Biomineralization of calcium phosphates functionalized ...

Abstract

Functionalization of calcium phosphates with biomimetic peptides is a promising strategy to increase cellular response for bone tissue repair. In this work, biphasic calcium phosphate pellets were functionalized with the hydroxyapatite-binding peptide pVTK by dropping a suspension of the peptide on the pellet surface. The bioactivity tests were performed in vitro by using McCoy culture medium. Cytotoxicity tests were also performed to assess cell viability. The material was characterized by X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FTIR) and scanning electron microscopy with field emission gun (FEG-SEM). The results showed that functionalization with the biomimetic peptide was most effective in inducing precipitation of bone-like apatite on the pellets surface, when compared to the control groups (two positive control groups and one negative control group). Cytotoxicity tests showed that all samples are biocompatible but the pellets with peptide showed the highest values of cell viability.

Introduction

Biomaterials are synthetic or natural structures that interact with biological tissues at physiological and biochemical levels. In To achieve this, they must be biocompatible, meaning they should function adequately for specific applications without triggering an adverse foreign body response (Nascimento, 2022). Notably, within the biomaterials class, bioceramics stand out for their potential in biomedical applications such as implants and grafts, mainly for the regeneration and repair of mineralized tissues (Ribeiro et al., 2020).

Hydroxyapatite (HA) and Calcium Phosphates (CaP) are classified as ceramic biomaterials that mimic the inorganic constituents of bone tissue, cartilage and the amelodentinary complex (enamel and dentin) (Moura et al., 2012). Moreover, these bioceramics satisfy the biocompatibility requirement by not induce inflammatory and/or immune response. This besides promoting crystallinity-dependent bioactivity (Fonseca et al., 2009). These materials can be designated as third generation's biomaterials in tissue engineering, where they have the technology of tissue regeneration systems through molecular biology and materials sciences (Zavaglia and Prado da Silva, 2016). However, although HA has biocompatibility and good conductivity, it still has a low potential for stimulating tissue growth (Dorozhkin, 2009, 2011).

Incorporation of other elements associated with the HA synthesis establishes a more dynamic form similar to tissue formation in natura. This in order to biomimicry organic apatites and achieve a greater potential for conduction and bioactivity (Dorozhkin, 2007; Prado da Silva and Navarro da Rocha, 2011). However, a strategy is to use HA-based and/or calcium phosphate-based main constituents as bone grafts (Ribeiro et al., 2020).

Hydroxyapatite-Binding Peptides (HBP) is biomimetic analogues that are established by mimicking the role of enamel matrix proteins and non-collagenous proteins. Thus, HBP can regulate the nucleation and growth of calcium phosphate particles as well as the transition phase to CaP (Prado da Silva and Navarro da Rocha, 2011; Ling et al., 2020).

The peptide pVTK (VTKHLNQISpQSpY) is a type of HBP in which it was noticed that, increasing the amount of phosphorylated amino acids (phosphorylated serines) would induce the nucleation of amorphous calcium phosphates and their differentiation into HA crystals. This peptide is unique in that it has four negatively charged phosphorylated amino acids and one non-phosphorylated, positively charged amino acid. The strategy is that: by increasing the oxidative phosphorylation capacity, the formation of amorphous calcium phosphate could be increased, subsequently the growth of HA. The pVTK exhibits a pronounced affinity towards HA, resulting in the formation of spherical nanoparticles (Ling et al., 2020).

Moreover, the charge density of the peptide mainly controls the surface binding affinity, while the secondary structure plays a minor role. HA binding affinity is correlated with inhibition of transformation into crystalline HA and formation of plate-like particles instead of needle-like particles. HBP templates possess mainly hydrophilic amino acid side chain residues, of which lysine, glutamic acid and phosphoserine are charged, while asparagine and serine are polar neutral amino acid residues. All these amino acids can interact with the calcium, phosphate and hydroxide ions on the surface of HA through electrostatic interactions or hydrogen bonding (Ling et al., 2020).

In this work, CaP pellet samples were used as representative models of amelodentinary tissues. The objective was to evaluate the biomineralization of calcium phosphates with hydroxyapatite-binding peptides.

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Section snippets

Calcium phosphate pellets synthesis

The CaP synthesis consisted of an aqueous precipitation route with pH control at ambient temperature (Prado da Silva and Navarro da Rocha, 2011). Thus, a precursor solution was prepared by mixing, under magnetic stirring, a 0.5 Mol suspension of [Ca(OH)2] (Isofar - Rio de Janeiro, Brazil), then a 1 Mol solution of [C3H6O3] (Isofar - Rio de Janeiro, Brazil) and, finally, a 0.3 Mol solution of [H3PO4] (Isofar - Rio de Janeiro, Brazil) was slowly added at a flow rate of 8 mL/min.

However, as CaP is

X-ray diffraction

The diffraction patterns shown in Fig. 1 were taken from CaPgreen (non-sintered) and CaPsintered (sintered at 1000 °C) samples. In addition, diffractograms from the groups that were used for bioactivity test, which are: CaP (CaP pellet in McCoy medium only), CaPPeptide (CaP pellet with solubilized peptide with PBS, in McCoy medium), and CaPPBS (CaP pellet in PBS only) are also shown. It should be noted that the samples of the bioactivity groups are based on the sintered samples (CaPsintered).

Discussion

In this study, we correlated the use of the pVTK peptide functionalizing CaP tablets to promote biomineralization through biological driving activity (bioactivity). Our results proved to be positive since the functionalization proved to be biocompatible (both the peptide and CaP), as well as, it was possible to promote bioactivity already in a shorter useful time with the presence of increased HA nucleation in CaP tablets.

The use of biomimetic peptides to functionalize CaP has been a strategy

Conclusion

In this study, the functionalization with HBP proved to be an effective way to increase bioactivity by fostering the precipitation of bone-like apatite in CaP samples. The HA content increase after the bioactivity tests, confirmed the biomineralization potential of the peptide. Furthermore, the biological effect of HBP on biomineralization was confirmed to be biocompatible and non-cytotoxic.

CRediT authorship contribution statement

Marvin do Nascimento: Writing – original draft. Aline Raybolt dos Santos Almeida: Supervision, Conceptualization. Mariah Cationi Hirata: Methodology, Investigation. Amal Elzubair: Writing – review & editing, Methodology, Investigation. Daniel Navarro da Rocha: Investigation, Formal analysis, Conceptualization. Marcelo Henrique Prado da Silva: Supervision, Project administration, Investigation, Formal analysis, Conceptualization.

Declaration of competing interest

I hereby declare that there is no financial interest involved in the submission of the study entitled “Biomineralization of Calcium Phosphates Functionalized with Hydroxyapatite-Binding Peptide”.

Acknowledgements

The authors are grateful to the agencies CAPES, FAPERJ for funding and endorsing this study and R Crio for cytotoxic test.

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