Development of a novel Ga-containing hydroxyapatite/chlorhexidine biomaterial with antibacterial properties for future application in bone tissue engineering: an experimental and theoretical study

Abstract

This study focuses on synthesizing gallium-containing hydroxyapatite (Ga-HA) with chlorhexidine (CLX) for potential use in bone and dental tissue restoration. The Ga-HA/CLX materials were prepared using a suspension-precipitation method and were surface-functionalized with varying CLX concentrations. X-ray diffraction analysis confirmed the hexagonal structure of Ga-HA with space group P63/m, while XPS revealed the presence of gallium and a Ca/P ratio ranging from 1.50 to 1.72. Infrared spectra exhibited characteristic bands for phosphate and CH₂ groups, indicating CLX incorporation. The Ga-HA/CLX materials demonstrated 100% inhibitory efficiency against Staphylococcus aureus and Escherichia coli bacterial strains. MTT assay indicated enhanced cell viability in the presence of gallium, with the Ga-HA/CLX-0.20 material classified as non-toxic with 81.0 ± 3% cell proliferation. Density Functional Theory calculations supported favorable thermodynamics in the interaction between hydroxyapatite and chlorhexidine. Overall, Ga-HA/CLX materials exhibit promising properties for biomedical applications.

Keywords:
Antibacterial properties; Bone repair; Chlorhexidine-functionalized biomaterials; Density Functional Theory (DFT); Surface functionalization

INTRODUCTION

Biomaterials are a class of compounds that induce an appropriate response when interacting with biologically active molecules and living organisms ¹. Among these biomaterials, calcium phosphate-based materials have been studied and used for over 60 years as restorative and filling materials for bone and dental tissues ².

To be classified as new products with potential for future applications in the field of bone tissue engineering, these biomaterials and their new formulations must have a set of adequate mechanical and biological properties that allow them to perform functions that will help, replace, and stimulate bone neoformation ^3)-(5. Hydroxyapatite (HA), Ca₁₀(PO₄)₆(OH)₂, is a bioceramic widely used in bone tissue repair processes because it is the main constituent of the inorganic part of the bone. In addition, it has excellent osteoinductive, osteoconductive, and osteogenic properties ^6)-(8.

This type of biomaterial can undergo punctual modifications in its physicochemical and biological properties, such as the potentiation of its antibacterial effects, cell proliferation and differentiation, non-cytotoxic effects and interaction with drugs, for example, modifications can be performed by increasing the dopant substituents: Ag^{+ (9}, Cu²⁺ and Zn^{2+ (10}, SeO₃ ^{2- (11}, Sr^{2+ (12} and representative metals (M³⁺ = Al³⁺, Ga³⁺, In³⁺ and Bi³⁺) ^13)-(15.

For example, gallium (Ga³⁺), a bioactive cation, can be present in inorganic biomaterials for orthopedic applications because it can potentiate and increase the physical, chemical, and biological effects on adjacent tissues ¹⁶. Furthermore, Ga is a non-toxic ion in living organisms, and even though gallium salts and complexes have no known physiological function in the human body, some of their characteristics allow their interaction with cellular processes and biologically important proteins, especially those involved in iron metabolism ¹⁷.

Although the details of the action of Ga in human bone are still uncertain, it is already well established that the mechanism that involves the insertion of Ga into the HA crystalline network causes greater protection relative to the reabsorption of this bioceramic and causes an improvement in its biomechanical properties when applied to living skeletal systems ¹⁸. On the other hand, cationic substitutions in the HA structure can, in addition to influencing crystal growth, increase the specific surface area, which in turn enhances adsorption capacity, promoting interaction with organic matrices possessing antibacterial properties ¹⁹.

It is also worth noting that the presence of ions constituting part of HA may also lead to punctual effects of changes in its crystallinity ¹⁹, increasing the level of interaction between HA and tissue biomineralization ²⁰, because the bone structure is not only composed of an inorganic mineral phase, but also by an organic matrix, constituted mainly by collagen and polypeptides ^21),(22. Therefore, the search for a new biomaterial with bacterial inhibitory efficiency and surface association of antiseptics for ceramic biocomposite formulations proves to be an excellent alternative. For example, the surface functionalization of HA enhances the biological and antibacterial characteristics of this new biomaterial ²³.

Thus, chlorhexidine (CLX), C₂₂H₃₀Cl₂N₁₀ (1,1-bis hexamethylene (5-p-chlorophenylbiguanide) di-D-gluconate), for example, a biocidal antiseptic with antimicrobial properties ²⁴, which is effective in controlling pathogenic bacteria in the oral cavity or as a topical agent ²⁵, usually in oral hygiene products for the treatment of periodontal disease and varnish as an alternative to constitute these types of formulations ²⁶, because smaller clusters of CLX salts are enough to delay the development or eliminate bacterial plaques, since when administered correctly and in adequate concentrations, CLX exhibits low toxic and corrosive effects ²⁷.

Regarding the interactions in CLX-HA systems, Soriano-Sousa et al.²⁹ investigated hydroxyapatite-based microspheres loaded with chlorhexidine and reported that the adsorption of chlorhexidine by hydroxyapatite follows a Langmuir mechanism at concentrations below 8.6 ± 1.3 µg CHX/mg HA. However, at higher concentrations, the process occurs through a Langmuir-Freundlich mechanism. Furthermore, strong molecular interactions were also observed on the HA surface at elevated CLX concentrations.

Some studies suggest the use of CLX as an antibacterial agent in bone implants because of its high response against a wide variety of Gram-positive and Gram-negative bacteria ²⁸. However, a great therapeutic challenge is to obtain an effective antimicrobial system that maintains inhibitory effects for a certain adequate period, reducing the incidence of local infection and adverse effects, in addition to stimulating bone tissue growth and rapid recovery of the patient.

Thus, this work aimed to synthesize and characterize gallium-containing hydroxyapatite (Ga-HA), followed by the production and characterization of a new biomaterial of the type Ga-HA/chlorhexidine (CLX), followed by the evaluation of its antibacterial properties against bacterial strains S. aureus and E. coli and their cytotoxic effects (in vitro) exerted by different concentrations of CLX adsorbed on the surface of Ga-HA; finally, demonstrate through DFT calculations (Density Functional Theory) some thermodynamic parameters of the possible surface interactions existing between Ga-HA and CLX.

EXPERIMENTAL

Calcium hydroxide (Vetec), Ca(OH)₂; dibasic ammonium phosphate (Neon), (NH₄)₂HPO₄, gallium nitrate (Sigma-Aldrich), Ga(NO₃)₃ and chlorhexidine, C₂₂H₃₀Cl₂N₁₀, 20% v/v (Polyorganic) were used as precursors. For cell viability experiments, the cell line GM07492 (human fibroblasts), acquired from the Coriell Cell Repository (CCR) bank, was used (1x10⁵ cells/well), DMEM culture medium (Gibco/Thermofisher), supplemented with fetal bovine serum (FBS) (Nutricell), DMSO (Sigma-Aldrich), penicillin and streptomycin 10 U/mL (Sigma-Aldrich). The reagents were not purified, and the water used for the synthesis was purified using a Milli-Q® system (Millipore Corporation).

The first stage (S_I) of this study was the synthesis of pure hydroxyapatite (HA) and gallium-containing hydroxyapatite (Ga-HA). The synthesis of HA and Ga-HA was performed by the suspension-precipitation method (SPM) ²⁹.

Initially, for HA synthesis, 0.3700 g of Ca(OH)₂ and 0.4473 g of (NH₄)₃PO₄ were dissolved in 50.0 mL of deionized water. Then, each solution containing the precursors was added to a 250.0 mL beaker to initiate the synthesis via SPM. The system was magnetically stirred for 3 h at 50 °C until a white precipitate was formed. The precipitate was then filtered, centrifuged (2500 rpm for 5 min), washed with distilled water (5x), and dried in an oven at 100 ºC for 12 h. The obtained powders were milled using a mortar and pestle and passed through a 35 VT sieve with an aperture of 425 µm.

The synthesis of the Ga-HA particles was processed according to the synthesis of HA, but with the addition of gallium (Ga(NO₃)₃) in solution, with an amount of 5.00% (m/m) about the substituent ion (Ca²⁺) (x mol Ga = 5.00 mol or 0.050 mol) following the stoichiometric relationship between Ca/P (Eq. A) chosen for this study (Ca_9.95Ga_0.05(PO₄)₆(OH)₂) ²⁹. The synthesis occurred in an open system, under magnetic stirring for about 3 hours, in basic medium (pH = 10-11), without the use of calcination after filtering and washing the obtained powders.

$$\begin{gathered} 9.95\mathrm{Ca}{\left(\mathrm{OH}\right)}_{2\left(\mathrm{s}\right)}+0.05\mathrm{Ga}{\left({\mathrm{NO}}_3\right)}_{3\left(\mathrm{aq}\right)}+6{\left({\mathrm{NH}}_4\right)}_2{\mathrm{NPO}}_{4\left(\mathrm{s}\right)} \\ \to {\mathrm{Ca}}_{9.95}{\mathrm{Ga}}_{0.05}{\left({\mathrm{PO}}_4\right)}_6{\left(\mathrm{OH}\right)}_2\downarrow +18{\mathrm{H}}_2{\mathrm{O}}_{\left(\mathrm{I}\right)}+12{\mathrm{NH}}_{4\left(\mathrm{aq}\right)}^{+}+3{\mathrm{NO}}_{3\left(\mathrm{aq}\right)}^{-} \end{gathered}$$ (A)

The second stage (S_II) was to carry out the incorporation of chlorhexidine (CLX) on the surface of the material through adsorption. The functionalization of the hydroxyapatites (HA and Ga-HA) occurred using approximately 500.0 mg of powders synthesized in the S_I of this study with 25.0 mL of solutions with varying concentrations of CLX. The concentration of CLX chosen for this study was based on work previously carried out by our research group ²³, who used CLX in a concentration range between 0.20% to 20% (v/v); thus, the concentration range of 0.20 to 1.20% (v/v) was chosen. The incorporation of CLX occurred via the solid-liquid interface process by rotational agitation (150 rpm), with a fixed temperature of 40 ºC, in a period of 8 hours. The supernatant solution was separated by centrifugation and quantified, in triplicate, through UV-Vis (λ = 255 nm) to determine the final concentration of the solutions through Eq. B:

$Qe=\frac{C_i-C_f}{m}xV$ (B)

in which, Qe is the amount of CLX incorporated into the hydroxyapatite in mg/g, c _i the initial concentration of the CLX solution in mg/L, c _f the concentration of the supernatant in mg/L, V is the volume that was used in the test of adsorption (mL) and m is the mass of HA that was used in the adsorption test (g). Figure 1 brings a summary scheme representing the S_I, S_II and the materials characterization step.

Figure 1:
Experimental arrangement representing the S_I (synthesis of Ga-HA) and S_II (synthesis of Ga-HA/CLX) as stages of characterization of the synthesized materials and the evaluation of their biological properties. (SPM = suspension-precipitation method, Ga-HA = Ca_9.95Ga_0.05(PO₄)₆(OH)2, CLX = chlorhexidine solutions, DCT = direct contact technique).

The synthesized powders were characterized by X-ray diffraction (XRD). The diffractograms were collected from 5° to 80° 2θ with a scan rate of 2°/min and a data collection time of 40 min. Monochromatic Cu-Kα radiation (λ = 1.5406 Å) was used on a LABX-XDR 600 by Shimadzu (Kyoto, Japan). In addition, the crystallographic information on the ICSD card n° 26,205 for pure HA was employed as the initial structural refinement model. Phases identification was performed by Rietveld method using GSAS EXPGUI 2012 software (2.0 version, University of California, Los Alamos, New Mexico, USA). For the Rietveld refinement analysis, XRD data were collected in the 2θ range from 10° to 110° with a scanning rate of 0.02°/min and an exposure time of 90 min. Calibration of the X-ray diffractometer was performed before XRD pattern acquisition. The NIST standard reference material LaB₆ (NIST-600b) was used in this calibration. The collected XRD pattern of the reference material was then used to obtain the initial input parameters for all the refinements ²⁹.

The X-ray photoemission spectra (XPS) were obtained using a spectrometer system (ESCA+, Scienta-Omicron, Taunusstein, Germany) equipped with a hemispherical analyzer (EA125) and a monochromatic Al Kα X-ray source (Xm 1000, 1486.7 eV). The X-ray source was used with a power of 280 W, as the spectrometer worked in a constant pass energy mode of 50 eV ²⁹.

Fourier-transform infrared spectroscopy (FT-IR) spectra were obtained using a Vertex 70 spectrophotometer (Brucker Optics, Billerica, MA, US). The KBr pellet method was used, which required 32 sweeps in the region of 4000 to 400 cm⁻¹ with a resolution of 4 cm^-1. The samples were ground in an agate mortar, and powders of approximately 1.0 mg were mixed with 100.0 mg of KBr. Thermogravimetric analyses (TGA) were carried out in the SDT Q600 V20.9 Build 20 apparatus from TA Instruments using approximately 5 mg of sample with a heating rate of 10 ºC/min in an argon atmosphere with a flow of 100 mL/min in a sample port of alumina in the temperature range of 25 to 1000 ºC. The micrographs of the samples were performed in a scanning electron microscope (FE-SEM) with a field emission cannon, FEI brand, model Quanta FEG 250, with acceleration voltage from 1 to 30 kV, equipped with SDD EDS (Silicon drift detectors), brand Ametek, model HX-1001, Apollo X-SDD detector. The conditions - energy, spot, and magnification - are recorded at the bottom of each photo, as scale and magnification (Notation: SE - ETD-SE secondary electron detector; BE - vCD backscattered electron detector). The samples were fixed on double-sided carbon adhesive tape and covered with Au in a metallizer, Quorum brand, model Q150R, for 30 s, at 20 mA, by plasma generated in an argon atmosphere. Regarding EDS microanalysis, the spectra of the samples were collected at 25 kV and spot 5.

Antibacterial activity was performed using Gram-positive (S. aureus) (ATCC 25,923) and Gram-negative strains (E. coli) (ATCC 25,922) by the direct contact technique (DCT). These strains were chosen because E. coli is a common Gram-negative bacterium associated with infections and biofilm formation, while S. aureus is a Gram-positive pathogen known for its role in hospital-acquired infections and antibiotic resistance. Cultures were obtained by transferring bacteria on nutrient agar into a falcon tube containing 3.0 mL of brain heart infusion (BHI) medium, followed by incubation at 37 °C for 24 h. From this BHI culture, a standardized bacterial suspension was prepared at a density equivalent to 0.5 on the McFarland scale, or approximately 1.5 × 10⁸ CFU/mL. Serial decimal dilutions were then performed in physiological saline solution, obtaining a suspension of 1.5 × 10⁴ CFU/mL for both bacteria. The antimicrobial test was performed according to the methodology of Zheng and Zhu (2003) [30], resulting in the counting of colony-forming units (CFUs). For the test, the standardized bacterial suspension was submitted to decimal serial dilutions until the 10⁻⁴ dilution (1.5 × 10⁴ CFU/mL) was obtained, upon which 2000 μL of this diluted suspension was transferred to a sterile falcon-type tube. Next, 2000 μg of the HA and Ga-HA were added to determine the inhibitory effect, and 100 μL of this suspension were transferred to Petri dishes containing the nutrient agar medium. Petri dishes were then seeded with the aid of a Drigalski loop by the spread plate method and were incubated at 37 °C for 24 h. Then, the counting of colony-forming units (CFUs) was performed. The inhibitory effect by each test solution was calculated according to the Eq. C:

$\eta =\left(N_1-N_2\right)/N_1$ (C)

where ƞ is defined as the inhibitory effect, N₁ is the arithmetic mean of the colony-forming units of the control plates, and N₂ is the average of the colony-forming units of each of the solutions tested and the results set out in percentages. As the positive control, nutrient agar plates containing bacterial suspension and saline solution (2000 μL) were used, as well as plaques containing the bacterial suspension. All tests were performed in triplicate ²⁹.

The in vitro cytotoxicity assay was performed by standard procedures (ISO 10993-5:2009). Cell viability was analyzed by MTT (3(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazoliumbromide) colorimetric assay. Human fibroblast cells (GM07492) were initially seeded (15 × 10³ cells/well) in 96-well culture dishes and maintained in Dulbecco’s modified Eagle’s medium (DMEM) enriched with fetal bovine serum (FBS-10% - v/v) and antibiotics (penicillin and streptomycin). The cells were then incubated (Panasonic-CO₂ incubator MOC-19 AIC-UV, Osaka, Japan) in a humidified atmosphere containing 5% CO₂ and 95% atmospheric air for cell adhesion. Simultaneously, samples were weighed, sterilized, immersed, and extracted in a culture medium (20 mg/mL, HA powders/DMEM free of bovine fetal serum) at 37 °C for 24 h, and the extracts were collected for the cytotoxicity assay. After that, the culture medium of the seeded wells was replaced by 100 μL of medium resulting from the extraction procedure of each sample. This experiment was carried out in triplicate and maintained under standard culture conditions for 24 h. Subsequently, the culture medium was removed from the wells, which were washed with phosphate buffered saline (PBS 3×). Aliquots of 50 μL of MTT were added to each well, and the cells were kept in culture conditions for 4 h. After this period, they were added by 100 μL of isopropyl alcohol was added, and the well content was mechanically homogenized until complete formazan solubilization. The absorbance optical density values of samples (OD) were obtained using a spectrophotometer at 570 nm wavelength. The cytotoxic effect was expressed in percentage using the following equation, where the control group was considered as 100% cell viability (Eq. D) ³¹:

$OD\left(\%\right)=\frac{\text{Absorbance of treated cells}}{\text{Absorbance of tcontrolled cells}}*100$ (D)

The OD value of each sample was converted into percentages of cell viability about the negative control group (DMEM + 10% FBS = 100% of cell viability). A positive control group was performed (cytotoxic effect reference) by the addition of dimethyl sulfoxide (DMSO 30% v/v) to the culture medium (DMEM + 10% FBS) ²⁹.

All calculations were performed using the Gaussian 09 software, through Density Functional Theory (DFT) ^32),(33 using the B3LYP method ^34)-(36 and CEP-31G(d) base set, as used in theory G3(MP2)//B3-SBK ³⁷, to obtain the geometries and vibrational frequencies. Calculations of total electronic energy and Gibbs energy, at 0 and 298.15 K respectively, were performed initially with optimization in the gas phase and then taking into account the influence of the solvent using the Polarizable Continuum Model (PCM) ³⁸ in aqueous medium to better approximate the experimental reality.

The data were statistically analyzed using the GraphPad Prism software version 5.0 (GraphPad Software, La Jolla, CA, USA). The groups were compared using one-way ANOVA, applying a post hoc Fisher’s test (level significance at a 95% CI, p ≤ 0.05 was considered for all tests).

RESULTS AND DISCUSSION

After synthesis, pure hydroxyapatite (HA) and 5.00% gallium (Ga-HA) materials were analyzed by XRD for effective phase confirmation and evaluation of material crystallinity (Figure 2). By diffractograms, the synthesized materials have good crystallinity, and intermediate phases were not identified. The specific planes that characterize the crystalline phase of hydroxyapatite: (1 1 1), (0 0 2), (1 0 2), (2 1 0), (2 1 1), (2 0 2), (3 1 0), (3 1 2), (2 1 3) and (5 1 1) were detected, being indexed with the JCPDS catalog file 001-1008; therefore, their detections suggest that the material was successfully obtained in its crystalline form ³⁹.

Figure 2:
(a) XRD pattern for HA and Ga-HA, (b) Rietveld refinement for the Ga-HA sample, and (c) representation of the unitary Ga-HA cells made in this study.

This XRD pattern calculated for the Ga-HA sample was similar to the pure hydroxyapatite pattern, with a hexagonal crystalline structure and space group P63/m. Normally, a refinement is qualified as satisfactory when the value of Chi² or χ² (convergence factor) is between 1 and 2 (χ² = 1 and 2) [40]. Rietveld refinement confirmed the existence of a crystalline profile similar to card number 26.205 (ICSD). For this refinement (Figure 2b), the value of χ² was equal to 1.966, indicating reliability in the refinement analyses. The structural parameters of the Ga-HA sample obtained by refinement are: a = b = 0.943 nm and c = 0.690 nm and V = 531.39 ų, while the values for synthesized HA samples are: a = b = 0.942 nm and c = 0.688 nm and V = 529.09 ų ²⁹. The values obtained in this study are close to those obtained in the studies by Oliveira et al. (2021) (a = b = 0.943 nm and c = 0.687 nm and V = 528.85 ų) ⁴¹.

According to Pereira-Rocha et al. ⁴², it is expected that the incorporation of Ga³⁺ in the HA structure causes a significant change in the lattice parameters since the ionic radius of the cations occupying the same coordination site in the HA structure are quite different. For example, Ga³⁺ and Ca²⁺ with coordination number (CN) 6 have ionic radii of rGa³⁺ = 0.62 Å and rCa²⁺ = 1.00 Å, respectively, while Ga³⁺ and P⁵⁺ with CN = 4 have ionic radii of rGa³⁺ = 0.47 Å and rP⁵⁺ = 0.17 Å, respectively.

In the present work, a slight increase in lattice parameter values was observed after the incorporation of Ga³⁺ into hydroxyapatite. Some authors have reported the synthesis of HA containing gallium, and most of them suggest that a possible substitution occurs by replacing Ca²⁺ ions with Ga^{3+ (43}. However, these authors fail to affirm the Ga³⁺ substitution site in the network due to the lack of appropriate experimental characterization.

Considering the ionic radius of the cations, the small expansion of the observed unit cell may be associated not only with the low concentration of Ga³⁺ but also with a probable substitution of the P⁵⁺ cations by Ga³⁺ and not by the substitution of Ca²⁺ by Ga^{3+ (14}. The same behavior has been evidenced in HA doped with Si^{4+ (44}, Fe^{3+ (45}, and Cu^{2+ (46}. The aforementioned authors observed, by Rietveld structural refinement, X-ray photoelectron spectroscopy (XPS), and X-ray absorption spectroscopy (XAS), that the entry of these ions occurs preferentially in tetrahedral sites located in the channels of the HA crystalline structure, leading to an expansion of the unit cell. On the other hand, some authors, using the ³¹P solid-state NMR technique, have suggested that gallium ions may be chemisorbed on the disordered surface of calcium-deficient apatite ⁴⁷. Based on the observations mentioned above, it is believed that the Ga³⁺ ions added during the synthesis may be occupying the tetrahedral sites of P⁵⁺ in the center of the channels formed by Ca²⁺ (see unit cell - Figure 2c) or even chemisorbed on the surface of the HA.

It is important to highlight that in the present case, although a Rietveld refinement analysis was used, it is difficult to accurately determine the exact amount of Ga ions and the precise location of incorporation of these cations in the structure since the synthesized material does not present a good crystallinity. Furthermore, the formation of a preserved phase with low crystallinity based on gallium oxides cannot be ruled out either.

Figure 3 shows the XPS spectra for the Ga-HA sample. Through the XPS technique, it was possible to observe, on the surface of the material, the main characteristic peaks of the chemical elements that constitute hydroxyapatite (Figure 3a), they are: 532 eV (O1s), 438 eV (Ca2s), 347 eV (Ca2p), 189 eV (P2s), 133 eV (P2p) and 43 eV (Ca3s). In Figure 3b, it is possible to observe the emission lines related to the electrons ejected from Ga in the region between 18 to 32 eV. The binding energy (B. E.) identified in the XPS around 24 eV reveals that the peak refers to the excited electrons of the 3d orbital of Ga.

Figure 3:
(a) Wide scan XPS spectrum for the Ga-HA sample and (b) High resolution XPS spectrum on the Ga 3d emission line

The most intensive set of XPS peaks for Ga3d is found in the region of 18 eV ⁴⁸. This BE belongs to the spin-orbital electronic state of the type Ga3d_5/2-Ga3d_3/2, which is characteristic for the chemical species gallium metal; however, this specific peak was not detected in the Ga-HA sample synthesized in our work. Zatsepin et al. (2018) ⁴⁹ observed emission lines with BEs between 20 and 25 eV, attributed to the overlapping of the O2s(II)+Ga3d lines, characterizing the formation of the Ga-O type bond.

The Ca/P ratio of these materials was also calculated using data provided by the XPS technique. The results show that the Ca/P ratio for HA was 1.50. The (Ca+Ga)/P ratio, which considers the total cation quantification, was 1.72. (Figure S1 - Supplementary material) shows the spectrum with the respective cation content values on the surface of the Ga-HA sample. Table 1 lists these values for the hydroxyapatite samples synthesized in this study in quantitative terms of mol percentage (% mol). This analysis was performed using the CasaXPS software version 2.3.19PR1.0.

Table I
Ca/P ratio calculated via XPS for synthesized HA and Ga-HA powders.

Sample	%molar			Ration
	O	Ca	P	Ga	Ca/P	(Ca+Ga)/P
HA	10.01	3.93	2.63	-	1.49	-
Ga-HA	4.90	3.02	2.32	0.98	-	1.72

The incorporation of CLX on the surface of Ga-HA through the solid-liquid interface was performed using a curve of variation of CLX concentrations (Figure 4). For comparative purposes of the possible positive or negative effects of the presence of Ga in the material, CLX was also incorporated into the HA sample following the same synthesis methodology for obtaining the material Ga-HA + CLX (Step II).

Figure 4:
Curve of concentrations of CLX incorporation into HA and Ga-HA.

Incorporation results (Table 2) were calculated using Eq. 2. For HA/CLX-0.80 and Ga-HA/CLX-0.80 materials, the average concentration of adsorbed CLX was 0.0112 mg/g and 0.0118 mg/g, respectively. Finally, for HA/CLX-1.20 and Ga-HA/CLX-1.20 materials, these concentrations were 0.0144 mg/g and 0.0114 mg/g, respectively. A linear and proportional trend in the drug adsorption curve when interacting with hydroxyapatite was observed, indicating that the proportions of CLX concentrations (0.20, 0.80, and 1.20 % - v/v) suggested in this study were followed.

Table II
Results of the incorporation of CLX into the Ga-HA surface.

Material	Theoretical concentration in the initial solution (v/v %)	Amount adsorbed (mg∕g)
HA/CLX-0.20	0.20	20.57
HA/CLX-0.80	0.80	83.96
HA/CLX-1.20	1.20	126.51
Ga-HA/CLX-0.20	0.20	20.63
Ga-HA/CLX-0.80	0.80	84.11
Ga-HA/CLX-1.20	1.20	126.66

The values of CLX adsorbed for each material show a clear correlation with the theoretical initial concentrations, confirming a linear and proportional trend in the adsorption curve. For both systems (HA/CLX and Ga-HA/CLX), the increase in the initial CLX concentration in the solution resulted in a consistent increase in the amount adsorbed, indicating that the experimental parameters employed allowed a predictable and efficient interaction between the material and the drug. These results highlight that both HA and Ga-HA exhibited adsorption capacity directly proportional to the initial CLX concentration, suggesting that both surfaces have accessible active sites with an affinity for the drug, even in the presence of gallium in the matrix.

The obtained Ga-HA/CLX material was characterized by XRD, FT-IR, TGA, and FE-SEM/EDS techniques. Figure 5 shows the results of the characterizations via XRD, FTIR, TGA, and FE-SEM/EDS for the Ga-HA/CLX material. The XRD (Figure 5a) of the HA sample showed a similar profile to the Ga-HA diffractogram, showing only a small decrease in the intensity peaks when compared to the HA sample. It is believed that the incorporation of CLX may have generated this decrease in the crystallinity of the material. The FT-IR spectra of the Ga-HA/CLX materials and both precursors (Ga-HA and CLX) are shown in Figure 5b. The Ga-HA spectrum shows a band at 3500 cm^-1 that is related to the hydroxyl O-H stretching characteristic of the hydroxyapatite phase. Bands at 3440 cm^-1 and 1640 cm^-1 correspond to stretching and deformations of the O-H type of water adsorbed on the surface of the materials. The bands between 1030 cm^-1 to 960 cm^-1 correspond to asymmetric and symmetric stretching vibration, related to PO₄ ^3- groups in HPO₄ ^2-. The band around 600 cm^-1 refers to the asymmetric P-O strain of the PO₄ ^3- group. In the region of 560 cm^-1, we have the P-OH type deformation, and at 470 cm^-1, bands corresponding to the vibrations of asymmetric deformations of the HPO₄ ^2- group (P-O-(H)) are shown ⁵⁰.

Figure 5:
(a) XRD for Ga-HA and Ga-HA/CLX (b) FTIR for HA and Ga-HA/CLX (0.20, 0.50, and 1.20 % v/v%), (c) and (d) TG curve for Ga-HA and Ga-HA/CLX, (e) qualitative chemical composition by EDS and (f) FE-SEM of the biomaterial Ga-HA/CLX.

For the spectra of materials containing CLX at different concentrations, in addition to the common points of the spectrum of the Ga-HA material, other modifications are perceived; for example, two discrete stretching bands were observed close to the region of 3330 cm^-1 and 2920 cm^-1 in the spectra of materials containing CLX. These bands are not seen in the Ga-HA spectrum. This fact must be the most flexible of the amine (N-H) and methylene (C-H) groups, respectively, coming from the CLX that was created with the (OH) groups present in the hydroxyapatite at 3500 cm^-1. Another observed change is the broadening of the band in the region from 1530 to 1330 cm^-1, which should become an angular deformation resulting from the incorporated CLX amino groups. Note also the intensification of the band at 830 cm^-1, referring to the out-of-plane angular deformation of the C-H group of the aromatic rings present in the structure of CLX ^51),(52.

Figures 5c and 5d show the TG curves of HAs and HAs containing CLX. The Ga-HA material (Figure 5c) showed a lower percentage of mass loss compared to the HA. The first thermal event occurred around 100 °C, which refers to physiorbed water. Between the initial temperature and close to 200 °C, a percentage loss of approximately 2.5% was observed, but for HA, this percentage was around 5.0%. These mass losses represent the output of water physically adsorbed on the surface of the material that was detected in the FT-IR analysis; this same thermal event is perceived in the Ga-HA/CLX samples.

It is also possible to verify that the presence of Ga increased the thermal stability of Ga-HA. This same trend was observed for the Ga-HA/CLX-1.20 material (Figure 6d), with a greater mass loss occurring for the HA/CLX-1.20 material between 200 ºC and 600 ºC. In other words, Ga is believed to provide greater stability to materials. This thermal event corresponds to the thermal degradation of the volatile organic groups present in the CLX ^53),(54.

Figure 6:
Images of antibacterial assay plates via DCT: (a and b) positive controls (saline); (c and d) inhibitory effect of HA and Ga-HA powders; and (e and f) inhibitory effect of HA/CLX-1.20 and Ga-HA/CLX-1.20 materials.

For example, between 100 and 200 °C, the HA/CLX and Ga-HA/CLX materials showed a weight loss of approximately 12% and 5%, respectively. Another thermal event in the region between 200 and 400 ºC, for both HA/CLX and Ga-HA/CLX materials, a loss of about 6% and 3%, respectively, was observed. This loss corresponds to the output of the other chemical groups of CLX. In the thermal room between 400 and 700 ºC, there was a weight loss of about 3% and 1% for HA/CLX and Ga-HA/CLX, respectively. This loss can be attributed to the other decomposition groups belonging to the chlorhexidine. Finally, the last thermal event was observed between 700 to 1000 ºC, which is related to the condensation of the OH groups belonging to the hydroxyapatite structure.

According to these results, a marked decrease in the thermal stability of the material absent from Ga can be verified. This statement is relevant because CLX is an organic molecule and, therefore, has a relatively low degradation temperature; however, this temperature is higher than 121 ºC, which is the temperature used in autoclaving processes. Therefore, thus ensuring the permanence of the drug in the material. Figure 5e provides information obtained by EDS regarding the qualitative chemical composition of the elements that constitute hydroxyapatite and chlorhexidine. The EDS spectrum obtained revealed the presence of chemical elements related to the composition of the Ga-HA/CLX material. In other words, the semi-qualitative presence of gallium, oxygen, and phosphorus that constitute hydroxyapatite and the semi-qualitative presence of carbon, nitrogen, and chlorine that constitute chlorhexhydin were detected. Additionally, Figure 5f provides FE-SEM images illustrating the morphological characteristics of Ga-HA/CLX particles. The observed formation of non-uniform clumps suggests a tendency for particle aggregation during synthesis or drying processes. This aggregation could be attributed to factors such as Van der Waals forces, hydrogen bonding, or electrostatic interactions among the particles, potentially driven by the HA and CLX components.

The irregular surface morphology of the clumps may reflect the inherent structural properties of the composite material. Such irregularities could arise from the heterogeneous distribution of CLX molecules within the Ga-HA matrix. These surface features might enhance the material’s specific surface area, which is advantageous for applications like drug delivery or bioactive coatings, where increased interaction with biological systems is desired.

The values in percentage of the inhibitory effect for the materials studied were obtained against the bacterial strains are shown in Table 3. For the evaluation and economy of materials, the two extremes of concentration of CLX adsorbed in the materials were chosen, that is, the materials developed with theoretical concentrations of 0.20% v/v and 1.20% v/v.

Table III
Results of the inhibitory effects of composites against bacterial strains via DCT.

Materials	Inhibitory effects (%)
	E. coli (ATCC 25,922)	S. aureus (ATCC 25,923)
HA	4±3	7±2
HA/CLX-0.20	51±4	70±4
HA/CLX-1.20	100	100
Ga-HA	45±3	74±4
Ga-HA/CLX-0.20	100	100
Ga-HA/CLX-1.20	100	100

The results showed that only the HA/CLX-0.20 material did not show 100% effectiveness in inhibiting bacterial growth against Staphylococcus aureus and Escherichia coli. Figure 6 shows the photos of the plaques obtained after bacterial inhibition assay via DCT.

It is noteworthy that the average amount in mg/g of CLX adsorbed for materials HA/CLX-0.20 and Ga-HA/CLX-0.20 were very close: 0.0130 mg/g and 0.0132 mg/g, respectively. Thus, the results of bacterial growth inhibition indicated that there is an improvement in antibacterial activity due to the presence of Ga and that the already known antibacterial effects of CLX are not acting with 100% effectiveness due to the low concentration of the antiseptic. For example, Moreira et al., 2011 ⁵⁵ demonstrated the efficacy of chlorhexidine at 0.12% v/v and 0.20% v/v; these two concentrations inhibited the growth of S. aureus, E. faecalis, E. aeruginosa, and S. mutans bacteria. According to Hortense et al., 2017 ⁵⁶ CLX is effective in controlling bacterial plaque, acting preventively and in the treatment of oral diseases, with low toxicity and minimal side effects. Therefore, it is believed that the presence of Ga may be a factor responsible for the degree of potentiation of the antibacterial inhibitory effects observed in the formulation by Ga-HA/CLX-0.20 (about 51% for E. coli and 70% for S. aureus inhibition).

These results can be explained by the antibacterial action of Ga³⁺, which is based on the replacement of Fe³⁺ ions and the consequent blockage of many important redox reactions and enzymes within bacterial cells. ^57),(58. Suenaga et al.,⁵⁹ pointed out that such a mechanism may occur between the interactions of Ga present in hydroxyapatite with the constituents of bacterial cell walls, with the occurrence of redox interactions and/or reactive oxygen species, thus promoting an imbalance related to cell permeability and, consequently, the occurrence of apoptosis.

The antimicrobial action of CLX can be attributed to probable interactions between the cationic groups that constitute the chemical structure of drugs. These groups are attracted by the negative charge of the bacterial surface, causing electrostatic interactions to occur, characterized by the adsorption of CLX on the surface of the bacterial cell wall. This phenomenon may be responsible for causing cell lysis, thus, consequently, the death of these microorganisms is caused ⁶⁰.

Figure 7 shows the results of cytotoxicity testing via the MTT colorimetric assay. The test was performed via an extract of the precursor (HA and Ga-HA) and Ga-HA/CLX-0.20 and Ga-HA/CLX-1.20 materials, with an incubation time of 24 h. Cell metabolism results were compared to the negative control (DMEM + FBS 10% v/v) (100% front) and groups were classified as having results above 80% are considered non-cytotoxic; those between 80 and 71%, weakly cytotoxic; those between 70 and 61% moderately cytotoxic; and those below 60%, strongly cytotoxic ⁶¹. The results obtained were expressed in optical density (OD)×100 (Eq. 4), obtained by a spectrophotometer at 570 nm.

Figure 7:
Results of in vitro cell viability of materials synthesized in this study. Results are expressed as a percentage of cell viability relative to the negative control group (C-): DMEM + 10% v/v FBS. The positive control group (C+) as a reference of cytotoxic effect is composed of: DMEM + FBS 10% v/v + DMSO 30% v/v. Statistical analysis: ONE WAY ANOVA and the post-hoc test. * p≤0.05.

The evaluated cell viability results indicated that the HA and Ga-HA materials had viability levels compatible with a non-cytotoxic effect (cell viability ≥ 80%). Statistical analysis showed that the HA and Ga-HA ceramic materials had cell viability levels of 89±1% and 98±6%, respectively, thus similar to the negative control group (100%), with significantly p ≤ 0 .05 observed.

The synthesized HA and Ga-HA materials did not interfere with cellular metabolic activity and, consequently, cell viability. In other words, HA and Ga-HA materials were classified as non-cytotoxic (inhibition less than 20% compared to the control group). The Ga-HA material exhibited a cell proliferation effect compared to HA of about 9% more. It is believed that this potentiation of the effects of inducing cell growth is due to the presence of Ga in the hydroxyapatite particles. Melnikov et al. (2008) ¹⁸ point out that gallium ions are clinically effective against bone resorption and for the treatment of osteoporosis and cancer-related hypercalcemia. It increases and induces growth in bone calcium and phosphorus content and has direct non-toxic effects on osteoclasts.

When evaluating the cytotoxic effects of materials functionalized with CLX, it was observed that Ga-HA/CLX-0.20 and Ga-HA/CLX-1.20 materials showed cell viability of 81±3% and 35±5%, respectively. The Ga-HA/CLX-0.20 material was classified as being non-cytotoxic, and the Ga-HA/CLX-1.20 was classified as being strongly cytotoxic (cell proliferation effect < 60%). Thus, the analysis of the curve of incorporation of CLX into Ga-HA (Figure 4), combined with tests of antimicrobial activity and cytotoxicity, showed that the concentration of 0.20% (v/v) of CLX is ideal to achieve an effective bactericidal effect and not to favor possible cytotoxicity effects of the materials obtained in this study.

According to Souza et al.. (2011) ⁶², the results of the in vitro tests signaled the potential of using the combination of hydroxyapatite + chlorhexidine for prophylactic or therapeutic procedures as an alternative to systemic antibiotics. The advantage of this combination is that it combined the bioactivity of Ga-HA as a hard tissue regeneration material with the antimicrobial activity of CLX. In addition, the effects of enhancing cell viability in the material containing Ga may have also influenced the minimization of probable toxic effects of CLX. Therefore, a possible biocompatible behavior was observed in the material Ga-HA/CLX-0.20 since the presence of CLX did not impair the viability of the cell host.

To investigate some thermodynamic parameters of the interaction between hydroxyapatite (HA or Ga-HA) and CLX, a brief computational study was also performed. The surface of hydroxyapatite, in the gas phase ⁶³ and aqueous medium ⁶⁴, has previously been studied through DFT calculations, which may justify the choice for this type of approach. It is also worth mentioning that the vibrational (infrared intensities) and structural properties of hydroxyapatite do not vary significantly with temperature ⁶⁵.

To represent the hydroxyapatite, the unit cell parameters were used, which were provided through refinement by the Rietveld method (parameter values set out in section 3.1 of this study). The ground state chlorhexidine geometry was obtained at the B3LYP/CEP-31G(d) level. Figure 8 shows a representative schematic of the hydroxyapatite and chlorhexidine structures used. Table 4 summarizes the results obtained at the B3LYP/CEP-31G(d) level.

Figure 8:
Representative scheme of the structures of hydroxyapatite (HA or Ga-HA) and chlorhexidine.

Table IV
Total electronic energy (0 K) and Gibbs energy (298.15 K) in arbitrary unit (a. u.), interaction energy (ΔE_int or ΔG_int) and relative stability (R.S.) of the n-configurations obtained in kcal mol^-1.

System	Eletronic energy (a.u.)	ΔEint (kcal mol-1)	R.S. (kcal mol-1)	Gibbs energy (a. u.)	ΔGint (kcal mol-1)	R.S. (kcal mol-1)
Gas phase
CLX	-271.16	-	-	-270.71	-	-
HA	-465.1	-	-	-465.11	-	-
conf(Int1)	-736.39	-37.2	68.1	-735.86	-23.3	65.8
conf(Int2)	-736.50	-105.3	0.0	-735.97	-89.1	0.0
Aqueous phase obtained with PCM
CLX	-271.20	-	-	-270.75	-	-
HA	-465.43	-	-	-465.38	-	-
conf(Int1)	-736.66	-17.1	41.3	-736.14	1.4	38.8
conf(Int2)	-736.72	-58.4	0.0	-736.20	-37.4	0.0

• R.S. = relative stability

The interaction energy is obtained through the energy $\Delta E_{int}=E_{conf\left(in{t}_n\right)}-E_{HA}+E_{chlorhexidine}]$ difference of a given configuration ($E_{conf\left(in{t}_n\right)}$ of the product, and the sum of the energies of isolated hydroxyapatite E_HA and chlorhexidine (E_{chlorhexidine}). Gibbs energy values and a parameter ΔG_int which is calculated analogously to ΔE_int were also shown. To simplify the discussion, the relative stability of the two most stable configurations found during the interaction between the HA + CLX systems was also calculated.

During the interaction of HA with CLX, which led to the formaion of conf(int₁) (Figure 9), , it was observed the existence of a coordination between the nitrogen atom of the group (-C=NH) in the CLX molecule with a of the calcium atoms on the HA surface, with an N-Ca bond distance of around 2.41 Å. However, as the chlorhexidine molecule is symmetrical, a new coordination of the opposite (-C=NH) group present in the chlorhexidine molecule with calcium atoms on the surface of the HA may also occur, which leads to the formaion of conf(int₂). The calculations performed considered only a single structure for both hydroxyapatite powders in this study; that is, the structure used in this stage represented HA and Ga-HA.

Figure 9:
More thermodynamically favorable configurations for the two configurations calculated between the interaction of CLX with HA or Ga-HA.

In the latter case, the non-simultaneous coordinations still occur with the migration of a hydrogen atom from one of the groups (-NH) neighboring the group (-C=NH), which is captured by one of the oxygen atoms on the HA surface. As can be seen in Table 4, a conf(int₂) (Figure 9) is approximately 68 kcal mol^-1 more stable in relation to the one that only occurs in a single N-Ca coordination and 66 kcal mol^-1 when analyzing the ΔG_int in gaseous phase. Thus, it can be stated that the interactions between hydroxyapatite and chlorhexidine are thermodynamically favorable and that the inclusion of the implicit solvent model significantly changes the energy of the system.

Figure 10 shows the calculated FT-IR theoretical spectrum of isolated HA and chlorhexidine, as well as conf(int₁) and conf(int₂) , and draws a parallel between the FT-IR spectra obtained experimentally for the materials (HA/CLX or Ga-HA/CLX) and the HA powder synthesized in this study.

Figure 10:
(a) Calculated FT-IR-theoretical spectra of isolated HA, HA/CLX and CLX, (b) FT-IR-experimental HA, HA/CLX or Ga-HA/CLX and CLX spectra and (c) spectra of theoretical FT-IR from conf (Int₁), conf (Int₂), experimental spectrum of Ga-HA/CLX and experimental spectrum of Ga-HA.

When calculating the theoretical CLX spectrum (Figure 10a), bands between the regions between 3800 to 2800 cm^-1 are observed. This region of the spectrum refers to the stretching vibrations of N-H groups present in the CLX structure. In the calculated spectrum of materials containing HA + CLX (Figure 10c), these bands were also observed but with a lower intensity since experimentally speaking, CLX is in lower concentrations about the adsorbent (HA or Ga-HA).

C=C vibrations of the aromatic and C-H rings that make up CLX also appeared in the theoretical and experimental spectra (Figure 10b) of CLX ^66),(67; however, in the latter, they appeared overlapping in this region. In the region between 1700 and 1300 cm^-1, in the conf(Int₁) e conf(Int₂) spectra, bands corresponding to the symmetric angular deformation of the plane as well as folding of the N-H and C-H groups were simulated and were also observed in the experimental spectrum of material ⁶⁷, but with peaks of very low intensity, once again justified by the amount of CLX in the system.

In this same region, now for the experimental spectrum of the material, peaks of very low intensity are observed, corresponding to the symmetrical angular deformation of the plane, as well as the bending of the amino and hydroxyl groups. In the region between 1550 and 1530 cm^-1, bands referring to the deformation of CH₂ groups appeared. These bands are present in all theoretical and experimental configurations that contain CLX and are absent in the theoretical and experimental spectra of hydroxyapatite. Finally, in the region between 960 and 820 cm^-1, bands related to the Cl-C vibration (chlorine bound to the aromatic ring) were observed, and bands from the para-disubstituted aromatic rings are present in all theoretical and experimental configurations that contain the CLX ⁶⁷.

Although the methodology employed is not the most rigorous for a more in-depth study involving hydroxyapatite, the results obtained at the B3LYP/CEP-31G(d) level show a favorable thermodynamic interaction with chlorhexidine that occurs through coordination of the N atom of the group (-C=NH) of CLX with calcium atoms from the surface of hydroxyapatite. A good agreement is also observed between the experimental and calculated spectra.

CONCLUSION

The study of the incorporation of CLX on the surface of HA and Ga-HA revealed the possibility of synthesizing a new biomaterial that can unify the antimicrobial properties of the drug, plus the applications of HA as a bioceramic in regions compatible with bone and dental tissues. The characterization techniques of the materials used in this study confirmed the success of the synthesis of the hydroxyapatite phase confirmed the presence of Ga, as well as the efficiency of the incorporation of CLX on the surface of the powders. The MTT results revealed that the presence of gallium in the HA potentiated the degree of cytocompatibility of the Ga-HA material. Direct contact antimicrobial tests against S. aureus and E. coli showed 100% efficacy for all materials containing Ga-HA/CLX. It is noteworthy that the amount of CLX (0.20% v/v) was 100% effective in the antimicrobial action against the strains studied and that the Ga-HA/CLX-0.20 material was classified, after 24 h of cell incubation, as being non-cytotoxic. The thermodynamic values and the similarity found between the theoretical and experimental FT-IR spectra in the computational simulations via DFT showed the existence of two configurations of possible interactions and favorable thermodynamics. The conf(int₂) was approximately 68 kcal mol^-1 more stable compared to conf(int₁), which occurs only with N CLX coordination with HA surface Ca. Thus, it was possible to conclude that the materials (HA/CLX and Ga-HA/CLX) are formed via chemical interaction through coordination of the N atom of the (-C=NH) group of CLX with calcium atoms on the HA surface. Therefore, the conf(int₂) is the most favorable for the system studied in this work.

ACKNOWLEDGEMENT

This work was partially supported by Brazilian agencies MCTIC/CNPq (Grant #306176/2019-0) by FAPEPI, and his study was financed in part by the Coordenação de Aperfeiçoamento de Pessoal de Nível Superior - Brasil (CAPES) - Finance Code 001.

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Edited by

• (AE: Daniel Z. de Florio)

Publication Dates

• Publication in this collection
  04 Apr 2025
• Date of issue
  2025

History

• Received
  11 Feb 2024
• Reviewed
  20 Nov 2024
• Reviewed
  23 Feb 2025
• Accepted
  28 Feb 2025