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The knowledge on human tissue is very important to recognize desirable properties of biomaterials. Host cells, extracellular matrix, integrated vessels and interstitial fluids create a complex and dynamic system able to regenerate and respond to environmental stimuli. Myoglobin is a protein with most of α-helices on its secondary structure, and responsible for oxygen binding and release in muscles, by the heme group. This work investigates the Mb adsorption process onto zinc-hydroxyapatite (ZnHA) surface by spectroscopic studies. To do so, ZnHA (0.05 g) was incubated with 4mL of 2mg Mb/mL on phosphate buffer solution pH 6.0 for 24h at 37°C. The FTIR analyses of ZnHA powders before and after protein adsorption provided information concerning the protein content. UV-Vis spectrocopy in the reflectance mode suggested a mixture of MbO2 and Met Mb on lyophilized solid Mb, and the prevalence of MetMb form when Mb was adsorbed on ZnHA sample. The decrease of UV-Vis secondary bands suggests interactions through the Mb heme group and the ZnHA surfaces. Circular Dichroism (CD) spectroscopy indicated the maintenance of the Mb α-helices secondary structure after the adsorption process on ZnHA powders.

The hydroxyapatite (HA) is a biocompatible and bioactive biomaterial used as bone substitute, however, the high crystallinity of HA and consequently its low solubility may be a limitation for its clinical use. In order to improve the biosorption of HA, the partial substitutions in the chemical structure and doping with small amounts of impurities have been study. The objective of this study was to evaluate the biocompatibility of 3% Zinc-containing nanostructured carbonated hydroxyapatite (ZncHA) compared with the carbonated hydroxyapatite (cHA), both synthesized at 37°C and non-sintered, using as control the stoichiometric HA microspheres in subcutaneous of mice. The X-ray Diffraction (XRD) and Vibrational Spectroscopy in Infra Red Fourier Transform (FTIR) were used to characterize the biomaterials. In vivo test was performed in BALB/c mice by implanting of HA, cHA and ZncHA spheres in the subcutaneous tissue for 1, and 9 weeks (n=5). The negative control consisted in incision without material implantation (Sham group). The samples were histological processed to descriptive analysis of biological effect. The microscopic analysis showed a similar granulation reaction between groups at the first experimental period. In 9 weeks there was a time dependent biosorption of cHA compared with other groups. In conclusion, the biomaterials tested were biocompatible and cHA group showed a significant biosorption in comparison with HA and ZncHA groups. The doping of zinc did not influence the biocompatibility of biomaterial, however, change the biosorption response

The hydroxyapatite (HA) is a biocompatible and bioactive biomaterial used as bone substitute, however, the high crystallinity of HA and consequently its low solubility may be a limitation for its clinical use. In order to improve the biosorption of HA, the partial substitutions in the chemical structure and doping with small amounts of impurities have been study. The objective of this study was to evaluate the biocompatibility of 3% Zinc-containing nanostructured carbonated hydroxyapatite (ZncHA) compared with the carbonated hydroxyapatite (cHA), both synthesized at 37°C and non-sintered, using as control the stoichiometric HA microspheres in subcutaneous of mice. The X-ray Diffraction (XRD) and Vibrational Spectroscopy in Infra Red Fourier Transform (FTIR) were used to characterize the biomaterials. In vivo test was performed in BALB/c mice by implanting of HA, cHA and ZncHA spheres in the subcutaneous tissue for 1, and 9 weeks (n=5). The negative control consisted in incision without material implantation (Sham group). The samples were histological processed to descriptive analysis of biological effect. The microscopic analysis showed a similar granulation reaction between groups at the first experimental period. In 9 weeks there was a time dependent biosorption of cHA compared with other groups. In conclusion, the biomaterials tested were biocompatible and cHA group showed a significant biosorption in comparison with HA and ZncHA groups. The doping of zinc did not influence the biocompatibility of biomaterial, however, change the biosorption response

Synthetic hydroxyapatite (HA) is a widely used ceramic biomaterial due to its well described biocompatibility. Some modifications in HA surface can be made to increase surface porosity. Likewise, HA can be modified by the coating with proteins, which may impact on biocompatibility. In this work, we aimed to evaluate the impact of two surface modifications – coating with albumin, a major serum protein, and augmented porosity - over osteoblast adhesion on stoichiometric HA discs. Dense HA discs were obtained by pressing HA powder at 30 KN and sinterization at 1000°C, while porous HA was molded after the addition of alginate (15:1), followed by thermal treatment. Protein adsorption was attained by incubation on 0.5mg/mL bovine serum albumin (BSA) for 24 h at 37°C. MC3T3 mouse preosteoblasts were seeded over both protein-coated and uncoated dense or porous tablets, and cell viability after 24 h was estimated by XTT and Neutral Red assays. Cell density was quantified by fluorescence microscopy. While both dense and porous discs presented altered surfaces after protein treatment, as observed by scanning electron microscopy, porous HA tablets presented significantly higher levels of adsorbed protein. There was a decrease in the concentration of calcium ions in all samples analyzed. Porous HA treated with protein presented significant higher mitochondrial dehydrogenase activity (XTT) than non treated tablets (p

Carbonated apatite (CHA) is commonly considered a promising synthetic material for biomedical applications in orthopedic and dental surgery due to its biocompatibility, bioresorption and bioactivity. CHA5, CHA37 and CHA90 powders were synthesized from wet method and the DRX patterns showed that the crystallinity and particle size of CHA samples increased proportionally with the synthesis temperature. Powder extracts medium were obtained from each sample to interact with MC3T3-E1 osteoblastics cells. It was evaluated morphology, citotoxicity, pH and Ca2+ concentration. Citotoxicity assays showed high metabolic activity on all samples when compared to control. The polygonal shaped and the confluent monolayer observed in control cells progressively changed according to the crystallinity increase of samples. Cells under mitosis and spindle-like shaped where the main alterations observed. In addition the cell viability could be sensitive to the acid reactivity and crystallinity of carbonated apatite samples.

Cell adhesion, proliferation and differentiation are important specific parameters to be evaluated on biocompatibility studies of candidate biomaterials for clinical applications. Several different methodologies have been employed to study, both qualitative and quantitatively, the direct interactions of ceramic materials with cultured mammal and human cells. However, while quantitatively evaluating cell density, viability and metabolic responses to test materials, several methodological challenges may arise, either by impairing the use of some widely applied techniques, or by generating false or conflicting results. In this work, we tested the inherent interference of different representative calcium phosphate ceramic surfaces (stoichiometric dense and porous hydroxyapatite (HA) and cation-substituted apatite tablets) on different tests for quantitative evaluation of osteoblast adhesion and metabolism, either based on direct cell counting after trypsinization, colorimetric assays (XTT, Neutral Red and Crystal Violet) and fluorescence microscopy. Cell adhesion estimation after trypsinization was highly dependent on the time of treatment, and the group with the highest level of estimated adhesion was inverted from 5 to 20 minutes of exposition to trypsin. Both dense and porous HA samples presented high levels of background adsorption of the Crystal Violet dye, impairing cell detection. HA surfaces also were able to adsorb high levels of fluorescent dyes (DAPI and phalloidin-TRITC), generating backgrounds which, in the case of porous HA, impaired cell detection and counting by image processing software (Image Pro Plus 6.0). We conclude that the choice for the most suitable method for cell detection and estimation is highly dependent on very specific characteristics of the studied material, and methodological adaptations on well established protocols must always be carefully taken on consideration.

In this work myoglobin (Mb) adsorption was carried out in a batch system using hydroxyapatite (HA) powder during 24 hours at 37°C. The HA samples were analyzed after protein adsorption by Fourier Transformed Infrared (FTIR) and UV-Vis Spectroscopy in reflectance mode. UV-Vis analyses showed that Soret and Q bands shifted to lower wavelengths when Mb is associated with HA surface. This result suggests that Mb Heme group is sensitive to the protein adsorption onto HA surface. HA disks coated with myoglobin and cultured with human osteoblastic cells during 7 days showed that cell adhesion and proliferation were not inhibited by the protein coating after 7 days in cell culture.

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

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.

Crystalline hydroxyapatite thin coatings have been prepared using a novel opposing RF magnetron sputtering approach at room temperature. X-ray diffraction (XRD) analysis shows that all the principal peaks are attributable to HA, and the as-deposited HA coatings are made up of crystallites in the size range of 50-100nm. Fourier transform infrared spectroscopy (FTIR) studies reveal the existence of phosphate, carbonate and hydroxyl groups, suggesting that HA coatings are highly crystalline. To study the biocompatibility of these coatings, murine osteoblast cells were seeded onto various substrates. Cell density counts using fluorescence microscopy show that the best osteoblast proliferation is achieved on an HA RAMS-coated titanium substrate. These experiments demonstrate that RAMS is a promising coating technique for biomedical applications.