nHAp Crystals Research Articles

Evaluation of Setting Time, Compressive Strength, Microleakage and Antibacterial Properties of Nano Hydroxyapatite Incorporated GIC: An In-vitro Study

Introduction: Glass Ionomer Cement (GIC) is widely used as a restorative material, but it still exhibits some limitations that pose significant drawbacks in clinical use. The composition of Hydroxyapatite (HA) mimics the inorganic fraction of mineralised tissues such as bones and teeth. HA possesses osteoconductive and bioactive properties, making it favourable for orthopaedic and dental applications. Aim: The aim of this study was to evaluate and compare the setting time, compressive strength, microleakage and antibacterial properties of nano-HA incorporated GIC (NHaGIC) at various concentrations with Type-II GIC. Materials and Methods: This in-vitro study was conducted in the Department of Paediatric and Preventive Dentistry at Sri Aurobindo College of Dentistry in Indore, Madhya Pradesh, India, for a duration of nine months, from May 2023 to January 2024. The study comprised five study groups as follows: Group-1- Type-II GIC (control), Group-2-NHA-GIC 4%, Group-3-NHA-GIC 8%, Group-4-NHA-GIC 10% and Group-5-NHA-GIC 15%. A total of 50 acrylic moulds containing the test material and TypeII GIC, with 10 in each group (n=10), were prepared to record the setting time, which was tested using a Gillmore needle. The compressive strength was checked using a Universal Testing Machine. Additionally, 50 primary molars were selected according to inclusion and exclusion criteria and randomly divided into five groups (n=10) to evaluate microleakage. Microleakage was assessed using the dye penetration method under a stereomicroscope. For antibacterial properties, 50 samples (n=10) were taken for each strain, i.e., Strain A: S. mutans and Strain B: L. fermentum and the disk diffusion method was employed. Thus, a total sample size of 250 was used. The preparation of GIC-NHAp was done by adding nano- HA to GIC at selected concentrations by weight/weight percentage (w/w%). The results were analysed using a one-way Analysis of Variance (ANOVA) test. Results: A statistically significant difference was observed (p<0.001) between Group-1 (GIC II) and Group-II (4% GICNHAp), III (8% GIC-NHAp), IV (10% GIC-NHAp) and V (15% GIC-NHAp) regarding setting time, compressive strength, microleakage and antibacterial properties. Conclusion: It can be concluded that adding NHAp crystals to GIC enhances its properties, such as compressive strength and antimicrobial efficiency, as the concentration increases. The microleakage property showed a consistent decrease with increasing concentration. However, the setting time increased with higher concentrations of NHAp in GIC.

Emission-tunable probes using terbium(III)-doped self-activated luminescent hydroxyapatite for in vitro bioimaging

Lanthanide ion (Ln3+)-doped nanoscale hydroxyapatites (nHAp) with tunable luminescence have attracted increasing attention due to their potential applications as useful biomedical tools (e.g., imaging and clinical therapy). In this study, we reported that doping Terbium (III) ions (Tb3+) in self-activated luminescent nHAp via a facile hydrothermal reaction, using trisodium citrate (Cit3−), generates unique emission-tunable probes known as Cit/Tb-nHAp. The morphology, crystal phase, and luminescence properties of these Cit/Tb-nHAp probes are studied in detail. Moreover, the results demonstrate that the luminescence of self-activated nHAp originates from the carbon dots trapped within the nHAp crystals, in which partial energy transfer occurs from carbon dots (CDs) to Tb3+. The color tunability is successfully achieved by regulating the addition of Cit3−. Biocompatibility study indicates that when co-cultured with C6 glioma cells in vitro for 3 days, ≤800 ppm Cit/Tb-nHAp is not cytotoxic for C6 glioma cells. We also present in vitro data showing efficient cytoplasmic localization of transferrin conjugated Cit/Tb-nHAp into C6 glioma cells by fluorescence cell imaging. We have successfully engineered Cit/Tb-nHAp, a promising biocompatible agent for future in vitro and in vivo fluorescence bioimaging.

Biomimetic mineralization of nanocrystalline hydroxyapatites on aminated modified polylactic acid microspheres to develop a novel drug delivery system for alendronate.

EPLA/nHAp composite microsphere, a novel drug delivery system potentially useful for the local delivery of alendronate (AL) to bone tissue was developed via the biomimetic mineralized deposition of nano-hydroxyapatite (nHAp) crystals on the surface of aminated modified polylactic acid (EPLA) microspheres. Scanning electron microscopy (SEM) observation showed that this system consisted of a polymer core with nanofiber network structure and inorganic coating composed of countless rod-like nanocrystalline particles, Fourier transform infrared spectroscopy (FTIR) and X-ray diffraction analysis (XRD) confirmed that these particles were nHAp crystals. An efficient AL-loading can be realized by facile impregnation-adsorption method under suitable conditions due to the high adsorption capacity of EPLA/nHAp composite microspheres. The drug loading efficiency of microspheres was detected by indirect ultraviolet spectrophotometry. It was found that the adsorption capacity of EPLA/nHAp composite microsphere towards AL was increased nearly 5-fold compared with that of bare EPLA microspheres owing to the strong interaction between alendronate and hydroxyapatite. Meanwhile, in vitro release study showed that AL-loaded EPLA/nHAp microspheres had a more sustained drug release than AL-loaded EPLA microspheres, all these results demonstrated that the as-prepared EPLA/nHAp composite microsphere is an efficient carrier for the delivery and sustained release of AL. Furthermore, an in vitro cell culture study revealed that these composite microspheres presented a good biocompatibility, showing great potential for the applications in the biomedical field.

Biomineralization improves mechanical and osteogenic properties of multilayer‐modified PLGA porous scaffolds

Poly-(lactide-co-glycolide acid) (PLGA) has been widely investigated as scaffold material for bone tissue engineering owing to its biosafety, biodegradability, and biocompatibility. However, the bioinert surface of PLGA may fail in regulating cellular behavior and directing osteointegration between the scaffold and the host tissue. In this article, oxidized chondroitin sulfate (oCS) and type I collagen (Col I) were assembled onto PLGA surface via layer by layer technique (LbL) as an adhesive coating for the attachment of inorganic minerals. The multilayer-modified PLGA scaffold was mineralized in vitro to ensure the deposition of nanohydroxyapatite (nHAP). It was found that nHAP crystals were more uniformly and firmly attached on the multilayer-modified PLGA as compared with the pure PLGA scaffold, which remarkably improved PLGA surface and mechanical properties. Additionally, in vitro biocompatibility of PLGA scaffold, in terms of bone mesenchymal stem cells (BMSCs) attachment, spreading and proliferation was greatly enhanced by nHAP coating and multilayer deposition. Furthermore, the fabricated composite scaffold also shows the ability to promote the osteogenic differentiation of BMSCs through the up-regulation of osteogenic marker genes. Thus, this novel biomimetic composite scaffold might achieve a desirable therapeutic result for bone tissue regeneration. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part A: 106A: 2714-2725, 2018.

Characterization of nano-hydroxyapatite silica gel and evaluation of its combined effect with Solcoseryl paste on bone formation: An experimental study in New Zealand rabbits

ObjectiveCharacterization of nano-hydroxyapatite (n-HAp) silica gel bone substitute and evaluation of its combined effect with Solcoseryl paste on bone formation experimentally in New Zealand rabbits. Materials and methodsCharacterization of the n-HAp silica gel granules (NanoBone®, Artoss GmbH, German) was done by the SEM, EDX analysis and TEM. Simulated bone defect (10 × 5 × 1 mm) was constructed from metallic mold to standardize the amount of the grafting materials. Two defects (10 × 5 × 1 mm) were created in the parietal bones of 12 New Zealand White rabbits. The rabbits were randomly divided into 4 groups with 3 rabbits/group (n = 6 defects/group). Group (C): The defects were left empty for natural bone healing (control group), group (S): The defects were filled with the pre-weighed Solcoseryl® paste (Legacy Pharmaceuticals Switzerland GmbH, Switzerland), group (N): the defects were filled with the pre-weighed NanoBone and group (SN): the defects were filled with Solcoseryl paste/NanoBone mixture (1:1 by Vol. %). The rabbits were sacrified on day 14; the defects were removed, prepared for histological assessment and histomorphometric analysis. The amount of bone formation was statistically analyzed by ANOVA and Post-hoc test (p < 0.05). ResultsThe SEM revealed the highly rough, porous n-HAp silica gel granules. The TEM revealed the interconnected porosities within the silica matrix and the platelet like shape of the n-HAp crystals. The Ca: P ratio of the n-HAp silica gel granules was 1.74. Histological evaluation revealed absence of new bone formation in group C while calcification in most of osteoid tissue areas was evident in all experimental groups with evident wide bone marrow cavities in S group. The NS group possessed the highest significant bone area percentage. ConclusionsThe interconnected porosities and the highly rough surface of n-HAp silica gel granules provided biomimetic bone substitute material that promotes new bone formation. Solcoseryl/NanoBone mixture (1:1 vol %) has a synergistic osteoinductive effect quantitatively and qualitatively on bone formation. Clinical significanceAcceleration of post-operative bone healing in certain clinical cases where physiologic or pathologic conditions could impair the vascularization process.

Spray drying as a viable process to produce nano-hydroxyapatite/chitosan (n-HAp/CS) hybrid microparticles mimicking bone composition

In this work nano-hydroxyapatite/chitosan (n-HAp/CS) hybrid microparticles were prepared by spray drying following a methodology where, in a first step, aqueous nanodispersions of n-HAp in the presence of chitosan were produced by fast stirring at pH values below and above chitosan pKa (5.5 and 7.0, respectively). The mixing system used allowed the production of homogeneous and stable nanodispersions, and the subsequent spray-dried microparticles, incorporating highly pure HAp nanoparticles of approximately 50nm, were successfully produced without degrading the organic component, chitosan. Comparatively with the n-HAp/CS-7.0, the n-HAp/CS-5.5 dispersions were characterized by a lower particle size and a higher zeta potential, being then more stable. Differences in the spray-dried microparticles were also evident from a morphological point of view. HApCS-5.5 microparticles, which present an average size in volume of 15.8μm and n-HAp crystals homogenously distributed, were found to be preferred over the HApCS-7.0 counterparts, which require an extra step in the productive process and presented a tendency to form large agglomerates. Both prepared hybrid particles presented similar composition to that one of natural bone (HAp/CS of 70/30) and no traces of KCl salts were observed if a washed n-HAp paste was used.

Bioactivity behaviour of nano-hydroxyapatite/freestanding aligned carbon nanotube oxide composite.

Bioactive and low cytotoxic three dimensional nano-hydroxyapatite (nHAp) and aligned carbon nanotube oxide (a-CNTO) composite has been investigated. First, freestanding aligned carbon nanotubes porous scaffold was prepared by large-scale thermal chemical vapour deposition and functionalized by oxygen plasma treatment, forming a-CNTO. The a-CNTO was covered with plate-like nHAp crystals prepared by in situ electrodeposition techniques, forming nHAp/a-CNTO composite. After that nHAp/a-CNTO composite was immersed in simulated body fluid for composite consolidation. This novel nanobiomaterial promotes mesenchymal stem cell adhesion with the active formation of membrane projections, cell monolayer formation and high cell viability.

Hydrothermal–electrochemical synthesis of nano-hydroxyapatite crystals on superhydrophilic vertically aligned carbon nanotubes

Nano-hydroxyapatite (nHap) is an attractive and biocompatible material due to its chemical likeness to bone and tooth. The nHAp has osteoconductivity and bioactive properties that promote regeneration and adherence to bone tissue, however, lack mechanical properties. Hence, there is an interest in associating this material with others to obtain composite with superior properties. Vertically aligned multi-walled carbon nanotubes (VAMWCNT-O) have outstanding mechanical, electrical, chemical and biocompatibility properties, which may boost typical nHAp properties. We presented for the first time the correlation between nHAp crystals-like and hydrothermal–electrochemical parameters collected during the electrodeposition process on VAMWCNT-O. Also, we show dependence between the nHAp crystals and bath temperature. Based on our results, we proposed and discussed a mechanism of formation of nHAp crystals on VAMWCNT-O electrodes.

In vitro and in vivo studies of a novel nanohydroxyapatite/superhydrophilic vertically aligned carbon nanotube nanocomposites

An association between in vitro and in vivo studies has been demonstrated for the first time, using a novel nanohydroxyapatite/superhydrophilic vertically aligned multiwalled carbon nanotube (nHAp/VAMWCNT-O2) nanocomposites. Human osteoblast cell culture and bone defects were used to evaluate the in vitro extracellular matrix (ECM) calcification process and bone regeneration, respectively. The in vitro ECM calcification process of nHAp/VAMWCNT-O2 nanocomposites were investigated using alkaline phosphatase assay. The in vivo biomineralization studies were carried out on bone defects of C57BL/6/JUnib mice. Scanning electron microscopy, micro-energy dispersive spectroscopy, X-ray photoelectron spectroscopy, and X-ray difractometry analyses confirmed the presence of the nHAp crystals. nHAp/VAMWCNT-O2 nanocomposites induced in vitro calcification of the ECM of human osteoblast cells in culture after only 24h. Bone regeneration with lamellar bone formation after 9weeks was found in the in vivo studies. Our findings make these new nanocomposites very attractive for application in bone tissue regeneration.

Proposed model for biomineralization of novel nanohydroxyapatite/vertically aligned multiwalled carbon nanotube scaffolds

For the first time, the growth mechanism of biominerals formed on plate-like nanohydroxyapatite (nHAp) electrodeposited on superhydrophilic vertically aligned multi-walled carbon nanotubes (VAMWCNT-O2) is presented and a model for the specific growth preference is discussed. VAMWCNT-O2 films were obtained by microwave-assisted chemical vapor deposition method and funcionalized by oxygen plasma. nHAp/VAMWCNT-O2 nanocomposites were fabricated with a direct electrodeposition of the thin nHAp films onto the VAMWCNT-O2 films. The biomineralized "scaffolds" were obtained by soaking nHAp/VAMWCNT-O2 in simulated body fluid for 7, 14 and 21 days. Results show that the carboxyl functional groups directly attached onto VAMWCNT tips after oxygen plasma treatment were essential for the acceleration of the OH- formation and the deposition of plate-like nHAp crystals.

Fast preparation of nano-hydroxyapatite/superhydrophilic reduced graphene oxide composites for bioactive applications.

A method for the direct electrodeposition of globular nano-hydroxyapatite (nHAp) onto reduced graphene oxide (RGO) is presented and a model for the specific growth preference is discussed. Results show that the carboxyl (carboxylic acid)/carboxylate functional groups attached directly to the RGO after oxygen plasma treatment were essential to accelerate the OH- formation and the deposition of globular nHAp crystals. High resolution scanning electron microscopy, energy dispersive X-ray and X-ray diffraction showed that homogeneous, highly crystalline, stoichiometric nHAp crystals, with preferential growth in the (002) plane direction, were formed without any thermal treatment. The nHAp/RGO composites were shown to be an appropriate surface for mesenchymal stem cell adhesion with active formation of membrane projections.

A molecular receptor targeted, hydroxyapatite nanocrystal based multi-modal contrast agent

Multi-modal molecular imaging can significantly improve the potential of non-invasive medical diagnosis by combining basic anatomical descriptions with in-depth phenotypic characteristics of disease. Contrast agents with multifunctional properties that can sense and enhance the signature of specific molecular markers, together with high biocompatibility are essential for combinatorial molecular imaging approaches. Here, we report a multi-modal contrast agent based on hydroxyapatite nanocrystals (nHAp), which is engineered to show simultaneous contrast enhancement for three major molecular imaging techniques such as magnetic resonance imaging (MRI), X-ray imaging and near-infrared (NIR) fluorescence imaging. Monodispersed nHAp crystals of average size ∼30 nm and hexagonal crystal structure were in situ doped with multiple rare-earth impurities by a surfactant-free, aqueous wet-chemical method at 100 °C. Doping of nHAp with Eu 3+ (3 at%) resulted bright near-infrared fluorescence (700 nm) due to efficient 5 D 0– 7 F 4 electronic transition and co-doping with Gd 3+ resulted enhanced paramagnetic longitudinal relaxivity ( r 1 ∼12 mM −1 s −1) suitable for T 1 weighted MR imaging together with ∼80% X-ray attenuation suitable for X-ray contrast imaging. Capability of MF-nHAp to specifically target and enhance the signature of molecular receptors (folate) in cancer cells was realized by carbodiimide grafting of cell-membrane receptor ligand folic acid (FA) on MF-nHAp surface aminized with dendrigraft polymer, polyethyleneimine (PEI). The FA-PEI-MF-nHAp conjugates showed specific aggregation on FR +ve cells while leaving the negative control cells untouched. Nanotoxicity evaluation of this multifunctional nHAp carried out on primary human endothelial cells (HUVEC), normal mouse lung fibroblast cell line (L929), human nasopharyngeal carcinoma (KB) and human lung cancer cell line (A549) revealed no apparent toxicity even upto relatively higher doses of 500 μg/mL and 48 h of incubation. Flow-cytometry based reactive oxygen species (ROS) analysis also showed no significant levels of ROS generation in the nHAp treated cells. The tri-modal contrast imaging functionality together with molecular receptor targeting capability and biocompatibility makes MF-nHAp a promising biomineral contrast agent for combinatorial molecular imaging.

Biodegradable Polyphosphazene-Nanohydroxyapatite Composite Nanofibers: Scaffolds for Bone Tissue Engineering

Bone is a natural composite comprised of hierarchically arranged collagen fibrils, hydroxyapatite and proteoglycans in the nanometer scale. This preliminary study reports the fabrication of biodegradable poly[bis(ethyl alanato)phosphazene]-nanohydroxyapatite (PNEA-nHAp) composite nanofiber matrices via electrospinning. Binary solvent compositions of THF and ethanol were used as a spinning solvent to attain better nanohydroxyapatite dispersibility in PNEA solution. These nanocomposites were characterized for morphology, nHAp distribution and content using spectroscopy and gravimetric estimations. Composite nanofibers fabricated in the diameter range of 100-310 nm could encapsulate 20-40 nm nHAp crystals. A better composite nanofiber yield was obtained for 50% (w/w) nHAp experimental loadings. Incremental experimental loading beyond 60% (w/w) hindered electrospinning due to polymer-nHAp phase separation. Composites nanofibers had a rougher surface and nodules along the length of the fibers suggesting nHAp encapsulation. Further, characterization via energy dispersive X-ray spectroscopy and X-ray mapping confirmed the nHAp encapsulation. Providing cells with a natural bone like environment with a fibrillar structure and natural hydroxyapatite can enhance bone tissue regeneration/repair.