Name: synthesis of graphene-hydroxyapatite nanocomposites for potential use in bone tissue engineering
Uploaded: 2022-07-27T13:50:00
Description: Watch this Scientific Journal Video about Synthesis of Graphene-Hydroxyapatite Nanocomposites for Potential Use in Bone Tissue Engineering at JoVE.com

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 t...

<ol>
	<li>Novoselov, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electric+field+effect+in+atomically+thin+carbon+films.">Electric field effect in atomically thin carbon films.</a> Science. 306 (5696), 666-669 (2004).</li><li>Novoselov, K. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unconventional+quantum+Hall+effect+and+Berry's+phase+of+2&#960;+in+bilayer+graphene.">Unconventional quantum Hall effect and Berry's phase of 2&#960; in bilayer graphene.</a> Nature Physics. 2 (3), 177-180 (2006).</li><li>Zhang, Y. B., Tan, Y. W., Stormer, H. 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S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanohydroxyapatite/graphene+nanoribbons+nanocomposites+induce+in+vitro+osteogenesis+and+promote+in+vivo+bone+neoformation.">Nanohydroxyapatite/graphene nanoribbons nanocomposites induce in vitro osteogenesis and promote in vivo bone neoformation.</a> ACS Biomaterials Science and Engineering. 4 (5), 1580-1590 (2018).</li><li>Faniyi, I. O., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+comparative+analyses+of+reduced+graphene+oxide+(RGO)+prepared+via+green,+mild+and+chemical+approaches.">The comparative analyses of reduced graphene oxide (RGO) prepared via green, mild and chemical approaches.</a> SN Applied Sciences. 1 (10), 1-7 (2019).</li><li>Neelgund, G. M., Oki, A., Luo, Z. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+situ+deposition+of+hydroxyapatite+on+graphene+nanosheets.">In situ deposition of hydroxyapatite on graphene nanosheets.</a> Materials Research Bulletin. 48 (2), 175-179 (2013).</li><li>Rajkumar, M., Sundaram, N. 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C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High+loads+of+nano-hydroxyapatite/graphene+nanoribbon+composites+guided+bone+regeneration+using+an+osteoporotic+animal+model.">High loads of nano-hydroxyapatite/graphene nanoribbon composites guided bone regeneration using an osteoporotic animal model.</a> International Journal of Nanomedicine. 14, 865-874 (2019).</li><li>Murugan, N., Sundaramurthy, A., Chen, S. -. M., Sundramoorthy, A. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graphene+oxide/oxidized+carbon+nanofiber/mineralized+hydroxyapatite+based+hybrid+composite+for+biomedical+applications.">Graphene oxide/oxidized carbon nanofiber/mineralized hydroxyapatite based hybrid composite for biomedical applications.</a> Materials Research Express. 4 (12), 124005 (2017).</li><li>Barbosa, M. C., Messmer, N. R., Brazil, T. R., Marciano, F. R., Lobo, A. O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effect+of+ultrasonic+irradiation+on+the+crystallinity+of+nano-hydroxyapatite+produced+via+the+wet+chemical+method.">The effect of ultrasonic irradiation on the crystallinity of nano-hydroxyapatite produced via the wet chemical method.</a> Materials Science and Engineering C. 33 (5), 2620-2625 (2013).</li><li>Rodrigues, B. V. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Graphene+oxide/multi-walled+carbon+nanotubes+as+nanofeatured+scaffolds+for+the+assisted+deposition+of+nanohydroxyapatite:+characterization+and+biological+evaluation.">Graphene oxide/multi-walled carbon nanotubes as nanofeatured scaffolds for the assisted deposition of nanohydroxyapatite: characterization and biological evaluation.</a> International Journal of Nanomedicine. 11, 2569-2585 (2016).</li><li>Sharma, M., Nagar, R., Meena, V. K., Singh, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Electro-deposition+of+bactericidal+and+corrosion-resistant+hydroxyapatite+nanoslabs.">Electro-deposition of bactericidal and corrosion-resistant hydroxyapatite nanoslabs.</a> RSC Advances. 9 (20), 11170-11178 (2019).</li><li>Kamrujjaman, M., Khandaker, J. I., Haque, M. M., Rahman, M. O., Rahman, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Study+of+the+dependency+of+pH+values+on+HAp+synthesis.">Study of the dependency of pH values on HAp synthesis.</a> Journal of Nanomaterials &amp; Molecular Nanotechnology. 7, 4 (2019).</li><li>Baradaran, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mechanical+properties+and+biomedical+applications+of+a+nanotube+hydroxyapatite-reduced+graphene+oxide+composite.">Mechanical properties and biomedical applications of a nanotube hydroxyapatite-reduced graphene oxide composite.</a> Carbon. 69, 32-45 (2014).</li><li>Sassoni, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydroxyapatite+and+other+calcium+phosphates+for+the+conservation+of+cultural+heritage:+A+review.">Hydroxyapatite and other calcium phosphates for the conservation of cultural heritage: A review.</a> Materials. 11 (4), 557 (2018).</li><li>Tang, H., Ehlert, G. 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A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enhanced+mechanical+properties+of+nanocomposites+at+low+graphene+content.">Enhanced mechanical properties of nanocomposites at low graphene content.</a> ACS Nano. 3 (12), 3884-3890 (2009).</li><li>Luque de Castro, M. D., Priego-Capote, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrasound-assisted+crystallization+(sonocrystallization).">Ultrasound-assisted crystallization (sonocrystallization).</a> Ultrasonics Sonochemistry. 14 (6), 717-724 (2007).</li><li>Azhari, A., Toyserkani, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Additive+manufacturing+of+graphene-hydroxyapatite+nanocomposite+structures.">Additive manufacturing of graphene-hydroxyapatite nanocomposite structures.</a> International Journal of Applied Ceramic Technology. 12 (1), 8-17 (2015).</li><li>Li, H., Wang, J., Bao, Y., Guo, Z., Zhang, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+sonocrystallization+in+the+salting-out+process.">Rapid sonocrystallization in the salting-out process.</a> Journal of Crystal Growth. 247 (1-2), 192-198 (2003).</li><li>Zou, Z., Lin, K., Chen, L., Chang, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Ultrafast+synthesis+and+characterization+of+carbonated+hydroxyapatite+nanopowders+via+sonochemistry-assisted+microwave+process.">Ultrafast synthesis and characterization of carbonated hydroxyapatite nanopowders via sonochemistry-assisted microwave process.</a> Ultrasonics Sonochemistry. 19 (6), 1174-1179 (2012).</li><li>Rouhani, P., Taghavinia, N., Rouhani, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+growth+of+hydroxyapatite+nanoparticles+using+ultrasonic+irradiation.">Rapid growth of hydroxyapatite nanoparticles using ultrasonic irradiation.</a> Ultrasonics Sonochemistry. 17 (5), 853-856 (2010).</li><li>Fan, Z., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=One-pot+synthesis+of+graphene/hydroxyapatite+nanorod+composite+for+tissue+engineering.">One-pot synthesis of graphene/hydroxyapatite nanorod composite for tissue engineering.</a> Carbon. 66, 407-416 (2014).</li><li>Ghosh, S., Mostafavi, E., Thorat, N., Webster, T. 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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 border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<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

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 &#181;m 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 t...

Watch this Scientific Journal Video about Synthesis of Graphene-Hydroxyapatite Nanocomposites for Potential Use in Bone Tissue Engineering at JoVE.com

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

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 ...

This work explains the strategy to fabricate novel composites of nano hydroxyapatite and graphene nanoribbons with opposite orientations. These biomaterials are significant for developing bone tissue engineering scaffolds. This is a simple one pot synthesis.It is a rapid, effective, and economical method. This work is extremely relevant with the context of treating bone injuries and associated ailments where more bone tissue regeneration will promote rapid healing. To begin, synthesize the pristine nanoparticles of hydroxyapatite using 50 milliliters of the reaction mixture.Dropwise, add 25%ammoniumhydroxide to maintain a pH of around 10. Thereafter, agitate the reaction mixture by ultrasound irradiation for 30 minutes. Allow the resulting solution to mature for 120 hours at room temperature until the white precipitate of nanoparticles of hydroxyapatite settles out.Recover the nanoparticles of hydroxyapatite by centrifugation at 1, 398 x g for five minutes at room temperature. Wash the precipitate with deionized water three times. To prepare the nHAP/GNR nanocomposite, use a cofunctionalization strategy, start with dissolving five milligrams of graphene nanoribbons in a mixture of one molar calcium nitrate tetrahydrate and 0.67 molar diammonium hydrogen phosphate to a 50 milliliter final volume.During this reaction, add 25%of ammonium hydroxide dropwise to maintain the pH at approximately 10. Agitate the resulting mixture by ultrasonication for 30 minutes. After completion of the reaction, leave the solution undisturbed for 120 hours at room temperature until maturation.Observe for the formation of a gelatinous precipitate of nanoparticles of hydroxyapatite, which coats the graphine nanoribbons, following which a white precipitate of nHAP/GNR settles. Wash the precipitate three times by centrifugation at 1, 398 x g for five minutes at room temperature followed by redispersion in deionized water. To synthesize GNR/nHAP nanocomposites, suspend commercially available nanoparticles of hydroxyapatite at a concentration of five milligrams per milliliter in 50 milliliters of deionized water, supplemented with five milligrams of graphine nanoribbons.Agitate the resulting mixture by ultrasonication for 30 minutes, and thereafter, leave the mixture undisturbed for 120 hours at room temperature. After maturation, recover the white precipitate of the resulting GNR/nHAP by centrifugation at 1, 398 x g for five minutes at room temperature. Wash the sample three times using deionized water.The HRTEM analysis of nHAP/GNR nanocomposite showed nHAP patches between 150 to 250 nanometers in length and width. Elemental mapping confirmed that the intermediated nodal patches on the GNRs were indeed nHAP due to the presence of elemental calcium and phosphorus. In GNR/nHAP nanocomposites, the GNR formed thin films on the surface of the spherical nHAP nanoparticles.The EDS analysis showed a clear increase in the carbon content due to GNRs, while the peaks specific to calcium and phosphorus were due to nHAP. The marked increase in the carbon content in the nHAP/GNR spectrum is due to the majority of the GNRs onto which only small patches of freshly synthesized nHAP were observed. XRD analysis confirmed the hexagonal crystal structure of the nHAP strong peaks corresponding to 002, 102, 211, 300, 202, 310, 113, 222, and 213 planes, respectively, confirmed the purity of the as-synthesized nHAP.TGA analysis showed a steady decrease in mass after 200 degrees Celsius up to 500 degrees Celsius due to the crystallization of nHAP. Further heating led to the decomposition of the complexes. Lost due to the presence of GNRs was found to be between 0.5 and 0.98%in GNR/nHAP and nHAP/GNR.The sequence of addition of reagents and reaction conditions are most critical to get the desired reverse orientation of the nanocomposites. Cell lines-based studies can be performed to check the potential of the nanocomposites to promote osteogenesis. This will open a new area of orientation-mediated modulation of cellular response for tissue engineering.

Research

JoVE Journal

Bioengineering

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