The authors would like to acknowledge the support from the New York Capital Region Research Alliance Grant.

1. Bioprinting Platform Establishment
The printer modification was based on a HP Deskjet 500 thermal inkjet printer and HP 51626a black ink cartridge.
<ol>
	<li>Remove the top plastic cover of the printer and carefully detach the control panel from the cover.</li>
	<li>Detach the 3&#160;cable connections between the printer top portion and base. Remove the printer top portion from the base.</li>
	<li>On the printer top portion, remove the small plastic and rubber accessories (printhead cleaning system) at the right hand side under the ink cartridge.</li>
	<li>Remove the base of the paper tray with springs.</li>
	<li>Remove the metallic plate covering the plastic paper feeding bar.</li>
	<li>Cut off the plastic paper feeding bar at the middle feeding wheel position using a hand saw or other cutting tool.</li>
	<li>Remove the 2&#160;paper feeding wheels exposed after the previous step. The wheel plastic is very hard and an electronic saw will be helpful.</li>
	<li>Clean the dust and debris using canned air and ethanol wipes.</li>
	<li>Attach the printer top portion to the base.</li>
	<li>UV sterilize the modified printer for at least 2 hr in a laminar flow hood before using.</li>
	<li>Cut off the cap of a HP 51626a ink cartridge using a hand saw or other cutting tool.</li>
	<li>Empty the ink and remove the filter that covers the bottom well reservoir of the cartilage.</li>
	<li>Rinse the cartridge thoroughly using running tap water.</li>
	<li>Ultrasonicate the cartridge in de-ionized (DI) water for 10 min to remove the residual ink.</li>
	<li>Examine the cartridge to make sure all the ink has been removed. Rinse or spray the cartridge thoroughly with 70% ethanol for sterilization, followed by sterilized DI water.</li>
	<li>Set up a long-wavelength ultraviolet lamp over the printing platform to provide simultaneous photopolymerization capacity.</li>
	<li>Measure UV intensity at the printing platform using a UV light meter. Adjust the distance between the UV lamp and the printer platform so the intensity at the printing subject is between 4-8 mW/cm2 (approximately 25 cm from lamp to the printer platform).</li>
</ol>
2. Bioink Preparation
<ol>
	<li>Monolayer chondrocyte expansion
	<ol>
		<li>Plate 5 million human chondrocytes into each T175 tissue culture flask for cell expansion in Dulbeccos Modified Eagles Medium (DMEM) supplemented with 10% calf serum and 1x penicillin-streptomycin-glutamine (PSG). Culture cells at 37 &#176;C with humidified air containing 5% CO2. Change the culture medium every 3 days until the flask is 85% confluence. Use cells from the same passage.</li>
	</ol>
	</li>
	<li>Dissolve PEGDA in PBS to a final concentration of 10% w/v. Add photoinitiator I-2959 to a final concentration of 0.05% w/v. Filter sterilize the solution.</li>
	<li>Suspend cultured human chondrocytes in the prepared PEGDA solution at 5 x 106 cells/ml.</li>
</ol>
3. Cartilage Tissue Printing
<ol>
	<li>Turn on the printer and laptop.</li>
	<li>Create a printing pattern of a solid circle with 4 mm in diameter using Microsoft Word or Adobe Photoshop.
	<ol>
		<li>Adjust the position of the pattern and make sure it will print exactly into the plastic mold.</li>
		<li>Calculate the number of prints needed to reach the desired thickness of scaffold. For 4 mm in height, 220 prints are required to create the desired scaffold.</li>
	</ol>
	</li>
	<li>Load the bioink into the ink cartridge. Cover the cartridge with aluminum foil to protect from the direct UV exposure during printing.</li>
	<li>Send printing command to the printer. Pull the paper sensor when the printer starts to print. The whole printing process should take less than 4 min for a scaffold with 4 mm in diameter and 4 mm in height.</li>
	<li>Transfer printed neocartilage to a 24-well plate and add 1.5 ml culture medium to each well.</li>
</ol>
4. Cell Viability Evaluation in 3D Scaffold
<ol>
	<li>Incubate the printed neocartilage in LIVE/DEAD Viability/Cytotoxicity working solution at room temperature for 15 min in dark.</li>
	<li>Cut the cell-laden hydrogel in half and take fluorescent images of the cutting area.</li>
	<li>Count live (green) and dead (red) cells by a blinded observer at five randomly taken images. Calculate cell viability by dividing the number of live cells by the total number of live and dead cells.</li>
</ol>

<ol>
	<li>Cui, X., Boland, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+microvasculature+fabrication+using+thermal+inkjet+printing+technology.">Human microvasculature fabrication using thermal inkjet printing technology.</a> Biomaterials. 30, 6221-6227 (2009).</li><li>Cui, X., Breitenkamp, K., Finn, M. G., Lotz, M., D'Lima, D. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+human+cartilage+repair+using+three-dimensional+bioprinting+technology.">Direct human cartilage repair using three-dimensional bioprinting technology.</a> Tissue Eng Part A. 18, 1304-1312 (2012).</li><li>Cui, X., Breitenkamp, K., Lotz, M., D'Lima, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synergistic+action+of+fibroblast+growth+factor-2+and+transforming+growth+factor-beta1+enhances+bioprinted+human+neocartilage+formation.">Synergistic action of fibroblast growth factor-2 and transforming growth factor-beta1 enhances bioprinted human neocartilage formation.</a> Biotechnol. Bioeng. 109, 2357-2368 (2012).</li><li>Cui, X., Breitenkamp, K., Finn, M. G., Lotz, M., Colwell, C. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Direct+human+cartilage+repair+using+thermal+inkjet+printing+technology.">Direct human cartilage repair using thermal inkjet printing technology.</a> Osteoarthritis and Cartilage. 19, (2011).</li><li>Cui, X., Boland, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Simultaneous+deposition+of+human+microvascular+endothelial+cells+and+biomaterials+for+human+microvasculature+fabrication+using+inkjet+printing.">Simultaneous deposition of human microvascular endothelial cells and biomaterials for human microvasculature fabrication using inkjet printing.</a> NIP24/digital Fabrication 2008: 24th International Conference on Digital Printing Technologies, Technical Program and Proceedings. 24, 480-483 (2008).</li><li>Cui, X., Dean, D., Ruggeri, Z. M., Boland, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+damage+evaluation+of+thermal+inkjet+printed+Chinese+hamster+ovary+cells.">Cell damage evaluation of thermal inkjet printed Chinese hamster ovary cells.</a> Biotechnol. Bioeng. 106, 963-969 (2010).</li><li>Cui, X., Hasegawa, A., Lotz, M., D'Lima, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structured+three-dimensional+co-culture+of+mesenchymal+stem+cells+with+meniscus+cells+promotes+meniscal+phenotype+without+hypertrophy.">Structured three-dimensional co-culture of mesenchymal stem cells with meniscus cells promotes meniscal phenotype without hypertrophy.</a> Biotechnol. Bioeng. 109, 2369-2380 (2012).</li><li>Cui, X., Gao, G., Qiu, Y. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accelerated+myotube+formation+using+bioprinting+technology+for+biosensor+applications.">Accelerated myotube formation using bioprinting technology for biosensor applications.</a> Biotechnol. Lett. 35, 315-321 (2013).</li><li>Cui, X., Boland, T., D'Lima, D. D., Lotz, M. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Thermal+inkjet+printing+in+tissue+engineering+and+regenerative+medicine.">Thermal inkjet printing in tissue engineering and regenerative medicine.</a> Recent Pat Drug Deliv Formul. 6, 149-155 (2012).</li><li>Bryant, S. J., Anseth, K. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrogel+properties+influence+ECM+production+by+chondrocytes+photoencapsulated+in+poly(ethylene+glycol)+hydrogels.">Hydrogel properties influence ECM production by chondrocytes photoencapsulated in poly(ethylene glycol) hydrogels.</a> Journal of Biomedical Materials Research. 59, 63-72 (2002).</li><li>Elisseeff, J., 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=Photoencapsulation+of+chondrocytes+in+poly(ethylene+oxide)-based+semi-interpenetrating+networks.">Photoencapsulation of chondrocytes in poly(ethylene oxide)-based semi-interpenetrating networks.</a> Journal of Biomedical Materials Research. 51, 164-171 (2000).</li><li>Buskirk, W. 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=Development+of+A+High-Resolution+Thermal+Inkjet+Printhead.">Development of A High-Resolution Thermal Inkjet Printhead.</a> Hewlett-Packard Journal. 39, 55-61 (1988).</li><li>Harmon, J. P., Widder, J. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Integrating+the+Printhead+Into+the+HP+Deskjet+Printer.">Integrating the Printhead Into the HP Deskjet Printer.</a> Hewlett-Packard Journal. 39, 62-66 (1988).</li><li>Kim, T. K., 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=Experimental+model+for+cartilage+tissue+engineering+to+regenerate+the+zonal+organization+of+articular+cartilage.">Experimental model for cartilage tissue engineering to regenerate the zonal organization of articular cartilage.</a> Osteoarthritis and Cartilage. 11, 653-664 (2003).</li><li>Sharma, B., 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=Designing+zonal+organization+into+tissue-engineered+cartilage.">Designing zonal organization into tissue-engineered cartilage.</a> Tissue Engineering. 13, 405-414 (2007).</li><li>Cohen, D. L., Lipton, J. I., Bonassar, L. J., Lipson, H. <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+for+in+situ+repair+of+osteochondral+defects.">Additive manufacturing for in situ repair of osteochondral defects.</a> Biofabrication. 2, (2010).</li></ol>

The authors have no financial interest in this study.

This 3D bioprinting system with simultaneous photopolymerization capacity provides a significantly greater printing resolution than the best previously reported method of in situ printing of osteochondral defects using syringe extruded a cellular alginate hydrogel16. Higher printing resolution is particularly critical for cartilage tissue engineering to restore the anatomic cartilage zonal organization. Simultaneous photopolymerization during layer-by-layer assembly is crucial to maintain precise depo...

Bioprinting based on thermal inkjet printing is one of the most promising enabling technologies in the field of tissue engineering and regenerative medicine. With digital control and high throughput printheads&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) positions rapidly. Many successful applications have been achieved using this technology in tissue engineering and regenerative medicine1-9. In this paper, a bioprinting platform was established with a modified Hewlett-Packard (HP) Deskjet 500 thermal inkjet printer and a simultaneous photopolymerization system. Synthetic hydrogels formulated from poly(ethylene glycol) (PEG) have shown the capacity of maintaining chondrocyte viability and promote chondrogenic ECM production10,11. In addition, photocrosslinkable PEG is highly soluble in water with low viscosity, which makes it ideal for simultaneous polymerization during 3D bioprinting. In this paper, human chondrocytes suspended in poly(ethylene) glycol diacrylate (PEGDA; MW 3,400) were precisely printed to construct neocartilage layer-by-layer with 1,400 dpi in 3D resolution. Homogeneous distribution of deposited cells in a 3D scaffold was observed, which generated cartilage tissue with excellent mechanical properties and enhanced ECM production. By contrast, in manual fabrication the cells accumulated at the bottom of the gel instead of their initially deposited positions due to slower scaffold polymerization, which led to inhomogeneous cartilage formation after culture2,3.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">HP Deskjet 500 thermal inkjet printer</td>
			<td style="width:221px;">Hewlett-Packard</td>
			<td style="width:195px;">C2106a</td>
			<td style="width:433px;">Discontinued. Purchased refurbished from internet vendor.</td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">HP black ink cartridge</td>
			<td style="width:221px;">Hewlett-Packard</td>
			<td style="width:195px;">51626a</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">Ultraviolet lamp</td>
			<td style="width:221px;">UVP</td>
			<td style="width:195px;">B-100AP</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">UV light meter</td>
			<td style="width:221px;">General Tools</td>
			<td style="width:195px;">UV513AB</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">Zeiss LSM 510 laser scanning microscope</td>
			<td style="width:221px;">Carl Zeiss</td>
			<td style="width:195px;">LSM 510</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">Dulbeccos Modified Eagles Medium (DMEM)</td>
			<td style="width:221px;">Mediatech</td>
			<td style="width:195px;">10-013</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">Penicillin-streptomycin-glutamine (PSG)</td>
			<td style="width:221px;">Invitrogen</td>
			<td style="width:195px;">10378-016</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">Accutase cell dissociation reagent</td>
			<td style="width:221px;">Invitrogen</td>
			<td style="width:195px;">A11105-01</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">Phosphate buffered saline (PBS)</td>
			<td style="width:221px;">Invitrogen</td>
			<td style="width:195px;">10010-023</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">Live/Dead viability/cytotoxicity Kit</td>
			<td style="width:221px;">Invitrogen</td>
			<td style="width:195px;">L-3224</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">Poly(ethylene glycol) diacrylate (PEGDA)</td>
			<td style="width:221px;">Glycosan Biosystems</td>
			<td style="width:195px;">GS700</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">Irgacure 2959</td>
			<td style="width:221px;">Ciba Specialty Chemicals</td>
			<td style="width:195px;">I-2959</td>
			<td style="width:433px;"></td>
		</tr>
		<tr height="22">
			<td height="22" style="height:22px;width:357px;">Human articular chondrocytes</td>
			<td style="width:221px;">Lonza</td>
			<td style="width:195px;">CC-2550</td>
			<td style="width:433px;"></td>
		</tr>
	</tbody>
</table>

human cartilage tissue fabrication using three-dimensional inkjet printing technology

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and regenerative medicine. With digital control&#160;cells, scaffolds, and growth factors can be precisely deposited to the desired two-dimensional (2D) and three-dimensional (3D) locations rapidly. Therefore, this technology is an ideal approach to fabricate tissues mimicking their native anatomic structures. In order to engineer cartilage with native zonal organization, extracellular matrix composition (ECM), and mechanical properties, we developed a bioprinting platform using a commercial inkjet printer with simultaneous photopolymerization capable for 3D cartilage tissue engineering. Human chondrocytes suspended in poly(ethylene glycol) diacrylate (PEGDA) were printed for 3D neocartilage construction via layer-by-layer assembly. The printed cells were fixed at their original deposited positions, supported by the surrounding scaffold in simultaneous photopolymerization. The mechanical properties of the printed tissue were similar to the native cartilage. Compared to conventional tissue fabrication, which requires longer UV exposure, the viability of the printed cells with simultaneous photopolymerization was significantly higher. Printed neocartilage demonstrated excellent glycosaminoglycan (GAG) and collagen type II production, which was consistent with gene expression. Therefore, this platform is ideal for accurate cell distribution and arrangement for anatomic tissue engineering.

The modified thermal inkjet printer was capable for cell and scaffold deposition at high throughput and excellent cell viability. Combining with simultaneous photopolymerization and photosensitive biomaterials, this technology is able to fix the cells and other printed substances to the initially deposited locations. According to the properties of the modified thermal inkjet printer, the 2D printing resolution was 300 dpi with a single ink drop volume of 130 pl. There are 50 firing nozzles in each printhead with 3.6 kHz ...

Watch this Scientific Journal Video about Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology at JoVE.com

Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

The methods described in this paper show how to convert a commercial inkjet printer into a bioprinter with simultaneous UV polymerization. The printer is capable of constructing 3D tissue structure with cells and biomaterials. The study demonstrated here constructed a 3D neocartilage.

Bioprinting, which is based on thermal inkjet printing, is one of the most attractive enabling technologies in the field of tissue engineering and ...

human-cartilage-tissue-fabrication-using-three-dimensional-inkjet

Research

JoVE Journal

Bioengineering

15.7K Views.  Rensselaer Polytechnic Institute.  I metodi descritti in questo documento mostrano come convertire una stampante a getto d'inchiostro di commercio in un bioprinter con polimerizzazione UV simultanea. La stampante è in grado di costruire la struttura del tessuto 3D con cellule e biomateriali. Lo studio ha dimostrato qui costruito un neocartilage 3D. 

Video: Human Cartilage Tissue Fabrication Using Three-dimensional Inkjet Printing Technology

Planar and Three-Dimensional Printing of Conductive Inks

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical devices1-3. In this paper, we focus on one- (1D), two- (2D), and three-dimensional (3D) printing of conductive metallic inks in the form of flexible, stretchable, and spanning microelectrodes.
Direct-write assembly4,5 is a 1-to-3D printing technique that enables the fabrication of features ranging from simple lines to complex structures by the deposition of concentrated inks through fine nozzles (~0.1 - 250 &mu;m). This printing method consists of a computer-controlled 3-axis translation stage, an ink reservoir and nozzle, and 10x telescopic lens for visualization. Unlike inkjet printing, a droplet-based process, direct-write assembly involves the extrusion of ink filaments either in- or out-of-plane. The printed filaments typically conform to the nozzle size. Hence, microscale features (&lt; 1 &mu;m) can be patterned and assembled into larger arrays and multidimensional architectures.
In this paper, we first synthesize a highly concentrated silver nanoparticle ink for planar and 3D printing via direct-write assembly. Next, a standard protocol for printing microelectrodes in multidimensional motifs is demonstrated. Finally, applications of printed microelectrodes for electrically small antennas, solar cells, and light-emitting diodes are highlighted.

Planar and three-dimensional printing of conductive metallic inks is described. Our approach provides new avenues for fabricating printed electronic, optoelectronic, and biomedical devices in unusual layouts at the microscale.

Printed electronics rely on low-cost, large-area fabrication routes to create flexible or multidimensional electronic, optoelectronic, and biomedical ...

Universal Hand-held Three-dimensional Optoacoustic Imaging Probe for Deep Tissue Human Angiography and Functional Preclinical Studies in Real Time

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously attaining anatomical, functional and molecular contrast in deep optically opaque tissues. While enormous potential has been recently demonstrated in the application of optoacoustics for small animal research, vast efforts have also been undertaken in translating this imaging technology into clinical practice. We present here a newly developed optoacoustic tomography approach capable of delivering high resolution and spectrally enriched volumetric images of tissue morphology and function in real time. A detailed description of the experimental protocol for operating with the imaging system in both hand-held and stationary modes is provided and showcased for different potential scenarios involving functional and molecular studies in murine models and humans. The possibility for real time visualization in three dimensions along with the versatile handheld design of the imaging probe make the newly developed approach unique among the pantheon of imaging modalities used in today&#8217;s preclinical research and clinical practice.

We provide herein a detailed description of the experimental protocol for imaging with a newly developed hand-held optoacoustic (photoacoustic) system for three-dimensional functional and molecular imaging in real time. The demonstrated powerful performance and versatility may define new application areas of the optoacoustic technology in preclinical research and clinical practice.

The exclusive combination of high optical contrast and excellent spatial resolution makes optoacoustics (photoacoustics) ideal for simultaneously ...

Generation of a Three-dimensional Full Thickness Skin Equivalent and Automated Wounding

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models reflect the human wound situation better than animal models. Although two-dimensional models are widely used to investigate processes such as cellular migration and proliferation, models that are more complex are required to gain a deeper knowledge about wound healing. Besides a suitable model system, the generation of precise and reproducible wounds is crucial to ensure comparable results between different test runs. In this study, the generation of a three-dimensional full thickness skin equivalent to study wound healing is shown. The dermal part of the models is comprised of human dermal fibroblast embedded in a rat-tail collagen type I hydrogel. Following the inoculation with human epidermal keratinocytes and consequent culture at the air-liquid interface, a multilayered epidermis is formed on top of the models. To study the wound healing process, we additionally developed an automated wounding device, which generates standardized wounds in a sterile atmosphere.

The goal of this protocol is to build up a three-dimensional full thickness skin equivalent, which resembles natural skin. With a specifically constructed automated wounding device, precise and reproducible wounds can be generated under maintenance of sterility.

In vitro models are a cost effective and ethical alternative to study cutaneous wound healing processes. Moreover, by using human cells, these models ...

Three-dimensional Biomimetic Technology: Novel Biorubber Creates Defined Micro- and Macro-scale Architectures in Collagen Hydrogels

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper composition, targeted modulus, and well-defined architectural features. Biomaterials that recapitulate the intrinsic architecture of in vivo tissue are vital for studying diseases as well as to facilitate the regeneration of lost and malformed soft tissue. A novel biofabrication technique was developed which combines state of the art imaging, three-dimensional (3D) printing, and selective enzymatic activity to create a new generation of biomaterials for research and clinical application. The developed material, Bovine Serum Albumin rubber, is reaction injected into a mold that upholds specific geometrical features. This sacrificial material allows the adequate transfer of architectural features to a natural scaffold material. The prototype consists of a 3D collagen scaffold with 4 and 3 mm channels that represent a branched architecture. This paper emphasizes the use of this biofabrication technique for the generation of natural constructs. This protocol utilizes a computer-aided software (CAD) to manufacture a solid mold which will be reaction injected with BSA rubber followed by the enzymatic digestion of the rubber, leaving its architectural features within the scaffold material.

An innovative biofabrication technique was developed to engineer three-dimensional constructs that resemble the architectural features, components, and mechanical properties of in vivo tissue. This technique features a newly developed sacrificial material, BSA rubber, which transfers detailed spatial features, reproducing the in vivo architectures of a wide variety of tissues.

Tissue scaffolds play a crucial role in the tissue regeneration process. The ideal scaffold must fulfill several requirements such as having proper ...

Fabrication of Inverted Colloidal Crystal Poly(ethylene glycol) Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing drugs targeted at the liver, requires a platform in which cells receive proper biochemical and mechanical cues. Recent liver tissue engineering systems have employed three-dimensional (3D) scaffolds composed of synthetic or natural hydrogels, given their high water retention and their ability to provide the mechanical stimuli needed by the cells. There has been growing interest in the inverted colloidal crystal (ICC) scaffold, a recent development, which allows high spatial organization, homotypic and heterotypic cell interaction, as well as cell-extracellular matrix (ECM) interaction. Herein, we describe a protocol to fabricate the ICC scaffold using poly (ethylene glycol) diacrylate (PEGDA) and the particle leaching method. Briefly, a lattice is made from microsphere particles, after which a pre-polymer solution is added, properly polymerized, and the particles are then removed, or leached, using an organic solvent (e.g., tetrahydrofuran). The dissolution of the lattice results in a highly porous scaffold with controlled pore sizes and interconnectivities that allow media to reach cells more easily. This unique structure allows high surface area for the cells to adhere to as well as easy communication between pores, and the ability to coat the PEGDA ICC scaffold with proteins also shows a marked effect on cell performance. We analyze the morphology of the scaffold as well as the hepatocarcinoma cell (Huh-7.5) behavior in terms of viability and function to explore the effect of ICC structure and ECM coatings. Overall, this paper provides a detailed protocol of an emerging scaffold that has wide applications in tissue engineering, especially liver tissue engineering.

Fabrication of Inverted Colloidal Crystal Polyethylene glycol Scaffold: A Three-dimensional Cell Culture Platform for Liver Tissue Engineering

This manuscript presents a detailed protocol for the fabrication of an emerging three-dimensional hepatocyte culture platform, the inverted colloidal crystal scaffold, and the concomitant techniques to assess hepatocyte behavior. The size-controllable pores, interconnectivity and ability to conjugate extracellular matrix proteins to the poly(ethylene glycol) (PEG) scaffold enhance Huh-7.5 cell performance.

The ability to maintain hepatocyte function in vitro, for the purpose of testing xenobiotics&#39; cytotoxicity, studying virus infection and developing ...

Fabrication of Three-dimensional Paper-based Microfluidic Devices for Immunoassays

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and directed within a layer of paper. Moreover, stacking multiple layers of patterned paper creates sophisticated three-dimensional microfluidic networks that can support the development of analytical and bioanalytical assays. Paper-based microfluidic devices are inexpensive, portable, easy to use, and require no external equipment to operate. As a result, they hold great promise as a platform for point-of-care diagnostics. In order to properly evaluate the utility and analytical performance of paper-based devices, suitable methods must be developed to ensure their manufacture is reproducible and at a scale that is appropriate for laboratory settings. In this manuscript, a method to fabricate a general device architecture that can be used for paper-based immunoassays is described. We use a form of additive manufacturing (multi-layer lamination) to prepare devices that comprise multiple layers of patterned paper and patterned adhesive. In addition to demonstrating the proper use of these three-dimensional paper-based microfluidic devices with an immunoassay for human chorionic gonadotropin (hCG), errors in the manufacturing process that may result in device failures are discussed. We expect this approach to manufacturing paper-based devices will find broad utility in the development of analytical applications designed specifically for limited-resource settings.

We detail a method to fabricate three-dimensional paper-based microfluidic devices for use in the development of immunoassays. Our approach to device assembly is a type of multilayer, additive manufacturing. We demonstrate a sandwich immunoassay to provide representative results for these types of paper-based devices.

Paper wicks fluids autonomously due to capillary action. By patterning paper with hydrophobic barriers, the transport of fluids can be controlled and ...

Melt Electrospinning Writing of Three-dimensional Poly(&#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology with great potential for biomedical applications. The technique facilitates the direct deposition of biocompatible polymer fibers to fabricate well-ordered scaffolds in the sub-micron to micro scale range. The establishment of a stable, viscoelastic, polymer jet between a spinneret and a collector is achieved using an applied voltage and can be direct-written. A significant benefit of a typical porous scaffold is a high surface-to-volume ratio which provides increased effective adhesion sites for cell attachment and growth. Controlling the printing process by fine-tuning the system parameters enables high reproducibility in the quality of the printed scaffolds. It also provides a flexible manufacturing platform for users to tailor the morphological structures of the scaffolds to their specific requirements. For this purpose, we present a protocol to obtain different fiber diameters using melt electrospinning writing (MEW) with a guided amendment of the parameters, including flow rate, voltage and collection speed. Furthermore, we demonstrate how to optimize the jet, discuss often experienced technical challenges, explain troubleshooting techniques and showcase a wide range of printable scaffold architectures.

Melt Electrospinning Writing of Three-dimensional Poly(&amp;#949;-caprolactone) Scaffolds with Controllable Morphologies for Tissue Engineering Applications

This protocol serves as a comprehensive guideline to fabricate scaffolds via electrospinning with polymer melts in a direct writing mode. We systematically outline the process and define the appropriate parameter settings for achieving targeted scaffold architectures.

This tutorial reflects on the fundamental principles and guidelines for electrospinning writing with polymer melts, an additive manufacturing technology ...

Culturing Mammalian Cells in Three-dimensional Peptide Scaffolds

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an environment that closely mimics the structural features of non-polarized tissue. Furthermore, the particular intrinsic nanofiber structure of the scaffold makes it transparent to visual light, which allows for easy visualization of the sample under microscopy. This advantage was largely used to study cell migration, organization, proliferation, and differentiation and thus any development of their particular cellular function by staining with specific dyes or probes. Furthermore, in this work, we describe the good performance of this system to easily study the redifferentiation of expanded human articular chondrocytes into cartilaginous tissue. Cells were encapsulated into self-assembling peptide scaffolds and cultured under specific conditions to promote chondrogenesis. Three-dimensional cultures showed good viability during the 4 weeks of the experiment. As expected, samples cultured with chondrogenic inducers (compared to non-induced controls) stained strongly positive for toluidine blue (which stains glycosaminoglycans (GAGs) that are highly present in cartilage extracellular matrix) and expressed specific molecular markers, including collagen type I, II and X, according to Western Blot analysis. This protocol is easy to perform and can be used at research laboratories, industries and for educational purposes in laboratory courses.

Here, we present a protocol to obtain 3D-culture systems in self-assembling peptide scaffolds to promote the differentiation of dedifferentiated human articular chondrocytes into cartilage-like tissue.

A useful technique for culturing cells in a self-assembling nanofiber three-dimensional (3D) scaffold is described. This culture system recreates an ...

Three-dimensional Printing of Thermoplastic Materials to Create Automated Syringe Pumps with Feedback Control for Microfluidic Applications

Microfluidics has become a critical tool in research across the biological, chemical, and physical sciences. One important component of microfluidic experimentation is a stable fluid handling system capable of accurately providing an inlet flow rate or inlet pressure. Here, we have developed a syringe pump system capable of controlling and regulating the inlet fluid pressure delivered to a microfluidic device. This system was designed using low-cost materials and additive manufacturing principles, leveraging three-dimensional (3D) printing of thermoplastic materials and off-the-shelf components whenever possible. This system is composed of three main components: a syringe pump, a pressure transducer, and a programmable microcontroller. Within this paper, we detail a set of protocols for fabricating, assembling, and programming this syringe pump system. Furthermore, we have included representative results that demonstrate high-fidelity, feedback control of inlet pressure using this system. We expect this protocol will allow researchers to fabricate low-cost syringe pump systems, lowering the entry barrier for the use of microfluidics in biomedical, chemical, and materials research.

Here we present a protocol to construct a pressure-controlled syringe pump to be used in microfluidic applications. This syringe pump is made from an&#160;additively manufactured body, off-the-shelf hardware, and open-source electronics. The resulting system is low-cost, straightforward to build, and delivers well-regulated fluid flow to enable rapid microfluidic research.

Microfluidics has become a critical tool in research across the biological, chemical, and physical sciences. One important component of microfluidic ...

Fabrication of Decellularized Cartilage-derived Matrix Scaffolds

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage research has focused on the development of regenerative scaffolds. This article describes the development of scaffolds that are completely derived from natural cartilage extracellular matrix, coming from an equine donor. Potential applications of the scaffolds include producing allografts for cartilage repair, serving as a scaffold for osteochondral tissue engineering, and providing in vitro models to study tissue formation. By decellularizing the tissue, the donor cells are removed, but many of the natural bioactive cues are thought to be retained. The main advantage of using such a natural scaffold in comparison to a synthetically produced scaffold is that no further functionalization of polymers is required to drive osteochondral tissue regeneration. The cartilage-derived matrix scaffolds can be used for bone and cartilage tissue regeneration in both in vivo and in vitro settings.

Decellularized cartilage-derived scaffolds can be used as a scaffold to guide cartilage repair and as a means to regenerate osteochondral tissue. This paper describes the decellularization process in detail and provides suggestions to use these scaffolds in in vitro settings.

Osteochondral defects lack sufficient intrinsic repair capacity to regenerate functionally sound bone and cartilage tissue. To this extent, cartilage ...