Nano Hydroxyapatite Research Articles

Transparent Nano Hydroxyapatite Coated Platform for Bone Cell Imaging

Hydroxyapatite is an essential inorganic component of bone tissue that is important for bone cell biology. In laboratory, bone cells are routinely cultured on glass or plastic plates with lacking of HA. Some commercial plates coated with HA are now available for investigating osteoclast bone resorption. However, the opaque nature of these plates limits further application to investigating bone cell biology. It's impossible to observe real-time interactions between hydroxyapatite and bone cells under microscope. In this study, glasses with HA nano-coatings were prepared by hydrothermal process. These nano-coating platform(NCP) offering a stage to directly image real-time interactions between hydroxyapatite and bone cells. The ease of fabrication and the repeatability of this platform approach could open a host of biomedical imaging applications in bone biology research as well as bone drug development. Different osteocytes were used to detect the biocompatibility and imaging advantages of the platform. Results showed that the platform could simulate the native bone environment and reflected the state of the bone cells accurately.

Recent trends in the application of widely used natural and synthetic polymer nanocomposites in bone tissue regeneration.

The goal of a biomaterial is to support the bone tissue regeneration process at the defect site and eventually degrade in situ and get replaced with the newly generated bone tissue. Nanocomposite biomaterials are a relatively new class of materials that incorporate a biopolymeric and biodegradable matrix structure with bioactive and easily resorbable fillers which are nano-sized. This article is a review of a few polymeric nanocomposite biomaterials which are potential candidates for bone tissue regeneration. These nanocomposites have been broadly classified into two groups viz. natural and synthetic polymer based. Natural polymer-based nanocomposites include materials fabricated through reinforcement of nanoparticles and/or nanofibers in a natural polymer matrix. Several widely used natural biopolymers, such as chitosan (CS), collagen (Col), cellulose, silk fibroin (SF), alginate, and fucoidan, have been reviewed regarding their present investigation on the incorporation of nanomaterial, biocompatibility, and tissue regeneration. Synthetic polymer-based nanocomposites that have been covered in this review include polycaprolactone (PCL), poly (lactic-co-glycolic) acid (PLGA), polyethylene glycol (PEG), poly (lactic acid) (PLA), and polyurethane (PU) based nanocomposites. An array of nanofillers, such as nano hydroxyapatite (nHA), nano zirconia (nZr), nano silica (nSi), silver nano particles (AgNPs), nano titanium dioxide (nTiO2), graphene oxide (GO), that is used widely across the bone tissue regeneration research platform are included in this review with respect to their incorporation into a natural and/or synthetic polymer matrix. The influence of nanofillers on cell viability, both in vitro and in vivo, along with cytocompatibility and new tissue generation has been encompassed in this review. Moreover, nanocomposite material characterization using some commonly used analytical techniques, such as electron microscopy, spectroscopy, diffraction patterns etc., has been highlighted in this review. Biomaterial physical properties, such as pore size, porosity, particle size, and mechanical strength which strongly influences cell attachment, proliferation, and subsequent tissue growth has been covered in this review. This review has been sculptured around a case by case basis of current research that is being undertaken in the field of bone regeneration engineering. The nanofillers induced into the polymeric matrix render important properties, such as large surface area, improved mechanical strength as well as stability, improved cell adhesion, proliferation, and cell differentiation. The selection of nanocomposites is thus crucial in the analysis of viable treatment strategies for bone tissue regeneration for specific bone defects such as craniofacial defects. The effects of growth factor incorporation on the nanocomposite for controlling new bone generation are also important during the biomaterial design phase.

Investigation of the physical, mechanical and thermal properties of nano and microsized particulate-filled dental composite material

Objective The purpose of this study was to fabricate bisphenol-a glycidyl methacrylate /tri-ethylene glycol dimethacrylate (TEGDMA)-based nano and micro-sized-filled dental composite and to study the effect of varying silane-treated nano hydroxyapatite on physical, mechanical and thermal properties. Methods The dental resin matrix was fabricated by bisphenol-a glycidyl (53 wt.%), TEGDMA (46 wt.%), camphorquinone (0.3 wt.%) and 2-(dimethylamino) ethyl methacrylate (0.7 wt.%). Silane-treated nano hydroxyapatite (nHA) filler content was varied from 0 to 8 wt.%, while zinc oxide (microsized) remains constant with 20 wt.%. A light curing unit was employed for polymerization of the resinous dental mixture. Results The result revealed that infrared spectra of untreated and treated nHA filler particles were confirmed by Fourier transform infrared spectroscopy. The dental composite filled with 6 wt.% nHA (DHZ6) exhibited maximum compressive strength (249 MPa) and Vickers hardness (49 Hv), while dental composite filled with 8 wt.% nHA (DHZ8) showed minimum depth of cure (5.12 mm), degree of conversion (54%) and polymerization shrinkage (1.12%). Significance Silane treatment of nano hydroxyapatite has a significant role in physical, mechanical, and thermal properties. Bonding strength of coupling agent has been seen between organic and inorganic materials.

A Comprehensive Research on Gamma-Ray Attenuation Characteristics of the Metal-Doped Hap and Natural Animal Bone

This investigation focuses on the gamma ray attenuation parameters of natural animal bone, iron, cobalt, copper and zinc doped Nano hydroxyapatite artificial bone powders and pure hydroxyapatite. The present specimens were excited by means of gamma ray photons with 59.5 keV energy radiated from a 241-Amercium annular radioactive source with 50 mCi activity by means of narrow beam transmission geometry. The transmitted gamma-rays were counted with using Ultra Low Energy Germanium (Ultra-LEGe) detector with a resolution 150 eV at 5.95 keV . The gamma-ray attenuation characteristics such as mass attenuation coefficient (MAC) ( µ/ρ ), linear attenuation coefficient (LAC) ( µ) , half value layer (HVL), tenth value layer (TVL) and mean free path (MFP), which has a significant role in the attenuation studies, are also calculated. The values obtained were checked with the predicted values from the XCOM NIST. The results show that the attenuation data of the metal-doped hydroxyapatite synthetic bone dust is very close to the value of the natural animal bone.

Effect of preheating and ultrasonic energy on penetration of low viscosity enamel resin infiltrant

Objectives: to determine the depth of resin infiltrated hybrid enamel after application of TTEMA/TEGDMA resin infiltration agent enriched with nano-hydroxy apatite applied on early carious enamel lesions.Materials and Methods: Sound maxillary centrals were coated with a nail varnish, leaving a window of 4 mm × 4 mm on buccal surface of sound and intact enamel. All specimens were subsequently immersed in a demineralizing solution to produce artificial enamel lesions then etched with a mixture of 2% chlorhexidine and 10% hydrochloric acid. After washing and drying the enamel was coated with a low viscosity TTEMA/TEGDMA resin infiltration agent followed by light polymerization. The effect of addition of 10% nano-hydroxy apatite, preheating at 45oC, and ultrasonic activation was tested in separate groups. Sectioned specimens were examined under a stereomicroscope equipped with a high-resolution digital camera for determining of the depth of penetration of the low viscosity resin in microns (α=0.05).Results: Data analysis revealed significant difference in depth of enamel hybrid layer between the tested groups (F=14.2, P<0.001). Average penetration depth was 185 ± 17 µm for TTEMA/TEGDMA resin infiltration agent. Addition of 10% nano-hydroxy apatite reduced penetration depth to 141± 19 µm. On the contrary, preheating and ultrasonic activation increased penetration depth to 211± 13 µm and 221 ± 12 µm respectively.Conclusions: Within the limitations of this study, the penetration depth of TTEMA/TEGDMA resin infiltration agent was significantly influenced by addition of nano-hydroxy apatite particles. Preheating and ultrasonic activation increased penetration depth significantly.

Comparative evaluation of remineralising efficacy of bioactive glass agent and nano-hydroxyapatite dentifrices on artificial carious lesion in primary teeth: An in vitro study

Introduction: The phenomenon of reversal of incipient or early enamel caries forms an important part of prevention leading to apparent repair of the lesion. Recently, novel biomaterials such as the bioactive glass (BAGs) and nano-hydroxyapatite (n-HAP) crystal have been introduced as remineralising agents. Aim: This study aimed to evaluate and compare the remineralising efficacy of BAG agent and nano-hydroxyapatite (NHA) dentifrices on artificial carious lesion in primary teeth. Methodology: Thirty human extracted primary teeth were selected for the study, and the specimens were randomly divided into Group A: BAGs agent toothpaste group and Group B: n-HAP toothpaste group. The surface microhardness of the teeth was measured using a Vickers microhardness tester at the baseline and after demineralisation and remineralisation. The statistical analysis was done using independent t-test for intergroup comparison and paired t-test for intragroup comparison. Results: On intergroup comparison, microhardness differences of baseline to exposure to dentifrice between the two groups showed that microhardness differences of baseline to exposure to dentifrice were higher in Group B with a t value of −2.158 and was statistically significant (P

Oxidation of pesticide (Coragen) using triple oxide coated titanium electrodes and nano hydroxyapatite as a sorbent

Oxidation of pesticide (Coragen) using triple oxide coated titanium electrodes and nano hydroxyapatite as a sorbent

In vitro study of Nano Hydroxyapatite/Streptomycin -Gelatin-Based Injectable Bone Substitute Associated- 3D printed Bone Scaffold for Spinal Tuberculosis Case

According to WHO (2018) Indonesia was placed in the third ranked of the highest number of TB sufferers worldwide. TB was caused by the Mycobacterium tuberculosis. This bacterial infection not only could affect the lungs, but also bone tissue, especially the spine, which then called as spinal tuberculosis. There had been many alternatives to overcome this case. One of them was by using Injectable Bone Substitutes (IBS) based on nano Hydroxyapatite (nanoHA), gelatin and streptomycin. The severe sponal tuberculosis case might cause the bone defect. Due to this bone defect, IBS alone would not be able to handle this issue. Thus, scaffold was needed to replace and support the infected bone. Besides, the combination between IBS paste and bone scaffold could help the bone tissue regeneration. The nanoHA and gelatin contents of the IBS paste provided the mineral which was need for bone tissue regeneration. While the bone scaffold would support those contents by providing the extracellular matrix for the new bone tissue attachment. The IBS associated- scaffold could be an alternative to deliver spinal tuberculosis’ drug locally. This study aimed to evaluate the performance of this combination through antibacterial study and drug release test using UVVis spectrophotometer. As well as the cytotoxicity test to know the risk of these two materials within the body. The IBS was synthesized by mixing nano hydroxyapatite and 20 w/v% gelatin with ratio of 65:35 and 10% streptomycin addition as antibiotic agent. The mixture was added by hydroxypropyl methylcellulose as suspending agent. FTIR test showed that there was a chemical reaction occurred in the mixture. The performance of this combination showed that the released- streptomycin, qualitatively, could kill the Staphylococcus aureus trough the antibacterial test. Furthermore, it also had been proven that the IBS associated- scaffold could release an increasing streptomycin concentration over time. In addition, the combination showed a good result in cytotoxicity test where the cell viability was more than 50%. According to the results mentioned before, the IBS paste associated- scaffold was a promising breakthrough to overcome spinal tuberculosis case as a local drug deliver.

Implantation of an Injectable Bone Substitute Material Enables Integration Following the Principles of Guided Bone Regeneration.

The present study investigates the in vivo tissue reaction and the integration behavior of an injectable bone substitute material (IBS) composed of a water-based gel combined with nano hydroxyapatite particles and biphasic calcium phosphate granules. The results of the IBS were compared to biphasic bone substitute granules (BBSM) of the same chemical composition. The subcutaneous implantation model in 40 female 5-week-old CD-1 mice up to 60 days after implantation was used for conduction of the in vivo experiments. Moreover, established histological, histopathological and histomorphometrical methods were applied. The results showed that the IBS was gradually invaded by cells and complex tissue elements. Thus, the implant bed could be distinguished in two areas, i.e. an outer and inner region. While the outer region started to interact with the peri-implant tissue by evoking multinucleated giant cells and at earlier time points by undergoing a continuous high vascularization, the inner part was free of peri-implant cells for at least 30 days, starting to undergo a similar tissue reaction at a later time point. The bone substitute granules allowed for a fast tissue influx between the interspaces of the granules starting at day 10. While the vessel density did not differ in both groups up to the end of the study, the amount of vascularization was significantly higher over the entire observation period in the BBSM group. Moreover, the amount of biomaterial-associated multinucleated giant cells (BMGCs) was significantly higher in the IBS group in the period of between 15 to 30 days after implantation, while comparable BMGC numbers were found in both groups towards the end of the study. IBS can build a barrier-like structure that is able to control the soft tissue influx into the central regions of the implantation bed, which could not be observed in other bone substitute granules of the same chemical composition. This directed integration behavior is assumed to be in accordance with the concept of Guided Bone Regeneration (GBR). Furthermore, BMGCs can significantly influence the process of angiogenesis within an implant bed of a biomaterial but not the maturity of blood vessels.

Biomineralization of 2304 duplex stainless steel with surface modification by electrophoretic deposition.

A hydroxyapatite (HAp) coating on 2304 duplex stainless steel (DSS) through an electrophoretic deposition process has been investigated in this work. The deposition voltage was 30 V with a time of 2 min. Field emission scanning electron microscopy and energy dispersive X-ray spectroscopy analyses were used for the microstructural and chemical examination of coatings, respectively. The Ca to P ratio for the nano HAp coating on 2304 DSS has been determined as equal to 1.642. It was observed from X-ray diffraction patterns that HAp nanoparticles were successfully combined into the substrate. The corrosion behavior of all samples was tested in simulated body fluid using a potentiodynamic polarization study. A homogeneous structure with a thin crack-free layer was obtained. Moreover, the porosity of this coating was very low leading to a high corrosion resistance, thus promoting good biocompatibility.

Radiation Synthesis of Magnesium Doped Nano Hydroxyapatite/(Acacia-Gelatin) Scaffold for Bone Tissue Regeneration: In Vitro Drug Release Study

Novel three-dimensional biodegradable porous nanocomposite bone scaffolds were fabricated using acacia gum and gelatin as the base polymer matrix and magnesium doped nano hydroxyapatite as cementing materials using gamma irradiation facility for crosslinking and sterilization processes. Mg-doped HAp nanoparticles were synthesized using wet chemical method. XRD studies verified the nano-scale size of the prepared HAp. In addition to Ca and P in the prepared n-HAp, the EDX analysis revealed the presence of Mg in the doped HAp samples. FTIR studies confirmed the existence of the characteristic functional groups of the scaffold constituents. The swelling behavior was found to be dependent on the quantity of embedded HAp nanoparticles. Nanocomposite scaffold porosity ranged from 26 to 39%, which increased with the inclusion of Mg ions. The developed scaffolds showed appropriate mechanical properties that enhanced by the existence of HAp nanoparticles. The incorporation of the Mg-doped HAp nanoparticles encourages the development of bone-like apatite layer. In vitro cytotoxicity assessment and blood compatibility demonstrated their biocompatibility. The developed scaffolds show promising antibacterial activity against Staphylococcus aureus and Escherichia coli. In vitro drug release study showed that the loaded Ketoprofen scaffolds were able to deliver the loaded drug sustainably.

Effect of anions on the structural,morphological and dielectric properties of hydrothermally synthesized hydroxyapatitenanoparticles

Synthetic nano hydroxyapatites (HA) have been considered aspotential biomaterials for bone tissue engineering applications because of itsexcellent biological properties. The present work deals with the synthesis of HAnanoparticles from different anion source materials via autoclave assistedhydrothermal method. All the prepared HA nanoparticles were characterized byX-ray diffraction (XRD), Fourier transformation infrared spectra, field emissionscanning electron microscopy, energy dispersive spectra and high resolutiontransmission electron microscopy. The XRD patterns reveal the pure and hexagonalphase structure with smaller crystallite size for HA obtained from variouscalcium salt precursors. HA particles prepared from nitrate precursors showspherical morphology with 32 nm grain size whereas those derived from theacetate, chloride and egg shell precursors respectively show needle-like,irregular and oval morphology. The effect of different anions on the dielectricproperties and alternating conductivity of HA is investigated, as a polarizedsurface can trigger biological reactions. For the particles obtained fromnitrate, acetate, chloride and egg shell precursors respectively give dielectricconstant (εʹ) values of 9.96, 13.22, 9.92 and 10.86 at 5 MHz. The εʹ anddielectric loss (εʹʹ) values for the HA nanoparticles decrease with increase inthe applied frequency as well. The alternating current conductivity valuesconfirm that the as-synthesized HA samples exhibit insulating behavior. In shortthis article provides the various applicability of HA particles inoptoelectronics and drug delivery.Graphic abstract

Regeneration of Intrabony Defects With Nano Hydroxyapatite Graft, and Periosteum as Barrier Membrane Under Magnification

Regeneration of Intrabony Defects With Nano Hydroxyapatite Graft, and Periosteum as Barrier Membrane Under Magnification

A biomimetic nano-hydroxyapatite/chitosan/poly(methyl vinyl ether-alt-maleic anhydride) composite with excellent biocompatibility

Nano-hydroxyapatite/chitosan/poly(methyl vinyl ether-alt-maleic anhydride) (NHA/CS/P(MVE-alt-MA)) composite was formed through electrostatic interaction. The results of characterization indicated that the nano-hydroxyapatite (NHA) in the composites was uniformly distributed in the polymer matrices (chitosan/poly(methyl vinyl ether-alt-maleic anhydride) (CS/P(MVE-alt-MA)). With the increase of NHA content, the porosity and strength of the composites increase first and then decrease. The mechanical properties of composite were better than those of the three kinds of single component, the maximum compressive strength of composite was up to 8.48 MPa. Moreover, this composite showed excellent biocompatibility through SD rat bone marrow mesenchymal stem cells (SDBMSCs) culture. Therefore, this composite which combined the advantages of various materials exhibited good mechanical properties and biocompatibility, it was expected to be applied in bone tissue repair.

Synthesis and characterization of hematite based calcium rich hydroxyapatite - A nano material from LD slag fines

Nano sized crystallites of hematite based calcium rich hydroxyapatite (CRHAP), a new product was synthesized by co-precipitation of the leach liquor obtained by hydrometallurgical treatment of non-metallic Linz and Donawitz (LD) slag with concentrated nitric acid. Leached liquor comprising mainly a mixture of iron and calcium as nitrates was taken for the synthesis of hematite based (CRHAP). The present study presents the innovative use of waste recycling plant (WRP) reject material mainly containing the non-metallic portion of LD Slag fines as a raw material for synthesis of the hematite based calcium rich hydroxyapatite nano material. The basis for this innovative synthesis process was the high calcium, iron and phosphorus content of the waste recycling plant material. In this hydrometallurgical process, the silica rich material can also be separated for further value addition. Elemental analysis of CRHAP was performed by using Inductively Coupled Plasma (ICP) Spectrometer. Characterization of the nano material was performed by using X-ray diffraction (XRD), Fourier transform infrared (FTIR) analysis, Raman spectroscopy and particle size analysis. XRD analysis revealed that the synthesized material was a hematite based calcium rich hydroxyapatite which was re-confirmed by FTIR analysis and Raman analysis. Scanning electron microscopy (SEM) analysis was also carried out to understand the morphology of the synthesized material. The average particle size d (0.5) of the nano material was about 24 μm and maximum size d (0.90) was 116 μm as determined by a laser particle size analyzer.

Effect of CPP ACP and Nano Hydroxyapatite Incorporated GIC on Remineralization of Dentin

The objective of this invitro study was to investigate, whether the remineralization potential of conventional GIC in human dentin could be improved by combining with CPP-ACP and nano hydroxyapatite. Class I cavities were prepared in sound human extracted third molars and they were randomly assigned to 3 groups according to the restorative materials. Group I: Conventional GIC, Group II: Conventional GIC and CPP-ACPand Group III: Conventional GIC powder & Nano hydroxy apatite. The samples were subjected to remineralizationand demineralization cycles for period of 2 weeks. Energy dispersive X-ray analysis was used to evaluate the remineralization. The elemental compositions were analyzed using One way ANOVA, Post Hoc and Mann Whitney tests. The calcium and phosphate concentration were statistically significantly greater in Group III than Group I and II (P<0.05). Nanohydroxy apatite with GIC remineralized the demineralized dentin almost equal to normal dentin.

Can Hydroxyapatite and Boron Oxide Nano-fertilizers Substitute Calcium Superphosphate and Boric Acid for Broccoli (Brassica oleracea var. italica) Grown on A Heavy Clay Soil?

Nano-fertilizers are effective substitutes for the traditional ones. However, to what extent can nano hydroxyapatite (NHA) and nano-boron oxide (NBO) be suitable substitutes for calcium superphosphate(CSP) and boric acid (BA)? The answer required conducting a field experiment on broccoli (Brassica oleraceavar. italica) cultivated on a heavy claysoil for two successive seasons of 2016/2017 and 2017/2018.The nano form of NHA surpassed CSP by 14.2 to 17.8 % for leaf area and 13.6 to 15.8% for total head yield. Foliar spray with NBO increased leaf area, head yield and vitamin C content in heads as compared with BA. P and B contents in broccoli shoots receiving the nano forms surpassed those of the conventional fertilizers. Combined application of the two nano-fertilizers surpassed the combined application of the two conventional fertilizers by 16.0 %. Values for yield and yield components were significantly positively correlated with P and B contents in plant shoots and heads.

SURFACE MORPHOLOGY AND MICROHARDNESS BEHAVIOR OF 316L IN HAP-PMEDM

The development of biomaterials for implants nowadays requires materials with superior mechanical and physical properties for enhanced osseointegration and sustained longevity. This research work was conducted to investigate the influence of nano hydroxyapatite (HAp) powder mixed electrical discharge machining (PMEDM) on surface morphology and microhardness of modified 316L stainless steel surface. The chosen process parameters were discharge current, pulse on/off duration and gap voltage in order to analyze the selected output responses. HAp concentration (15 g/l) along with reverse polarity was kept constant for current experimentation. The experimental results testified that surface morphology of PMEDM surface was significantly improved along with augmentation of 79% in microhardness (HV) of HAp modified surface of medical grade stainless steel. Furthermore, XRD and SEM characterization confirmed the deposition of calcium, phosphorous and inter-metallic compounds on HA-PMEDMed surface. The surface thus produced is expected to facilitate better bone-implant adhesion and bioactivity.

Nano-hydroxyapatite enhanced double network hydrogels with excellent mechanical properties for potential application in cartilage repair

Hydrogels with desirable characteristics have been supposed to be potential materials for cartilage repair. However, the biomechanical, biotribological and biocompatible properties of hydrogels remain some crucial challenges. To address these challenges, we developed a dual physically cross-linked poly (vinyl alcohol)-(nano hydroxyapatite)/(2-hydroxypropyltrimethyl ammonium chloride chitosan) (PVA-HA/HACC-Cit) hydrogels with double-network (DN) through a simply freezing/thawing technique and an immersing process. The DN hydrogel with an optimized HA concentration exhibited outstanding fracture tensile stress (2.70 ± 0.24 MPa), toughness (14.09 ± 2.06 MJ/m3) and compressive modulus (0.88 ± 0.09 MPa). In addition, the PVA-HA/HACC-Cit DN hydrogels demonstrated remarkable anti-fatigue property, extraordinary self-recovery and energy dissipation ability due to their unique dual physically cross-linked structures. Moreover, the low friction coefficient, the predominant wear resistance property, as well as the excellent cytocompatibility were realized for the DN hydrogels because of the existence of nano-hydroxyapatite. Thus, this work puts forward a new strategy in the preparation of DN hydrogels for promising applications in cartilage repair.

Maxillary sinus floor elevation using hydroxyapatite nano particles vs tenting technique with simultaneous implant placement: A randomized clinical trial.

The present study was conducted to evaluate the amount of bone height gain, density values, and implant stability after maxillary sinus floor elevation using graftless tenting technique or the use of Hydroxyapatite Nanoparticles bone substitute for augmentation with simultaneous implant placement. A total of 20 sinuses with a residual alveolar bone height ranging from 4-6 mm were divided into two groups and underwent sinus augmentation using nano hydroxyapatite bone substitute material and the graftless tenting technique with simultaneous implant placement. Computed tomography CT scans and ISQ measurements were conducted to evaluate bone quality, quantity, and implant stability. Radiographic analysis revealed that the mean bone height gain of the nano group was (7.0 ± 0.8 mm) compared to (5.0 ± 1.5 mm) in the tent group, which was statistically significant (P = .002) being higher in the nano group. The mean bone density value of the nano group was (548 ± 25 HU) compared to (420 ± 23 HU) in the tent group, which was statistically significant (P < .001) being higher in the nano group. The mean ISQ value after 6 months in the nano group was (78 ± 5) compared to (77 ± 5) in the tent group, which was statistically nonsignificant (P = .901). Nano hydroxyapatite bone graft offered superior results in terms of the bone height gain and the relative bone density as compared to graftless tenting technique. However, both techniques showed accepted results regarding implant stability.