The biocompatibility and bioactivity properties of hydroxyapatites (HAs) modified through lithium addition were investigated. Hydroxyapatites obtained from bovine bone were mixed with lithium carbonate (Li), in the proportions of 0.25, 0.50, 1.00, and 2.00% wt, and sintered at 900°, 1000°, 1100°, 1200°, and 1300°C, creating LiHA samples. The osteoblast culture behavior was assessed in the presence of these LiHA compositions. The cellular interactions were analyzed by evaluating the viability and cellular proliferation, ALP production and collagen secretion. The cytotoxic potential was investigated through measurement of apoptosis and necrosis induction. The process of cellular attachment in the presence of the product of dissolution of LiHA, was evaluated trough fluorescence analysis. The physical characteristics of these materials and their cellular interactions were examined with SEM and EDS. The results of this study indicate that the LiHA ceramics are biocompatible and have variable bioactivities, which can be tailored by different combinations of the concentration of lithium carbonate and the sintering temperature. Our findings suggest that LiHA 0.25% wt, sintered at 1300°C, combines the necessary physical and structural qualities with favorable biocompatibility characteristics, achieving a bioactivity that seems to be adequate for use as a bone implant material.
Bones provide mechanical protection for internal organs and the blood-forming marrow, and they facilitate locomotion and serve as a reservoir for calcium, magnesium, and phosphate minerals [
BHA (bone hydroxyapatite) powder was derived from bovine bone with a calcination method at 850°C, as described in a previous study [
Osteoblasts were isolated from the calvaria of 1-to 5-day-old neonatal Wistar rats [
Bioceramics were suspended in culture medium RPMI (GIBCO), 0.5 g in 50 mL, shaked for 5 h at 37°C and filtered twice. The second filtration was conducted in a 0.22 mm filter for sterilization. This medium, containing ionic products from the bioceramics dissolution which had the pH adjusted to 7.0, was supplemented with 10% of FBS (GIBCO) and 1% antibiotic-antimycotic and was used to stimulate the osteoblasts.
Osteoblasts were plated in 24 well plates at a density of 5 × 104 cells/mL, and after 72 h, the medium was changed to medium containing ionic products from the dissolution of the samples. After 72 h of incubation, osteoblasts were tested. Control cells were submitted to the same process using only medium, without ionic products.
Osteoblasts were seeded in 24 well plates at a density of 5 × 104 cells/mL with medium containing each type of LiHA powder. After adhesion, the LiHA powders were placed in the cell cultures at a rate of 10 cells/1 particle. Control cells were osteoblasts in pure medium.
The viability of the cells in culture with medium containing each type of LiHA powder was evaluated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Briefly, MTT (5 mg/mL) was added to each well. The culture was then incubated in a humidified 5% CO2 incubator at 37°C. Two hours later, the cell morphology and formazan salts were visualized by inverted optical microscopy. The formazan salts were dissolved with 10% SDS-HCl overnight, and the optical density measurement was conducted at 595 nm. The values of all samples were calculated relative to the value of the control and expressed as percentages. The control consisted of nonstimulated cells.
Alkaline phosphatase production by the cells cultured with medium containing each type of LiHA powder was evaluated after 72 h by BCIP-NBT assay (GIBCO). Briefly, the BCIP-NBT solution was prepared per manufacturer’s protocol. The supernatant of each well was removed and placed in a tube. Next, 300
Collagen production by the cells cultured in the presence of LiHA samples was evaluated by using a SIRCOL Collagen Assay (Biocolor Ltda, Ireland), according to the manufacturer’s instructions. Briefly, the collagen present in the supernatant, precipitated by the dye Sirius red, was solubilized and measured by an optical density analysis at 595 nm. The amount of collagen was calculated based on a standard curve of previously known concentrations of collagen and their optical density measurements. We used cultures without samples as controls. The experiment was performed in triplicate, and data are presented as the mean ± standard deviations (SD).
The apoptotic parameters were analyzed by flow cytometry using a BD LSR II flow cytometer, and data were analyzed using BD FACSDiva software (Becton Dickinson, San Jose, CA). The samples were analyzed, and in all cases 1 × 104 cells were recorded. Fluorophores were diluted in Me2SO or dimethylformamide (DMF), preloaded just prior to FACS analysis, and incubated at 37°C and 5% CO2 in cell culture medium, unless otherwise indicated. Control cells were incubated with vehicles, which never exceeded 0.1% in the final concentration. Propidium iodide (PI) was added at a final concentration of 10
Confocal microscopy (Zeiss LSM 510 Meta) was used to characterize focal adhesion and cytoskeleton structure of control cells and cells incubated in the presence of the ionic product from the dissolution of each sample. Cells were seeded on glass cover slips, fixed with 3.7% formalin for 20 min, permeabilized with 0.5% Triton X-100 in PBS for 30 min, and incubated with the following antibodies in sequence: (1) primary antibodies: mouse monoclonal antibodies against rat vinculin (Abcam-USA, ab11194) diluted 1 : 400 with PBS containing 1% BSA, or with rabbit antibodies against rat actin (Abcan-USA, ab1801), diluted 1 : 100 with PBS containing 1% BSA; (2) secondary antibodies: goat antibodies against mouse IgG labeled with Alexa Fluor 594 (Molecular Probes), diluted 1 : 600 with PBS containing 1% BSA, or goat antibodies against rabbit IgG labeled with FITC (Abcam-USA, ab6717), diluted 1 : 500 with PBS containing 1% BSA. Then, the samples were incubated with DAPI (Sigma) diluted 1 : 10000 with PBS for nucleus staining.
The LiHA samples with osteoblasts were fixed with a solution containing 2.5% glutaraldehyde and 0.5% paraformaldehyde in phosphate buffer (pH 7.4) for 2 hours, postfixed in 1% osmium tetroxide for 2 hours, dehydrated in increasing concentrations of ethanol (from 30%, 40%, 50%, 60%, 70%, 80% 90%, 95% to 100%) and were critical-point-dried. The scaffolds were coated with gold (Sputter Coater—SPI Supplies) for 90 s at 13 mA. Images were taken in the Department of Metallurgy and Materials Engineering, Federal University of Minas Gerais, Brazil, using the scanning electronic microscope (JEOL 6360 LV), at 15 kV and 750 mA.
The results were analyzed using Prism 4.0 software, and the chosen method was a one-way ANOVA test and a Bonferroni’s posttest.
Considering that cellular viability and secretion capability are the first parameters used to evaluate the cytotoxicity of a given material, we performed the MTT assay, which allows the analysis of viability as well as, indirectly, the cellular proliferation. We cultured the osteoblasts in the presence of the different biomaterial powders. The cellular viability was measured and compared to control osteoblasts cultured in pure medium. The results showed that the viability of osteoblasts incubated in the presence of 0.25% LiHA was similar to control or commercial HA in all sintering temperatures (Figure
Cellular viability of osteoblasts cultured in the presence of different concentrations of LiHA. (a) 0.25% LiHA, (b) 0.50% LiHA, (c) 1.0% LiHA, and (d) 2.0% LiHA. The results are expressed as the means ± SD, (*) indicating a significant difference comparing to control with
Alkaline Phosphatase (ALP) production of osteoblasts cultured in the presence of different concentrations of LiHA. Panel (A): ALP production measured inside the cells. (a) 0.25% LiHA, (b) 0.50% LiHA, (c) 1.0% LiHA, and (d) 2.0% LiHA. Panel (B): ALP secretion measured from culture supernatant. (a) 0.25% LiHA, (b) 0.50% LiHA, (c) 1.0% LiHA, and (d) 2.0% LiHA. The results are expressed as the means ± SD, (*) indicating a significant difference comparing to control with
Collagen secretion of osteoblasts cultured in the presence of different concentrations of LiHA. (a) 0.25% LiHA, (b) 0.50% LiHA, (c) 1.0% LiHA, and (d) 2.0% LiHA. The results are expressed as the means ± SD, (*) indicating a significant difference comparing to control with
Li has been proposed for use as an addictive to ceramic composites [
Flow cytometry analysis of apoptotic cells. (a–c) Representative histograms, the M1 marker represents the subdiploid content. (a) Control, (b) Commercial hydroxyapatite, and (c) LiHA sample. (d) Graphic summarizing the percentage of death cells. The results are expressed as the means ± SD.
It is known that Lithium alters the signaling and differentiation of central nervous system cells [
Actin and Vinculin expression of osteoblasts in the presence of LiHA. Magnification 1000x. (a) 0.25% LiHA at 900°C, (b) 0.25% LiHA at 1300°C, (c) 2.00% LiHA at 900°C, and (d) 2.00% LiHA 1300°C.
During all the tests, the morphology of the cells was observed under light microscopy. No alteration was observed. However, considering the difference of porosity in the samples sintered at different temperatures, and knowing that this difference can alter cell morphology, we decided to investigate, using electronic microscopy, the osteoblast adhesion and filopodia emission when osteoblasts were cultured on the LiHA sample surface. We used two time points, 72 hours and 120 hours after incubation with LiHa. Naturally the cell density at 120 hours of culture was higher, and the demonstrative images shown here were chosen from this time point. However, in both time points we had good results. The osteoblasts adhered to all porous samples, spread, and showed normal elongation and interconnections between cells. Figures
SEM micrographs of the morphology of osteoblast cultured on LiHA matrix for 120 hours. Images demonstrated osteoblast adhesion to the matrix surface and interaction between cells. (a) Scale bar: 10
SEM micrographs of the morphology of osteoblast cultured on LiHA matrix for 120 hours. (a) Detail of cell surface showing secretion vesicles indicating normal physiology. Cells are flattened and elongated. Scale bar: 2
EDS analysis of osteoblast cultured on LiHA matrix for 120 hours. (a) Selected site for EDS analysis. Scale bar: 10
The results of this study indicate that the LiHA ceramics are biocompatible and have variable bioactivities, which can be tailored by different combinations of the concentration of lithium carbonate and the sintering temperature. Our findings suggest that LiHA 0.25% wt, sintered at 1300°C, combines the necessary physical and structural qualities with favorable biocompatibility characteristics, achieving a bioactivity that seems to be adequate for use as a bone implant material. However, we propose that in vivo experiments should be conducted to validate its use.
This study was partly carried out with the financial support of the Turkish Republic Government Planning Organization in the framework of the Project “Manufacturing and Characterization of Electro-Conductive Bioceramics” (no. 2003 K120810). Dr. F. N. Oktar acknowledges the support of the Portuguese Science and Technology Foundation (FCT Fundaçao para a Ciência e a Tecnologia) under the program “Compromisso com a Ciência,” Reference C2008-UA/TEMA/01. Financial support of biological studies was provided by CNPq (Brazil), CAPES (Brazil), and FAPEMIG (Brazil). The authors also acknowledge the kind scientific support of Dr. Gultekin Goller from Istanbul Technical University, Turkey and Dr. M. A. Valente from Physic Department of Aveiro University, Portugal.