieved a maximum photodegradation efficiency of 97.49% with a rate constant of 0.0494 min- 1 for GV dye. Additionally, it exhibited an efficiency greater than 70% against other emerging pollutants via advanced oxidation processes (AOP). The enhanced photocatalytic activity was attributed to the formation of a Z-Scheme heterojunction between SnS2 and HAp, which enhanced the charge separation efficiency and delayed the charge recombination. The study also demonstrated the photocatalyst's remarkable reusability, maintaining high performance over five cycles and across various water environments. This highlights its potential as a sustainable solution for the removal of organic pollutants from aqueous streams. Finally, a Z-scheme electron transport mechanism is proposed to explain the photodegradation process of GV dye using the SnS2/HAp photocatalyst.

Enhancing craniofacial bone tissue engineering strategy: integrating rapid wet chemically synthesised bioactive glass with photopolymerized resins.

BACKGROUND: Craniofacial bone regeneration represents a dynamic area within tissue engineering and regenerative medicine. Central to this field, is the continual exploration of new methodologies for template fabrication, leveraging established bio ceramic materials, with the objective of restoring bone integrity and facilitating successful implant placements. METHODS: Photopolymerized templates were prepared using three distinct bio ceramic materials, specifically a wet chemically synthesized bioactive glass and two commercially sourced hydroxyapatite variants. These templates underwent comprehensive characterization to assess their physicochemical and mechanical attributes, employing techniques including Fourier transform infrared spectroscopy, scanning electron microscopy, and nano-computed tomography. Evaluation of their biocompatibility was conducted through interaction with primary human osteoblasts (hOB) and subsequent examination using scanning electron microscopy. RESULTS: The results demonstrated that composite showed intramolecular hydrogen bonding interactions with the photopolymer, while computerized tomography unveiled the porous morphology and distribution within the templates. A relatively higher porosity percentage (31.55 ± 8.70%) and compressive strength (1.53 ± 0.11 MPa) was noted for bioactive glass templates. Human osteoblast cultured on bioactive glass showed higher viability compared to other specimens. Scanning micrographs of human osteoblast on templated showed cellular adhesion and the presence of filopodia and lamellipodia. CONCLUSION: In summary these templates have the potential to be used for alveolar bone regeneration in critical size defect. Photopolymerization of bioceramics may be an interesting technique for scaffolds fabrication for bone tissue engineering application but needs more optimization to overcome existing issues like the ideal ratio of the photopolymer to bioceramics.

The effect of nano-hydroxyapatite and casein phosphopeptide-amorphous calcium phosphate with and without laser irradiation on the microhardness and surface morphology of demineralized primary enamel: An in vitro experimental study.

Background: Various topical gels, varnishes, and fluoride gels are being used by dentists for the treatment of White spot lesions (WSLs). The remineralizing effect of casein phosphopeptide-amorphous calcium phosphate (CPP-ACP), nano-hydroxyapatite (nHAp), and lasers has been proven earlier. This study was designed to evaluate the remineralizing effect of nHAp and CPP-ACP with and without erbium-doped yttrium aluminum garnet (Er: YAG) laser irradiation on demineralized primary enamel. The aim of this study was to evaluate the effect of CPP-ACP and nHAp with and without Er:YAG laser irradiation on the microhardness and surface morphology of demineralized primary enamel. Materials and Methods: The present study is an experimental in vitro study. Fifty extracted primary incisors were selected for the study. Following cleaning and sectioning, teeth were embedded in acrylic. The tooth models were divided into four groups randomly - Group 1 (CPP-ACP), Group 2 (nHAp), Group 3 (CPP-ACP + laser), and Group 4 (nHAp + laser). The baseline, postdemineralization, and postremineralization Vickers hardness testing was performed. One sample from each group was analyzed by scanning electron microscopy. Descriptive statistics such as frequencies and percentages for categorical data, mean and standard deviation for numerical data were depicted. The normality of numerical data was checked using the Shapiro-Wilk test. The level of significance was kept at 5%. Intergroup comparison (>2 groups) was done using one-way analysis of variance followed by pair-wise comparison using the post hoc test. Results: There was a statistically significant increase in surface microhardness in each group after remineralization. The highest increase in microhardness value was seen in Group 4 (nHAp + laser) followed by Group 3 (CPP-ACP + laser) and the least in Group 1 (CPP-ACP). Similar observations were made in scanning electron microscopic images. This indicated that nHAp has a comparable, if not better ability for remineralization than CPP-ACP. The remineralizing capacity of both the remineralizing agents was seen to be improved in this study when simultaneous laser application was employed. Conclusion: Currently, the evidence supporting the efficacy of nHAp dentifrices and laser in primary teeth is limited. Additional long-term in vivo studies employing standardized protocols and large sample sizes are necessary to draw definitive findings about the effect of remineralizing agents and lasers on primary enamel.

Regenerative treatment of canine osteogenic lesions with Platelet-Rich Plasma and hydroxyapatite: a case report.

Introduction: This study examined the efficacy of a therapy based on a combination of Platelet Rich Plasma and hydroxyapatite nanoparticles in a severe clinical case involving a young Rottweiler with a complex spiral fracture of the tibia. Method: Following a worsening of the lesion after traditional surgical intervention, the subject was treated with the combined therapy. X-rays were taken at the following stages: immediately post-surgery, four weeks post-surgery, and 10 days post-treatment. Fracture gap and callus density measurements were obtained using ImageJ analysis, allowing for a detailed quantitative assessment of bone regeneration over time. Results: Post-operative radiographs indicated a clinical worsening of the fracture, revealing an increased fracture gap due to bone loss. However, significant improvements were observed ten days following the treatment, with a marked reduction in fracture gaps and increased callus density. These results demonstrated a notable acceleration in bone healing and callus formation compared to typical recovery times for similar lesions. Conclusion: The method showed potential for enhancing osteogenic regeneration, facilitating faster healing of serious orthopedic injuries compared to traditional methods.

Effect of Hydroxyapatite Nanoparticle Crystallinity and Colloidal Stability on Cytotoxicity.

Hydroxyapatite nanoparticles (nHA) have gained attention as potential intracellular drug delivery vehicles due to their high binding affinity for various biomolecules and pH-dependent solubility. Yet, the dependence of nHA cytocompatibility on their physicochemical properties remains unclear since numerous studies have revealed starkly contrasting results. These discrepancies may be attributed to differences in size, shape, crystallinity, and aggregation state of nHA, which complicates fundamental understanding of the factors driving nHA cytotoxicity. Here, we hypothesize that nHA cytotoxicity is primarily driven by intracellular calcium levels following the internalization of nHA nanoparticles. By investigating the cytotoxicity of spherical nHA with different crystallinity and dispersity, we find that both lower crystallinity and increased agglomeration of nHA raise cytotoxicity, with nanoparticle agglomeration being the more dominant factor. We show that the internalization of nHA enhances intracellular calcium levels and increases the production of reactive oxygen species (ROS). However, only subtle changes in intracellular calcium are observed, and their physiological relevance remains to be confirmed. In conclusion, we show that nHA agglomeration enhances ROS production and the associated cytotoxicity. These findings provide important guidelines for the future design of nHA-containing formulations for biomedical applications, implying that nHA crystallinity and especially agglomeration should be carefully controlled to optimize biocompatibility and therapeutic efficacy.

Effect of pH and hydroxyapatite-like layer formation on the antibacterial properties of borophosphate bioactive glass incorporated poly(methyl methacrylate) bone cement.

Infection is a leading cause of total joint arthroplasty failure. Current preventative measures incorporate antibiotics into the poly (methyl methacrylate) (PMMA) bone cement that anchors the implant into the natural bone. With bacterial resistance to antibiotics on the rise, the development of alternative antibacterial materials is crucial to mitigate infection. Borate bioactive glass, 13-93-B3, has been studied previously for use in orthopedic applications due to its ability to be incorporated into bone cements and other scaffolds, convert into hydroxyapatite (HA)-like layer, and enhance the osseointegration and antibacterial properties of the material. The purpose of this study is to better understand how glass composition and change in surrounding pH effects the composite's antibacterial characteristics by comparing the incorporation of 30% wt/wt 13-93-B3 glass and pH neutral borophosphate bioactive glass into PMMA bone cement. We also aim to elucidate how HA-like layer formation on the cement's surface may affect bacterial adhesion. These studies showed that 13-93-B3 incorporated cements had significant reduction of bacterial growth surrounding the composite beyond 24 h of exposure when compared to a neutral borate bioactive glass incorporated cement (p < 0.01) and cement only (p < 0.0001). Additionally, through soaking cement composites in simulated body fluid and then exposing them to a bioluminescent strand of staphylococcus aureus, we found that the presence of a HA-like layer on the 13-93-B3 or pH neutral glass incorporated cement disks resulted in an increase in bacterial attachment on the composite cement's surface, where p < 0.001, and p < 0.05 respectively. Overall, our studies demonstrated that borate bioactive glass incorporated PMMA bone cement has innate antimicrobial properties that make it a promising material to prevent infection in total joint arthroplasties.

Electrospun polyvinyl alcohol nanofiber scaffolds incorporated strontium-substituted hydroxyapatite from sand lobster shells: synthesis, characterization, and in vitro biological properties.

The paper describes the synthesis of hydroxyapatite (HAp) and strontium-substituted hydroxyapatite (SrHAp) from sand lobster shells by a hydrothermal method. The HAp and SrHAp were incorporated into the polyvinyl alcohol (PVA) nanofiber scaffold through the eletrospinning method. The scaffolds were incorporated with 5wt% of hydroxyapatite (HAp), 5wt%, 10wt%, and 15% of SrHAp. The physicochemical, mechanical, and in vitro biological properties of the scaffold were evaluated. The incorporation of HAp or SrHAp was evidenced by the diffraction patterns and the phosphate functional groups related to HAp. The morphological results showed the decrement of fiber diameter in line with the increased SrHAp concentration. A tensile test was conducted to investigate the mechanical properties of the scaffolds, and the results showed that the scaffolds perform poorly at a higher SrHAp concentration because of exceeding agglomeration levels. The PVA/SrHAp15 performed the best antibacterial activity against E. coli and S. aureus with an inhibition zone of (15.2 ± 0.2) and (14.5 ± 0.8), respectively. The apatite formation was more abundant in PVA/SrHAp10 after immersion in a simulated body fluid (SBF). Cell viability results showed that the scaffold enabled the osteoblast cells to grow and proliferate. The biocompatibility of HAp and SrHAp resulted in the enhancement of cell adhesion. Based on all tests, the PVA/SrHAp 10 scaffold shows a strong candidate for further in vivo studies.

Evaluating various properties of nanohydroxyapatite synthesized from eggshells and dual-doped with Si4+ and Zn2+: An in vitro study.

Background: This study aimed to evaluate morphological, chemical and biocompatible properties of nanohydroxyapatite (N-HA) synthesized from eggshells and dual-doped with Si4+ and Zn2+. Methods: In the current study, N-HA was synthesized from chicken eggshells using the wet chemical precipitation method and doped with Si4+ and Zn2+. The physical assessment was carried out using field emission scanning electron microscopy (FE-SEM), energy dispersive X-ray (EDX) analysis, and X-ray diffraction (XRD) analysis. Crystal size was calculated using the Scherrer equation. Cytotoxicity was studied in vitro using the MTT (3-(4,5-Dimethylthiazol-2-yl)-2,5-Diphenyltetrazolium Bromide) cytotoxicity assay. The optical density (OD) of each well was obtained and recorded at 570 nm for 24 h (t1), 48 h (t2), 72 h (t3), and 5 days (t4) using a microplate reader. Results: The results of Si-Zn-doped HA showed a high specific surface area with an irregular nano-sized spherical particle structure. The atomic percentage provided the ratio of calcium to phosphate; for non-doped HA, the atomic Ca/P ratio was 1.6, but for Si-Zn-doped HA, where Zn+2 Ca and Si + replaced 4 substituted P, the atomic ratio (Ca + Zn)/(P + Si) was 1.76. The average crystal size of Si-Zn-doped HA was 46 nm, while for non-doped HA it was 61 nm. both samples were non-toxic and statistically significantly less viable than the control group After 5 days, the mean cell viability of Si-Zn-doped HA (79.17 ± 2.18) was higher than that of non-doped HA (76.26 ± 1.71) (P = 0.091). Conclusions: The MTT assay results showed that Si-Zn-doped HA is biocompatible. In addition, it showed characteristic physiochemical properties of a large surface area with interconnected porosity.

Direct Immobilization of Folic Acid Molecules on Hydroxyapatite Nanoparticles with Substitution and Coordination Phenomena.

We successfully synthesized folic acid (FA) immobilized hydroxyapatite (HA) nanoparticles without using a mediative reagent (e.g., silane coupling agent), and the immobilization states were evaluated and discussed. The HA nanoparticles with higher biocompatibility have two different planes, namely, c- and m-planes. These plane surfaces are rich in phosphate groups (P-site) and Ca2+ ions (C-site), respectively. We suggested that during the synthesis of the HA nanoparticles, the P-site substitution and C-site coordination with the addition of organic molecules containing -COO- ions can occur. Thus, it is possible to simultaneously immobilize two molecules to one HA nanoparticle. In this study, we successfully synthesized FA-immobilized HA nanoparticles by P-site substitution and C-site coordination reactions, which were named as substitution type and coordination type. In the substitution type, when FA was reacted with HA during the nucleation stage, the PO43- ions of HA decreased as the FA ratio of coverage surface area increased, and the crystalline phase was changed significantly from the Ca deficient HA to the carbonated HA phase. Accordingly, it was indicated that FA was immobilized on HA by the P-site substitution. In the coordination type, since FA was reacted with HA after the completion of crystal growth, the crystalline phase was changed slightly as the FA ratio of coverage surface area increased, indicating that FA was immobilized on HA by the C-site coordination. From the above, we controlled the FA immobilization states on the HA nanoparticles by the P-site substitution and the C-site coordination through the FA addition timing in the synthesis. Since the -COO- ions in FA could be selectively substituted with the P-site in HA, it is possible to directly coordinate the foreign organic molecules to the Ca2+ ions in HA. Therefore, the immobilization technique of this study is expected to achieve two different drug molecules with diagnosis and therapy functions (i.e., theranostics) on one nanoparticle.

Use of Zebrafish (Danio rerio) for Biosafety Evaluation of Strontium Nanostructured Hydroxyapatite.

Despite the numerous studies on biocompatibility with nano-biomaterials, the biological effects of strontium-substituted HA nanoparticles (nSrHA) need to be better understood. So, we conducted an embryotoxicity test using zebrafish (Danio rerio) according to the OECD 236 guideline, a model that represents a viable alternative that bridges the gap between in vitro and mammalian models. Zebrafish embryos were exposed for 120 h to microspheres containing nSrHA nanoparticles with low and high crystallinity, synthesized at temperatures of 5°C (nSrHA5) and 90°C (nSrHA90). We evaluated lethality, developmental parameters, and reactive oxygen species (ROS) production. The larval behavior was assessed at 168 hpf to determine if the biomaterials affected motor responses and anxiety-like behavior. The results showed that the survival rate decreased significantly for the nSrHA5 group (low crystalline particles), and an increase in ROS was also observed in this group. However, none of the biomaterials caused morphological changes indicative of toxicity during larval development. Additionally, the behavioral tests did not reveal any alterations in all experimental groups, indicating the absence of neurotoxic effects from exposure to the tested biomaterials. These findings provide valuable insights into the biosafety of modified HA-based nanostructured biomaterials, making them a promising strategy for bone tissue repair. As the use of hydroxyapatite-based biomaterials continues to grow, it is crucial to ensure rigorous control over the quality, reliability, and traceability of these materials.

Hyperactivation of crosslinked lipases in elastic hydroxyapatite microgel and their properties.

Effective enzyme stabilization through immobilization is essential for the functional usage of enzymatic reactions. We propose a new method for synthesizing elastic hydroxyapatite microgel (E-HAp-M) materials and immobilizing lipase using this mesoporous mineral via the ship-in-a-bottle-neck strategy. The physicochemical parameters of E-HAp-M were thoroughly studied, revealing that E-HAp-M provides efficient space for enzyme immobilization. As a model enzyme, lipase (LP) was entrapped and then cross-linked enzyme structure, preventing leaching from mesopores, resulting in highly active and stable LP/E-HAp-M composites. By comparing LP activity under different temperature and pH conditions, it was observed that the cross-linked LP exhibited improved thermal stability and pH resistance compared to the free enzyme. In addition, they demonstrated a 156% increase in catalytic activity compared with free LP in hydrolysis reactions at room temperature. The immobilized LP maintained 45% of its initial activity after 10 cycles of recycling and remained stable for over 160 days. This report presents the first demonstration of a stabilized cross-linked LP in E-HAp-M, suggesting its potential application in enzyme-catalyzed processes within biocatalysis technology.