Zinc-containing Hydroxyapatite Research Articles

Zinc-doped Calcium Phosphate Coating on Titanium Surface Using Ostrich Eggshell as a Ca2+ Ions Source

Natural calcium source materials can be thermochemically processed and directly transformed into hydroxyapatite. In this work, ostrich eggshell was used as Ca2+ ions source in the preparation of zinc-calcium phosphate coatings on Ti substrates promoted by a two-step deposition method. The obtained coatings were characterized by X-ray diffraction (XRD) analysis, scanning electron microscopy with field emission gun (FEG-SEM), bioactivity using McCoy medium and toxicity with chicken embryo chorioallantoic membrane (CAM). The results showed the presence of Znbrushite and Zn-hydroxyapatite as coating phases. Bioactivity was observed in all samples showing bone-like apatite on their surface. The toxicity test showed inflammatory response between mild to moderate confirming the material’s biocompatibility. The use of ostrich eggshells was shown to be effective as a Ca2+ ions source to obtain zinc-containing hydroxyapatite coatings on Ti substrates.

Zinc-Containing Hydroxyapatite Enhances Cold-Light-Activated Tooth Bleaching Treatment In Vitro.

Cold-light bleaching treatment has grown to be a popular tooth whitening procedure in recent years, but its side effect of dental enamel demineralization is a widespread problem. The aim of this study was to synthesize zinc-substituted hydroxyapatite as an effective biomaterial to inhibit demineralization or increase remineralization. We synthesized zinc-substituted hydroxyapatite containing different zinc concentrations and analysed the product using X-ray diffraction (XRD), Fourier transform infrared (FTIR) spectroscopy, and energy dispersive spectrometer (EDS). The biological assessment of Zn-HA was conducted by CCK-8 assay and bacterial inhibition tests. pH cycling was performed to estimate the effect of Zn-HA on the enamel surface after cold-light bleaching treatment. The XRD, FTIR, and EDS results illustrated that zinc ions and hydroxyapatite combined in two forms: (1) Zn2+ absorbed on the surface of HA crystal and (2) Zn2+ incorporated into the lattice of HA. The results indicated that 2% Zn-HA, 4% Zn-HA, and 8% Zn-HA effectively inhibited the growth of bacteria yet showed poor biocompatibility, whereas 1% Zn-HA positively affected osteoblast proliferation. The XRD and scanning electron microscopy (SEM) results showed that the use of Zn-HA in pH cycling is obviously beneficial for enamel remineralization. Zinc-substituted hydroxyapatite could be a promising biomaterial for use in cold-light bleaching to prevent enamel demineralization.

Physico-chemical and Histomorphometric Evaluation of Zinc-containing Hydroxyapatite in Rabbits Calvaria.

The aim of this study was to characterize the physico-chemical properties and bone repair after implantation of zinc-containing nanostructured porous hydroxyapatite scaffold (nZnHA) in rabbits' calvaria. nZnHA powder containing 2% wt/wt zinc and stoichiometric nanostructured porous hydroxyapatite (nHA - control group) were shaped into disc (8 mm) and calcined at 550 °C. Two surgical defects were created in the calvaria of six rabbits (nZnHA and nHA). After 12 weeks, the animals were euthanized and the grafted area was removed, fixed in 10% formalin with 0.1 M phosphate buffered saline and embedded in paraffin (n=10) for histomorphometric evaluation. In addition, one sample from each group (n=2) was embedded in methylmethacrylate for the SEM and EDS analyses. The thermal treatment transformed the nZnHA disc into a biphasic implant composed of Zn-containing HA and Zn-containing β-tricalcium phosphate (ZnHA/βZnTCP). The XRD patterns for the nHA disc were highly crystalline compared to the ZnHA disc. Histological analysis revealed that both materials were biologically compatible and promoted osteoconduction. X-ray fluorescence and MEV-EDS of nZnHA confirmed zinc in the samples. Histomorphometric evaluation revealed the presence of new bone formation in both frameworks but without statistically significant differences (p>0.05), based on the Wilcoxon test. The current study confirmed that both biomaterials improve bone repair, are biocompatible and osteoconductive, and that zinc (2wt%) did not increase the bone repair. Additional in vivo studies are required to investigate the effect of doping hydroxyapatite with a higher Zn concentration.

Osteoblastic Cells Response to Albumin Coatings on Zinc Containing Hydroxyapatite

In this work, zinc-containing hydroxyapatite (ZnHA) and hydroxyapatite (HA) discs were coated with native bovine serum albumin (BSA) using 100 μg/ml BSA solution at 37°C. Changes on BSA secondary structure from α-helix to β-turn were detected by deconvolution analyses of FTIR amide I band. Fluorescence images of MC3T3-E1 osteoblast cultured on HA and ZnHA discs revealed cells with elongated morphology, typical of 2D experiments, after 24 hours. An intense cytoskeleton organization was observed on samples coated with BSA.

Short-term in vivo evaluation of zinc-containing calcium phosphate using a normalized procedure

The effect of zinc-substituted calcium phosphate (CaP) on bone osteogenesis was evaluated using an in vivo normalized ISO 10993-6 protocol. Zinc-containing hydroxyapatite (ZnHA) powder with 0.3% by wt zinc (experimental group) and stoichiometric hydroxyapatite (control group) were shaped into cylindrical implants (2×6mm) and were sintered at 1000°C. Thermal treatment transformed the ZnHA cylinder into a biphasic implant that was composed of Zn-substituted HA and Zn-substituted β-tricalcium phosphate (ZnHA/βZnTCP); the hydroxyapatite cylinder was a highly crystalline and poorly soluble HA implant. In vivo tests were performed in New Zealand White rabbits by implanting two cylinders of ZnHA/βZnTCP in the left tibia and two cylinders of HA in the right tibia for 7, 14 and 28days. Incorporation of 0.3% by wt zinc into CaP increased the rate of Zn release to the biological medium. Microfluorescence analyses (μXRF-SR) using synchrotron radiation suggested that some of the Zn released from the biomaterial was incorporated into new bone near the implanted region. In contrast with previous studies, histomorphometric analysis did not show significant differences between the newly formed bone around ZnHA/βZnTCP and HA due to the dissolution profile of Zn-doped CaP. Despite the great potential of Zn-containing CaP matrices for future use in bone regeneration, additional in vivo studies must be conducted to explain the mobility of zinc at the CaP surface and its interactions with a biological medium.

Surface modification of zinc-containing hydroxyapatite by tartaric acid

The surface of Zn-substituted hydroxyapatites (Ca(10−x)Znx(PO4)6(OH)2, with 0≤x≤2, CaxZnxHA) modified by tartaric acid was investigated by various techniques. XRD powder patterns revealed that synthesized samples were single phase of HA with broadened XRD reflections. FTIR and (13C, 1H and 31P) MAS NMR measurements confirmed the grafting of the tartaric acid on the apatite surface. Chemical analysis showed that for ZnHA the grafted acid amount increases with the increase of the acid concentration in the starting solutions, while for HA, it remained constant, indicating more reactivity for ZnHA surface. A mechanism of surface modification is proposed based on the obtained results.

Long-term biocompatibility evaluation of 0.5 % zinc containing hydroxyapatite in rabbits

This study investigates the long-term biocompatibility of 0.5% zinc-containing hydroxyapatite compared with hydroxyapatite. Spheres (425<∅<550) of both materials were produced by extrusion of ceramic slurry in calcium chloride and characterized by FTIR, XRD, XRF and SEM. Fifteen White New Zealand rabbits were submitted to general anesthesia, and an perforation (2mm), was made in each tibia, one for zinc-containing hydroxyapatite sphere implantation and one for hydroxyapatite sphere implantation. After 26, 52 and 78weeks, the animals were euthanized, and the fragment containing the biomaterial was harvested. A 30-50μm section was obtained for histological analysis in bright field and polarized light. SEM images revealed similar morphologies between the tested biomaterials. Histological analysis showed that there was no difference between the test groups. The morphometric analysis, however, indicates that there was a greater absorption. The materials are biocompatible, promote osteogenesis and that the zinc-containing hydroxyapatite microspheres were absorbed more quickly.

Cytocompatibility and Structural Arrangement of the Collagen Fibers: An &lt;i&gt;In Vitro&lt;/i&gt; and &lt;i&gt;In Vivo&lt;/i&gt; Evaluation of 5% Zinc Containing Hydroxyapatite Granules

Hydroxyapatite (HA) is one of the most employed materials for bone therapy due to its structural similarity with bone, its biocompatibility and physicochemical properties. Additionally, HA performance may be improved by ionic substitution of calcium with divalent bioactive metallic cations such as zinc. In this context, zinc incorporation into HA have been well studied, in spite of conflicting results regarding its biocompatibility: while previous reports on in vitro cytocompatibility have described 5% zinc containing HA (ZnHA) as slightly cytotoxic, this material presented an excellent response on in vivo studies. In order to bring more information on ZnHA biocompatibility, we performed a multiparametric assay evaluating sequentially on the same cells three different viability parameters: mitochondrial activity (XTT), membrane integrity (Neutral Red) and cell density (Crystal Violet Dye Exclusion test). Additionally, we intended to complement the existing data on ZnHA in vivo performance, by assessing its ability to affect the arrangement of collagen fibers on the grafted area, an important indicative of bone maturation. MC3T3-E1 cells were exposed to 24-hours extracts of ZnHA or stoichiometric HA on culture medium (DMEM) and cell viability was assayed. ZnHA was very cytocompatible, since the levels of viable cells on all 3 tests were similar to the HA and polystyrene (negative control) extracts, but significantly higher than cells treated with 4% phenol (positive control). For the in vivo studies, critical size defects in rats calvaria were filled with HA or ZnHA granules. The histological evaluation after 30 and 180 days revealed an increase along time. Event tough ZnHA is cytocompatible the presence of Zn was unable to alter the interaction between collagen fibers and the mineral bone phase, as compared to stoichiometric HA.

Osseoinduction Evaluation of Hydroxyapatite and Zinc Containing Hydroxyapatite Granules in Rabbits

This study investigated the osteoinductive potential of granules of stoichiometric hydroxyapatite (HA) and 0.5% zinc containing hydroxyapatite (ZnHA) in intramuscular (IM) site of rabbit’s abdomen. The biomaterials were both used in granular form, with 75% porosity and particle diameter between 450 and 500μm, sintered at 1100°C. Both materials performed adequately on a multiparametric in vitro cytocompatibility assay, indicating their suitability for in vivo testing. After approval by the Ethics Commission on Teaching and Research in Animals, fifteen rabbits were submitted to general anesthesia, incision and tissue dilatation, and a small site was created for HA (right incision) and ZnHA (left incision) intramuscular implantation. The animals were killed after 2, 4 and 12 weeks for biomaterials and surrounding tissues removal. Histological analysis after 2 weeks revealed the presence of granulation tissue surrounding biomaterials with multinucleated giant cells and no newly formed bone for both materials. After 4 weeks there was fibrous tissue involving the material and few inflammatory cells. Following 12 weeks it was observed the presence of connective tissue surrounding the biomaterial, cellularized enough for the two experimental groups, but it was not observed the presence of bone matrix associated with the biomaterials. We conclude that both biomaterials are cytocompatible and did not present the property of osseoinduction after 12 weeks of implantation.

Sustained Efficacy of Erythropoietin with a Hydroxyapatite Carrier Administered in Mice

For chronic kidney disease patients with renal anemia, recombinant human erythropoietin (rHuEPO) is a very effective drug; however, the treatment regime is troublesome, requiring multiple administrations each week. In the present study, we examined the efficiency of hydroxyapatite (HAp) as a drug delivery carrier for the sustained release of erythropoietin (EPO) to reduce the frequency of administration. Spray-dried HAp microparticles, formed from zinc-containing HAp (Zn-HAp) and Zn-HAp calcined at 400 degrees C, were used as carriers of EPO, and five Zn-HAp formulation samples incorporating EPO were prepared; no formulation, poly-L-lactic acid (PLA) formulation, zinc (Zn) formulation, Zn/PLA formulation, and calcined/Zn/PLA formulation. ICR mice were administered these samples or commercial rHuEPO (Epogin) as a control from dorsal neck subcutaneous, and hematological and histopathological analyses, including enzyme-linked immunosorbent assay for plasma EPO concentration, were performed. An increase in the blood EPO level was detected on days 3 and 8 post-administration. Peak hematopoiesis was delayed and higher hematological values were obtained on day 14 post-administration with no serious adverse reactions compared with the control. The Zn/PLA formulation sample was found to be most effective in reducing the initial peak while sustaining the delayed release of EPO. In conclusion, the Zn-HAp formulation samples were considered to be useful carriers for the sustained release of EPO, and the Zn/PLA formulation appears to be the most effective of five Zn-HAp formulation samples in sustaining EPO release.

Histomorphometric Analysis of Bone Repair in Critical Size Defect in Rats Calvaria Treated with Hydroxyapatite and Zinc-Containing Hydroxyapatite 5%

Biomaterials for treatment of bone defects have been studied for a long time. Alloplastic materials, mainly hydroxyapatite (HA), are under intense investigation due to its biocompatibility and osteoconductive properties. The HA can be modified by the incorporation of bivalent cations as Zn2+ known as a positive effectors for bone repair. The purpose of this study was to evaluate comparatively the effectiveness of 5% zinc-containing hydroxyapatite (ZnHA) in the treatment of critical size defect (CSD) in rat’s calvaria. CSD (8mm diameter) created in the skull of forty-five Wistar rats were filled with autogenous bone, HA and ZnHA. Skulls harvested after 30, 90 and 180 days were submitted to histological processing for paraffin embedding. Sections of 5 µm-thick stained with hematoxylin and eosin (HE) allowed histomorphometric analysis. The area of neoformed bone increased (p&lt;0.001) from 30 to 180 days irrespective to treatment groups. ZnHA and the control group showed a large at 180 days but no significant difference compared to HA. Therefore, we concluded that both biomaterials are biocompatibles and osteoconductors, promoting new bone formation and apposition of bone on the surface throughout the periods and the addition of zinc improved the osteogenesis.

Effect of Hydroxyapatite and Zinc-Containing Hydroxyapatite on Osseous Repair of Critical Size Defect in the Rat Calvaria

Hydroxyapatite (HA), widely used as bone graft, can be modified by the incorporation of bivalent cations (Mg2+ and Zn2+) and its gradual release could favor the bone repair. The purpose of this research was to evaluate the effect of the HA and zinc-containing hydroxyapatite (Zn-HA) in the bone repair in rat calvaria in comparison to autogenous bone. Critical size defect in the calvaria was filled with the graft material and the samples were harvested at the 30, 90 and 180 days. The light microcopy observations showed the biocompatibility of the graft materials. In the Zn-HA group the area of neoformed bone was larger than in the HA group, but smaller than in the autograft. A fibrous connective tissue was more evident around HA granules. It could be conclude that the presence of zinc ions in HA crystal accelerated the osteogenesis and increased the area of newly formed bone in relation to HA.

Antibacterial Effects of Sol-Gel Derived Zinc-Containing Hydroxyapatite

In order to modify the biological properties of pure hydroxyapatite (HAp), two kinds of zinc-containing HAp, which had zinc content of 5.10wt% and 2.54wt%, respectively, were synthesized via sol-gel technique. The obtained coatings were characterized by X-ray diffraction (XRD) and scanning electron microscope (SEM). Then the antibacterial effects were evaluated through bacteriostatic test on Streptococcus mutans (S.mutans) in vitro and zinc ions releasing ability was investigated through detecting the zinc ions concentration by inductively coupled plasma-atomic emission spectroscopy (ICP-AES). The obtained coatings were observed to possess typical apatite peaks in XRD patterns and high homogeneous and porous surfaces in SEM morphology. The coating layer demonstrated good releasing ability in Brain Heart Infusion (BHI) liquid and obviously inhibitory effects to the growth and proliferation of S. mutans. Based on the results obtained above, it is concluded that the sol-gel derived zinc-containing hydroxyapatite could be applied as an antibacterial effective biomaterial.

Development and characterization of 5% mol Zn bioceramic in granular form

Hydroxyapatite (HA) is capable of accepting substitute ions within its lattice, including zinc ions. Zinc is a trace element that activates the osteogenesis of osteoblastic cells and therefore plays an important role in the activity of alkaline phosphatase enzyme. The purpose of this work was to produce and characterize 5% mol Zn bioceramic in granular form (Zn-granules) for clinical applications and compare it with granules made from HA by using the same production route. Granules with addition of porogen agents were produced from powders of HA and zinc-containing HA by uniaxial pressing and heat treatment. The granules were subsequently ground and sieved. The results indicated that zinc contributed to the reduction of sample crystallinity and formed a biphasic structure after calcination at 1200 °C. Additionally, zinc release from granular material may have clinical applications as bone graft.

Selective protein adsorption property and characterization of nano-crystalline zinc-containing hydroxyapatite

Nano-crystalline Zn-containing hydroxyapatite (ZnHAp) was prepared by the wet-chemical method and the selective adsorption of essential proteins was examined, taking bovine serum albumin (BSA) and pathogenic protein such as β 2-microglobulin (β 2-MG) as model proteins. Transmission electron microscopy observation and X-ray diffraction analysis indicated that the increase of Zn content led to smaller crystallites and their specific surface area of ZnHAps increased with increasing Zn content, accordingly. Furthermore, the amounts of BSA adsorption on ZnHAp particles decreased with increasing Zn content in spite of the increase in the specific surface area. It is thus concluded that nano-crystalline ZnHAps had a highly selective adsorption property with regard to β 2-MG.

X‐Ray absorption fine structure analysis of the local environment of zinc in dentine treated with zinc compounds

It has been reported that zinc oxide (ZnO) inhibits dentine demineralization. By using the X-ray absorption fine structure (XAFS) technique, our aims in this study were to provide information about the local environment of zinc atoms in dentine that had been treated with zinc compounds. We measured the Zn K-edge X-ray absorption near-edge structure (XANES) and the extended X-ray absorption fine structure (EXAFS) of dentine specimens treated with zinc chloride or ZnO. In XAFS analyses, the spectra of dentine specimens treated with ZnO (D-ZO) or with zinc chloride (D-ZC) were similar and obviously different from the reference ZnO spectrum. This suggests that most of the zinc atoms detected in D-ZO are not derived from particles of ZnO. The spectra of D-ZO and D-ZC were similar to the spectrum of the synthetic, zinc-containing hydroxyapatite, but were not similar to that of zinc in ZnCl2-treated collagen. The results of this study suggest that most of the zinc atoms detected were attached to hydroxyapatite and not to collagen.