Bio-inspired synthesis of hybrid tube-like structures based on CaCO₃ and type I-collagen

Abstract

Although the exciting features exhibited by tube-like structures make them potential candidates for biomedical applications, few studies have dealt with the combination of tubular geometries and the biological responses of biominerals. This study reports on the synthesis of tube-like hybrid structures based on calcium carbonate, a biocompatible mineral, and collagen, the most abundant protein in bone tissue. A biomimetic model aided the synthesis of uniform tubular structures with controlled shape and diameter in the range of 100, 200, and 400 nm. Immersion of the particles in simulated body fluid (SBF) at 37 °C for 48 h allowed us to evaluate the bioactivity of the structures. Infrared spectroscopy revealed that biological apatite emerged in the material, confirming its bioactivity. Zeta-potential measurements attested to the high colloidal stability of the nanoparticles in the presence and absence of collagen. Confocal laser microscopy images confirmed that osteoblastic cells cultured in the presence of collagen-modified CaCO₃ tube-like nanoparticles proliferated. The 100 nm particles presented special behavior—they adsorbed a larger amount of collagen due to their higher surface area.

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Introduction

Over the last few years, tube-like nanostructures have attracted scientists' attention. Their geometry and composition can be tailored, to give rise to materials with unique physical and chemical properties, high surface area, and great mechanical strength. The resulting materials can be applied in several fields such as microelectronics;¹ catalysis;² optical;³ electronic;⁴ and magnetic⁵ devices; high-performance ceramics;⁶ sensors;⁷ and biomaterials.⁸ In particular, properties like high surface area; high mechanical, thermal, and chemical resistance; excellent flexibility; and low density make the tube-like structures potential candidates for biomedical research and application.

Among the uncountable biomedical uses of these materials, drug delivery systems,⁹ molecular detection,¹⁰ agents for imaging,¹¹ and osteoblastic cell proliferation enhancement stand out.¹² Studies have shown that tube-like structures mimic the nanoscale bone tissue features, which has a nanofiber arrangement.^13,14 As a consequence, osteoblasts proliferation, attachment, and migration are enhanced¹⁵ in the presence of these structures in comparison to conventional surfaces.^16,17 Despite these exciting features, application of these materials in vivo is limited by lack of knowledge about their toxicity, biocompatibility, and bioactivity, so additional efforts to improve their specific biological responses are welcome. The idea of combining tubular geometries with the biological responses of biominerals seems to be a good strategy to develop new biocompatible and bioactive materials.

Materials consisting of CaCO₃, which is one of the most abundant minerals in natural systems and displays biocompatibility and osteoconductivity,¹⁸ have many biomedical applications due to their suitable biocompatibility as compared with many other materials previously employed for similar purposes.¹⁹ Furthermore, CaCO₃ exhibits high mechanical resistance when it precipitates in the presence of organic matrixes. This property results from the morphology and interactions between the organic and inorganic parts of the system.²⁰ CaCO₃ can also function as a potential precursor to induce formation of bone minerals such as carbonated hydroxyapatite (HAp).²¹ One of the best examples of natural hybrid systems is the bone tissue—this tissue combines carbonated-hydroxyapatite and collagen fibrils in a highly organized network at the nano and micro levels, providing the bone with excellent physical and biological properties.²² Researchers have developed collagen-based biomedical devices in several physical forms; e.g., fibers, films, hydrogels, and membranes, to serve mainly as bone substitute.

Inclusion of collagen on nanoparticle surfaces can improve cell attachment and proliferation because the resulting nanoparticle microenvironment²³ should mimic the bone matrix. Therefore, insertion of proteins in biomaterials can be an effective strategy to boost their biomolecular recognition by body cells.

The present study describes the synthesis and characterization of tube-like hybrid particles based on CaCO₃ and collagen, the most abundant protein in the bone tissue, aiming to fabricate a novel biomaterial that offers suitable conditions for osteoblastic cells to grow and proliferate. The resulting nanoparticles combine bioactive compounds with tube-like morphology giving rise to materials which represent the next generation of bone tissue engineering.

Experimental

Synthesis of tube-like CaCO₃ nanoparticles

Poly(acrylic acid) (PAA) (0.1 wt%, Sigma MW 1800 g mol⁻¹) was dissolved in 0.05 mol L⁻¹ CaCl₂ (Merck P.A.) aqueous solution. After 12 h under stirring, the final solution was filtered through a Millipore® cellulose ester membrane (0.45 μm pore size). Polycarbonate membranes (Millipore®) with 100, 200, and 400 nm pore size were used as template to grow the tubular CaCO₃ particles by an approach modified from Meldrum et al.²⁴ Briefly, the polycarbonate membranes were cleaned in a plasma cleaner system (Harrick Plasma PDC-32G) for 30 s, to unblock their pores. Next, the membranes were immersed in the PAA/CaCl₂ solution and left under vacuum for 30 s to help the polymer chains bound to Ca²⁺ ions diffuse into the pores, to generate nucleation centers for the mineralization process. The membranes were then maintained in the solution for 12 h. All the solutions and washing steps were carried out with Milli-Q® water (18.2 MΩ cm and surface tension 72.3 mN m⁻¹). The modified membranes were placed between two glass slides, and some drops of PAA/CaCl₂ solution were added between the slides and the membrane as represented in Scheme 1. This system was placed in a sealed desiccator containing (NH₄)₂CO_3(s) (Vetec) and left to stand at room temperature for 12 h. (NH₄)₂CO₃ decomposition at room temperature produced CO_2(g), which reacted with Ca²⁺ ions and the water present in the membrane pores, to give CaCO₃. Then, the membrane surfaces were cleaned with filter paper to remove the crystals precipitated on their surfaces. The templates were removed by washing with CHCl₃ (Sigma), which was followed by five-minute centrifugation (11 000 rpm). The supernatant was discarded, and the precipitate was isolated. This procedure was repeated three times.

Scheme 1 Schematic representation of the experimental procedure used to deposit CaCO₃ in the pores of the polycarbonate membranes. (NH₄)₂CO₃ decomposition at room temperature produced CO_2(g), which reacted with Ca²⁺ ions and the water present in the membrane pores, to give CaCO₃.

Collagen adsorption

Type-I collagen (Sigma®-from calf skin suitable for cell culture) was used to modify nanoparticle surface. To this end, the particles removed from the polycarbonate membrane template were immersed in a positively charged collagen solution (pH 3.5) for 12 h, under stirring. Next, the particles were centrifuged at 11 000 rpm for 3 min and washed with water. The procedure used to prepare the collagen solution is described elsewhere.²⁵

Bioactivity evaluation

Immersion in simulated body fluid (SBF) at 37 °C for 48 h helped us to evaluate the bioactivity of the particles. This well-known approach mimics the processes taking place at the first contact of a material with the human blood plasma.²⁶ Samples that generate HAp after this procedure are considered bioactive. The reactants used to prepare the SBF (calcium chloride, sodium chloride, sodium bicarbonate, potassium chloride, magnesium chloride, sodium phosphate dibasic, sodium sulfate, and tris-hydroxymethyl amine methane) were all analytical grade; the concentrations of the reactants and the procedure used to prepare the solution are described elsewhere.²⁷

Nanoparticles characterization

The morphology of the gold-coated particles was investigated by scanning electron microscopy (SEM) on a Zeiss-EVO 50 microscope by using the acceleration voltage at 20 kV. The chemical groups were identified by Fourier-transform infrared spectroscopy (FTIR) coupled with an attenuated total reflectance (ATR) accessory (Shimadzu-IRPrestige-21), with resolution of 2 cm⁻¹. X-ray diffraction (XRD) (Bruker AXS D5005 diffractometer) with Cu Kα radiation helped to analyze the mineral phase. The colloidal stability of the nanoparticles was evaluated by zeta-potential (ζ) measurements on a Zetasizer Nano ZS (Malvern Instruments).

Cell proliferation

All cell experiments were performed in accordance with the Ethical Principles in Animal Research adopted by the National Council for the Control of Animal Experimentation, approved by the Local Animal Ethical Committee from the School of Medicine of Ribeirão Preto of the University of São Paulo-Brazil (protocol number: 011/2014-1). Cells were obtained and cultured according to Maniatopoulos et al.²⁸ with modifications standardized by Simão et al.²⁹ Bone marrow was obtained from the femora of young adult male rats of the Wistar strain weighing 110–120 g. Released bone marrow cells were collected in a 75 cm² plastic culture flask containing 10 mL of osteogenic culture medium. The cells were cultured until subconfluence was achieved. Then, the cells were enzymatically released, and first-passage cells were cultured in the same medium, at a concentration of 2 × 10⁴ cells per well, in 24-well microplates (Falcon, Franklin Lakes, NJ, USA). Cells were cultured at 37 °C for up to 21 days, under humidified atmosphere of 5% CO₂ and 95% air; the medium was changed every 48 h. The osteogenic culture medium, which allowed osteoblasts to differentiate, consisted of α-MEM supplemented with 15% fetal bovine serum, 50 μg mL⁻¹ gentamicin, 0.3 μg mL⁻¹ fungizone, 10⁻⁷ M dexamethasone, 5 μg mL⁻¹ ascorbic acid, and 2.16 mg mL⁻¹ β-glycerophosphate;²⁹ it was used throughout the experimental procedure. The osteoblasts were cultured in a 24-well microplate (Thermo Scientific) at 37 °C. The assayed samples were divided into three groups: control cultured in the absence of nanotubes, sample cultured in the presence of nanotubes with collagen, and sample cultured in the presence of the nanotubes but without collagen. The amount of particles dispersed in each well of the cell culture was controlled by addition of particles extracted from five membranes.

Confocal microscopy

Osteoblasts were fixed with formaldehyde (4%) and stained with acridine orange (0.005 mg mL⁻¹), to reveal the morphology of the culture (cell size, number of cells, cell attachment, cell proliferation, and mineralization nodules). At the 14th day of culture, the stained cells were analyzed on a Leica TCS SP5 microscope.

To visualize RNA, the sample was excited with the argon laser 458 nm line; the emission spectrum was recorded from 642 to 682 nm. To visualize DNA, the sample was excited with the argon laser 488 nm line; the emission spectrum was recorded from 499 to 541 nm.

Results and Discussion

CaCO₃ particles composition, morphology, and bioactivity

Fig. 1A–C show the SEM images of the hybrid CaCO₃ tube-like structures with diameter of 100 (NT-100), 200 (NT-200), and 400 nm (NT-400), respectively. The particles are monodispersed and exhibit well-defined tube-like morphology. Fig. 1D (NT-100 + col), Fig. 1E (NT-200 + col), and Fig. 1F (NT-400 + col) display the hybrid structures obtained after immersion of the particles in type I-collagen solution for 12 h. The SEM images revealed the presence of fibrillar structures (indicated by the blue arrows) on the particle surface; these structures resembled the structure of collagen. Incorporation of osteogenic proteins into the surface of materials creates a biomimetic environment that improves the recognition ability of biological systems, thus decreasing rejection of dental implants.³⁰ This is an efficient approach to build scaffolds that mimic the bone extracellular matrix, providing suitable biological conditions to guide new bone formation.³¹ Other studies have also described that protein adsorption onto the surface of biomaterials improves the osteogenic properties of these materials. For example, Hennessy and collaborators reported that collagen, which mimics peptides, enhanced osseointegration of hydroxyapatite (HAp) supports due to osteoblastic differentiation.³² Hence, addition of collagen to CaCO₃ tube-like particles is an important starting point to improve their application as biomaterials. Fig. 1G (NT-100 + SBF), Fig. 1H (NT-200 + SBF), and Fig. 1I (NT-400 + SBF) correspond to the SEM images obtained after immersion of NT-100 + col, NT-200 + col, and NT-400 + col in SBF. Homogenous formation of nanoparticles (indicated by the red arrows and characterized as biomimetic HAp, see next sections) took place after 48 h of immersion. Formation of HAp spherulites in SBF solution has been documented.³³ In this aqueous medium, ion nucleation generates phosphate clusters. These clusters later aggregate to give calcium phosphate spheres, which may bind onto the biomaterial surface or continue to grow in the solution.³⁴ The implanted material first makes contact with the aqueous medium consisting of the body fluid, followed by contact with proteins and eventually with cells.³⁵ HAp formation on the surface of a biomaterial that is in contact with the body is an essential requirement for an artificial material to bind to a living bone because the apatite layer at the interface mediates their integration with the bone.³⁶ Additionally, HAp-based materials chemically bind to the bone, to conduct bone regeneration.^37,38 The SBF test is a well-known procedure that simulates the first contact of a material with the body, which will determine the bioactivity of the material.³⁹ Surfaces that promote HAp growth after this first contact are called bioactive, which is the case of the CaCO₃ particles synthesized in this study.

Fig. 1 SEM images of the as-prepared CaCO₃ nanoparticles (A) NT-100, (B) NT-200, (C) NT-400. SEM images of the as-prepared CaCO₃ nanoparticles after immersion in collagen solution—(D) NT-100 + col, (E) NT-200 + col, (F) NT-400 + col—and after exposure to SBF solution for 48 h (G) NT-100 + SBF, (H) NT-200 + SBF, and (I) NT-400 + SBF.

Fig. 2A–C depict the ATR-FTIR spectra of NT-100, NT-200, and NT-400. The numbers (1), (2), and (3) refer to the as-prepared, collagen-modified, and SBF-exposed samples, respectively. All the spectra displayed bands related to the presence of the CO₃²⁻ group at 1400 cm⁻¹ (ν₂CO₃²⁻, asymmetric stretching) and 860 cm⁻¹ (γCO₃²⁻ out-of-plane bending vibration), confirming CaCO₃ formation. The ATR-FTIR spectra presented in Fig. 2A-2, B-2 and C-2, obtained for the particles immersed in collagen solution for 12 h, contain the typical bands of collagen: amide I (1600–1700 cm⁻¹), amide II (1300–1590 cm⁻¹), and amide III (1200–1290 cm⁻¹).⁴⁰ Additionally, the bands at 3450 and 2850 cm⁻¹ correspond to the O–H stretching overlapping with the N–H stretching in the amide groups and the C–H stretching in the CH₃ groups, respectively. These bands attest to collagen incorporation at the particle surface and corroborate the presence of the fibrillar structures observed in the SEM images (Fig. 1D–F).

Fig. 2 ATR-FTIR spectra of the CaCO₃ nanoparticles (A) NT-100, (B) NT-200, and (C) NT-400 as-prepared (1), modified with collagen (2), and after exposure to SBF solution for 48 h (3).

The FTIR spectra of all the particles obtained after exposure to SBF for 48 h (Fig. 2A-3, B-3 and C-3) displayed a band close to 1030 cm⁻¹, assigned to asymmetric stretching (ν₃) of the PO₄³⁻ group.⁴¹ The presence of this band confirmed that a new mineral phase emerged after the bioactivity test. CaCO₃ conversion to phosphate stems from a dissolution–recrystallization mechanism. In SBF solution, the CaCO₃ surface dissolves and subsequently reprecipitates via an ion exchange mechanism in which phosphate ions from the surrounding medium replace the carbonate ions.⁴² The phosphate bound to the surface interacts with the Ca²⁺ ions present in the matrix, to give an apatite layer on the biomaterial surface.⁴³ Several features such as composition, structure, micro- and macroporosity, crystal structure, degree of crystallinity, and chemical composition of the materials govern this process.⁴⁴

Zeta-potential: colloidal stability and collagen incorporation

The surface charge greatly influences the interaction of a biomaterial with biomolecules and proteins in the biological fluid.⁴⁵ Therefore, the zeta-potential is an essential parameter to evaluate the properties of a biomaterial at the nano-bio level and consequently its potential in vivo applications. Besides, suitable colloidal stability is a remarkable issue regarding the biomedical applications of nanoparticles—aggregation avoids the biodistribution process control.⁴⁶ In this way, measuring ζ for the dispersed particles as a function of the pH helped to evaluate the colloidal stability of the nanoparticles and the effect of collagen adhesion on surface charge. Fig. 3 illustrates ζ as a function of the pH for a pure collagen aqueous solution (black curve); for the NT-100, NT-200, and NT-400 particles (red curve); and for the NT-100 + col, NT-200 + col, and NT-400 + col particles (green curve). In neutral pH, the as-prepared particles presented ζ values close to −30 mV independent of particle diameter, which indicated high colloidal stability (Fig. 3A–C, red lines). These negative charges were due to hydrolysis of the CO₃²⁻ surface groups, which were deficient in Ca²⁺ ions according to the reaction: CO₃²⁻_(surface) + H₂O ↔ HCO₃⁻ + OH⁻.⁴⁷ Moreover, incorporation of HCO₃⁻ into the nanoparticle surface was possible due to exchange with CO₃²⁻ during the precipitation process.⁴⁸ Increasing pH made the ζ-potential more negative because it shifted the equilibrium reaction. Determination of the zero charge point (ZCP, indicated by the red line) helped to evaluate collagen adhesion onto the nanoparticle surface. The ZCP of NT-100 and NT-200 (Fig. 3A and B, black curve) was achieved at pH close to 2.5. This value drastically increased in the presence of collagen (Fig. 3A and B, green curve) and was close to the value obtained for the pure collagen solution (Fig. 3A and B, green line). This result corroborated collagen adsorption onto the particle surface, as previously indicated by the SEM and FTIR results.

Fig. 3 Zeta potential versus pH curves of ● CaCO₃ nanoparticles (A) NT-100, (B) NT-200, and (C) NT-400, ▲ modified with collagen and ■ collagen.

Interaction between the nanoparticles and collagen resulted mainly from electrostatic interactions—the nanoparticles were negatively charged, whereas the protein was positively charged through almost all the pH range. For NT-400 (Fig. 3C, red line), changes in ZCP were less remarkable after immersion of the particles in the collagen solution (Fig. 3C, green line). The obtained value lay between the value obtained for the as-prepared particles (Fig. 3C, red line) and for pure collagen (Fig. 3C, black line). This happened because the larger particle diameter of NT-400 gave rise to smaller surface area, which led to lower collagen adsorption onto the nanoparticle surface. This result was consistent with data obtained by Clemments et al.,⁴⁵ who described that smaller particles adsorbed a higher amount of proteins due to their larger external surface area. After collagen adsorption, all the particles exhibited high ζ values at physiological pH, which resulted in remarkable colloidal stability and should pave the way for their application as biomaterials in suspensions.

Crystal phase identification

The higher amount of collagen adsorbed onto the surface of NT-100 structures as demonstrated by ζ measurements prompted us to analyze the crystalline structure of this sample by X-ray powder diffraction (XRD). Fig. 4 brings the XRD patterns obtained for NT-100 before and after exposure to SBF.

Fig. 4 X-ray diffraction patterns of NT-100 before (black line) and after exposure to SBF (red line).

The X-ray pattern obtained before exposure of NT-100 to SBF exhibited peaks related to the calcite-CaCO₃ polymorph (JCPDS 00-005-0586) and to the vaterite polymorph (JCPDS 01-074-1867). After exposure to SBF, diffraction peaks characteristic of HAp (JCPDS 00-055-0592) emerged, attesting to the bioactivity of the particles. This result agreed with the ATR-FTIR data, which had shown that the particles induced phosphate mineral (now assigned to HAp) formation after exposure to SBF. Less intense peaks related to the calcite polymorph were also evident, suggesting that the HAp phase originated from calcite conversion.⁴⁹ CaCO₃ nanoparticles have been used to induce HAp growth after their immersion in phosphate solutions. Sungjin et al.⁵⁰ observed conversion of CaCO₃ microspheres into HAp after their immersion in SBF solution for 48 h. According to these authors, the precursor mineral dissolved in the medium, to give Ca²⁺ out to SBF and induce local supersaturation of Ca²⁺ with consequent hydroxyapatite nucleation. Moreover, previous studies had shown that materials consisting mainly of CaCO₃ such as nacre, coral, and calcite can initiate bone formation in vivo^51,52 and in vitro.^53,54

Osteoblast morphology and proliferation on CaCO₃: confocal laser microscopy

Confocal laser microscopy is useful to study cell behavior on biomaterials⁵⁵—this technique generates high-resolution 3D images from relatively thick cell cultures or tissues.⁵⁶ Literature papers have reported that cytotoxicity assays are essential to evaluate the biological responses of a given biomaterial.⁵⁷ The cell toxicity studies using MTT did not reveal any toxic effects related to the presence of CaCO₃ nanoparticles after 21 days of culture as compared to the control (data not shown). Fig. 5 illustrates the confocal laser microscopy images of the osteoblasts cultured in the presence of the as-prepared nanoparticles NT-100 (Fig. 5A), NT-200 (Fig. 5B), and NT-400 (Fig. 5C) and of the nanoparticles modified with collagen NT-100 + col (Fig. 5D), NT-200 + col (Fig. 5E), and NT-400 + col (Fig. 5F) after 14 days of culture. The insert in Fig. 5A shows the image obtained for the osteoblasts cultured in the absence of the nanoparticles (control). The cells cultured in the absence of collagen were small and exhibited the same circular shape observed in the control sample (Fig. 5A–C). Increased proliferation of the cells cultured in the presence of the collagen-modified CaCO₃ nanoparticles, especially the 100 nm-particles, was remarkable (Fig. 5D). Osteoblast morphology also changed in the presence of the protein. The long branches and prolongations emerging from the central body associated with the elevated intra-cellular link predicted good cell adhesion on the material surface (Fig. 5D–F). The polygonal wide shape was consistent with the early stages of adhesion.⁵⁸ Literature works have described higher adhesion of osteoblasts tailored by the presence of proteins.⁵⁹ Increased cell proliferation in the case of NT 100 + col may be related to the higher surface area of this material and to the higher collagen adsorption on this sample as evidenced by the ζ measurements (Fig. 3A).

Fig. 5 Confocal laser scanning microscopy images of osteoblasts adhered to (A) NT-100 control and CaCO₃ nanoparticles, (B) NT-200, (C) NT-400, (D) NT-100 + col, (E) NT 200 + col, and (F) NT 400 + col after 14 days of incubation. The insert shows the image obtained for the osteoblasts cultured in the absence of the nanoparticles (control).

Conclusions

Tube-like structures based on CaCO₃/collagen were successfully obtained. The FTIR and SEM results attested that the particles induced formation of biomimetic HAp after exposure to SBF for 48 h, proving their bioactivity. Zeta-potential measurements evidenced the high colloidal stability of the tube-like structures in the presence and absence of collagen. Confocal laser microscopy images showed increased proliferation of osteoblasts cultured in the presence of the collagen-modified CaCO₃ nanoparticles, especially for the 100 nm-particles. This special behavior was related to the higher amount of collagen adsorbed onto the NT-100 particles. The overall results indicated the potential application of the particles in bone-tissue biomimetic matrixes to support cell proliferation. Moreover, these particles can be used as filling agents to enhance the mechanical properties of organic scaffolds.

Acknowledgements

The authors thank the São Paulo Research Foundation (FAPESP—grant 2013/12615-0, 2014/24249-0 and 2012/20946-3) and the National Council of Technological and Scientific Development (CNPq- grant 44283412014-4) for financial support. Camila B. Tovani thanks FAPESP for the PhD scholarship. Ana P. Ramos and Pietro Ciancaglini are CNPq researchers.

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