3.1. Composition Porous HA Frameworks and Composites HA-PLGA
When obtaining porous HA frameworks, each stage was carried out with control of the phase composition. The diffractogram of the initial mixture of HA + pore former (
Figure 1) shows the presence of two phases in its composition—halite (NaCl) and hydroxyapatite (Ca
10(PO
4)
6(OH)
2), no foreign phases were found in the mixture.
The content of NaCl in the samples ranged from 10 to 50 wt.%. The HA-NaCl ratios are shown in
Table 1.
Controlling the phase composition of the resulting frameworks showed that the phase composition of the HA material (75) changed after calcination and removal of NaCl; the main phase was hydroxyapatite with the composition Ca
10(PO
4)
5,55(HPO
4)
0,45(O
0,53(OH)
1,39) (
Figure 2). In the composition of HA (90) and HA (50), the phase of hydroxyapatite was registered Ca
10(PO
4)
6(OH)
2.
With an increase in the amount of NaCl, the lattice parameters and coherent scattering region (CSR) of the samples change (
Table 2). Depending on the amount of NaCl, the phase ratio changes, which leads to a different final structure of the hydroxyapatite framework.
We found that in the course of investigating the intermediate stage of preparation after calcining the framework in the process of partial incorporation of NaCl into the HA structure, a new phase of chlorine-substituted HA Ca
9.7(P
6O
23.81)Cl
2.35(OH)
2.01 is formed (
Figure 3), Ca
10(PO
4)
6(OH)
2 and NaCl were also observed in the composition.
After calcination, sodium chloride in the composition of the ceramic was subjected to dissolution and observed the formation of a new phase Ca
10(PO
4)
5.55(HPO
4)
0.45(O
0.53(OH)
1.39). It can be assumed that the process of the formation of a new phase occurs according to the scheme:
Since the final phase contains hydrophosphate acid residues, we can assume that during the dissolution of NaCl, the hydrolytic decomposition of the compound Ca9.7(P6O23.81)Cl2.35(OH)2.01 occurs with the formation of a nonstoichiometric HA of the composition Ca10(PO4)5.55(HPO4)0.45(O0.53(OH)1.39), the crystallite size remains practically unchanged.
By the method of X-ray photoelectron spectroscopy, the chemical composition of the surface of samples of porous frameworks with different NaCl content was studied.
Figure 4 shows the XPS spectra of Ca2p, P2p, and O1s of the porous ceramic surface ready for impregnation. The binding energies of Ca2p and P2p electrons and their ratios practically do not change, while the oxygen fraction and the ratio change in HA (75), which can be explained by its phase composition that is different from other materials.
The difference in the elemental composition of the surface between HA (90)–HA (50) does not go beyond 1% (
Table 3). The HA sample (75) has the closest to the literary Ca/P ratio for natural HA, which is also the closest to natural bone. The Ca/P ratios of real minerals differ significantly from the calculated one, since the calculation was performed minus the carbonates that are formed on the surface during the calcination of ceramics and are also adsorbed in the air atmosphere.
To obtain composite materials, porous HA frameworks were impregnated with an PLGA solution. The concentration of PLGA was selected with the condition of determining the maximum weight gain of PLGA after impregnation (
Figure 5). The concentration of PLGA affected the polymer content of the final composite (
Figure 5). With an increase in the concentration of the solution, the proportion of PLGA in the materials increases, however, with an increase in the concentration above 0.1 g/mL, the proportion of PLGA in the materials decreases, which is associated with an increase in the viscosity of the solutions. The concentration at which occurs a change in slope of the curve inherent viscosity (η
i) upward coincides with the point of the maximum mass fraction of PLGA in materials. The observed effect is explained by the fact that at concentrations above 0.1 g/mL the intermolecular interaction of the polymer molecules is amplified, high viscosity prevents the solution to penetrate more deeply into PLGA and secured in the frame HA. The porosity of materials also affects the amount of polymer in the material; with an increase in the amount of a pore former, an increase in the amount of PLGA in composites occurs.
In summary, it should be noted that the amount of PLGA in the final composites increases from the HA (90) to the HA (50). This fact is associated with an increase in the porosity of the frameworks of the composites (
Table 4). Ultrasonic treatment provides an increase in the proportion of PLGA in the material due to the sound capillary effect.
The use of ultrasound leads to a decrease in time costs by ≈72 times and an improvement in the quality of impregnation with a polymer by ≈40% relative to ordinary impregnation without ultrasound.
The IR spectra (
Figure 6) of the composites contain bands corresponding to the vibrations of the νC=O and δC–C bonds in the PLGA structure, as well as δPO
43− included in the HA structure [
20].
Due to the fact that the mass fraction of the copolymer is small, the intensity of the νCH and νCH
3 bands is extremely low. In the spectra of composites, a slight shift of the carbonyl group band to shorter wavelengths occurs, which is usually associated with the geometry of the molecule, the mass of atoms associated with the carbonyl group, induction and mesomeric effects, steric factors [
21], which suggests the formation of low-energy bonds between the PLGA and the surface HA.
3.2. Porosity of HA-PLGA Composites
The study of the specific surface area of the samples (
Figure 7a) showed that after sonication there is a significant decrease in Ssp ≈ 50% relative to untreated samples, which is associated with the displacement of air that fills the ceramic frame. Probably, there is a partial overlap of micro- and mesopores (
Figure 7b), while macroporosity is retained in the samples. After removing the material from the solution, the solvent evaporates and the surface of the ceramic is lined with a polymer to form a film that overlaps the microporosity but leaves the macroporosity (empty space capable of forming channels in the ceramic through which liquids penetrate into the material).
This statement is confirmed by scanning electron microscopy (SEM) images of sample fractures (
Figure 8). The sections are covered with pores from 5 to 70 μm. There are also single pores up to 150 μm in size.
The apparent (open) porosity of the samples was determined by the liquid absorption method. Based on the results obtained, the open porosity was calculated for composites and initial non-impregnated frameworks (
Figure 9). The volume of open porosity decreases after the ceramic is impregnated with PLGA due to the filling of a part of the pores. Moreover, the higher the initial porosity, the better the penetration of the polymer into the interior of the porous ceramic.
To confirm that the porosity of the samples is open, the diffusion coefficient was calculated using the Franz cell (
Figure 9). Composites and frameworks were used as a membrane. This approach allows us to conclude that three structures were obtained in which open porosity is formed in different ratios. The data obtained are in direct relationship—with an increase in the porosity of materials, there is a regular increase in the diffusion coefficient D.
3.3. Surface Properties of Composite Materials HA-PLGA
SEM images were obtained for the composites (
Figure 10). All three composites are characterized by different surface polymer coating. HA (90)-PLGA has an uneven deficient coating with single polymer aggregates with a coverage volume of 10–50% of the surface in different areas. HA (75)-PLGA has a uniform island coverage of HA particles with a polymer over the entire surface area and the coverage volume is ≈68–70% in different areas, such a coating forms a large number of interphases. HA (50)-PLGA is completely covered by the PLGA, the coating is nonuniform, as evidenced by large PLGA aggregates on the ceramic surface.
The chemical composition of composite samples was studied by X-ray photoelectron spectroscopy (
Figure 11). The binding energies of electrons Ca2p and P2p practically do not change when comparing the composites with each other, as in the case of pure ceramics, however, the O1s ratio changes in HA (75)-PLGA, which can be explained by its phase composition that differs from other materials.
The XPS results (
Table 5) showed that on the surface of HA (75)-PLGA, the carbon content is significantly less than on the surface of HA (90)-PLGA and HA (50)-PLGA. This is probably due to the fact that the HA (75)-PLGA sample has a different phase composition from the others, which led to a change in the nature of the coating.
Comparing the composition of the surface after coating with a polymer with pure ceramics, it can be observed that the coating with a PLGA layer leads to a change in the Ca/P ratio on the surface; it significantly decreases with an increase in the amount of PLGA.
The ratio of chemical bonds of carbon in composites changes (
Table 6). In the HA (75)-PLGA samples, an increase in the area of the C1s peak corresponding to the C–C/C–H (1) bond and a decrease in the area of the C1s peak for the C–O/COOR (2) and (COOH) bond (3) are observed. It can be assumed that the decrease in the proportion of C–O/COOR (2) and (COOH) (3) bonds in the HA (75)-PLGA sample is due to the fact that the bonds are associated with HA at the interphases (according to SEM data,
Figure 10). Since the Ca/P ratio in the initial HA ceramics (75) is somewhat lower than in the other samples, this suggests that a larger number of P atoms, which is more electronegative than Ca, allows a stronger association of PLGA molecules on the HA surface, as a result of which functional groups CO/COOR (2) and (COOH) (3) are oriented “inward” to HA (
Figure 12). This fact leads to a significant decrease in the area of C2 and C3.
The analysis of functional centers on the surface of composite materials was carried out by the Hammett indicator method. The diagrams (
Figure 13) show that pure HA has pronounced acid–base centers (ABC) with pK
a = −4.4; −0.29; 9.15; 16.8, represented by calcium and the oxygen lone pair of phosphate groups. The profile of the composites looks different from HA and PLGA. They also contain acid–base centers corresponding to HA and PLGA, but at the same time, intensely expressed new centers appear in the region of pK
a = 6.4–6.9, pK
a = 9.45, and pK
a = 12. The appearance of new centers is associated with weak interactions between the components of composite materials. Due to the orientation of PLGA relative to the HA centers in composite materials, their acid–base properties are enhanced. In composites, the concentration of acidic and basic centers increases, and there is a correlation between the results and the Ca/P ratio on the surface. The presence of a large number of interphases of composite materials promotes the formation of new centers, while the specific distribution of PLGA enhances this effect.
For all composites and initial components, the contact angle (θ, °) was determined by the lying drop method. All materials and components are hydrophilic in nature, since θ
water < 90° (
Table 7).
Calculation of the surface energy of the samples (
Table 8) showed that HA (75)-PLGA also has the highest surface energy among composites, this is facilitated by the composition of its surface, on which the number of ABC in the region pK
a = 6.4 prevails, the centers are formed behind due to the greater amount of oxygen on the surface of the material, as well as due to the island coating of the ceramic with a polymer.
There is a codependency between σ
P and the oxygen concentration on the surface of materials—with an increase in the amount of oxygen, the polar component of the surface energy naturally increases (
Figure 14).
The study of the acid–base properties of the surface showed that in the composites there is an increase in the concentration of acid–base centers, there is a codependency of the results with the Ca/P ratio. Calcium-deficient HA on the surface of materials promotes the formation of new centers, while the specific distribution of PLGA enhances this effect.
3.4. Viability, Pro- and Anti-Inflammatory Properties of Composite HA-PLGA Materials
Important functional properties such as biocompatibility and anti-inflammatory properties of HA-PLGA composites were studied by analyzing the developmental features of the cell-mediated immune response of individual donors in vitro by the nature of the production of pro- and anti-inflammatory cytokines by primary monocytic macrophages in the studied samples, depending on the direction of their differentiation in the process culturing CD14+ blood monocytes.
The Enzyme-linked immunosorbent assay (ELISA) results showed that in the presence of PLGA, a significant increase in the secretion of IL-1β was recorded in the culture of M0-macrophages from donors 1 and 2 (
Figure 15a). In the presence of HA, the production of IL-1β by M0-macrophages increases 4.5-fold relative to the control only in donor 1. M2 macrophages obtained by stimulating monocytes with IL-4 secrete IL-1β in large amounts on the sixth day of cultivation in the presence of PLGA in all donors and in the control in donors 1 and 2 (cells cultured on plastic). Upon stimulation of macrophages with IFNγ, the expression of IL-1β is similar, but it is more pronounced than in macrophages M2 (
Figure 15a). As for the samples of HA (75)-PLGA and HA (50)-PLGA, no statistically significant parameters of IL-1β secretion were recorded in the presence of these composites, with the exception of donor 3, whose cells showed a proinflammatory cytokine response in the M0 culture in the presence of the sample. HA (75)-PLGA and culture M2, in the presence of HA (50)-PLGA, respectively, and in the control cells of this donor IL-1β did not secrete (
Figure 15a).
The ELISA results showed that in the presence of the HA (75)-PLGA composite, an increase in the secretion of IL-6 in the culture of M0-macrophages (not activated macrophages) of donor 5 is observed, which may indicate the potential readiness of M0-macrophages for differentiation in the “proinflammatory” direction of M1 (
Figure 15b). Increased production of IL-6 in comparison with control values was recorded in M0-macrophages of donor 5 in the presence of HA (90)-PLGA and PLGA. In donor 6, on the contrary, in the culture of M0-macrophages, a decrease in the production of IL-6 in the presence of PLGA was observed in comparison with the control values. In the presence of hydroxyapatite, IL-6 secretion in M0 macrophages was not detected in any of the donors, which indicates the absence of an acute inflammatory immune response (
Figure 15b). During M2 activation of macrophages, statistically significant changes in IL-6 secretion were not found, regardless of the composition of the test sample (
Figure 15b). With M1 activation, only donors 5 in the presence of HA and 6 in the presence of the sample HA (75)-PLGA show a significant decrease in the concentration of IL-6 in comparison with the control values. In donor 7, on the contrary, an increase in the production of IL-6 in M1 macrophages in the presence of PLGA was recorded both in comparison with the control and in comparison with its concentration in M0 macrophages.
Enzyme immunoassay showed that expression of CCL18 occurs in control in almost all donors with M2 activation of macrophages; insignificant secretion of this cytokine is also observed in control samples in M0 and M1 macrophages. Consequently, the cytokine-producing physiological function of M2 macrophages is preserved and is fully realized. In the presence of hydroxyapatite, a statistically significant level of CCL18 production was observed only in donor 7 with alternative M2 activation of macrophages. In the presence of composites and pure PLGA, expression of CCL18 was not observed regardless of the direction of differentiation of macrophages in any of the studied samples. This fact, on the one hand, indicates the absence of an active anti-inflammatory cytokine response during the interaction of innate immunity cells with samples of polymer materials. On the other hand, the cytokine network functions in such a way that the biological role of CCL18 in this case can be duplicated by other anti-inflammatory mediators. In addition, minimal cytokine production or its absence in a particular donor, regardless of the direction of differentiation of macrophages, may be due to the epigenetic mechanisms of the peculiarities of cytokine gene expression.
The viability test showed (
Figure 16) that the cells in the samples with PLGA and composites were generally comparable to the control sample. Cells in pure HA samples show low viability. Despite the fact that pure HA has pronounced cytotoxic properties, the addition of PLGA can significantly reduce the surface energy and improve the viability of cells.
The values of the viability correlate well with the value of the surface energy of materials and the concentration of acid–base centers in the pKa region of 6.4 and 6.9; this effect may indicate that it is these surface properties that are most important for these composites. These properties are determined by the phase composition of the initial HA ceramics and the nature of the PLGA distribution over its surface.