Basic calcium phosphate (BCP) crystals have been associated with many diseases due to their activation of signaling pathways that lead to their mineralization and deposition in intra-articular and periarticular locations in the bones. In this study, hydroxyapatite (HAp) has been placed in a polysaccharide network as a strategy to minimize this deposition. This research consisted of the evaluation of varying proportions of the polysaccharide network, cellulose nanocrystals (CNCs), and HAp synthesized via a simple sol-gel method. The resulting biocompatible composites were extensively characterized by means of thermogravimetric analysis (TGA), powder X-ray diffraction (XRD), Fourier transform infrared spectroscopy (FT-IR), dynamic light scattering (DLS), zeta potential, and scanning electron microscopy (SEM). It was found that an nHAp = CNC ratio presented greater homogeneity in the size and distribution of the nanoparticles without compromising the crystalline structure. Also, incorporation of bone morphogenetic protein 2 (BMP-2) was performed to evaluate the effects that this interaction would have in the constructs. Finally, the osteoblast cell (hFOB 1.19) viability assay was executed and it showed that all of the materials promoted greater cell proliferation while the nHAp > CNC proportion with the inclusion of the BMP-2 protein was the best composite for the purpose of this study.
Advances in Bone Tissue Engineering (BTE) hold promise for the development of new functional coatings for bone regeneration. The integration of novel bionanomaterials that induce bone regeneration can contribute to the field of BTE and help settle the incidence of bone disorders and conditions [
Overall, the use of basic calcium phosphate (BCP) crystals, such as HAp, has been associated with many diseases due to their deposition in intra-articular and periarticular locations site [
There are many polysaccharides that have been used for tissue engineering applications such as chitosan and gelatin [
Due to all of the above, herein we propose to synthesize nanohydroxyapatite (nHAp) − nanocellulose (CNC) composites via the sol-gel route in order to develop uniform molecular-level mixing of the calcium and phosphorus precursors. This strategy might provide several advantages such as improving chemical homogeneity of the resulting HAp, low synthesis temperature, and promotion of controlled growth of spherical nanoparticles [
Cellulose nanocrystals (CNCs) 11.8% aqueous solution was purchased from the University of Maine Process and Development Center (Orono, USA). Hydroxyapatite (HAp), ethanol, acetic acid, phosphate buffered solution 1x, pH = 7.2 (PBS), and bone morphogenetic protein 2 (BMP-2) were purchased from Sigma-Aldrich. All chemicals and solvents were used as received without further purification. Deionized water (18.3 MW, MilliQ Direct 16) was used at all times.
In order to compare the effects of polysaccharide to BCD crystals, three composites were prepared with different proportions of HAp and CNC. The three compounds were prepared, following the proportions of Hap > CNC, Hap = CNC, and Hap < CNC. The same ratio of ethanol and acetic acid was used to produce nanohydroxyapatite (nHAp), following a similar procedure to that of Monreal Romero et al. [
For the inclusion of BMP-2 to the composites, 10
Human osteoblast cell line (hFOB 1.19) was purchased from the American Type Culture Collection (Manassas, VA, USA). Osteoblast cells were cultured in 1 : 1 mixture of Ham’s F12 Medium/Dulbecco’s Modified Eagle’s Medium (Gibco by Life Technologies) supplemented with 2.5 mM L-glutamine, 10% fetal bovine serum (Gibco by Life Technologies), and 0.3 mg/mL G148 (Gibco by Life Technologies) at 34°C with 5% CO2. To assess the cell viability effect of HAp, CNC, Hap > CNC, Hap = CNC, Hap > CNC + BMP-2, and Hap = CNC + BMP-2 nanoparticles, the MTS CellTiter 96 AQueous Solution Cell proliferation Assay (Promega) was used. In brief, cells were plated at 7.5 × 104 cells/mL in 96-well plates (Falcon) in contact with 5 mg/mL of the different nanoconstructs and incubated for 24 hrs. Then, 20
In order to fully characterize the composite material, several physical characterization techniques were employed in this study.
A Perkin-Elmer STA 6000 simultaneous thermal analyzer was used to measure the changes in weight and heat flow as a function of temperature. Approximately 10 mg of each sample was added to ceramic crucible and heating of 30–750°C at a ramp of 20°C/min in air atmosphere at a flow rate of 20 mL/min.
Powder X-ray diffraction measurements were conducted over 10 to 90° 2
Malvern ZetaSizer Nano Series with 4 mW 632.8 nm laser was used to determine the average diameter of composite suspensions. First, suspensions were sufficiently diluted with deionized water to avoid agglomeration; then approximately 1 mL of suspension was added to a disposable plastic cuvette. The backscattering mode was used in triplicate for all the samples and the
Scanning electron microscopy (SEM) images were recorded using a JEOL 5800LV Scanning Microscope with electron beam energy of 20 kV. The samples were freeze-dried before measurement and, to avoid charge accumulation, a thin film of gold (15 nm) was added to the surface.
Infrared spectra of freeze-dried samples were recorded on a Bruker tensor 27 Fourier transform infrared using Attenuated Total Reflectance (ATR). Sample was placed in a glass slide and then pressed using a diamond probe. The spectral width ranged from 400 to 4000 cm−1, with 4 cm−1 resolution and accumulation of 64 scans.
The main objective of this work was to prepare and characterize biocompatible constructs for Bone Tissue Engineering and repair. Nevertheless, it was of interest to extensively characterize the prepared materials to account for any structural or chemical changes in the products. In this sense, thermal stability of the material provides an indication of the interactions between particles and also about the critical transition temperatures in the material. In order to determine this, a dual thermal gravimetric analyzer with differential scanning calorimetry functions was utilized and results are shown in Figure
TGA and DSC analysis of nHAp > CNC (red), nHAp = CNC (black), and nHAp < CNC (blue), in air atmosphere at 20 mL/min and a ramp of 20°C/min.
After observing the variations in thermal stability of the constructs, it was important to determine if any crystallinity loss is observed for the prepared materials. In order to assess this, XRD was carried out and the resulting patterns are shown in Figure
Powder XRD analysis of nHAp > CNC (red), nHAp = CNC (black), and nHAp < CNC (blue).
Powder XRD analysis of CNC phase in nHAp > CNC (red), nHAp = CNC (black), and nHAp < CNC (blue).
Moreover, even when the crystallinity of the samples was conserved, it was of our interest to determine any chemical changes in the surface of the composites after the synthesis. FTIR was carried out from 400 to 4000 cm−1 (Figure
FTIR analysis of nHAp > CNC (red), nHAp = CNC (black), and nHAp < CNC (blue).
It has been noted that the composite size and homogeneity of the particles can influence the adherence of tissue to the host implant. As a strategy to account for the composite size, DLS analyses were conducted and are shown in Table
DLS analyses for HAp, CNC, and nHAp-CNC at a 1% w/v.
Sample |
|
PDI |
---|---|---|
nHAp > CNC (3.2%) | 311 ± 7 | 0.21 ± 0.02 |
nHAp = CNC (3.2%) | 289 ± 3 | 0.21 ± 0.01 |
nHAp < CNC (3.2%) | 307 ± 5 | 0.43 ± 0.00 |
Natural HAp | 1965 ± 170 | 0.29 ± 0.04 |
CNC | 142.3 ± 0.72 | 0.30 ± .007 |
DLS analyses for BMP-2, nHAp > CNC, and nHAp = CNC composites with protein at 1% w/v.
Sample |
|
PDI |
---|---|---|
nHAp > CNC (3.2%) + BMP-2 | 333.4 ± 11 | 0.18 ± 0.01 |
nHAp = CNC (3.2%) + BMP-2 | 364.6 ± 2 | 0.30 ± 0.02 |
BMP-2 | 59.97 ± 0.9 | 0.46 ± 0.04 |
Representation of the interaction between nanohydroxyapatite (nHAp) and cellulose nanocrystals (CNCs) polymer network.
Representation of the interaction between the nHAp/CNC nanocomposites with the BMP-2.
SEM analyses of nHAp > CNC (a and b), nHAp = CNC (c and d), and nHAp < CNC (e and f).
After thorough characterization of the composites,
Zeta potential measurements of nHAp > CNC and nHAp = CNC with BMP-2.
Sample | Zeta potential (mV) |
---|---|
Natural HAp | −20 ± 2 |
BMP-2 | −11.4 ± 0.7 |
nHAp > CNC | −18 ± 1 |
nHAp = CNC | −11.0 ± 0.6 |
nHAp > CNC & BMP-2 | −17.3 ± 0.6 |
nHAp = CNC & BMP-2 | −20 ± 2 |
In recent years, a great interest has been dedicated to the development of biocompatible and nontoxic nanoparticles that can be used in bone implants and biomedical devices. For instance, studies with carbon nanotubes have been shown to support the osteoblastic cells grow. However, the hydrophobic nature of carbon nanotubes requires extensive functionalization with hydrophilic conjugates in order to afford its biocompatibility [
Cell viability in human osteoblasts exposed to the different nanoconstructs. The cells were exposed to 1 mg/mL of HAp, CNC, Hap > CNC, HAP = CNC, Hap > CNC + BMP-2, and HAP = CNC + BMP-2 nanoconstructs for 24 h and cell viability was determined with the MTS reduction assay. Values represent mean ± standard error of the mean (SEM) from three replicates. An asterisk indicates statistical significance in comparison with the untreated samples (
Recent studies have shown that the use of hydroxyapatite- (HA-) 2,2,6,6-tetramethylpiperidine-1-oxyl- (TEMPO-) oxidized bacterial cellulose- (TOBC-) GEL composites in calvarial osteoblasts from Sprague-Dawley rats did not compromise the cell viability and promote cell proliferation [
The proportion of the nHAp = CNC ratio appeared to be the most suitable ratio for this methodology as it provides homogeneity of the particle’s size and distribution that was shown by the DLS and SEM analyses. We conclude that the CNC did work as expected by the sol-gel method to include HAp in its network without affecting its degree of crystallinity, as shown by the XRD analyses, or the thermal stability as shown by the TGA. Neither of the properties of either CNC or HAp changed due to the chemical interaction between them. The inclusion of BMP-2 does not appear to have a negative effect on the particle size and the charge reduction indicates that there is an interaction between the protein and the composite. The cell proliferation and viability assays demonstrated that all of our composites promoted an increased cell growth, but the nHAp > CNC ratio with BMP-2 had a higher growth percentage than the other composites. Therefore it appears to be the best nanocomposite for the fabrication of scaffolds or bioactive layers that can be used in the Bone Tissue Engineering field.
The authors declare no competing financial interests.
The paper was written through contributions of all authors. All authors have given approval to the final version of the paper. Claudia S. Herdocia-Lluberes and Simara Laboy-López contributed equally to this work.
Claudia S. Herdocia-Lluberes is grateful for the support of the UPR-RP Honors Program. The authors acknowledge the UPR Materials Characterization Center (MCC) for the provided support during the attainment of this work. Also, Carlos García and Karlene Vega are acknowledged for their help with all osteoblast cells related experiments. Institutional resources as seed funds were received during the attainment of this work. This work was also supported in part by the NASA Experimental Program to Stimulate Competitive Research (EPSCoR) under Grant no. NNX14AN18A.