Naturally-Derived Biphasic Calcium Phosphates through Increased Phosphorus-Based Reagent Amounts for Biomedical Applications

Abstract

Calcium carbonate from marble and seashells is an eco-friendly, sustainable, and largely available bioresource for producing natural bone-like calcium phosphates (CaPs). Based on three main objectives, this research targeted the: (i) adaptation of an indirect synthesis route by modulating the amount of phosphorus used in the chemical reaction, (ii) comprehensive structural, morphological, and surface characterization, and (iii) biocompatibility assessment of the synthesized powdered samples. The morphological characterization was performed on digitally processed scanning electron microscopy (SEM) images. The complementary 3D image augmentation of SEM results also allowed the quantification of roughness parameters. The results revealed that both morphology and roughness were modulated through the induced variation of the synthesis parameters. Structural investigation of the samples was performed by Fourier transform infrared spectroscopy and X-ray diffraction. Depending on the phosphorus amount from the chemical reaction, the structural studies revealed the formation of biphasic CaPs based on hydroxyapatite/brushite or brushite/monetite. The in vitro assessment of the powdered samples demonstrated their capacity to support MC3T3-E1 pre-osteoblast viability and proliferation at comparable levels to the negative cytotoxicity control and the reference material (commercial hydroxyapatite). Therefore, these samples hold great promise for biomedical applications.

Keywords: CaCO3 derived-calcium phosphates; SEM; dolomitic marble; image analysis; modulated synthesis set-up; pre-osteoblasts; seashell.

Figure 1

Comparative XRD patterns of the calcium phosphate powders synthesized using natural resources (i.e., (a) marble and (b) seashells) as calcium precursors and phosphoric acid amounts situated in the range 0–90%.

Figure 2

Comparative FTIR-ATR (attenuated total reflectance) spectra of the calcium phosphate powders synthesized using natural resources (i.e., (a,c,e,g) marble and (b,d,f,h) seashells) as calcium precursors and phosphoric acid amounts situated in the range 0–90%, collected in the four relevant wave numbers regions: (a,b) 700–500 cm⁻¹; (c,d) 1300–700 cm⁻¹; (e,f) 1800–1300 cm⁻¹; and (g,h) 3750–2650 cm⁻¹. To facilitate a better visual evaluation, the FTIR-ATR spectra were normalized to the intensity of the most prominent band region situated at ~1100–900 cm⁻¹.

Figure 3

Ca/P molar ratio of the powders synthesized from both natural precursors (M—marble, S—seashell) with acid addition in the 0–90% range.

Figure 4

3D surface topology reconstruction on the basis of SEM micrographs processing via Mountains Map software and roughness profiles assessment. Images were recorded at 500× magnification. Top center image: Scale bar (50 µm) for all micrographs. For all roughness profiles, the scale bar is provided on the right side (up and down) of the figure.

Figure 5

Roughness parameters quantification for marble (M) and seashell (S) derived samples: R_sk—profile skewness, R_a—profile arithmetic mean deviation, R_ku—profile kurtosis, R_p—maximum profile peak height, R_v—maximum profile valley depth, R_z—maximum height of profile, R_t—total height of profile.

Figure 6

Fluorescence micrographs of the MC3T3-E1 pre-osteoblasts grown in the extracts of marble and seashell-derived powdered samples for 1 day and 3 days. Cell staining with the LIVE/DEAD Cell Viability/Cytotoxicity Assay Kit (green fluorescence: live cells; red fluorescence: dead cells). Scale bar: 100 µm.

Figure 7

Cell viability/ proliferation of MC3T3-E1 pre-osteoblasts grown in the extraction media of marble- (a) and seashell- (b) derived powdered samples, as assessed by MTT assay (n = 3, mean ± SD).