Cytotoxicity Evaluation of High-Temperature Annealed Nanohydroxyapatite in Contact with Fibroblast Cells

Biomaterials are substances manufactured for medical purposes in direct contact with the tissues of organisms. Prior to their use, they are tested to determine their usefulness and safety of application. Hydroxyapatites are used in medicine as a bony complement because of their similarity to the natural apatite therein. Thanks to their bioactivity, biocompatibility, stability and non-toxicity hydroxyapatite are the most commonly used materials in osteoimplantology. The use of materials at the nanoscale in medicine or biology may carry the risk of undesirable effects. The aim of the study was to evaluate the cytotoxic effect of high-temperature annealed nanohydroxyapatites on the L929 murine fibroblasts. Nanohydroxyapatites in powder form were obtained by the wet chemistry method: in the temperature range of 800–1000 °C and used for the study. Based on performed studies evaluating the morphology and fibroblast viability, it was found that nanohydroxyapatites show no cytotoxic effects on the L929 cell line.


Introduction
The demand for new biomaterials continues to grow due to the increasing average life expectancy, traumatism, lowering the age of the users, and the expectation of continuous improvements in the quality of life [1][2][3][4]. The range of biomaterial use is wide. Depending on the intended use of the material, it must have specific properties. Biomaterial used for bone substitution should be a safe material with a composition as close as possible to the natural bone tissue, which it is meant to replace. In analyzing the chemical composition of the bone system, it was found that the bones are composed of 30% organic and 70% inorganic substance, such as apatite (approx. 65% bone temperature, as a result of the well-known Ostwald ripening process. Depending on the sintering temperature, the crystallize size was estimated to be 45 nm for the sample sintered at 800 • C, 63 nm for the sample obtained at 900 • C and 82 nm for the sample annealed at 1000 • C, indicating progressive grain growth.
Materials 2017, 10, 590 3 of 13 temperature, the crystallize size was estimated to be 45 nm for the sample sintered at 800 °C, 63 nm for the sample obtained at 900 °C and 82 nm for the sample annealed at 1000 °C, indicating progressive grain growth.

TEM Microscopy
The final confirmation of particle size of the nHAP powders was done utilizing HRTEM microscopy (High Resolution Transmission Electron Microscopy, Philips CM-20 Super Twin microscope, Eindhoven, The Netherlands). In accordance with the TEM study (see Figure 2), the particles of Ca10(PO4)6(OH)2 sintered at 800 °C are regular with a mean particle size of 40 nm. Analysis of SAED (Selected Area Diffraction) pattern revealed the appearance of well developed rings with clear reflections at positions corresponding with a reference standard of calcium hydroxyapatite.

TEM Microscopy
The final confirmation of particle size of the nHAP powders was done utilizing HRTEM microscopy (High Resolution Transmission Electron Microscopy, Philips CM-20 Super Twin microscope, Eindhoven, The Netherlands). In accordance with the TEM study (see Figure 2), the particles of Ca 10 (PO 4 ) 6 (OH) 2 sintered at 800 • C are regular with a mean particle size of 40 nm. Analysis of SAED (Selected Area Diffraction) pattern revealed the appearance of well developed rings with clear reflections at positions corresponding with a reference standard of calcium hydroxyapatite.  , the crystallize size was estimated to be 45 nm for the sample sintered at 800 °C, 63 nm  for the sample obtained at 900 °C and 82 nm for the sample annealed at 1000 °C, indicating progressive grain growth.

TEM Microscopy
The final confirmation of particle size of the nHAP powders was done utilizing HRTEM microscopy (High Resolution Transmission Electron Microscopy, Philips CM-20 Super Twin microscope, Eindhoven, The Netherlands). In accordance with the TEM study (see Figure 2), the particles of Ca10(PO4)6(OH)2 sintered at 800 °C are regular with a mean particle size of 40 nm. Analysis of SAED (Selected Area Diffraction) pattern revealed the appearance of well developed rings with clear reflections at positions corresponding with a reference standard of calcium hydroxyapatite.

ICP-OES Analysis
Element analysis was done using the ICP-OES (Inductively Coupled Plasma-Optical Emission Spectrometers, Agilent 720 apparatus, Santa Clara, CA, USA)) technique (Table 1) in order to confirm the composition and homogenous distribution of the Ca and P. All of the constituting elements were in a proper molar ratio confirming right stoichiometry of the final material. The ratio of the Ca 2+ cation to the P 5+ was about 1.67 for all samples, well matching with the theoretical ratio of Ca/P in calcium hydroxyapatite.

Cytotoxicity
The percentage of survival and evaluation of changes in the morphology of mouse fibroblast cells L-929 ( Figure 3) after contact with suspensions from the control materials (HDPE, SLS) and test materials from nHAP-800, nHAP-900 and nHAP-1000 are given in Tables 2 and 3. Microscopic images of the morphology of cell cultures treated with the suspensions from nanohydroxyapatites and the control are shown in Figures 4-8. Evaluation of nanohydroxyapatite, regardless of the temperature used in the production process, that is, nHAP-800, nHAP-900 and nHAP-1000, did not reduce L-929 cell survival below 80%. No significant differences were observed in the survival of cells in the suspensions 100% between nHAP-800, nHAP-900 and nHAP-1000. L-929 cell survival after contact with suspension from material nHAP-800 was 87.64%. In the case of materials nHAP-900 and nHAP-1000, the percentage of survival rate was, respectively, 88.87% for nHAP-900 and 88.30% for nHAP-1000. With a reduced concentration of suspensions, increase in cell survival was observed. The highest values were observed for nHAP-1000; cell survival increased on average by 21%. Table 2. The cell viability (MTT assay; 3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide) and the evaluation of the morphology of cells treated with the control (contrast reverse-phase microscope CKX41 (Olympus, Tokyo, Japan), mag. 10×).   In a microscopic image of L-929, cells treated with the suspension 100% from nHAP-800, the cytoplasmic granules were observed inside. Approximately 10% of the cells in the culture shrunk and became detached from the substrate ( Figure 6); those changes were classified by a criterion for degree 1-weak toxicity. Similar changes in cell morphology were observed in systems with 100% of the applied suspension-from nHAP-900 and nHAP-1000. Cells in the range of 10%-15% shrunk and became detached from the substrate, and the appearance of fine granules inside cytoplasmic cells was observed (Figures 7a and 8a). With reduced concentration of suspension from nanohydroxyapatite single intraplasmic granules, culture density comparable to the density of the control culture was observed. There were a lot of cells in the divisions, single cells shrunk, and no cell lysis was observed. Reducing the concentration of the suspension from nanohydroxyapatite to 25%, single intra cytoplasmatic granulations were observed, and culture density was comparable to the densities of the control culture. There were a lot of cells in the divisions, single cells were shrunk, and no cell lysis was observed. Regardless of the material used, there was no significant change in the amount of L-929 cells in comparison to the control.   In a microscopic image of L-929, cells treated with the suspension 100% from nHAP-800, the cytoplasmic granules were observed inside. Approximately 10% of the cells in the culture shrunk and became detached from the substrate ( Figure 6); those changes were classified by a criterion for degree 1-weak toxicity. Similar changes in cell morphology were observed in systems with 100% of the applied suspension-from nHAP-900 and nHAP-1000. Cells in the range of 10%-15% shrunk and became detached from the substrate, and the appearance of fine granules inside cytoplasmic cells was observed (Figures 7a and 8a). With reduced concentration of suspension from nanohydroxyapatite single intraplasmic granules, culture density comparable to the density of the control culture was observed. There were a lot of cells in the divisions, single cells shrunk, and no cell lysis was observed. Reducing the concentration of the suspension from nanohydroxyapatite to 25%, single intra cytoplasmatic granulations were observed, and culture density was comparable to the densities of the control culture. There were a lot of cells in the divisions, single cells were shrunk, and no cell lysis was observed. Regardless of the material used, there was no significant change in the amount of L-929 cells in comparison to the control. In a microscopic image of L-929, cells treated with the suspension 100% from nHAP-800, the cytoplasmic granules were observed inside. Approximately 10% of the cells in the culture shrunk and became detached from the substrate ( Figure 6); those changes were classified by a criterion for degree 1-weak toxicity. Similar changes in cell morphology were observed in systems with 100% of the applied suspension-from nHAP-900 and nHAP-1000. Cells in the range of 10-15% shrunk and became detached from the substrate, and the appearance of fine granules inside cytoplasmic cells was observed (Figures 7a and 8a). With reduced concentration of suspension from nanohydroxyapatite single intraplasmic granules, culture density comparable to the density of the control culture was observed. There were a lot of cells in the divisions, single cells shrunk, and no cell lysis was observed. Reducing the concentration of the suspension from nanohydroxyapatite to 25%, single intra cytoplasmatic granulations were observed, and culture density was comparable to the densities of the control culture. There were a lot of cells in the divisions, single cells were shrunk, and no cell lysis was observed. Regardless of the material used, there was no significant change in the amount of L-929 cells in comparison to the control. In surgical practice, for the reconstruction of bone defects synthetic hydroxyapatite in chemical formula Ca 10 (PO 4 ) 6 (OH) 2 , which acts as a substitute for bone tissue, is used on an increasing scale. The chemical composition and crystalline structure of synthetic hydroxyapatite are similar to the mineral component of bone. The hydroxyapatite is used in the powder form (or paste) to fill bone defects, in the form of ceramic fittings, or in the form of coatings applied to metal implants (e.g., hip or knee endoprostheses made from titanium alloys). Hydroxyapatite implanted into a living organism is not resorbable by this organism; however, it fills an existing gap in the bone or forms a bioactive layer between the metal implant and the bone. The bioactivity of hydroxyapatite consists of that on its surface, and, as a result of activity of bone-forming cells (osteoblasts), a new natural bone tissue will grow.

Control
Many studies of in vitro cytotoxicity of hydroxyapatite are examined. The hydroxyapatite in the form of granules was tested with neutral red uptake assay NRU (neutral red solution) and the colorimetric MTT assay (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide). In the study, it showed no toxicity to human fetal hFOB osteoblasts, and their survival rate was about 70% in relation to the control after 24 h and after 48 h of the study. In the MTT, significant differences in the results were observed depending on the prepared suspension from hydroxyapatite. The suspension from highly porous granules showed no toxicity, and cell viability was 70.6% and 60.3% compared with the control assay after 24 and 48 h of incubation. In the case of suspension from microporous hydroxyapatite, granules showed a decrease in cell viability to 52.4% after 24 h and 37.7% after 48 h of incubation [19]. The authors assumed that the result was due to suspension ion, during which changes to the ions concentrations occurred in the culture medium, which was not without an effect on the metabolism of the cultured cells. Analysis of the results confirmed that the addition of hydroxyapatite to other materials has an impact on cell viability and metabolism [19]. Another form of the hydroxyapatite matrices (scaffolds) was also studied, on which the cells were applied. Scaffolds were made of composite PLGA (poly(lactic-co-glicolic acid) or composite PLGA-HAP. The MTT assay showed that the viability of cells cultured on the scaffold from PLGA was lower than in the case of the PLGA-HAP scaffold [20].
Hydroxyapatite has very good biological properties. For this reason, more and more biological research is conducted on nanohydroxyapatite. In studies carried out by Chen et al., the assay MTT and LDH (lactate dehydrogenase) test to evaluate the activity of lactate dehydrogenase on murine cells MC3T3 E1 preosteoblasts [21]. The test material was nanohydroxyapatite positively, negatively charged and neutral. The tests showed improved survival and proliferation of cells in relation to the control system-polystyrene. Positively charged nanoparticles of hydroxyapatite were characterized with the highest improvement in survival and proliferation of cells [21]. Lack of cytotoxic activity in relation to cells of mouse fibroblasts of line L-929, demonstrated in our own study in vitro, allows for qualifying the evaluated nanohydroxyapatites to further stages of biological evaluation. Depending on the future use of nanohydroxyapatite, it is necessary to conduct, among others, a study of allergenic and irritating reactions, as well as an evaluation of reactions after implantation. Obtaining negative results in tests, in vitro proves that the developed nanomaterials have no toxic effect and can be used in the future as materials for biomedical applications. In surgical practice, for the reconstruction of bone defects synthetic hydroxyapatite in chemical formula Ca10(PO4)6(OH)2, which acts as a substitute for bone tissue, is used on an increasing scale. The chemical composition and crystalline structure of synthetic hydroxyapatite are similar to the mineral component of bone. The hydroxyapatite is used in the powder form (or paste) to fill bone defects, in the form of ceramic fittings, or in the form of coatings applied to metal implants (e.g., hip or knee endoprostheses made from titanium alloys). Hydroxyapatite implanted into a living mineral component of bone. The hydroxyapatite is used in the powder form (or paste) to fill bone defects, in the form of ceramic fittings, or in the form of coatings applied to metal implants (e.g., hip or knee endoprostheses made from titanium alloys). Hydroxyapatite implanted into a living organism is not resorbable by this organism; however, it fills an existing gap in the bone or forms a bioactive layer between the metal implant and the bone. The bioactivity of hydroxyapatite consists of that on its surface, and, as a result of activity of bone-forming cells (osteoblasts), a new natural bone tissue will grow. Many studies of in vitro cytotoxicity of hydroxyapatite are examined. The hydroxyapatite in the form of granules was tested with neutral red uptake assay NRU (neutral red solution) and the colorimetric MTT assay (3-[4 ,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide). In the study, it showed no toxicity to human fetal hFOB osteoblasts, and their survival rate was about 70% in relation to the control after 24 h and after 48 h of the study. In the MTT, significant differences in the results were observed depending on the prepared suspension from hydroxyapatite. The suspension from highly porous granules showed no toxicity, and cell viability was 70.6% and 60.3% compared with the control assay after 24 and 48 h of incubation. In the case of suspension from microporous hydroxyapatite, granules showed a decrease in cell viability to 52.4% after 24 h and 37.7% after 48 h of incubation [19]. The authors assumed that the result was due to suspension ion, during which changes to the ions concentrations occurred in the culture medium, which was not without an effect on the metabolism of the cultured cells. Analysis of the results confirmed that the addition of hydroxyapatite to other materials has an impact on cell viability and metabolism [19]. Another form of the hydroxyapatite matrices (scaffolds) was also studied, on which the cells were applied. Scaffolds were made of composite PLGA (poly(lactic-co-glicolic acid) or composite PLGA-HAP. The MTT assay showed that the viability of cells cultured on the scaffold from PLGA was lower than in the case of the PLGA-HAP scaffold [20].
(a) (b) Many studies of in vitro cytotoxicity of hydroxyapatite are examined. The hydroxyapatite in the form of granules was tested with neutral red uptake assay NRU (neutral red solution) and the colorimetric MTT assay (3-[4 ,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide). In the study, it showed no toxicity to human fetal hFOB osteoblasts, and their survival rate was about 70% in relation to the control after 24 h and after 48 h of the study. In the MTT, significant differences in the results were observed depending on the prepared suspension from hydroxyapatite. The suspension from highly porous granules showed no toxicity, and cell viability was 70.6% and 60.3% compared with the control assay after 24 and 48 h of incubation. In the case of suspension from microporous hydroxyapatite, granules showed a decrease in cell viability to 52.4% after 24 h and 37.7% after 48 h of incubation [19]. The authors assumed that the result was due to suspension ion, during which changes to the ions concentrations occurred in the culture medium, which was not without an effect on the metabolism of the cultured cells. Analysis of the results confirmed that the addition of hydroxyapatite to other materials has an impact on cell viability and metabolism [19]. Another form of the hydroxyapatite matrices (scaffolds) was also studied, on which the cells were applied. Scaffolds were made of composite PLGA (poly(lactic-co-glicolic acid) or composite PLGA-HAP. The MTT assay showed that the viability of cells cultured on the scaffold from PLGA was lower than in the case of the PLGA-HAP scaffold [20].  relation to cells of mouse fibroblasts of line L-929, demonstrated in our own study in vitro, allows for qualifying the evaluated nanohydroxyapatites to further stages of biological evaluation. Depending on the future use of nanohydroxyapatite, it is necessary to conduct, among others, a study of allergenic and irritating reactions, as well as an evaluation of reactions after implantation. Obtaining negative results in tests, in vitro proves that the developed nanomaterials have no toxic effect and can be used in the future as materials for biomedical applications.

Materials and Methods
Nanohydroxyapatites in powder form, nHAP-800, nHAP-900 and nHAP-1000, obtained by the wet chemistry method at temperatures of 800 • C, 900 • C, and 1000 • C, respectively, were used for the study [7]. Materials were designed and manufactured in the Institute of Low Temperature and Structure Research, Polish Academy of Sciences, Wroclaw, Poland. The cytotoxicity tests were carried out on the reference mouse fibroblast cell line L-929 ( Figure 3). The investigation was conducted at the Department of Experimental Surgery and Biomaterials Research, Wroclaw Medical University, Poland [12][13][14].

Apparatus
Evolution of crystal structure was followed using the XRD technique by collecting patterns in 2θ range of 5 • -120 • with X'Pert PRO X-ray diffractometer (Cu, Kα1: 1.54060 Å) (PANalytical, Almelo, The Netherlands). The morphology and microstructure of nanoparticles were investigated by high-resolution transmission electron microscopy (HRTEM) using a Philips CM-20 Super Twin microscope (Eindhoven, The Netherlands), operated at 200 kV. Samples for measurements were prepared by dispersing of powders in methanol. Afterwards, a droplet of suspension was deposited on a copper microscope grid covered with perforated carbon. ICP-OES elemental analysis was done using an Agilent 720 apparatus (Santa Clara, CA, USA). Calibration curves were measured using ICP standard solution for determination of the Ca 2+ and P 5+ ions content. Prior to elemental analysis, the nHAP samples were digested using HNO 3 with spectral purity.
In this method, stoichiometric amounts of Ca(NO 3 ) 2 ·4H 2 O and (NH 4 ) 2 HPO 4 were dissolved in MQ-water separately. Subsequently, the prepared solutions were mixed. As a result, there was fast precipitation of the by-product. The solution pH was adjusted to 10 with NH 4 OH under constant and vigorous stirring at 90 • C for 2 h. Finally, the by-product was dried for 24 h at 90 • C and thermally treated at the temperature range of 800-1000 • C for 3 h, resulting in formation of white, fine-grained powders.

Suspension Preparation
Suspensions of the materials investigated (nHAP-800, nHAP-900 and nHAP-1000) and negative control materials were prepared under sterile conditions in proportion: samples 20 g/mL in culture medium. As a negative control, high density polyethylene (HDPE U.S. Pharmacopeia-Rockville, MD, USA) was used, and, as a positive control sample, a solution of sodium lauryl sulfate (SLS, Sigma-Aldrich ® , St. Louis, MO, USA) with indicated concentrations (0.1, 0.15, 0.2) mg/mL was used. To evaluate the cytotoxicity, the following concentrations of suspensions were used: 100%, 50%, 25% and 12.5%. Blank (complete cell culture medium without any samples) was also included.

Cytotoxicity Tests
Murine fibroblast cells L-929 (NCTC clone 929) were obtained from ATCC were cultured in medium Minimal Essential Eagle's Medium (MEM) (Lonza) supplemented with 10% FBS (Fetal Bovine Serum) and L-glutamine with streptomycin and penicillin solution (Sigma-Aldrich ® ). Cells were removed from culture flasks with the use of 0.25% trypsin-EDTA (ethylenediaminetetraacetic acid) solution (Sigma-Aldrich ® ) and seeded in 96-well flat-bottomed plates (Nunc, Nunclon ™ Surface Roskilde, Denmark) in a concentration of 1 × 10 4 cells per well (1 × 10 5 cells per mL). After 24 h of incubation in standard conditions, the cells medium was discarded and replaced with 100 µL of suspension or control. The evaluation of the cytotoxic effect was provided after 24 h incubation at (37 ± 1) • C in a humidified atmosphere containing 5% CO 2 with suspension and included the determination of morphological changes and viability of the cells. The changes in cell morphology were evaluated with the use of the contrast reverse-phase microscope CKX41 (Olympus, Tokyo, Japan), according to the criteria given in Table 4, According to the 4-grade scale, changes in the cell cultures higher than 2 degrees and a reduction of cell viability greater than 30% are considered to be caused by the cytotoxic effect [12]. The cell viability was determined by MTT assay with concentration of 3-4,5-dimethylthiazol-2-yl]-2,5-diphenyl tetrazolium bromide (MTT) (Sigma-Aldrich ® ) 1 mg/mL in EMEM (ATCC ® ). To provide the MTT assay, the suspensions of samples were discarded and 50 µL of MTT solution was added to each well and plates were incubated for 3 h at (37 ± 1) • C in 5% CO 2 . Then, the MTT solution was discarded and 100 µL of isopropanol, analytical grade (Stanlab ® Lublin, Poland) were added in each well. After 30 min, the absorbance values were recorded at 570 nm using an Epoch microplate spectrophotometer (BioTek ® Highland Park, MI, USA). Cell viability was calculated according to formula (1): V = (Ab:As) 100%, where V is cell viability percentage, Ab is mean absorbance of the test sample, and As is mean absorbance of the blank. Table 4. Criteria of toxicity effect based on changes in cell morphology [12].

Conclusions
The high-temperature annealed nanohydroxyapatites (nHAP) were obtained using a co-precipitation technique and heat-treated in the temperature range of 800-1000 • C. The detailed study of the hydroxyapatite structures presented complete crystallization and was confirmed by X-ray diffraction and TEM analysis. All of the samples caused only a slight reduction of cell viability. The cell viability (MTT test) and the evaluation of the morphology of cells that were treated with the extracts of the nHAPs showed the low (grade 1) toxicity of the tested materials. In accordance with the norm ISO 10993-5, changes in the culture above the second degree of toxicity reduction of cell viability by more than 30% and morphological changes in cells (intracytoplasmic granules, not more 50% growth inhibition, 50% of cells are round) are considered a cytotoxic effect. Taken together, investigated materials on a 4-grade scale of cytotoxic effect cause 1 degree and are considered a slight cytotoxic. The examined nanohydroxyapatites have not shown cytotoxic effects on L-929 murine fibroblasts.