Synthesis of Graphene-Hydroxyapatite Nanocomposites for Potential Use in Bone Tissue Engineering

Summary

Novel nanocomposites of graphene nanoribbons and hydroxyapatite nanoparticles were prepared using solution-phase synthesis. These hybrids when employed in bioactive scaffolds can exhibit potential applications in tissue engineering and bone regeneration.

Abstract

Developing novel materials for bone tissue engineering is one of the most important thrust areas of nanomedicine. Several nanocomposites have been fabricated with hydroxyapatite to facilitate cell adherence, proliferation, and osteogenesis. In this study, hybrid nanocomposites were successfully developed using graphene nanoribbons (GNRs) and nanoparticles of hydroxyapatite (nHAPs), that when employed in bioactive scaffolds may potentially improve bone tissue regeneration. These nanostructures can be biocompatible. Here, two approaches were used for preparing the novel materials. In one approach, a co-functionalization strategy was used where nHAP was synthesized and conjugated to GNRs simultaneously, resulting in nanohybrids of nHAP on GNR surfaces (denoted as nHAP/GNR). High-resolution transmission electron microscopy (HRTEM) confirmed that the nHAP/GNR composite is comprised of slender, thin structures of GNRs (maximum length of 1.8 µm) with discrete patches (150-250 nm) of needle-like nHAP (40-50 nm in length). In the other approach, commercially available nHAP was conjugated with GNRs forming GNR-coated nHAP (denoted as GNR/nHAP) (i.e., with an opposite orientation relative to the nHAP/GNR nanohybrid). The nanohybrid formed using the latter method exhibited nHAP nanospheres with a diameter ranging from 50 nm to 70 nm covered with a network of GNRs on the surface. Energy dispersive spectra, elemental mapping, and Fourier transform infrared (FTIR) spectra confirmed the successful integration of nHAP and GNRs in both nanohybrids. Thermogravimetric analysis (TGA) indicated that the loss at elevated heating temperatures due to the presence of GNRs was 0.5% and 0.98% for GNR/nHAP and nHAP/GNR, respectively. The nHAP-GNR nanohybrids with opposite orientations represent significant materials for use in bioactive scaffolds to potentially promote cellular functions for improving bone tissue engineering applications.

Introduction

Graphene has sheet-like two-dimensional structures composed of sp-hybridized carbon. Several other allotropes can be attributed to the extended honeycomb network of graphene (e.g., the stacking of graphene sheets forms 3D graphite while rolling off the same material results in the formation of 1D nanotubes¹). Likewise, 0D fullerenes are formed due to wrapping². Graphene has attractive physicochemical and optoelectronic properties that include an ambipolar field-effect and a quantum Hall effect at room temperature^3,4. Detection of single-molecule adsorption events and extremely high carrier mobility add to the attractive attributes of graphene^5,6. Further, graphene nanoribbons (GNRs) with narrow widths and a large mean free path, low resistivity with a high current density, and high electron mobility are considered promising interconnecting materials⁷. Hence, GNRs are being explored for applications in a myriad of devices, and more recently in nanomedicine, particularly tissue engineering and drug delivery⁸.

Among various traumatic ailments, bone injuries are considered one of the most challenging due to difficulties in stabilizing the fracture, regeneration and replacement with new bone, resisting infection, and re-aligning bone non-unions^9,10. Surgical procedures remain the only alternative for femoral shaft fractures. It should be noted that almost $52 million is spent every year on treating bone injuries in Central America and Europe¹¹.

Bioactive scaffolds for bone tissue engineering applications can be more effective by incorporating nano-hydroxyapatite (nHAP), as they resemble the micro and nano architectural properties of the bone itself¹². HAP, chemically represented as Ca₁₀(PO₄)₆(OH)₂ with a Ca/P molar ratio of 1.67, is the most preferred for biomedical applications, particularly for treating periodontal defects, the substitution of hard tissues, and fabricating implants for orthopedic surgeries^13,14. Thus, the fabrication of nHAP-based biomaterials reinforced with GNRs can possess superior biocompatibility and may be advantageous due to their ability to promote osseointegration and be osteoconductive^15,16. Such hybrid composite scaffolds can preserve biological properties such as cell adherence, spreading, proliferation, and differentiation¹⁷. Herein, we report the fabrication of two new nanocomposites for bone tissue engineering by rationally altering the spatial arrangement of nHAP and GNRs as illustrated in Figure 1. The chemical and structural properties of the two different nHAP-GNRs arrangements were evaluated here.

Protocol

1. Synthesis of nHAP by precipitation

1. Synthesize the pristine nHAP using 50 mL of the reaction mixture containing 1 M Ca(NO₃)₂∙4H₂O and 0.67 M (NH₄)H₂PO₄ followed by the dropwise addition of NH₄OH (25%) to maintain a pH around 10¹⁸.
2. Thereafter, agitate the reaction mixture by ultrasound irradiation (UI) for 30 min (500 W power and 20 kHz ultrasound frequency).
3. Allow the resulting solution to mature for 120 h at room temperature until the white precipitate of nHAP settles out. Recover the nHAP by centrifugation at 1398 x g for 5 min at room temperature.
4. Wash the precipitate with deionized (DI) water 3x and lyophilize for 48 h. Store the dry powder at 4 °C.

2. Preparation of nHAP/GNR nanocomposites

NOTE: The following describes two approaches for fabricating nHAP/GNR (i.e., nHAP on GNR surfaces) and GNR/nHAP (GNR-coated nHAP) nanocomposites that represent two different spatial arrangements of nHAP and GNRs (Figure 1).

1. Synthesis of nHAP/GNR
   1. To prepare the nHAP/GNRs nanocomposite, use a co-functionalization strategy where nHAP can be synthesized and conjugated to GNRs simultaneously, as follows.
   2. Dissolve 5 mg of GNRs (Table of Materials) in a mixture of 1 M calcium nitrate tetrahydrate [Ca(NO₃)₂·4H₂O] and 0.67 M diammonium hydrogen phosphate [(NH₄)₂HPO₄] to a 50 mL final volume¹⁹.
   3. During this reaction, add 25% of NH₄OH dropwise to maintain the pH at ~10. Agitate the resulting mixture by UI for 30 min.
   4. After completion of the reaction, leave the solution undisturbed for 120 h at room temperature until maturation.
   5. Observe the formation of a gelatinous precipitate of nHAP which coats the GNRs, following which a white precipitate of nHAP/GNRs settles.
   6. Wash the precipitate 3x by centrifugation at 1398 x g for 5 min at room temperature followed by re-dispersion in DI water.
   7. Lyophilize the recovered washed precipitate for 48 h. Store the dry powder at 4 °C.
   8. Use pristine nHAP and GNRs as control samples.
2. Synthesis of GNR/nHAP nanocomposite
   1. Suspend commercially available nHAP at a concentration of 5 mg/mL in 50 mL of DI water supplemented with 5 mg of GNRs.
   2. Agitate the resulting mixture by UI for 30 min and thereafter leave the mixture undisturbed for 120 h at room temperature.
   3. After maturation, recover the white precipitate of the resulting GNR/nHAP by centrifugation at 1398 x g for 5 min at room temperature.
   4. Wash the sample 3x using DI water, lyophilize it for 48 h, and store the dry powder at 4 °C for further use.

3. Characterization of nHAP, nHAP/GNR, and GNR/nHAP

1. Use a high-resolution transmission electron microscope (HRTEM) (see Table of Materials) to characterize the morphology and size of the nanocomposites¹¹.
2. Analyze the elemental composition of the nanocomposites employing energy dispersive spectroscopy (EDS) and perform elemental mapping using the scanning transmission electron microscope (STEM)¹¹.
3. Perform Fourier transform infrared (FTIR) spectroscopy for the neat samples at wavenumbers of 500-4000 cm⁻¹ to analyze the chemical groups in the nanocomposite¹⁶.
4. Perform powder X-ray diffraction (XRD) analysis of the as-synthesized nHAP using an X-ray wavelength of 1.5406 Å, current and voltage settings of 40 mA and 40 kV, respectively, and 2θ ranging from 20° to 90°.
5. Evaluate the percent loading of GNR in the nanocomposite using thermogravimetric analysis (TGA) by heating the samples from room temperature to 1000 °C at a rate of 10 °C/min under nitrogen flow.

Results

HRTEM analysis
Individually, GNRs were slender bamboo-like structures with some bends at some distance as observed in Figure 2. The longest GNR was 1.841 µm while the smallest bent GNR was 497 nm. The nanoribbons often showed a visible variation in width that might be attributed to twisting to form helical configurations in many places. Such unidirectional alignment of GNRs may help to obtain attractive features such as magnetic properties, conductivity, or heat transpor...

Discussion

Although various metals, polymers, ceramics, and their combinations have been researched as orthopedic implants and fixation accessories, HAP is considered to be one of the most preferable materials due to its chemical similarity to the bone itself and consequent high cytocompatibility^20,21,22. In this study, the orientation of HAP was varied, which can have a significant impact on its unique properties, such as promotion of ost...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ammonium phosphate monobasic	Sigma-Aldrich	216003-100G	Synthesis
Calcium nitrate tetrahydrate	Sigma-Aldrich	237124	Synthesis
Centrifuge	Hettich	EBA 200S	Recovery
Fourier transform infrared spectrometer	Brucker	Vertex 70	Characterization
Graphene nanoribbon	Sigma-Aldrich	922714	Synthesis
High resolution transmission electron microscope	Thermo Fisher Scientific	Themis Titan 300	Characterization
Magnetic stirrer	IKA	C-MAG HS7 S68	Functionalization
Micropipettes	TreffLab	06H35687	Reagent preparation
pH meter	Eutech pH5+	ECPH503PLUSK	Reagent preparation
Thermogravimetric analyzer	TA Instruments	SDT Q600	Characterization
Ultrasonic bath	Bandelin	DT100	Functionalization
Universal Oven	Memmert	UF55	Functionalization
Weighing balance	Precisa	XB220A	Reagent preparation
X-ray diffractometer	Brucker	D8-Advanced	Characterization