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.

synthesis-graphene-hydroxyapatite-nanocomposites-for-potential-use

Research

JoVE Journal

Bioengineering

Elastomeric PGS Scaffolds in Arterial Tissue Engineering

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and increasing obesity1. Currently, bypass surgery using autologous vessels, allografts, and synthetic grafts are known as a commonly used for arterial substitutes2. However, these grafts have limited applications when an inner diameter of arteries is less than 6 mm due to low availability, thrombotic complications, compliance mismatch, and late intimal hyperplasia3,4. To overcome these limitations, tissue engineering has been successfully applied as a promising alternative to develop small-diameter arterial constructs that are nonthrombogenic, robust, and compliant. Several previous studies have developed small-diameter arterial constructs with tri-lamellar structure, excellent mechanical properties and burst pressure comparable to native arteries5,6. While high tensile strength and burst pressure by increasing collagen production from a rigid material or cell sheet scaffold, these constructs still had low elastin production and compliance, which is a major problem to cause graft failure after implantation. Considering these issues, we hypothesized that an elastometric biomaterial combined with mechanical conditioning would provide elasticity and conduct mechanical signals more efficiently to vascular cells, which increase extracellular matrix production and support cellular orientation.
The objective of this report is to introduce a fabrication technique of porous tubular scaffolds and a dynamic mechanical conditioning for applying them to arterial tissue engineering. We used a biodegradable elastomer, poly (glycerol sebacate) (PGS)7 for fabricating porous tubular scaffolds from the salt fusion method. Adult primary baboon smooth muscle cells (SMCs) were seeded on the lumen of scaffolds, which cultured in our designed pulsatile flow bioreactor for 3 weeks. PGS scaffolds had consistent thickness and randomly distributed macro- and micro-pores. Mechanical conditioning from pulsatile flow bioreactor supported SMC orientation and enhanced ECM production in scaffolds. These results suggest that elastomeric scaffolds and mechanical conditioning of bioreactor culture may be a promising method for arterial tissue engineering.

Elastomeric PGS scaffolds with vascular smooth muscle cells cultured in a pulsatile flow bioreactor may lead to promising small-diameter arterial constructs with native ECM production in a relatively short culture period.

Cardiovascular disease is one of the leading cause of mortality in the US and especially, coronary artery disease increases with an aging population and ...

Use of Human Perivascular Stem Cells for Bone Regeneration

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are a FACS-sorted population of 'pericytes' (CD146+CD34-CD45-) and 'adventitial cells' (CD146-CD34+CD45-), each of which we have previously reported to have properties of mesenchymal stem cells. PSCs, like MSCs, are able to undergo osteogenic differentiation, as well as secrete pro-osteogenic cytokines1,2. In the present protocol, we demonstrate the osteogenicity of PSCs in several animal models including a muscle pouch implantation in SCID (severe combined immunodeficient) mice, a SCID mouse calvarial defect and a femoral segmental defect (FSD) in athymic rats. The thigh muscle pouch model is used to assess ectopic bone formation. Calvarial defects are centered on the parietal bone and are standardly 4 mm in diameter (critically sized)8. FSDs are bicortical and are stabilized with a polyethylene bar and K-wires4. The FSD described is also a critical size defect, which does not significantly heal on its own4. In contrast, if stem cells or growth factors are added to the defect site, significant bone regeneration can be appreciated. The overall goal of PSC xenografting is to demonstrate the osteogenic capability of this cell type in both ectopic and orthotopic bone regeneration models.

Human perivascular stem cells (PSCs) are a novel stem cell class for skeletal tissue regeneration similar to mesenchymal stem cells (MSCs). PSCs can be isolated by FACS (fluorescence activated cell sorting) from adipose tissue procured during standard liposuction procedures, then combined with an osteoinductive scaffold to achieve bone formation in vivo.

Human perivascular stem cells (PSCs) can be isolated in sufficient numbers from multiple tissues for purposes of skeletal tissue engineering1-3. PSCs are ...

Encapsulation of Cardiomyocytes in a Fibrin Hydrogel for Cardiac Tissue Engineering

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying cellular responses under various culture conditions in vitro. The three dimensional environment more closely mimics what the cells observe in vivo due to the application of mechanical and chemical stimuli in all dimensions 1. Three-dimensional hydrogels can either be made from synthetic polymers such as PEG-DA 2 and PLGA 3 or a number of naturally occurring proteins such as collagen 4, hyaluronic acid 5 or fibrin 6,7. Hydrogels created from fibrin, a naturally occurring blood clotting protein, can polymerize to form a mesh that is part of the body's natural wound healing processes 8. Fibrin is cell-degradable and potentially autologous 9, making it an ideal temporary scaffold for tissue engineering.
Here we describe in detail the isolation of neonatal cardiomyocytes from three day old rat pups and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. Neonatal myocytes are a common cell source used for in vitro studies in cardiac tissue formation and engineering 4. Fibrin gel is created by mixing fibrinogen with the enzyme thrombin. Thrombin cleaves fibrinopeptides FpA and FpB from fibrinogen, revealing binding sites that interact with other monomers 10. These interactions cause the monomers to self-assemble into fibers that form the hydrogel mesh. Because the timing of this enzymatic reaction can be adjusted by altering the ratio of thrombin to fibrinogen, or the ratio of calcium to thrombin, one can injection mold constructs with a number of different geometries 11,12. Further we can generate alignment of the resulting tissue by how we constrain the gel during culture 13.
After culturing the engineered cardiac tissue constructs for two weeks under static conditions, the cardiac cells have begun to remodel the construct and can generate a contraction force under electrical pacing conditions 6. As part of this protocol, we also describe methods for analyzing the tissue engineered myocardium after the culture period including functional analysis of the active force generated by the cardiac muscle construct upon electrical stimulation, as well as methods for determining final cell viability (Live-Dead assay) and immunohistological staining to examine the expression and morphology of typical proteins important for contraction (Myosin Heavy Chain or MHC) and cellular coupling (Connexin 43 or Cx43) between myocytes.

We describe the isolation of neonatal cardiomyocytes and the preparation of the cells for encapsulation in fibrin hydrogel constructs for tissue engineering. We describe methods for analyzing the tissue engineered myocardium after the culture period including active force generated upon electrical stimulation and cell viability and immunohistological staining.

Culturing cells in a three dimensional hydrogel environment is an important technique for developing constructs for tissue engineering as well as studying ...

Postproduction Processing of Electrospun Fibres for Tissue Engineering

Electrospinning is a commonly used and versatile method to produce scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo undergo biaxial distension to varying extents such as skin, bladder, pelvic floor and even the hard palate as children grow. In producing scaffolds for these purposes there is a need to develop scaffolds of appropriate biomechanical properties (whether achieved without or with cells) and which are sterile for clinical use. The focus of this paper is not how to establish basic electrospinning parameters (as there is extensive literature on electrospinning) but on how to modify spun scaffolds post production to make them fit for tissue engineering purposes - here thickness, mechanical properties and sterilisation (required for clinical use) are considered and we also describe how cells can be cultured on scaffolds and subjected to biaxial strain to condition them for specific applications.
Electrospinning tends to produce thin sheets; as the electrospinning collector becomes coated with insulating fibres it becomes a poor conductor such that fibres no longer deposit on it. Hence we describe approaches to produce thicker structures by heat or vapour annealing increasing the strength of scaffolds but not necessarily the elasticity. Sequential spinning of scaffolds of different polymers to achieve complex scaffolds is also described. Sterilisation methodologies can adversely affect strength and elasticity of scaffolds. We compare three methods for their effects on the biomechanical properties on electrospun scaffolds of poly lactic-co-glycolic acid (PLGA).
Imaging of cells on scaffolds and assessment of production of extracellular matrix (ECM) proteins by cells on scaffolds is described. Culturing cells on scaffolds in vitro can improve scaffold strength and elasticity but the tissue engineering literature shows that cells often fail to produce appropriate ECM when cultured under static conditions. There are few commercial systems available that allow one to culture cells on scaffolds under dynamic conditioning regimes - one example is the Bose Electroforce 3100 which can be used to exert a conditioning programme on cells in scaffolds held using mechanical grips within a media filled chamber.4 An approach to a budget cell culture bioreactor for controlled distortion in 2 dimensions is described. We show that cells can be induced to produce elastin under these conditions. Finally assessment of the biomechanical properties of processed scaffolds cultured with or without cells is described.

Electrospun scaffolds can be processed post production for tissue engineering applications. Here we describe methods for spinning complex scaffolds (by consecutive spinning), for making thicker scaffolds (by multi-layering using heat or vapour annealing), for achieving sterility (aseptic production or sterilisation post production) and for achieving appropriate biomechanical properties.

Electrospinning is a commonly used and versatile method to produce  scaffolds (often biodegradable) for 3D tissue engineering.1, 2, 3 Many tissues in vivo ...

Capillary Force Lithography for Cardiac Tissue Engineering

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical discoveries with the aims of developing functional tissues for cardiac regeneration as well as in vitro screening assays. However, the ability to create high-fidelity models of heart tissue has proven difficult. The heart&#8217;s extracellular matrix (ECM) is a complex structure consisting of both biochemical and biomechanical signals ranging from the micro- to the nanometer scale2. Local mechanical loading conditions and cell-ECM interactions have recently been recognized as vital components in cardiac tissue engineering3-5.
A large portion of the cardiac ECM is composed of aligned collagen fibers with nano-scale diameters that significantly influences tissue architecture and electromechanical coupling2. Unfortunately, few methods have been able to mimic the organization of ECM fibers down to the nanometer scale. Recent advancements in nanofabrication techniques, however, have enabled the design and fabrication of scalable scaffolds that mimic the in vivo structural and substrate stiffness cues of the ECM in the heart6-9.
Here we present the development of two reproducible, cost-effective, and scalable nanopatterning processes for the functional alignment of cardiac cells using the biocompatible polymer poly(lactide-co-glycolide) (PLGA)8 and a polyurethane (PU) based polymer. These anisotropically nanofabricated substrata (ANFS) mimic the underlying ECM of well-organized, aligned tissues and can be used to investigate the role of nanotopography on cell morphology and function10-14.
Using a nanopatterned (NP) silicon master as a template, a polyurethane acrylate (PUA) mold is fabricated. This PUA mold is then used to pattern the PU or PLGA hydrogel via UV-assisted or solvent-mediated capillary force lithography (CFL), respectively15,16. Briefly, PU or PLGA pre-polymer is drop dispensed onto a glass coverslip and the PUA mold is placed on top. For UV-assisted CFL, the PU is then exposed to UV radiation (&#955; = 250-400 nm) for curing. For solvent-mediated CFL, the PLGA is embossed using heat (120 &#176;C) and pressure (100 kPa). After curing, the PUA mold is peeled off, leaving behind an ANFS for cell culture. Primary cells, such as neonatal rat ventricular myocytes, as well as human pluripotent stem cell-derived cardiomyocytes, can be maintained on the ANFS2.

In this protocol, we demonstrate the fabrication of biomimetic cardiac cell culture substrata made from two distinct polymeric materials using capillary force lithography. The described methods provide a scalable, cost-effective technique to engineer the structure and function of macroscopic cardiac tissues for in vitro and in vivo applications.

Cardiovascular disease remains the leading cause of death worldwide1. Cardiac tissue engineering holds much promise to deliver groundbreaking medical ...

Tissue Engineering of a Human 3D in vitro Tumor Test System

Cancer is one of the leading causes of death worldwide. Current therapeutic strategies are predominantly developed in 2D culture systems, which inadequately reflect physiological conditions in vivo. Biological 3D matrices provide cells an environment in which cells can self-organize, allowing the study of tissue organization and cell differentiation. Such scaffolds can be seeded with a mixture of different cell types to study direct 3D cell-cell-interactions. To mimic the 3D complexity of cancer tumors, our group has developed a 3D in vitro tumor test system.	
	Our 3D tissue test system models the in vivo situation of malignant peripheral nerve sheath tumors (MPNSTs), which we established with our decellularized porcine jejunal segment derived biological vascularized scaffold (BioVaSc). In our model, we reseeded a modified BioVaSc matrix with primary fibroblasts, microvascular endothelial cells (mvECs) and the S462 tumor cell line. For static culture, the vascular structure of the BioVaSc is removed and the remaining scaffold is cut open on one side (Small Intestinal Submucosa SIS-Muc). The resulting matrix is then fixed between two metal rings (cell crowns).	
	Another option is to culture the cell-seeded SIS-Muc in a flow bioreactor system that exposes the cells to shear stress. Here, the bioreactor is connected to a peristaltic pump in a self-constructed incubator. A computer regulates the arterial oxygen and nutrient supply via parameters such as blood pressure, temperature, and flow rate. This setup allows for a dynamic culture with either pressure-regulated pulsatile or constant flow.	
	In this study, we could successfully establish both a static and dynamic 3D culture system for MPNSTs. The ability to model cancer tumors in a more natural 3D environment will enable the discovery, testing, and validation of future pharmaceuticals in a human-like model.

Tissue Engineering of a Human 3D in vitro Tumor Test System

Methods to create human 3D tumor tissues as test systems are described. These technologies are based on a decellularized Biological Vascularized Scaffold (BioVaSc), primary human cells and a tumor cell line, which can be cultured under static as well as under dynamic conditions in a flow bioreactor.

Cancer is one of the leading causes of death worldwide.  Current therapeutic strategies are predominantly developed in 2D culture  systems, which ...

Production, Characterization and Potential Uses of a 3D Tissue-engineered Human Esophageal Mucosal Model

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore esophageal adenocarcinoma generally has a poor prognosis, with little improvement in survival rates in recent years. These are difficult conditions to study and there has been a lack of suitable experimental platforms to investigate disorders of the esophageal mucosa.
A model of the human esophageal mucosa has been developed in the MacNeil laboratory which, unlike conventional 2D cell culture systems, recapitulates the cell-cell and cell-matrix interactions present in vivo and produces a mature, stratified epithelium similar to that of the normal human esophagus. Briefly, the model utilizes non-transformed normal primary human esophageal fibroblasts and epithelial cells grown within a porcine-derived acellular esophageal scaffold. Immunohistochemical characterization of this model by CK4, CK14, Ki67 and involucrin staining demonstrates appropriate recapitulation of the histology of the normal human esophageal mucosa.
This model provides a robust, biologically relevant experimental model of the human esophageal mucosa. It can easily be manipulated to investigate a number of research questions including the effectiveness of pharmacological agents and the impact of exposure to environmental factors such as alcohol, toxins, high temperature or gastro-esophageal refluxate components. The model also facilitates extended culture periods not achievable with conventional 2D cell culture, enabling, inter alia, the study of the impact of repeated exposure of a mature epithelium to the agent of interest for up to 20 days. Furthermore, a variety of cell lines, such as those derived from esophageal tumors or Barrett&#8217;s Metaplasia, can be incorporated into the model to investigate processes such as tumor invasion and drug responsiveness in a more biologically relevant environment.

This manuscript describes the production, characterization and potential uses of a tissue engineered 3D esophageal construct prepared from normal primary human esophageal fibroblast and squamous epithelial cells seeded within a de-cellularized porcine scaffold. The results demonstrate the formation of a mature stratified epithelium similar to the normal human esophagus.

The incidence of both esophageal adenocarcinoma and its precursor, Barrett&#8217;s Metaplasia, are rising rapidly in the western world. Furthermore ...

Engineering 3D Cellularized Collagen Gels for Vascular Tissue Regeneration

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. Collagen has been extensively used for a wide range of biomedical applications and is considered a valid alternative to synthetic materials due to its inherent biocompatibility (i.e., low antigenicity, inflammation, and cytotoxic responses). However, the limited mechanical properties and the related low hand-ability of collagen gels have hampered their use as scaffold materials for vascular tissue engineering. Therefore, the rationale behind this work was first to engineer cellularized collagen gels into a tubular-shaped geometry and second to enhance smooth muscle cells driven reorganization of collagen matrix to obtain tissues stiff enough to be handled.
The strategy described here is based on the direct assembling of collagen and smooth muscle cells (construct) in a 3D cylindrical geometry with the use of a molding technique. This process requires a maturation period, during which the constructs are cultured in a bioreactor under static conditions (without applied external dynamic mechanical constraints) for 1 or 2 weeks. The &#8220;static bioreactor&#8221; provides a monitored and controlled sterile environment (pH, temperature, gas exchange, nutrient supply and waste removal) to the constructs. During culture period, thickness measurements were performed to evaluate the cells-driven remodeling of the collagen matrix, and glucose consumption and lactate production rates were measured to monitor the cells metabolic activity. Finally, mechanical and viscoelastic properties were assessed for the resulting tubular constructs. To this end, specific protocols and a focused know-how (manipulation, gripping, working in hydrated environment, and so on) were developed to characterize the engineered tissues.

In this work, we present a technique for the rapid fabrication of living vascular tissues by direct culturing of collagen, smooth muscle cells and endothelial cells. In addition, a new protocol for the mechanical characterization of engineered vascular tissues is described.

Synthetic materials are known to initiate clinical complications such as inflammation, stenosis, and infections when implanted as vascular substitutes. ...

Sandwich-like Microenvironments to Harness Cell/Material Interactions

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. However in vivo, most of the cells are completely surrounded by the extracellular matrix (ECM), resulting in a three-dimensional (3D) distribution of receptors. This may trigger differences in the outside-in signaling pathways and thus in cell behavior.
This article shows that stimulating the dorsal receptors of cells already adhered to a 2D substrate by overlaying a film of a new material (a sandwich-like culture) triggers important changes with respect to standard 2D cultures. Furthermore, the simultaneous excitation of ventral and dorsal receptors shifts cell behavior closer to that found in 3D environments. Additionally, due to the nature of the system, a sandwich-like culture is a versatile tool that allows the study of different parameters in cell/material interactions, e.g., topography, stiffness and different protein coatings at both the ventral and dorsal sides. Finally, since sandwich-like cultures are based on 2D substrates, several analysis procedures already developed for standard 2D cultures can be used normally, overcoming more complex procedures needed for 3D systems.

The following protocol describes the procedure to assemble sandwich-like cultures to be used as an intermediate stage between bi-dimensional (2D) and three-dimensional (3D) cellular environments. The engineered systems can have applications in microscopy, biomechanics, biochemistry and cell biology assays.

Cell culture has been traditionally carried out on bi-dimensional (2D) substrates where cells adhere using ventral receptors to the biomaterial surface. ...

Synthesis of Keratin-based Nanofiber for Biomedical Engineering

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of these porous nanofibers is of great interest due to their unique physiochemical properties. Here we elaborate on the fabrication of keratin containing poly (&#949;-caprolactone) (PCL) nanofibers (i.e., PCL/keratin composite fiber). Water soluble keratin was first extracted from human hair and mixed with PCL in different ratios. The blended solution of PCL/keratin was transformed into nanofibrous membranes using a laboratory designed electrospinning set up. Fiber morphology and mechanical properties of the obtained nanofiber were observed and measured using scanning electron microscopy and tensile tester. Furthermore, degradability and chemical properties of the nanofiber were studied by FTIR. SEM images showed uniform surface morphology for PCL/keratin fibers of different compositions. These PCL/keratin fibers also showed excellent mechanical properties such as Young&#39;s modulus and failure point. Fibroblast cells were able to attach and proliferate thus proving good cell viability. Based on the characteristics discussed above, we can strongly argue that the blended nanofibers of natural and synthetic polymers can represent an excellent development of composite materials that can be used for different biomedical applications.

Electrospun nanofibers have a high surface area to weight ratio, excellent mechanical integrity, and support cell growth and proliferation. These nanofibers have a wide range of biomedical applications. Here we fabricate keratin/ PCL nanofibers, using the electrospinning technique, and characterize the fibers for possible applications in tissue engineering.

Electrospinning, due to its versatility and potential for applications in various fields, is being frequently used to fabricate nanofibers. Production of ...