Characterization of calcium phosphate phases
In this study, the choice of the sol-gel method for the CaP´s synthesis was based on the simplicity of the method and the ability to obtain particles with nanometric structure, aiming at the in vivo biomineralization process. Using the same chemical precursors and different temperatures, it is possible to obtain two different CaP´s phases, namely: CPP and HA, with properties needed as biomaterial, such as osteoinductivity, bioactivity and nontoxic.
Figure 1A shows the FTIR analysis for CPP and HA. The CPP spectrum presents characteristic bands of OH stretching and H-OH deformation of H2O at 3434 cm− 1 and 1646 cm− 1, respectively. Several bands observed at 1131, 1018 and 926 cm− 1 were attributed to the symmetric P-O stretching of PO43− groups. The peak at 555 cm− 1 corresponds of the O-P-O deformation (PO43−). The existence of all these bands indicates the CPP formation [17]. In the HA spectrum, the water absorption appears around 3638 cm− 1 as stretching vibrations of OH groups. We observed the characteristic absorbance of the PO43− asymmetric deformation between 1029 cm− 1 and 1100 cm− 1 and around 874 cm− 1, associated with the P-OH stretch in HPO42−. The band near 600 cm− 1 corresponds to the asymmetric deformation of P-O bond in the phosphate network. Whereas the peak at 562 cm− 1 is related to the asymmetric deformation of P-O (H) bond [21]. The XRD patterns (Fig. 1B) confirmed the existence of the CaP´s phases obtained in each heat treatment performed in the sol-gel precipitate [17]. Both samples revealed broad peaks at 2θ = 20‒40°, characteristic of nanocrystalline phases. The intense peaks at 2θ = 25°, 32° and 35° indicated that product obtained at 400°C was Ca2P2O7 base on CSM card #96-100-1557. The thermal treatment at 600°C confirmed the formation the Ca5(PO4)3OH, identified by the peaks 26°, 31°and 33° (card #96-900-1234). In addition, morphological characterization was carried out (Fig. 1C). The CPP micrographs showed filiform particles grouped in the form of agglomerates with a size range of 170‒650 nm, while the particles of HA presented sizes between 110‒620 nm and hexagonal shape.
Calcium phosphate incorporation into the NRL membranes
The NRL membranes were prepared according to the Sect. 2.3. The proteins responsible for the allergic reactions of NRL have a molecular weight of about 14 kDa, and due to that, they can be easily separated by clarification unit (centrifugation),[22] where reduction of 27% of the proteins were achieved.
The incorporation process of CaP´s phases was fulfilled by precipitation, unlike the casting method commonly used in the literature [23–25]. Figure 2 (A-C) shows SEM micrographs of the surfaces of the NRL membranes without and with the inclusion of the phases containing CPP and HA. The pure NRL membrane (NRL-m) exhibited a smooth surface, with evidence of some irregularities (Fig. 2A), which can be explained by the fact that, in solution, some of the NRL proteins are released and, after drying, they are precipitated on the surface [26]. With a magnification of 2000x, it was possible to observe that the CPP particles are homogeneously dispersed in the NRL matrix (CPP-m) (Fig. 2B). In the micrograph of the membrane incorporated with HA (HA-m), a heterogeneous layer of HA was denoted on the material surface (Fig. 2C). The lower uniformity of the HA particles in the HA-m membrane in relation to the CPP-m membranes may be related to the different solubility of each CaP´s phase, because the higher the Ca/P ratio, the lower the solubility in water [27]. HA has the highest Ca/P ratio with a value of 1.67, while CPP has a ratio of 1.0. Thus, this large difference in solubility makes CPP easier to be adsorbed by the polymeric membrane. To confirm the presence of CPP and HA particles in the membranes, we used the EDX technique coupled to the SEM. In Fig. 2 (D-E), the EDX spectra are demonstrated and the presence of both chemical elements Ca and P, constituents of CPP and HA, can be observed on the membranes surface.
The obtaining of CaP´s-NRL composites with different CaP´s has been the object of study by different researchers [28, 29]. However, there are few studies that have precisely explained the molecular interactions between the non-rubber constituents of NRL and inorganic particles. However, it is known that, on the CaP´s particles surface, there are Ca2+ ions that are attracted by the negative charges found in the NRL proteins and lipids.
In vitro toxicity assays
Evaluation of hemolytic activity
Hemolytic activity assay was accomplished to assess whether the components released by the samples NRL-e24, NRL-e120, CPP-e24, CPP-e120, HA-e24 and HA-e120 are able to rupture the red blood cell membrane, causing hemoglobin release [16, 30]. Figure 3 shows the positive control with red color, indicating that hemolysis occurred due to the release of hemoglobin, and the transparent samples (negative control and other samples), where there was no hemolysis. According to Fischer et al. [31] rates below 10% of hemolysis suggest that the samples are considered non-toxic. The released hemoglobin percentages were calculated and it was proved that the eluates at 24 and 120 h exhibited hemolysis levels less than 3%, confirming that there was no damage to the red blood cell membrane and the type of CaP´s did not influence hemolytic activity. Our results are complemented by those obtained by De Barros et al. [32] who tested the hemolytic activity of the NRL membranes with oxytocin, and values below 5% were achieved. Floriano et al. [33] also verified that the NRL membranes coated with CaP´s and with ketoprofen respectively, did not present hemolytic effect.
Cytotoxicity assay in osteoblasts
The in vitro test was performed with MC3T3 osteoblasts. The samples NRL-e24, NRL-e120, CPP-e24, CPP-e120, HA-e24 and HA-e120 were placed in indirect contact with the cells for 24 and 48 h. After 24 h of plating, it was found that the cells were adhered and the culture medium was replaced. The MTT assay was carried out at 48 h and it was observed that all samples, except the positive control (100%), demonstrated percentage values of cell viability less than 50%. This result indicates that the eluates tested showed cytotoxic behavior, which is in contradiction with the countless studies in the literature that demonstrate the excellent biocompatibility and bioactivity in vivo of NRL [9, 10, 34, 35].
However, in this study, biocompatibility assays were evaluated in the serum (eluates) not in the NRL membranes. It is known that NRL contains proteolytic enzymes, such as serine and cysteine proteases, [16, 36–38] which can influence the cell viability assay. To prove this hypothesis, a cell culture test was accomplished using only NRL serum previously centrifuged (at 19000 rpm), and after 5 min of incubation, it was microscopically verified that there was no evidence of cell adhesion and the MC3T3 osteoblast cell body was dissociated. This result indicated that the proteolytic enzymes present in NRL may be able to dissociate the MC3T3 cells, causing the low values of cell viability obtained in the MTT assays. Borges et al. [20] obtained similar results when studying the eluate of the incubation of the NRL membrane without dilution, exhibiting a cell viability of approximately 48%.
From these results, a new cell adhesion test was realized. At this time, to eliminate the enzymes that prevent adhesion, the membranes were prepared according to Sect. 2.7. The micrographs of the membranes surface after 48 h of culture (Fig. 4(A-C)) revealed that the cell line established points of adhesion and the cells did not undergo morphological changes. The existence of lamellipodia and philopodia of osteoblasts (red arrows), as well as intercellular connections (blue arrows), indicated that there was a good adhesion of osteoblasts to the membranes surface [20]. In tissue engineering, in addition to cell adhesion, cell proliferation and differentiation are necessary to guarantee greater bioactivity of the material [39]. In this study, it was possible to observe the cell division in the NRL-m, CPP-m and HA-m membranes (Fig. 4 (D-F)) through the FEG-SEM analysis, suggesting the cell proliferation qualitatively.
According to Deligianni et al. [40] osteoblasts present better proliferation, adhesion and mineralization on rough and sandy surfaces. Thus, the CaP´s phases are excellent candidates for polymer coating, because, in addition to these characteristics, CaP´s are formed by the main bone constituents, bonding chemically and directly to the bone, resulting in a better bone repair. Figure 4 (D-F) exhibits a close connection between clusters of CPP and HA and osteoblasts on the NRL membranes surface (green arrows). These results suggest good cell viability on the membranes. Finally, the in vitro biocompatibility of the prepared membranes did not show appreciable differences between them.
In vivo toxicity assays
Toxicity assay in Caenorhabditis elegans
The toxicity assay using C. elegans followed the methodology described in Sect. 2.4.1 where all membranes were incubated separately in PBS solution for 24 and 120 h. The eluates resulting from these incubations were placed in contact with the worms and analyzed after 24 and 48 h. Figure 5 (A-C) reveals the percentages of survival, where in the first 24 h of the experiment, all samples did not significantly reduce the survival of C. elegans worms. After 48 h of assay, the survival rates of the animals exposed to the samples were analyzed again and the eluates NRL-e24, NRL-e120, CPP-e24, CPP-e120 and HA-e24 did not show mortality. However, the worms exposed to the HA-e120 sample presented toxic effects, with only 42% survival.
In this study, the toxicity also was verified by the worm development and by monitoring the body shape. Live worms (sinusoidal shape) were observed for all samples and a predominant amount of dead worms were identified by their rod-shape in the HA-e120 sample (Fig. 5 (D-F)). The toxicity of this sample may be related to the high presence of Ca2+ ions in the HA-e120 eluate (5 days). As previously mentioned, the difference in the Ca/P ratio between the two CaP´s resulted in a lower solubility for HA, which caused the HA particles to not be fully adsorbed on the NRL membranes surface (Fig. 2C), being easily released in the HA-e-120 eluate. Ca2+ plays an essential role in physiological processes and in cell proliferation or differentiation. Alvarez et al. [41] summarized evidence from various experiments on C. elegans worms that helped to understand the role of Ca2+ in living systems. However, their results showed that the system is quite complex and there is only a fine line that separates the levels of calcium flows that can be considered normal from those that can lead to pathology. Therefore, an excess of Ca2+ ions in the HA-e120 eluates (after 5 days of incubation) may have induced the death of a greater number of worms. Nematode reproduction was also evaluated (Fig. 5E) and in all samples, except HA-e120, the presence of the 1st larval stage (L1) from the 4th larval stage (L4) reproduction indicates the non-toxic character of these samples.
Embryotoxicity assay in Danio rerio (Zebrafish)
A second in vivo model was used to investigate the biocompatibility of the eluates incubated for 5 days (NRL-e120, CPP-e120 and HA-e120). Recently, Zebrafish proved to be an ideal model for studying biocompatibility and bone disease [42]. Zebrafish embryos were analyzed by determining different physiological parameters, such as percentage of viability and phenotypic morphological alteration. Figure 6A shows the survival rate of the three samples and the control in relation to the contact time with the embryos. The samples were obtained employing the same methodology used in the C. elegans assay and the eggs were exposed for 24, 48, 72 and 96 hpf. Quantitatively, all samples, including the control, exhibited dead embryos from 24 hpf. The survival rate in the solvent control was approximately 76%, which was reduced to nearly 60% when the embryos were incubated in the studied eluates. It was noted that the reduction was not dependent on the exposure time or the type of eluate. These results suggest low embryotoxicity, as the values obtained by the eluates were near to those of the control sample (containing only embryonic medium, without embryos), which is widely used as control in this type of assay [43].
In view of these results and knowing that the embryonic stages are extremely sensitive for the determination of toxicity, [15] the number of live, dead, delay in development and deformed embryos was counted. This study showed only the existence of live or dead animals (more than 50% of Zebrafish embryos were found alive) and none of the tested samples induced deformation or delay in embryonic development (Fig. 6B). This result was supported by the analysis of the images obtained from the development of live embryos. Through the transparency of the embryos, it was possible to observe different stages of normal development, according to the phases of gastrula, pharynx, early larvae and larva (Fig. 7(A-D)), which were found in all samples [44]. The certification that the embryos were alive was carried out through microscopic observation of the sequence of development and movement of the embryos. The existence of worms with curvature of the tail and pericardial edema were considered dead.
These results are in line with those reported by Makkar et al. [45] who used the Zebrafish model to study the biocompatibility of two dental bioceramics, one of which is the mineral trioxide aggregate (MTA) composed mainly of inorganic elements containing calcium. The eluates of MTA contain silicates, aluminates and calcium oxides, which facilitated the increase in abnormal metabolic activity in embryonic cells, causing a percentage of embryonic deaths from oxidative stress and cellular apoptosis.