The influence of carbonated hydroxyapatite on pre osteogenics promoters on a bone cementABSTRACT
Objective The aim of this study was to synthesize nanoparticulate carbonate-substituted hydroxyapatite. Physicochemical properties and cytotoxicity were evaluated. Subsequently, carbonated hydroxyapatite-based cement was developed and characterized, and its in vitro biological performance was assessed to investigate its potential for bone regeneration applications on pre osteogenics promoters.
Material and Methods The powders/cements were physicochemical characterized and in vitro biological tests will be assessed in L929 and MC3T3 cell cocultures. For the study, a series of cements of pure hydroxyapatite cement (CHAP), CHAC with 5% carbonate (CHAC5), CHAC with 10% (CHAC10), and a control group (CONT) were prepared in weight by mixing a solid phase and one liquid phase and were investigated by scanning electron microscopy (SEM), x-ray diffraction (XRD), FT-IR, contact angle, pH test and setting time test. Biological assays were performed using the MTT assay and Alizarin Red S for mineralization.
Results The CHAC samples' characterization results showed shorter crystallinity peaks than pure hydroxyapatite, indicating increased sample solubility but no cement breakdown when in solution. Furthermore, this cement was hydrophilic and had a pH appropriate for use in bone regeneration. These findings and setting time tests support further research into the biomaterial created. In biological experiments, CHAC increased cell survival and promoted mineralization.
Conclusion The inclusion of CHAC was beneficial in promoting osteogenic features in bone cement by enhancing cell survival and driving mineralization, resulting in promising findings for bone regeneration.
KEYWORDS
Bone regeneration; Biocompatible materials; Bone cements; Calcium phosphates; Hydroxyapatite
RESUMO
Objetivo Sintetizar hidroxiapatita carbonatada nanoparticulada. As propriedades físico-químicas e citotoxicidade foram avaliadas. Posteriormente, um cimento à base de hidroxiapatita carbonatada foi desenvolvido e caracterizado, e seu desempenho biológico in vitro foi avaliado a fim de investigar seu potencial para aplicações em regeneração óssea, com ênfase em promotores pré-osteogênicos.
Material e Métodos Os pós/cimentos foram caracterizados físico-quimicamente e testes biológicos in vitro foram avaliados em Cocultura de células L929 e MC3T3. Para o estudo, uma série de cimentos de hidroxiapatita pura (CHAP), CHAC com 5% de carbonato (CHAC5), CHAC com 10% (CHAC10) e um grupo controle (CONT) foram preparados, misturando uma fase sólida e uma fase líquida, e foram investigados por microscopia eletrônica de varredura (MEV), difração de raios-X (DRX), FT-IR, ângulo de contato, teste de pH e tempo de presa. Os ensaios biológicos foram realizados utilizando o ensaio MTT e Alizarin Red S para mineralização.
Resultados Os resultados da caracterização das amostras de CHAC mostraram picos de cristalinidade mais curtos do que a hidroxiapatita pura, indicando maior solubilidade da amostra, mas sem degradação do cimento quando em solução. Além disso, este cimento foi hidrofílico e apresentou um pH apropriado para uso em regeneração óssea. Estes achados e os testes de tempo de presa apoiam pesquisas futuras sobre o biomaterial criado. Em experimentos biológicos, o CHAC aumentou a sobrevivência celular e promoveu a mineralização.
Conclusão A inclusão de HAC foi benéfica na promoção de características osteogênicas em cimentos ósseos, melhorando a sobrevivência celular e impulsionando a mineralização, resultando em achados promissores para a regeneração óssea.
PALAVRAS-CHAVE:
Regeneração óssea; Materiais biocompatíveis; Cimentos ósseos; Fosfato de cálcio; Hidroxiapatita
INTRODUCTION
Bone substitutes are biomaterials that have been shown to help promote bone healing. They attempt to stimulate bone tissue regeneration by stimulating self-degradation, which allows it to be replaced with newly formed bone. In recent years, many biomaterials have been studied in an attempt to influence cellular activity, including those involved in bone tissue regeneration. As a result, various bone cements using synthetic materials with mineral compositions similar to bone and high osteoconductive qualities have recently become popular for filling bone defects [1-3]. Calcium phosphate has a unique composition called hydroxyapatite (HA). To promote bone regeneration, functional ions such as strontium, magnesium, zinc, and carbonate have been added into hydroxyapatite's structure [4-6]. These highlights the significance of utilizing hydroxyapatite with carbonate.
Biological apatite contains carbonate ions in its natural composition, which are recognized to play an important mechanical and biological role. Carbonated Hydroxyapatite (CHA) can change and form granules with a wide range of well-defined geometries and sizes, adjusting to the morphologies of bone defects. However, there have been reports that employing CHA causes a significant rate of issues (>32%), which is due to the fact that traditional CHA has restricted porosity, inhibited more new bone formation sites and making remodeling difficult. That is why it is crucial to obtain a nanometric-sized CHA to allow bone neoformation between the granules. Furthermore, the ability to identify the physicochemical characteristics of calcium carbonate particles has grown in prominence because it has the potential to increase drug distribution in the body, as particle geometry has a substantial impact on cellular activity [7-11]. As a result of their ubiquitous availability, low cost, and ease of use, HA-based bone cements are becoming increasingly popular in surgical bone replacement and regeneration applications. However, it is biocompatible, safe, pH-sensitive, and biodegradable [11,12].
Thus, the aim of this study was to synthesize nanoparticulate carbonate-substituted hydroxyapatite using an aqueous precipitation method at low temperature. Physicochemical properties and cytotoxicity were evaluated. Subsequently, carbonated hydroxyapatite-based cement was developed and characterized, and its in vitro biological performance was assessed to investigate its potential for bone regeneration applications.
MATERIAL AND METHODS
Synthesis of hydroxyapatite
Pure hydroxyapatite was manufactured using the method disclosed by Cox et al. [13]. Briefly, two aqueous solutions were prepared: 4.3 g of Na2HPO4 (0.030 M) dissolved in 45 mL of deionized water, and 6.5 g of Ca(NO3)2·4H2O (0.04 M) dissolved in 50 mL of deionized water. The pH of the mixed solutions was adjusted to 9 by the addition of NaOH solution. The resulting HA precipitate was collected, washed, and dried in an oven at 85 °C overnight. The same method was used to produce nanostructured CHA. The solutions of (NH4)2CO3 (Sigma-Aldrich, USA), 0.05 mol/L and 0.10 mol/L, were added to solution B and then mixed with solution A at a flow rate of 4mL/min. Three groups were developed: HAP, HAC5, and HAC10 [14].
Synthesis of hydroxyapatite bone cements
Hydroxyapatite bone cements have two phases: powder and liquid. The powder phase was enriched with 90% HA and 10% anhydrous dibasic calcium phosphate (Synth, Brazil). The liquid was made of an aqueous solution containing 2% sodium hydrogen phosphate (Vetec, Brazil), 2% calcium chloride dihydrate (Vetec, Brazil), 2% beta-glycerophosphate (Sigma-Aldrich, USA), and 5% hydroxypropylcellulose (Sigma-Aldrich, USA). For the study, a series of cements of pure hydroxyapatite cement (CHAP), CHAC with 5% carbonate (CHAC5), CHAC with 10% (CHAC10), and a control group (CONT) were prepared in weight by mixing a solid phase and one liquid phase
Physico-chemical Characterization
Scanning Electron Microscopy (SEM)
The morphological examination of the CHA particles and cements were performed using scanning electron microscopy (SEM) on a scanning electron microscope (JEOL model JSM 6360 LV) with an acceleration voltage of 15 kV and a magnification power of 1000x and 15000x. The samples were metallized with 3 mm of gold before analysis.
X-Ray Diffrraction
The crystallinity of the produced CHAs was assessed by X-ray diffraction. The Shimadzu device, model XRD-7000 X-RAY (DQ-UFMG), acquired the diffractograms using a copper tube and Cu Kα radiation (λ=1.54051 Å) at 30 kV and 30 mA. The analyses were conducted at 2θ angles between 4 and 60 degrees, with a scanning velocity of 4θ min−1.
FT-IR
The FT-IR was utilized to determine the functional groups in the HAC, CHAC samples. The data was recorded using a Perkin Elmer GX spectrophotometer with a 4 cm-1 resolution from UFMG's Department of Chemistry. The samples, manufactured in KBr tablets, was dried first and then examined in the 4000 - 400 cm-1 range. The data was handled by the ACD/SpecManager 6.0 application and ACD/labs.
Contact angle
The contact angle, which is produced by the tangent of the liquid-vapor interface to the solid surface at the three-phase contact line, determines wettability. The liquid side was traditionally used to measure the contact angle. The SEO Phoenix 300 Touch Contact Angle Measurement System will be used to conduct the experiment.
Setting time test
The setting time test was determined by measuring the initial and ultimate setting durations of the cements using Gillmore needles in line with the ASTM C266-08 Standard [15]. The cement powder and liquid phases were mixed for 120 seconds to ensure homogeneity. The modified paste was put onto metal discs measuring 10 mm by 3 mm. After 120 seconds, the light needle (113.4g and 2.12 mm) was put on the paste's surface for 5 seconds and repeated every 1 minute. The initial setup time was determined when the needle left no mark on the surface. The ultimate setting time was determined when the heavy needle (453.6g and 1.06mm) was unable to mark the cement surface. The measurements were taken in triplicate.
pH measurements
The pH measurements were studied by placing the samples in falcon tubes containing 10 ml of MilliQ® (Merck, Darmstadt, Germany) water and keeping them at room temperature. The measurements were performed using the MS TECNOPON, mPA-210 from Brazil. Values were collected at 5, 10, 20, 30, 60, and 120-minute intervals, as well as 1, 2, 3, 4, and 5 days later. MilliQ water is refilled every day. The test was carried out three times.
In vitro biological testing
Cells obtained of Rio de Janeiro - Cell Bank were used: L929 ATCC no. CCL-1, a mouse fibroblast cell line, and a preosteoblast cell line MC3t3-E1 ATCC no. CRL-2594 cells were exposed to culture medium samples high-glucose Dulbecco’s Modified Eagle Medium (DMEM; Gibco) supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic/antimycotic solution (100U/mL penicillin and 100 μg/mL streptomycin), and maintained at 37 °C in a humidified atmosphere. 200 μg/mL of the CHA powder was plated in 24-well plates to evaluate direct or indirect cytotoxicity by using the MTT test (3-(4,5-di methyl thiazol-2-yl)-2,5-diphenyltetrazolium bromide to assess the direct cytotoxicity of the cements
Direct MTT cytotoxicity assay
The samples measured 6x4mm and were produced in duplicate. The CHA samples were thereafter disinfected for one hour using UV light. The designated durations were 7 and 14 days.
Indirect MTT cytotoxicity assay
The indirect cytotoxicity of HA powder was evaluated utilizing the MTT assay and its eluate. Preparation of Cements eluates: To obtain the eluates, 2 grams from each group were weighed and subjected to UV light for 1 hour for disinfection. The samples were thereafter placed into plates containing 2 mL of the suitable growth media for each utilized cell line and incubated at 37°C for 24 hours. 200 μL of eluate was deposited onto a 96-well plate at 24, 48, and 72 hours. The experiments were performed in hexaplicates, and absorbance was recorded at 570 nm using a Thermo Scientific Multiskan spectrophotometer (MCC/340 model).
Alizarin S Red assay
The Alizarin S Red test was employed to visually assess the mineralization resulting from calcification nodules within the cells. Two millimeter cubic cement samples were positioned in a 24-well plate. The culture media was substituted bi-daily. Following 7 and 14 days, the cell cultures were subjected to two washes in PBS, fixed with 4% paraformaldehyde for 30 minutes, and subsequently rinsed twice with PBS. Following the second wash, 0.2% Alizarin red was introduced to the wells and allowed to incubate for 5 minutes. Subsequently, thorough washing with PBS was performed. The plates were examined and photographed utilizing the Citation 5 apparatus.
Statistical analysis
The results are presented as the mean SD, and the analysis is provided using the OriginPro Lab 2024b. Two- way ANOVA was used to evaluate the comparison between the groups for cell viability. The post-test Bonferroni for multiple comparisons was used to analyze significant differences between pairs among groups. For all experiments, a 5% significance level was used.
RESULTS
The microscopy findings were presented in Figure 1. Panels A and B illustrate the morphology and the presence of agglomerated particles smaller than 50 micrometers, leading to an irregular surface. This exemplifies the production process of HA nanoparticle precipitation method. The micrographic images of HAP and HAC10 (C, D) illustrate the presence of agglomerated particles measuring < 50 micrometers and a heterogeneous, porous surface (Figures 1B and 1D). Upon scrutinizing the micrographs of the cements in the CHAP (panels E, F) and CHAC10 cements (G, H), one can observe a granular surface. Notwithstanding the incorporation of pure and carbonated hydroxyapatite particles in both samples, the surface roughness exhibits greater homogeneity attributable to the presence of polymer from the liquid phase.
The XRD imaging standards for hydroxyapatites and cements (Figure 2) align with ICCD standards for hydroxyapatite. The diffractogram peaks were more dispersed, indicating low crystallinity. The results indicated diffraction peaks equivalent to those of stoichiometric HA at values of 2θ: 25.68º, 28.85º, 31.80º, 32.81º, 33.85º, 39.77º, 46.71º, and 49.40º. It was also noticed that when the amount of carbonate ions increased, the strength of the peaks decreased.
XRD Patterns of powders and bone cements. (A) HAP, HAC5 and HAC10 powders; (B) CHAP, CHAC5 and CHAC10 bone cements.
The IR spectra of HAC and CHAC contained HA-specific absorption regions. The absorption band characteristic of symmetrical stretching of the OH- group of HA may be seen at around 3560 cm-1. The bands at 870 cm-1 and 1460 cm-1 are typical of the CO3-2 group. PO4-3 ions are responsible for the bands observed at 1030 cm-1 (Figure 3). 3400 – 3450 cm−1: A broad and intense peak representing the stretching vibrations of the hydroxyl (-OH) groups.2974 – 2980 cm−1: Strong C-H stretching vibrations from the methyl groups (-CH3) present in the hydroxypropyl substituents.1460 – 1464 cm−1: C-H deformation and asymmetric bending vibrations of methyl groups.1050 – 1100 cm−1:
(A) FTIR spectra of HAP, HAC5 and HAC10 powders; (B) FTIR spectra of CHAP, CHAC5 and CHAC10 bone cements.
The strongest band cluster, which shows vibrations of the glucose pyranose ring structure, C-O stretching, and the C-O-C ether linkage. Analyzing the IR hydroxypropyl cellulose spectra one could find the 3400 – 3450 cm−1The stretching vibrations of the hydroxyl (-OH) groups are represented as a wide, strong peak. C-H stretching vibrations from the hydroxypropyl substituents' methyl groups (-CH3) at 2974–2980 cm−1. At 1460–1464 cm−1 and 1050–1100 cm−1, C–H deformation and asymmetric bending vibrations of methyl groups were detected: The strongest band cluster, which shows vibrations of the glucose pyranose ring structure, C-O stretching, and the C-O-C ether linkage. Other substances including beta-glycerophosphate, calcium chloride dihydrate, and sodium hydrogen phosphate were unable to produce variations in the spectra.
The wettability test for contact angle at a contact period of 1 second, all cements exhibited hydrophilic properties. However, CHAC5 and CHAC10 shown superior hydrophilic potential compared to CHAP. After a duration of 30 seconds, the hydrophilicity of the materials was confirmed through a reduction in angles (Table I).
The setting time test measured the ratio of powder to liquid was established at 0.6 for the purpose of handling the cements and conducting the tests. The initial setup time was comparable among all samples, with a small elevation observed in the CHAP sample. In contrast, the CHAC5 and CHAC10 groups exhibited a significant reduction in the final setup time compared to the CHAP group (Table II).
The pH levels of all samples decreased within the initial 10-minute period. Following a duration of 20 minutes, the CHAP and CHAC5 groups exhibited a progressive increase in values, whereas the CHAC10 group-maintained stability until the 60-minute mark. Within 24 hours, there was a decline in the values of all groups, with the CHAC5 and CHAC10 groups maintaining a value of 6.6 until day 3. Following a 4-day assessment period, the pH levels rose to 6.9 (CHAP), 7.0 (CHAC5), and 7.1 (CHAC10), as shown on Figure 4.
In vitro biological tests
Cytotoxicity
Analysis of the cytotoxicity date after direct contact of cement MTT assay results revealed that the CHAP, CHAC5, and CHAC10 samples demonstrated a significant enhancement in cell viability (Figure 5). After 14 days, the viability of CHAC10 was 102.05%, while the CHAC5 group exhibited a viability of 86.87%. The CHAC10 group had cell viability above 100%. Consequently, the cements had no detrimental effects on the MC3T3 and L929 cells during co-culture.
Direct contact MTT experiment on coculture cells of L929 and MC3T3 in the CHAP, CHAC5, CHAC10, and control groups. The assay was conducted for 7 days and 14 days.
The indirect cytotoxicity test, assessed using the MTT method, indicated that the groups did not exhibit any cellular cytotoxicity (Figure 6). In the MTT assay conducted using L929 cells, the experimental groups exhibited outcomes that were comparable to those of the control group during a 24-h timeframe. The outcomes of CHAP, CHAC5, and CHAC10 were superior to those of the control group at 48 and 72 h. When exposed to MC3T3 cells, the eluates from the HAC5 group exhibited superior cell viability consistently, but both HAP and HAC10 had comparable outcomes to the control group. Upon examining the findings of MTT in Coculture, it was observed that the outcomes were consistent across all groups.
Indirect contact MTT assay of HAP, HAC5, HAC10 on (A) L929 cells, (B) MC3T3 cells and (C) Coculture cells.
Alizarin Red S mineralization test
Figure 7 shows the Alizarin Red S staining used to detect mineralization. The calcification nodules had a pinkish-reddish hue in the microscopic images. During the 7-day study, the CHAC5 group exhibited a higher quantity of nodules, which occupied a large area of mineralization as shown in Figure 7B. Only two calcification nodules were found in the control group, however a larger quantity was observed in the CHAP, CHAC5, and CHAC10 groups.
Alizarin Red S staining for mineralization. (A) CHAP – 7 days; (B) CHAC5 – 7 days; (C) CHAC10 – 7 days; (D) Control group – 7 days; (E) CHAP – 14 days; (F) CHAC5 – 14 days; (G) CHAC10 – 14 days; (H) Control Group – 14 days.
The findings from the 14-day experiment revealed a greater extent of calcification in all groups. Nevertheless, the CHAP, CHAC5, and CHAC10 groups had a more intense hue with 4x magnification in comparison to the control group. Furthermore, the control group had a greater extent of non-calcified region.
DISCUSSION
The alteration of the crystalline grating parameter induces a modification in the powder's morphology and thermal behavior, which is closely correlated with the amount of carbonate incorporated into the composition. Moreover, a heightened concentration of substituted carbonate ions in hydroxyapatite results in augmented dissolution, reduced crystallinity, and smaller particle sizes. The findings corroborate previous observations, indicating the existence of particle clusters smaller than 2 μm and favorable results in the biological evaluations [16-18]. The incorporation of carbonate within the structure of biological apatites accounts for their diminished crystallinity, a commonly recognized phenomenon. Thus, the presence of these ions in HA is essential for the biomaterial's development, leading to the generation of nanoscale and potentially amorphous crystals [19,20].
The incorporation of HPMC polymer in the cement improves cohesiveness. This polymer has been previously reported in the literature for these specific purposes, and its application enhances the control over the mechanical properties of cement. The incorporation of hydroxypropyl methylcellulose polymer boosts cohesion, setting time, and mechanical characteristics [21-23].
The development of irregular surfaces resulting from the accumulation of HAP and HAC particles is a prevalent characteristic noted in the precipitation process of HA nanoparticles. Previous research indicated that the SEM examination produced a microscopic image akin to that of the present study, wherein hydroxyapatite (HA) particles were noted in clusters of 2μm. These clusters generated several chains of nanometric-sized hydroxyapatite. A separate study successfully synthesized hydroxyapatite (HA) at a temperature of 100ºC for 12 hours. This technique led to the creation of agglomerates exhibiting various forms, sizes, and crystallite structures. Furthermore, the HA demonstrated a significant ability for agglomeration, resulting in the formation of micropores among the particles. The micropores enable the infiltration of physiological fluids and improve the adherence of the biomaterial to the freshly created bone tissue, thus fostering osseointegration. Another investigation revealed the presence of carbonated hydroxyapatite clusters within microspheres of 425-600μm, aligning with our research findings. The incorporation of Hydroxypropyl methylcellulose (HPMC) produced a more homogeneous cement surface, hence improving cell deposition and osseointegration. Composites selectively facilitate the degradation of their constituents at different rates, leading to the creation of holes that enhance angiogenesis and osteogenesis [24-26].
The observed diffractograms corroborate the findings of Li et al. [27], demonstrating that the peaks of the substituted nHAs are somewhat smaller than those of HAP. The reduction in particle crystallinity may be ascribed to the inclusion of carbonate ions. The peaks of the substituted nHAs demonstrate a slight decline, attributable to the intrinsic characteristics of the HAP synthesis method and the substitution process. As the concentration of carbonate ions rose, a progressive decline in crystallinity was visible. Dias et al. [28] observed a decrease in crystallinity upon the addition of Sr, Mg, and Zn ions, leading to a reduction in diffractogram peaks [13,27,28].
The FT-IR spectrum seen in powders and cements corresponds with the bands discovered in the studies by Dias et al. [28] and Ruiz et al. [29]. These spectra display resemblances to those of HA, albeit with changes in peak strength and minor shifts in bands. The alterations noted in the spectra of powders and cements can be ascribed to the elevated concentration of carbonate ions inside the functional group network [29].
The cements demonstrated hydrophilic characteristics, enhancing cell adhesion by offering an appropriate surface. The composites attain an optimal surface equilibrium by creating a region that is neither overly hydrophilic nor overly hydrophobic. This facilitates protein adsorption and encourages the proliferation of osteoblasts. In an in vivo investigation on animals, it was noted that groups utilizing composites for alveolar defect regeneration exhibited more mature and organized trabeculae than those who did not. This resulted in an enhancement of the quality of the freshly formed bone [30].
The incorporation of carbonate ions in hydroxyapatite-based cements, together with the use of HPMC polymer, led to a significant reduction in the final solidification time. The initial setting time was slightly reduced, with CHAC5 taking 39 minutes and CHAC10 lasting 38 minutes. This discovery corroborates existing literature, demonstrating that elevated carbonate concentrations decrease the solidification period of the material. This is ascribed to the processes of dissolution and precipitation occurring during the setting period. The ideal duration for the setting time process should be established between 15 and 25 minutes. The evidence indicated that the carbonated hydroxyapatite-based cements exhibited prolonged setting periods beyond the optimal duration. This was presumably attributable to the presence of the HPMC polymer in the liquid phase. The cements exhibited commendable resistance to degradation, inhibiting their disintegration upon exposure to physiological fluids. The quick setting time limits the working period, as the cement becomes unfit for application before the task is finished. An extended setting time may result in a postponement of the procedure, as completion is contingent upon reaching the ultimate setting time [22,29,31].
The pH shown a minor decrease in the initial minutes and predominantly stayed neutral thereafter, which is essential for the biocompatibility of the composite in contact with cells. The pH levels of the CHAP, CHAC5, and CHAC10 samples consistently remained above 6.3, reaching a maximum of 7.1. This outcome is more promising compared to the findings documented in the current literature, which suggest that a pH level over 4.2 correlates with apatite cements. The slight decrease in pH noted in the initial minutes of the CHAC5 and CHAC10 groups may be ascribed to the release of carbonate and phosphate ions during cement consolidation. The decline in value can be attributed to the presence of unreacted carbonate ions, but the alkaline characteristics of the phosphate ions preserved these values within a neutral pH range. The observed pH increase over 120 minutes can be considered advantageous for the samples, likely linked to improved cell adhesion, activity, and homeostasis [28,29,32].
The cell models employed in the biological assays were chosen for their influence on tissue repair and wound healing mechanisms. The L929 cell line was utilized to examine its impact on wound healing, whereas the MC3T3 cells were employed to analyze their capacity to enhance bone cell proliferation. Cell proliferation is a dependable measure of a material's biocompatibility in laboratory environments [33]. Previous studies on carbonated hydroxyapatite have shown its remarkable biocompatibility and its ability to promote the proliferation of fibroblasts and osteoblasts. Despite several investigations on carbonated hydroxyapatite, only a limited number have employed biological assays involving osteoblasts and fibroblasts in coculture. Coculture has been shown to successfully imitate tissue settings, facilitating natural regeneration by attracting resident cells and activating the self-repair mechanism. The amalgamation of these cells yields a synergistic effect on bone development and production, augmenting intercellular communication and promoting the formation of the extracellular matrix. The extracellular matrix, synthesized by various cells in tissues and organs, constitutes a fibrous network comprised of glycosaminoglycans and proteoglycans. These proteins are organized in a highly ordered manner, forming a three-dimensional architecture that creates a distinct local environment. An experiment employing flavonoid-doped nanohydroxyapatite in a cell triculture of osteoblasts, osteoclasts, and endothelial cells exhibited an enhancement of the bone regeneration microenvironment and an improvement in the micro-angiogenesis process, essential for new bone development [27,34,35].
The MTT experiment demonstrated that the carbonated hydroxyapatite-based cement showed positive outcomes regarding cellular response to the evaluated compounds. The use of calcium phosphate cement infused with elevated levels of strontium and magnesium led to a reduction in cell viability. A distinct study performed the MTT assay on MC3T3 cells subjected to silver-doped hydroxyapatite and noted a reduction in cell viability relative to our findings. A separate study performed a cytotoxicity assessment utilizing HAC, and the outcomes corroborated the current study's findings, demonstrating a notable enhancement in the viability of MC3T3 cells upon interaction with the material's scaffolds [36-39].
The mineral component of bone tissue, consisting of biological apatite, is essential for linking the physicochemical and metabolic processes required for the production of precursor minerals. These precursor minerals are vital for the nucleation of phosphate and calcium ions, and for promoting the production and accumulation of collagen and hydroxyapatite in bone defects. Analysis of the results from the mineralization tests revealed that both CHAC5 and CHAC10 had a markedly reduced mineralization time relative to the control group. A previous study demonstrated an increase in calcium deposition when utilizing hydroxyapatite doped with carbonate and magnesium. The Alizarin Red test illustrated the importance of calcium phosphate cements, especially in the presence of carbonated hydroxyapatite, in facilitating mineralization in a laboratory environment. The result may be influenced by the variety in crystallinity observed in the different forms of calcium phosphate [28,40-42]. The favorable results derived from the biomaterial analysis establish a foundation for further in vitro biological studies in this study.
CONCLUSION
Carbonated hydroxyapatite-based cements demonstrated promising results for bone regeneration, improving cell viability and facilitating mineralization in vitro. The findings jointly delineate the critical physicochemical and biological characteristics necessary for the optimal functionality of the novel composite, facilitating and providing a basis for its future in vivo application in research and the advancement of materials for tissue engineering.
Acknowledgements
The authors thank the Brazilian Agencies: FAPEMIG, CNPq, and CAPES for the grants, and to the Microscopy Center at UFMG (CM-UFMG) and LabCri - Laboratório de Cristalografia, ICEx - UFMG - Campus Pampulha for the technical support and access to the electron microscopy facilities.
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Conflict of Interest
Maria Esperanza Cortés (Corresponding address)
Universidade Federal de Minas Gerais, Faculdade de Odontologia, Departamento de Odontologia Restauradora, Belo Horizonte, MG, Brazil. Email: mecortes@yahoo.com, mecortes@ufmg.brEditor-in-chief:
Editor:
