Name: Synthesis of Graphene-Hydroxyapatite Nanocomposites for Potential Use in Bone Tissue Engineering (Video) | JoVE Journal
Uploaded: 2022-07-27T13:50:00.000+00:00
Duration: 7 min 14 s
Description: Learn a simple one-pot synthesis method for graphene-hydroxyapatite nanocomposites for bone tissue engineering. This rapid, economical approach creates bioactive scaffolds that promote rapid healing of bone injuries.

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5 minutes

Dr. Sougata Ghosh acknowledges the Department of Science and Technology (DST), Ministry of Science and Technology, Government of India, and Jawaharlal Nehru Centre for Advanced Scientific Research, India for funding under the Post-Doctoral Overseas Fellowship in Nano Science and Technology (Ref. JNC/AO/A.0610.1(4) 2019-2260 dated August 19, 2019). Dr. Sougata Ghosh acknowledges Kasetsart University, Bangkok, Thailand for a Post-Doctoral Fellowship, and funding under the Reinventing University Program (Ref. No. 6501.0207/10870 dated November 9, 2021). The authors would like to thank the Kostas Advanced Nano-Characterization Facility (KANCF) for assistance with the characterization experiments. KANCF is a shared multidisciplinary research and educational facility within the Kostas Research Institute (KRI) at Northeastern University.

1. Synthesis of nHAP by precipitation
<ol>
	<li>Synthesize the pristine nHAP using 50 mL of the reaction mixture containing 1 M Ca(NO3)2&#8729;4H2O and 0.67 M (NH4)H2PO4 followed by the dropwise addition of NH4OH (25%) to maintain a pH around 1018.</li>
	<li>Thereafter, agitate the reaction mixture by ultrasound irradiation (UI) for 30 min (500 W power and 20 kHz ultrasound frequency).</li>
	<li>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.</li>
	<li>Wash the precipitate with deionized (DI) water 3x and lyophilize for 48 h. Store the dry powder at 4 &#176;C.</li>
</ol>
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).
<ol>
	<li>Synthesis of nHAP/GNR
	<ol>
		<li>To prepare the nHAP/GNRs nanocomposite, use a co-functionalization strategy where nHAP can be synthesized and conjugated to GNRs simultaneously, as follows.</li>
		<li>Dissolve 5 mg of GNRs (Table of Materials) in a mixture of 1 M calcium nitrate tetrahydrate [Ca(NO3)2&#183;4H2O] and 0.67 M diammonium hydrogen phosphate [(NH4)2HPO4] to a 50 mL final volume19.</li>
		<li>During this reaction, add 25% of NH4OH dropwise to maintain the pH at ~10. Agitate the resulting mixture by UI for 30 min.</li>
		<li>After completion of the reaction, leave the solution undisturbed for 120 h at room temperature until maturation.</li>
		<li>Observe the formation of a gelatinous precipitate of nHAP which coats the GNRs, following which a white precipitate of nHAP/GNRs settles.</li>
		<li>Wash the precipitate 3x by centrifugation at 1398 x g for 5 min at room temperature followed by re-dispersion in DI water.</li>
		<li>Lyophilize the recovered washed precipitate for 48 h. Store the dry powder at 4 &#176;C.</li>
		<li>Use pristine nHAP and GNRs as control samples.</li>
	</ol>
	</li>
	<li>Synthesis of GNR/nHAP nanocomposite
	<ol>
		<li>Suspend commercially available nHAP at a concentration of 5 mg/mL in 50 mL of DI water supplemented with 5 mg of GNRs.</li>
		<li>Agitate the resulting mixture by UI for 30 min and thereafter leave the mixture undisturbed for 120 h at room temperature.</li>
		<li>After maturation, recover the white precipitate of the resulting GNR/nHAP by centrifugation at 1398 x g for 5 min at room temperature.</li>
		<li>Wash the sample 3x using DI water, lyophilize it for 48 h, and store the dry powder at 4 &#176;C for further use.</li>
	</ol>
	</li>
</ol>
3. Characterization of nHAP, nHAP/GNR, and GNR/nHAP
<ol>
	<li>Use a high-resolution transmission electron microscope (HRTEM) (see Table of Materials) to characterize the morphology and size of the nanocomposites11.</li>
	<li>Analyze the elemental composition of the nanocomposites employing energy dispersive spectroscopy (EDS) and perform elemental mapping using the scanning transmission electron microscope&#160;(STEM)11.</li>
	<li>Perform Fourier transform infrared (FTIR) spectroscopy for the neat samples at wavenumbers of 500-4000 cm&#8722;1 to analyze the chemical groups in the nanocomposite16.</li>
	<li>Perform powder X-ray diffraction (XRD) analysis of the as-synthesized nHAP using an X-ray wavelength of 1.5406 &#197;, current and voltage settings of 40 mA and 40 kV, respectively, and 2&#952; ranging from 20&#176; to 90&#176;.</li>
	<li>Evaluate the percent loading of GNR in the nanocomposite using thermogravimetric analysis (TGA) by heating the samples from room temperature to 1000 &#176;C at a rate of 10 &#176;C/min under nitrogen flow.</li>
</ol>

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</ol>

The authors have no conflicts of interest.

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 cytocompatibility20,21,22. In this study, the orientation of HAP was varied, which can have a significant impact on its unique properties, such as promotion of ost...

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 temperature3,4. Detection of single-molecule adsorption events and extremely high carrier mobility add to the attractive attributes of graphene5,6. 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-unions9,10. 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 surgeries13,14. 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 osteoconductive15,16. 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.

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

synthesis of graphene-hydroxyapatite nanocomposites for potential use in bone tissue engineering

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

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 &#181;m) 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.

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...

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.

Developing novel materials for bone tissue engineering is one of the most important thrust areas of nanomedicine. Several nanocomposites have been ...

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JoVE Journal

Bioengineering

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Learn a simple one-pot synthesis method for graphene-hydroxyapatite nanocomposites for bone tissue engineering. This rapid, economical approach creates bioactive scaffolds that promote rapid healing of bone injuries.

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

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