Calcium silicate-based bone cement incorporated with carbon nanotubes (CNT) : In vitro and in vivo

Abstract

Calcium silicate cements (CaSiO₃) are widely used in bone repair treatments for both medical and dental applications. To meet the demands of tissue engineering, three calcium silicate cements were developed: a control group without carbon nanotubes (CNT) and two experimental groups incorporating CNT nanoparticles at concentrations of 0.2% and 0.5%. The surface topography of the calcium silicate-based cements was analyzed using field emission scanning electron microscopy (FEG-SEM) and X-ray diffraction. Additionally, an in vitro cell viability assay was performed to assess cytotoxicity. An in vivo study was also conducted using 24 Wistar rats, where critical bone defects of 3.0 mm in diameter were surgically created in both tibiae using a trephine drill. A clot group was included as a control. Following euthanasia, the samples were evaluated through histological and histomorphometric analyses, and a three-point flexural biomechanical test was performed. Statistical analysis was conducted using one- and two-way ANOVA, with a significance level set at 5%. The results indicated that none of the cements exhibited cytotoxicity. Regarding bone neoformation, the clot group showed significantly lower values compared to the SiCa and SiCa+0.5%CNT (mass) groups (p < 0.05), while the SiCa+0.2%CNT group did not differ statistically from the others (p > 0.05). The biomechanical test revealed a statistically significant difference between the SiCa+0.2%CNT group and the SiCa and SiCa+0.5%CNT groups, with the SiCa+0.2%CNT group exhibiting lower values (p < 0.05), whereas the clot group showed no statistical difference from the other groups (p > 0.05). These findings indicate that the incorporation of carbon nanotubes (CNT) into calcium silicate cements did not result in significant differences in bone tissue regeneration when compared to cements without CNT.

Keywords

bone repair carbon nanotubes calcium silicate cement biomechanics cytotoxicity

Introduction

Bone remodeling can be compromised under certain conditions, leading to imbalances in the regeneration process.¹ In such cases, correcting these issues can be complex and significantly impact a patient’s quality of life.^2,3 As a result, research efforts have been directed toward developing novel biomaterials capable of mimicking bone tissue properties, accelerating healing, and providing mechanical strength comparable to natural bone.^4,5 Various biomaterials are already used as bone grafts to stimulate osteogenesis or to enhance bone healing in a more efficient and controlled manner.⁶ However, to achieve satisfactory outcomes, these materials must meet strict criteria, including inertness, non-toxicity, malleability during surgery, and minimal additional surgical time.⁷

Calcium silicate-based cements have emerged as key materials in bone tissue engineering due to their antibacterial, anti-inflammatory, and biocompatible properties.⁸ Furthermore, their in vivo degradation releases silicate and calcium ions, promoting the proliferation and differentiation of human bone marrow mesenchymal stem cells, facilitating apatite deposition, and playing a crucial role in bone tissue reconstruction.^9–11 Despite their benefits, calcium silicate-based cements present limitations such as rapid degradation and low mechanical strength, which make them unsuitable for load-bearing areas or complex bone defects.^12,13 Other challenges include prolonged setting time, which can hinder surgical handling,¹⁴ and the high alkalinity generated during hydration, which may cause tissue irritation and local inflammation.¹⁵ Accelerated degradation before complete bone regeneration also represents a major limitation, as it can result in structural failure and compromise graft stability.¹⁶ The incorporation of carbon nanotubes (CNTs) and other carbon nanostructures into calcium silicate-based cements has emerged as a promising strategy to overcome these limitations. CNTs provide enhanced mechanical strength, chemical stability, and biocompatibility, while also promoting osteoconduction and cellular proliferation.^14,15 Additionally, these nanostructures modulate ion release, improving cement integration with bone tissue and prolonging its in vivo durability.^16,17

In this context, nanotechnology has emerged as a promising field in materials science, offering innovative biomaterials designed to overcome these limitations. Carbon nanotubes (CNTs), in particular, stand out for their exceptional mechanical and biological properties, making them ideal for applications in synthetic bone tissue. Their chemical stability, nanoscale size, high surface area, and functionalization capacity allow them to interact with various molecules.

Therefore, this study aims to investigate in detail the bioactive properties, regenerative potential, and biomechanical performance of neogenerated bone tissue by incorporating different concentrations of CNTs (0.2% and 0.5%) into calcium silicate-based cements. The ultimate goal is to develop a cement with optimized mechanical and biological characteristics for applications in both orthopedic and dental fields. Based on the treatments evaluated, we hypothesized that there was no difference in bone regeneration between the treated groups and the control group (null hypothesis, H₀).

Materials and methods

Fabrication SiCa+0.2%CNT and SiCa+0.5%CNT

The biomaterial used in this study was developed and provided by Thim at the Nanotechnology Laboratory of the Physics Department of the Technological Institute of Aeronautics. Two compositions based on silicate cement functionalized with different concentrations (mass) of carbon nanotubes (CNTs) were investigated: 0.2% CNT and 0.5% CNT. The cements were prepared from calcium silicate in the α-wollastonite phase, which was activated with an NH₄⁺ solution (pH ≈ 7). The resulting specimens measured 3 mm × 3 mm after shrinkage during the cement hardening process. The choice of CNT concentrations (0.2% and 0.5%) was based on our previous study (Ribas et al., 2025), in which the incorporation of 0.2% CNT promoted better dispersion within the silicate matrix, improving the physicochemical homogeneity of the composite, whereas higher concentrations (>0.5%) resulted in CNT agglomeration and negatively affected the material’s performance.

Functionalization of carbon nanotubes (CNTs)

The carbon nanotubes used in this study were multi-walled carbon nanotubes (MWCNTs), acquired from Nanostructured & Amorphous Materials Inc. (Texas, USA), with lengths ranging from 2 to 5 μm and diameters between 20 and 30 nm. The as-received, non-functionalized nanotubes were referred to as CNT-NF.

Oxidative functionalization was performed using acid treatment. For this, 1 g of CNT-NF was dispersed in 120 mL of a mixed acid solution composed of H₂SO₄ (98%, Merck) and HNO₃ (70%, Vetec) in a 3:1 volume ratio (v/v), in a 500 mL Erlenmeyer flask. The mixture was magnetically stirred at 400 rpm for 6 h at room temperature. Subsequently, the suspension was centrifuged (SL-701, Solab) at 3500 rpm for 1 h at −10°C.

The resulting precipitate was vacuum-filtered using a cellulose acetate membrane (0.45 µm pore size) and washed with distilled water until the filtrate reached neutral pH (pH 7). The filtered material was then dried in an oven at 80°C for 15 h. After drying, the CNTs were ground using a mortar and pestle, sieved through a 200 mesh sieve (75 µm), and subjected to an additional drying step at 80°C for 8 h. The final powder of the functionalized CNTs was then incorporated into the calcium silicate to fabricate the cements molded into 3 mm by 3 mm pieces.

Characterizations

Characterization of specimens

The samples were characterized using scanning electron microscopy (SEM) (JEOL/JSM-5310, Tokyo, Japan) with a Field Emission Gun (FEG) (Tescan/Vega 3, Brno, Czech Republic). For this, the samples were mounted on an aluminum stub with the aid of double-sided carbon tape (3M, Sumaré SP, Brazil) and coated with a thin layer of gold (80 Å). SEM analysis was performed using a secondary electron detector (SE), which provides surface imaging, and a backscattered electron detector (BSE). The magnifications used were 1,000X, 5,000X, and 10,000X.

X-ray diffractometry

To perform a qualitative analysis of the diffraction pattern, the analyzed scaffolds were prepared in a 3 × 3 cm format and placed in the XRD device (Shimadzu XRD7000, CuKα radiation, 2θ = 20–80°, 30 mA, 40 kV) at the Technological Institute of Aeronautics (ITA), São José dos Campos.

In vitro test

In vitro studies

The biological procedure for in vitro cell viability was developed and carried out at the Laboratory of Interdisciplinary Studies in Cells (LEIC), located in the Department of Biosciences and Oral Diagnosis at the Institute of Science and Technology of São Paulo State University “Júlio de Mesquita Filho” - Unesp, São José dos Campos Campus.

Cell culture

For the in vitro tests, MG63 osteoblast-like cell lines (Rio de Janeiro Cell Bank, APABCAM, Rio de Janeiro, RJ, Brazil) were cultured in α-modified Eagle’s Medium (DMEM; Gibco-Life Technologies, NY, USA), supplemented with 10% fetal bovine serum (Gibco-Life Technologies, NY, USA), 100 U/mL penicillin (Gibco-Life Technologies, NY, USA), and 100 μg/mL streptomycin (Gibco-Life Technologies, NY, USA) in 75 cm² flasks (TPP, Biosystems, Curitiba, Brazil). The cells were maintained in a humidified atmosphere with 5% CO₂ and 95% air at 37°C. The medium was changed every 2 days. Cultures were monitored daily using an inverted optical microscope (Carl Zeiss Microlimaging GmbH – Axiovert 40C, Germany). When confluent, cells were enzymatically detached and plated on the sterile samples in 24-well polystyrene plates (TTP, Biosystems, Curitiba, Brazil) at a density of 2 × 10⁴ cells/well for up to 14 days. Cement samples, measuring 3 mm by 3 mm, were added to the wells containing culture medium and cells for the cellular tests. Prior to use, the samples were sterilized in absolute ethanol and under ultraviolet light for 3 h. All tests were performed following the ISO 10,993-5 standard, as described in 18. For all in vitro experiments, three independent experiments were conducted (n = 5).

Analyzing cell viability in vitro

Cell viability was assessed by the MTT assay (3–4, 5-dimethylthiazol-2-yl-2,5-diphenyltetrazolium bromide; Sigma-Aldrich, St Louis, USA) at 3 and 7 days after cell plating. The culture medium was removed, and MTT solution (0.5 mg/mL) was added to each well. The cells were then incubated in a 5% CO₂ atmosphere. After incubation, the formed formazan crystals were dissolved using dimethyl sulfoxide (DMSO; Sigma-Aldrich, St Louis, USA). Viability was quantified using a spectrophotometer (Biotek Instruments, EL808IU, Winooski, USA) at 570 nm, and the results were expressed as optical density units of absorbance.

In vivo studies

This study was approved by the Animal Studies Review Committee of the Institute of Science and Technology, UNESP – Universidade Estadual Paulista, São José dos Campos, Brazil (17/2016) – and adhered to the ARRIVE guidelines for reporting in vivo animal experiments. Twenty-four male Wistar rats (Rattus norvegicus, Albinus), weighing between 250 and 300 g and aged 3 months, were housed in the vivarium of the Institute of Science and Technology under a 12-h light/dark cycle. The animals were fed GuabiNutrilabor® (Mogiana Alimentos; São Paulo, SP, Brazil) and had access to water ad libitum, both provided by qualified personnel in the vivarium.

For the animal experiment, the sample size calculation was based on previously published studies. A total of 24 animals were randomly allocated into four experimental groups (n = 6 per group) using a computer-generated allocation table: Control group – the defect was filled with blood clot only; SiCa group – the defect was filled with calcium silicate cement (SiCa); SiCa+0.5%CNT group – the defect was filled with calcium silicate cement containing 0.5% carbon nanotubes (SiCa+0.5%CNT); and SiCa+0.2%CNT group – the defect was filled with calcium silicate cement containing 0.2% carbon nanotubes (SiCa+0.2%CNT). Each animal received two defects, one in each tibia, in accordance with the 3 Rs principle (“Reduction”). The biological number (n = 6) was considered for all analyses.

Surgical procedure

The animals were weighed, and general anesthesia was administered using a solution of 14.2 mg/kg of 2-(2,6 xylidine)-5,6-dihydro-4H-1,16-thiazine chlorhydrate (Anasedan® - Vetbrands, Jacareí, Brazil) and 8.5 mg/kg of ketamine base (Dopalen® – Vetbrands, Jacareí, Brazil) via intramuscular injection.

Next, the animals underwent trichotomy and antisepsis with an iodine alcohol solution. An incision was made, and the total flap was displaced. A critical size defect of 3 mm diameter was created in the right and left tibiae using a trephine drill, with abundant irrigation using 0.9% sodium chloride. The right tibia was filled with SiCa, and the left tibia was filled with a blood clot. The animals in the other groups underwent the same procedure, but the right tibia was filled with SiCa+0.2%CNT and the left tibia with SiCa+0.5%CNT. Both critical defects were filled with the predetermined material, maintaining standardized volume and diameter. The flap was repositioned and sutured with silk thread (Ethicon, São José dos Campos, SP, Brazil). The animals were euthanized after 4 weeks, and the samples were submitted to radiographic analysis, the three-point bending test, and qualitative histological and histomorphometric analysis of bone neoformation. The experimental workflow, including the in vitro and in vivo steps, is shown in Figure 1.

Histologic and histomorphometric analysis

The samples were fixed and decalcified with formic acid for 40 days. After decalcification, the samples were washed, dehydrated, and embedded in paraffin blocks. Subsequently, 5 μm semi-serial sections were prepared and stained with Hematoxylin and Eosin. The bone repair process was assessed through qualitative histological analysis by evaluating aspects such as granulation tissue formation, bone neoformation, arrangement of immature bone trabeculae, and bone maturation.

The sections were photographed using a Zeiss Axiophot 2 microscope (Carl Zeiss, Oberkochen, Germany) with a 10× ocular and a 2.5× objective, coupled to an Axiocam MRC5 digital camera (Carl Zeiss, Oberkochen, Germany). The obtained digital images (JPEG format) were used for the quantitative analysis of bone repair tissue using LasPhase software (Leica, Wetzlar, Germany), which allowed for automatic measurement of the newly formed bone in the defect area. The control group values were considered 100% healing, and the values for the other groups were calculated as percentages.

Biomechanical properties

After euthanasia, the tibiae were preserved in Ringer’s solution at −20°C. For biomechanical testing, each specimen was placed centrally along its length on a support system containing two supports 15 mm apart, with its anterior face facing downward. A transverse load was applied along the long axis of the tibia at a midpoint between the two supports, on its posterior face. Both the load application support and the supports were cylindrical with a diameter of 3 mm. The test was conducted using a universal testing machine (Emic®—model DL 200 MF, Testing Equipment and Systems Ltd, São José dos Pinhais, Brazil), with a force of 50 kg/F applied at a constant speed of 5.08 mm/min until specimen failure.¹⁹

Statistical analysis

Data were plotted and analyzed using GraphPad Prism software. The normality of the data was assessed using the Shapiro-Wilk test. Once normality was confirmed, comparisons among groups were performed using one-way ANOVA, followed by Tukey’s post-hoc test for multiple comparisons. The null hypothesis (H₀) was that there would be no difference in bone regeneration between the treated groups and the control group. Differences were considered statistically significant at p < 0.05.

Results

Characterization of designed materials

The structural architecture of the specimens was examined using SEM-FEG. The surface morphology revealed spherulitic-like structures, typically associated with hydroxyapatite crystal growth, along with the presence of a porous network (Figure 2). In the SiCa+0.2%CNT group, sharper and more pointed protrusions were identified, suggesting alterations in nucleation or crystal growth patterns induced by CNT incorporation. For accurate comparison, all SEM micrographs were acquired at the same magnification, and scale bars were included in each image.

X-ray diffractometry

Figure 3 shows the standard diffractograms of HAp (ICSD 169,499), simulated with a FWHM of 1, alongside the diffractograms of SiCa, SiCa+0.2%CNT, and SiCa+0.5%CNT cements. The same peaks observed in the control cement (without reinforcement) were also present in the experimental cements reinforced with 0.2% and 0.5% CNT.

Using HighScoreX software and the Rietveld method, the proportion of HAp present in the cements was estimated to be approximately 58%, 63%, and 62% in the SiCa, SiCa+0.2%CNT, and SiCa+0.5%CNT specimens, respectively. These values are relatively close but show a tendency for the percentage of HAp formation to increase when the cement is reinforced with either 0.2% or 0.5% CNT. The nanotubes likely act as nucleating agents, facilitating the precipitation of HAp, leading to a higher percentage of phase formation.

The presence of minor HAp even without prior treatment has been reported in previous studies on calcium silicate cements, including Ribas et al. (Influence of Different Ions on the Production of Wollastonite Cements for Bone Regeneration), supporting the possibility of a mixture of α-wollastonite and HAp phases in these materials.

Biological tests

Prior to the statistical analysis, the statistical hypotheses were evaluated. The results indicated that the data followed a normal distribution. Graphical representation confirmed uniformity, and none of the ANOVA assumptions were violated.

The data from the in vitro and in vivo tests were analyzed using one-factor ANOVA to compare the groups with each other and with the control group. Tukey’s multiple comparison test was applied when necessary (p < 0.05).

Cell viability analysis of test specimens In vitro

The cell viability data revealed that the materials were not cytotoxic. After 3 days, the SiCa group exhibited the highest cell viability, which was statistically different from the experimental groups (p < 0.05). The experimental groups showed lower values with no significant differences between them (p > 0.05). After 7 days, the SiCa+0.2%CNT group showed the highest cell viability, but there were no significant differences between the groups (p > 0.05). The results are shown in Figure 4.

Histological results

Using an optical microscope (OM), bone neoformation at the interface with different concentrations of CNTs was observed. The tissue exhibited a normal appearance, as seen in Figure 5, including a clot. In the descriptive histological analysis, aspects of bone repair development were noted, including the formation of bone tissue and the arrangement of immature bone trabeculae, with osteoblasts lining the periphery and osteocytes filling the gaps. Reverse lines, indicative of bone remodeling activity, were also observed.

Histomorphometric results

Histomorphometric analysis was performed on the neoformed tissue that proliferated in the region of the critical defect, where the cements with different activating solutions were applied. Statistical data showed that bone neoformation was lower in the Clot group, with bone neoformation values showing a statistically significant difference when compared to the SiCa experimental group (p < 0.005). However, the SiCa group did not differ statistically from the SiCa+0.2%CNT and SiCa+0.5%CNT groups (p > 0.005) (Figure 6) indicating that the incorporation of CNTs did not significantly alter bone neoformation under the conditions tested.

Biomechanical properties

The three-point bending test was used to assess bending strength (load/Newton) and stiffness (N/mm). The data obtained were used to evaluate the characteristics of the newly formed bone tissue and were subjected to one-factor ANOVA statistical analysis, followed by Tukey’s multiple comparison test. The maximum force (N) and stiffness values showed no statistically significant differences between the groups (p > 0.005) (Figure 7).

The breaking strength of the neoformed bone was also evaluated. The maximum strength obtained in the test was related to the intrinsic properties of the bone, such as its diameter and width. The results showed that the SiCa+0.2%CNT group had the lowest values, which were statistically significantly different from the SiCa and SiCa+0.5%CNT groups (p < 0.0005), but did not differ from the Clot group (p > 0.0005), as shown in Figure 6.

Discussion

Calcium silicate-based cements incorporating nanoparticles are actively being developed to investigate their biomechanical properties, self-repair capabilities, and apatite mineralization potential.²⁰ Given the limited understanding of the effects of carbon nanotubes (CNTs) in biomaterial compositions, this study aimed to evaluate calcium silicate cements with varying CNT concentrations for use in bone grafts, focusing on their influence on bone neoformation in critical defects in rat tibias, as well as on the mechanical strength and rigidity of the newly formed tissue.

The histological analysis revealed bone deposition in the defect region, and histomorphometric analysis showed greater bone neoformation in the calcium silicate and CNT-containing groups (SiCa+0.2%CNT and SiCa+0.5%CNT), with no statistically significant difference between the latter two groups (p > 0.05). Notably, the SiCa group showed a statistically significant difference compared to the clot control group (p < 0.05). Additionally, the SiCa and SiCa+0.5%CNT groups exhibited higher rupture force values, which differed significantly from the SiCa+0.2%CNT and clot groups (p < 0.05). Our results provide valuable insights into the effects of CNT incorporation in calcium silicate cements. While the null hypothesis (H₀) that there would be no difference in bone regeneration between the treated groups and the control group is rejected for some comparisons, these findings highlight the potential of CNTs to influence bone repair, supporting further exploration in future studies. These findings align with the study by Gong et al.,²¹ which demonstrated that silicate-based cements possess excellent osteoconductive properties, promoting bone formation. This reinforces our study’s results, particularly considering that the 0.2%CNT concentration did not yield significant benefits for the biomechanics of bone formation.

One hypothesis worth considering is that CNTs, due to their high rigidity, should be incorporated into biomaterials in controlled proportions (1–10% by weight). While they enhance mechanical properties, CNTs alone cannot replicate the mechanical characteristics of host tissues. These proportions can be adjusted during the manufacturing process to tailor the stiffness of the nanocomposites.

X-ray diffraction analysis indicated an increase in the hydroxyapatite content in the CNT-reinforced groups. Scanning electron microscopy (SEM) images revealed spherulitic structures on the surface of the biomaterial, similar to hydroxyapatite, along with the development of a porous structure. These results are consistent with Huang et al.,²² who observed the pore volume and morphology of mesoporous calcium silicate nanoparticles, suggesting a surface area that supports osteogenic tissue regeneration.²³

In vitro cell viability tests showed satisfactory results, with the SiCa group exhibiting the highest cell viability after 3 days, statistically differing from the other groups. After 7 days, the SiCa+0.2%CNT group had the highest cell viability values, though no statistical differences were observed between the groups. These findings meet the ISO 10,993-5 standard, which recommends cell viability greater than 70%.

Overall, the in vitro results suggest that CNT-reinforced calcium silicate cement positively impacts osteoblastic cell viability. Additionally, bone neoformation and biomechanical properties of the newly formed tissue were enhanced in the experimental group with 0.5% CNT reinforcement in critical bone defects in rat tibias. However, due to the potential for CNT migration in the bloodstream,²⁴ it is recommended that their application be restricted to topical use in tissue engineering, avoiding areas such as the abdominal cavity, liver, and cardiovascular system.²⁵ Further in vivo studies are needed to investigate the chemical functionalization and cellular uptake of these materials.

Although the present study demonstrates promising results, a few limitations should be acknowledged to provide context and guide future work. First, while the rat model is widely adopted in bone regeneration research due to its reproducibility and cost-effectiveness, it may not fully replicate human bone physiology and healing mechanisms.^26,27 Moreover, the sample size was selected to be consistent with previous in vivo biomaterial studies, but it may limit the power to detect more subtle differences between groups, suggesting that future investigations would benefit from a priori power analyses. We also evaluated only two concentrations of carbon nanotubes (CNTs) in the cement composite; expanding the concentration range could optimise outcomes and enhance understanding of dose–response behavior.²⁸ Additionally, although histological, histomorphometric and biomechanical assessments were performed, complementary molecular or immunohistochemical analyses could provide deeper mechanistic insight. Finally, given the potential concerns around CNT biodistribution and long-term safety, which have been raised in pulmonary and systemic exposure studies,²⁹ further investigations are clearly needed before clinical translation. Nonetheless, the consistency of our in vitro and in vivo findings provides a strong foundation for future refinement and supports the relevance of CNT-reinforced calcium silicate cements in bone tissue engineering.

Conclusions

Calcium silicate cement, with or without CNT incorporation, demonstrated non-cytotoxicity, favorable mechanical properties, and the ability to promote bone neoformation in vivo. While no statistically significant differences were observed between the experimental groups (0.2% and 0.5% CNT) and the cement without CNT, the formulations containing CNT exhibited trends that indicate potential enhancement, supporting their promise for applications in repairing critical bone defects.

Footnotes

ORCID iD

Letícia Adrielly Dias Grisante

Author contributions

The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript. These authors contributed equally

Funding

The authors disclosed receipt of the following financial support for the research, authorship, and/or publication of this article: This project was supported by two distinct, both granted by FAPESP (São Paulo Research Foundation), Brazil: Doctoral fellowship in Brazil – Process n° 17/27079-7 and Undergraduate research fellowship in Brazil – Process n° 2020/07092-1.

Declaration of conflicting interests

The authors declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article.

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