This study was supported by the Ministry of National Defense (MAB-105-070; MAB-106-077; MAB-107-032; MAB-107-065), the Ministry of Science and Technology (MOST 107-2320-B016-016), Tri-Service General Hospital, the National Defense Medical Center, Taiwan (TSGH-C106-046; TSGH-C106-115; TSGH-C107-041), and Cheng-Hsin General Hospital and National Defense Medical Center Cooperation (CH-NDMC-107-8).

The fat tissues were obtained from orthopedic surgeries as certified by the Institutional Review Board of Tri-Service General Hospital, Taipei, Taiwan, R.O.C. Procedures involving animal subjects have been approved by the Animal Care Committee at National Defense Medical Center, Taiwan (R.O.C).
1. Wet Spinning Process
<ol>
	<li>Solution preparation
	<ol>
		<li>Dissolve 5 g of gelatin powder in 100 mL of double-distilled water to obtain 5% (w/v) solution concentration.</li>
		<li>Stir the mixture slowly at 60 - 70 &#176;C overnight to achieve a completely homogeneous dispersion without any bubbles.</li>
	</ol>
	</li>
	<li>Wet spinning
	<ol>
		<li>Fiber formation 
		NOTE: A schematic of the method is shown in Figure 1A. The spinning setup is equipped with a peristaltic pump machine (see Table of Materials) that provides high-precision speed and controls the smooth delivery of the flow.
		<ol>
			<li>Plug a 26 G x 1/2 inch (0.45 mm x 12 mm) syringe as the solution injector into clear vinyl tubing.</li>
			<li>Prepare 100 mL of 99.5% acetone solution in a beaker glass to be used as the coagulation bath.</li>
			<li>Run the peristaltic pump machine at 21 rpm (3.25 mL/min) and let the gelatin solution stand for several seconds in acetone solution before rolling it up.</li>
			<li>Take out the gelatin fibers from the acetone solution and immerse them into 2.5% (w/v) of polycaprolactone/dichloromethane (PCL/DCM) solution with 1:20 (w/w).</li>
			<li>Let the gelatin fibers in the PCL/DCM solution dry overnight, under the hood at room temperature.</li>
		</ol>
		</li>
		<li>Tube formation 
		NOTE: A schematic of the method is shown in Figure 1B.
		<ol>
			<li>Use a peripheral venous catheter of 24 G x 3/4 inch (0.7 mm x 19 mm) as tube mold.</li>
			<li>Load the catheter into the gelatin solution and hold it there for 3 s.</li>
			<li>Load the catheter into the acetone solution and hold for 1 min.</li>
			<li>Load the catheter again into the gelatin solution and hold it there for 3 s.</li>
			<li>Repeat this alternate procedure 20x.</li>
			<li>Take out the catheter from the acetone solution and let the molded tube dry at room temperature for 5 min.</li>
			<li>Gently take out the gelatin tube from the catheter by using a hemostat and carefully transfer the gelatin tube into a glass Pasteur pipette with the immersion of 2.5% (w/v) of the PCL/DCM solution.</li>
			<li>Let the Pasteur pipette containing the gelatin tube and PCL/DCM solution dry overnight, under the hood at room temperature.</li>
		</ol>
		</li>
	</ol>
	</li>
</ol>
2. Morphology of the Gelatin Tube
<ol>
	<li>Mount the piece of dried gelatin tube on a carbon stub.</li>
	<li>Put the sample into the ion sputter coater machine (see Table of Materials) and coat the sample with gold for 60 s; then, observe its morphology by scanning electron microscopy(see Table of Materials).</li>
</ol>
3. Culture of Human Adipose Stem Cells
<ol>
	<li>Place the fat tissues (obtained from oral adipose tissue by clinical surgery) into a Petri dish containing 10 mL of transfer solution consisting of 0.1 M phosphate-buffered saline (PBS), 1% penicillin/streptomycin, and 0.1% glucose.</li>
	<li>Cut the tissues into small pieces (less than 1 mm2) with a scalpel blade.</li>
	<li>Transfer the tissues into a 15 mL plastic tube and centrifuge at 500 x g for 5 min.</li>
	<li>Remove the supernatant and incubate the sediment in a plastic tube containing 10 mL of Dulbecco&#39;s modified Eagle&#39;s medium (DMEM, consisting of 4 mM L-glutamine, 1 mM sodium pyruvate, and 15 mg/L phenol red) with 0.1% collagenase at 37 &#176;C, in a humidified atmosphere containing 95% air and 5% CO2, for 1 day.</li>
	<li>Centrifuge the plastic tube at 500 x g for 5 min, discard the supernatant carefully (do not touch the sediment), and then, suspend the sediment gently by adding 10 mL of DMEM containing 10% fetal bovine serum (FBS) and let it stand for 1 day at 37 &#176;C, in a humidified atmosphere containing 95% air and 5% CO2.</li>
	<li>Centrifuge the plastic tube at 500 x g for 5 min and discard the supernatant carefully; then, add 1 mL of stem cell medium (consisting of keratinocyte serum-free medium (K-SFM), 5% of FBS, N-acetyl-L-cysteine, ascorbic acid-2-phosphate, streptomycin, and amphotericin) to suspend the sediment, transfer it into a T25 culture flask containing 4 mL of stem cell medium, and let it stand for 3 days at 37 &#176;C, in a humidified atmosphere containing 95% air and 5% CO2.</li>
	<li>Discard the supernatant, add 1 mL of 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA), and incubate it at 37 &#176;C, in a humidified atmosphere containing 95% air and 5% CO2 for 3.5 min, to detach the cells from the bottom of the culture flask.</li>
	<li>Add 1 mL of FBS, suspend the mixture, and transfer it to a microcentrifuge tube.</li>
	<li>Centrifuge the suspension at 500 x g for 3.5 min, suspend the sediment with 1 mL of stem cell medium, and transfer it to the culture flask containing 5 mL of stem cell medium; then, keep it at 37 &#176;C in a humidified atmosphere containing 95% air and 5% CO2.</li>
	<li>Maintain the subculture by changing the stem cell medium every 3 days.</li>
</ol>
4. Cultivation of Cells on the Gelatin Tube
<ol>
	<li>Sterilize the gelatin tube with UV light for 2 h; then, immerse it in 75% (v/v) ethanol and wash it 2x with stem cell medium to remove the residual ethanol.</li>
	<li>Collect the human adipose stem cells (hASCs) from the culture flask.
	<ol>
		<li>Remove the medium from the culture flask.</li>
		<li>Add 1 mL of 0.25% trypsin-EDTA and incubate at 37 &#176;C in a humidified atmosphere containing 95% air and 5% CO2 for 3.5 min, to detach hASCs from the bottom of the culture flask.</li>
		<li>Add 1 mL of FBS, suspend the mixture, and transfer it to a microcentrifuge tube.</li>
		<li>Centrifuge the microcentrifuge tube at 500 x g for 3.5 min; then, discard the supernatant carefully, and suspend the sediment with 1 mL of stem cell medium.</li>
	</ol>
	</li>
	<li>Place the gelatin tube in a 6-well plate containing 3 mL of stem cell medium and seed 4 x 104 of hASCs to the tube; incubate them for 2 weeks at 37 &#176;C, in a humidified atmosphere containing 95% air and 5% CO2.</li>
	<li>Change the stem cell medium every 3 days to provide enough nutrition for cell growth.</li>
</ol>
5. Immunocytochemistry
<ol>
	<li>Remove the stem cell medium and wash the well with PBS.</li>
	<li>Fix the tube with cells with 0.1% acetic acid glacial for 30 min at room temperature.</li>
	<li>Wash the well 2x with PBS.</li>
	<li>Add a surfactant (NP-40, 0.05%) and incubate for 10 min at room temperature.</li>
	<li>Wash the well 3x with PBS.</li>
	<li>Add 2% normal goat serum and incubate at room temperature for 30 min.</li>
	<li>Decant the solution, add primary antibody nestin (neural progenitor cell marker), and incubate overnight at 4 &#176;C.</li>
	<li>Wash the well 3x with PBS.</li>
	<li>Add secondary antibody donkey anti-mouse-fluorescein isothiocyanate (FITC) and keep the sample in the dark for 2 h.</li>
	<li>Wash the well 3x with PBS.</li>
	<li>Add 1 mL of Hoechst 33342 to stain the nuclei and incubate for 10 min at room temperature.</li>
	<li>Wash the well gently and observe it under a fluorescence microscope.</li>
</ol>
6. In Vivo Biocompatibility Test
NOTE: Rats with a weight between 201 - 225 g have been successfully tested using this protocol.
<ol>
	<li>Anesthesia
	<ol>
		<li>Induce and maintain anesthesia; preferably, anesthetize a rat (8-week-old Sprague Dawley rat, female) via an intramuscular injection of tiletamine and zolazepam (25 mg/kg + 25 mg/kg, respectively) and xylazine (5 mg/kg). Perform a pain stimulation test by pressing the rat&#39;s fingers to confirm the anesthetization.</li>
	</ol>
	</li>
	<li>Sterilization of the surgical site
	<ol>
		<li>Shave and clean the skin of rat&#8217;s trapezius area (over the back of the neck) and wipe it with povidone-iodine lotion to prepare the aseptic condition of skin (100 mg/mL).</li>
		<li>Cover the rat with sterile surgical drapes to reduce bacterial transfer and subsequent contamination of the surgical site.</li>
	</ol>
	</li>
	<li>Implantation of the gelatin tube
	<ol>
		<li>Gently cut the skin of rat&#8217;s trapezius area with a surgical scissor.</li>
		<li>Create a wound of 2 cm and place the gelatin tube directly on the layer between the fascia and the muscle.</li>
	</ol>
	</li>
	<li>Postimplantation surgery
	<ol>
		<li>Close the wound with nylon suture and wipe it with povidone-iodine solution (100 mg/mL).</li>
		<li>Induce the rat via an intramuscular injection of ketoprofen (2.5 mg/kg) and cefazolin (15 mg/kg).</li>
		<li>Keep the rat under aseptic conditions for 7 days.</li>
	</ol>
	</li>
	<li>Euthanasia
	<ol>
		<li>Rat is euthanized via CO2 asphyxiation in accordance with AVMA Guidelines for Euthanasia. Confirm the death of rat by toe and tail pinch, followed by chest touching to ensure the heartbeat.</li>
		<li>Cut the rat gently with a scalpel blade, remove the implanted tissue, and take photos for observation.</li>
	</ol>
	</li>
</ol>

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The authors have nothing to disclose.

We presented the development of gelatin-based biomaterials by using a simple wet spinning technique that can be applied in the study of natural polymers for tissue regeneration. This work demonstrated the possibility of gelatin fabrication as a great protein source without the addition of other sources, with the aim to optimize the properties of gelatin itself. The development of gelatin-based biomaterials was entirely carried out in room temperature (22 - 26 &#176;C). A gentle solution preparation is a critical step wit...

The latest development in tissue regeneration involves the application of tissue engineering, which represents a challenge for the improvement of new therapeutic strategies in medical treatments. For example, the limited potential of nervous system regeneration, following injury or disease, poses a significant health problem worldwide. Due to the complexity of pathophysiological processes associated with the nervous system, the use of traditional autograft or the implementation of stabilization surgery has been shown to offer benefits in functional outcomes, but there is no strong evidence for the effects of spinal fixation surgery1,2. The tissue at the damaged area is lost and replaced with hypertrophically induced astrocytes3, eventually forming a dense glial scar4,5. This matrix acts as a barrier that blocks the recovery of nerve function6,7 and is, thus, greatly hinders regeneration. Therefore, a suitable filling gap material is expected to prevent the loss of tissue and reduce the formation of scar-associated connective tissue by maintaining the integrity of the damaged area, as well as by providing the direct replacement of neural cells and circuitry to promote axon regeneration.
Polymeric biomaterials have been preferred as scaffolds for tissue regeneration therapy, based on the regulation of cell or axon behavior and tissue progression through natural extracellular matrix (ECM) support. The fiber format is commonly considered as a building block for various materials, owing to its one-dimensional structure8. The fibers can generally be obtained by melt extrusion or wet spinning method; however, the large size and cost of the equipment and the difficulty to perform these methods are challenging. In addition, the majority of the work related to polymer fibers has been focused on synthetic or composite materials. Natural polymers as a source of biomaterial offer better biocompatibility properties for the human body. Nonetheless, to obtain the alignment of natural polymer fibers is relatively more difficult than of synthetic polymer sources9. Hence, the conversion of a natural polymer as a rich source of protein into biomaterial fibers is an important strategy &#8212; not only can the biomaterial fibers be directly isolated from the raw material, thus avoiding an unnecessary transformation to monomers, but the protein fibers also have a good appearance and favorable characteristics10.
In this regard, we describe an inexpensive processing method for the manufacturing of natural polymer fibers through the basic concept of wet spinning, that can be implemented on the laboratory scale for tissue engineering. Wet spinning is performed by the extrusion and coagulation of a polymer solution into a suitable polymer nonsolvent. An appropriate, viscous solution doped into coagulation medium causes the polymer molecules to dissolve. Through the phase transition, the filaments then lose their solubility and are precipitated in the form of a solid polymer phase11. Referring to this concept, we then expanded the development of gelatin into the tube form by a molding process, which is considered proper for tissue regeneration application. In addition, intrinsically, we can also develop any shape of material from gelatin fibers (e.g., gelatin conduit rolled up from several gelatin fibers), for other desired applications.
Gelatin, a biodegradable natural polymer, is formed from denatured and hydrolyzed collagen, including any semicrystalline, amorphous, or triple helical state of collagen12. It is well known that collagen is the essential structural protein in all connective tissues of vertebrates and invertebrates13,14, which is similar to the protein structure of the main ECM that induces nerve growth and, simultaneously, replaces a large amount of glycosaminoglycan secreted during spinal cord injuries. Therefore, the use of gelatin as a source would be a great choice for any medical vehicle. Besides being an inexpensive source, gelatin is also biodegradable and cytocompatible and clinically proven to be a temporary defect filler15. Developed into a tube form, in vitro and in vivo tests described here demonstrate that gelatin has an excellent biocompatibility and suitability for future tissue engineering applications. Cultured with human adipose stem cells, gelatin tubes improve cell differentiation into neural progenitor cells by using positive nestin staining as a neural cell marker. Furthermore, gelatin as filling gap material, as produced by the method established in this study, is expected to be manageable and safe and to greatly benefit tissue engineers who are currently developing correlative natural polymers for the enhancement of tissue regeneration strategies.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td colspan="4">Solution preparation:</td>
		</tr>
		<tr>
			<td>Gelatin type B (porcine)</td>
			<td>Ferak</td>
			<td>Art. -Nr. 10733</td>
			<td>500 g vial</td>
		</tr>
		<tr>
			<td>Wet spinning process:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Peristaltic pump</td>
			<td>Gilson</td>
			<td>Model M312</td>
			<td>Minipuls*3</td>
		</tr>
		<tr>
			<td>Plastic tube connector</td>
			<td>World Precision Instruments</td>
			<td>14011</td>
			<td>1 box</td>
		</tr>
		<tr>
			<td>Syringe</td>
			<td>Sterican</td>
			<td>5A06258541</td>
			<td>26Gx1/2&#34;(0.45 x 12mm)</td>
		</tr>
		<tr>
			<td>Acetone</td>
			<td>Ferak</td>
			<td>Art. -Nr. 00010</td>
			<td>2.5 L vial</td>
		</tr>
		<tr>
			<td>Polycaprolactone CAPA 6500</td>
			<td>Perstorp</td>
			<td>24980-41-4</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Dichloromethane&#160;</td>
			<td>Scharlau</td>
			<td>CL03421000</td>
			<td>1 L vial</td>
		</tr>
		<tr>
			<td>Glass Pasteur pipette</td>
			<td>Fisher Scientific</td>
			<td>13-678-20A</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Hemostat</td>
			<td>Shinetec instruments</td>
			<td>ST-B021</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Peripheral venous catheter (Introcan Certo)</td>
			<td>B. Braun</td>
			<td>1B03258241</td>
			<td>24Gx3/4&#34;(0.7 x 19mm)</td>
		</tr>
		<tr>
			<td colspan="4">Morphology of the gelatin tube:</td>
		</tr>
		<tr>
			<td>Ion sputter coater machine&#160;</td>
			<td>Hitachi</td>
			<td>e1010</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Scanning electron microscopy</td>
			<td>Hitachi</td>
			<td>S-3000N</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Cultivation of cells on the gelatin tube:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Trypsin-EDTA</td>
			<td>Gibco</td>
			<td>488625</td>
			<td>100 mL vial</td>
		</tr>
		<tr>
			<td>Fetal bovine serum</td>
			<td>Gibco</td>
			<td>923119</td>
			<td>500 mL vial</td>
		</tr>
		<tr>
			<td>Dulbecco&#39;s modified Eagle&#39;s medium&#160;</td>
			<td>Gibco</td>
			<td>31600-034</td>
			<td>Powder</td>
		</tr>
		<tr>
			<td>Keratinocyte-SFM medium</td>
			<td>Gibco</td>
			<td>10744-019</td>
			<td>500 mL vial</td>
		</tr>
		<tr>
			<td>T25 culture flask</td>
			<td>TPP</td>
			<td>90025</td>
			<td>VENT type</td>
		</tr>
		<tr>
			<td>6-well plate</td>
			<td>Falcon</td>
			<td>1209938</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Immunocytochemistry:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Phospate-buffered saline</td>
			<td>Gibco</td>
			<td>654471</td>
			<td>500 mL vial</td>
		</tr>
		<tr>
			<td>Acetic acid glacial</td>
			<td>Ferak</td>
			<td>Art. -Nr. 00697</td>
			<td>500 mL vial</td>
		</tr>
		<tr>
			<td>NP-40 surfactant (Tergitol solution)</td>
			<td>Sigma</td>
			<td>056K0151</td>
			<td>500 mL vial</td>
		</tr>
		<tr>
			<td>Normal goat serum</td>
			<td>Vector Laboratories</td>
			<td>S-1000-20</td>
			<td>20 mL vial, concentrate</td>
		</tr>
		<tr>
			<td>Nestin (primary antibody)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-23927</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Donkey anti-mouse-fluorescein isothiocyanate (secondary antibody)</td>
			<td>Santa Cruz Biotechnology</td>
			<td>SC-2099</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Hoechst 33342</td>
			<td>Anaspec</td>
			<td>AS-83218</td>
			<td>5 mL vial</td>
		</tr>
		<tr>
			<td>In vivo biocompatibility test:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Tiletamine+zolazepam&#160;</td>
			<td>Virbac</td>
			<td>BC91</td>
			<td>5 mL vial</td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Bayer korea</td>
			<td>KR03227</td>
			<td>10 mL vial</td>
		</tr>
		<tr>
			<td>Ketoprofen</td>
			<td>Astar</td>
			<td>1406232</td>
			<td>2 mL vial</td>
		</tr>
		<tr>
			<td>Povidone-iodine solution</td>
			<td>Everstar</td>
			<td>HA161202</td>
			<td>4 L barrel</td>
		</tr>
		<tr>
			<td>Cefazolin</td>
			<td>China Chemical &#38; Pharmaceutical</td>
			<td>18P909</td>
			<td>1 g vial</td>
		</tr>
		<tr>
			<td>Scalpel blade</td>
			<td>Shinetec instruments</td>
			<td>ST-B021</td>
			<td>-</td>
		</tr>
		<tr>
			<td>Surgical scissor</td>
			<td>Shinetec instruments</td>
			<td>ST-B021</td>
			<td>-</td>
		</tr>
	</tbody>
</table>

wet-spinning-based molding process of gelatin for tissue regeneration

This article presents an inexpensive method to fabricate gelatin, as a natural polymer, into monofilament fibers or other appropriate forms. Through the wet spinning method, gelatin fibers are produced by smooth extrusion in a suitable coagulation medium. To increase the functional surface of these gelatin fibers and their ability to mimic the features of tissues, gelatin can be molded into a tube form by referring to this concept. Examined by in vitro and in vivo tests, the gelatin tubes demonstrate a great potential for application in tissue engineering. Acting as a suitable filling gap material, gelatin tubes can be used to substitute the tissue in the damaged area (e.g., in the nervous or cardiovascular system), as well as to promote regeneration by providing a direct replacement of stem cells and neural circuitry. This protocol provides a detailed procedure for creating a biomaterial based on a natural polymer, and its implementation is expected to greatly benefit the development of correlative natural polymers, which help to realize tissue regeneration strategies.

In this study, we successfully developed the gelatin into fibers (Figure 2A) and tubes (Figure 2B,C) through the user-friendly wet spinning concept. These gelatin-based materials can be utilized as any medical tool, depending on their shapes. Considering that the functional surface and frame of such materials are more suitable for tissue regeneration, we examined the biocompatibility of gelatin t...

Watch this Scientific Journal Video about Wet-spinning-based Molding Process of Gelatin for Tissue Regeneration at JoVE.com

Wet-spinning-based Molding Process of Gelatin for Tissue Regeneration

We developed and describe a protocol based on the wet spinning concept, for the construction of gelatin-based biomaterials used for the application of tissue engineering.

This article presents an inexpensive method to fabricate gelatin, as a natural polymer, into monofilament fibers or other appropriate forms. Through the ...

wet-spinning-based-molding-process-of-gelatin-for-tissue-regeneration

Research

JoVE Journal

Bioengineering

8.6K Views.  National Taiwan University of Science and Technology. We developed and describe a protocol based on the wet spinning concept, for the construction of gelatin-based biomaterials used for the application of tissue engineering.

Video: Wet-spinning-based Molding Process of Gelatin for Tissue Regeneration

Expression, Isolation, and Purification of Soluble and Insoluble Biotinylated Proteins for Nerve Tissue Regeneration

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most widely used host organism. The flexibility of the system allows for the addition of moieties such as a biotin tag (for streptavidin interactions) and larger functional proteins like green fluorescent protein or cherry red protein. Also, the integration of unnatural amino acids like metal ion chelators, uniquely reactive functional groups, spectroscopic probes, and molecules imparting post-translational modifications has enabled better manipulation of protein properties and functionalities. As a result this technique creates customizable fusion proteins that offer significant utility for various fields of research. More specifically, the biotinylatable protein sequence has been incorporated into many target proteins because of the high affinity interaction between biotin with avidin and streptavidin. This addition has aided in enhancing detection and purification of tagged proteins as well as opening the way for secondary applications such as cell sorting. Thus, biotin-labeled molecules show an increasing and widespread influence in bioindustrial and biomedical fields. For the purpose of our research we have engineered recombinant biotinylated fusion proteins containing nerve growth factor (NGF) and semaphorin3A (Sema3A) functional regions. We have reported previously how these biotinylated fusion proteins, along with other active protein sequences, can be tethered to biomaterials for tissue engineering and regenerative purposes. This protocol outlines the basics of engineering biotinylatable proteins at the milligram scale, utilizing&#160; a T7 lac inducible vector and E. coli expression hosts, starting from transformation to scale-up and purification.

Developing biotinylatable fusion proteins has many potential applications in various fields of research. Recombinant protein engineering is a straight forward procedure that is cost-effective, providing high yields of custom-designed proteins.

Recombinant protein engineering has utilized Escherichia coli (E. coli) expression systems for nearly 4 decades, and today E. coli is still the most ...

Rapid and Low-cost Prototyping of Medical Devices Using 3D Printed Molds for Liquid Injection Molding

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using traditional liquid injection molding processes can be an expensive process due to tooling and equipment costs. As a result, it has traditionally been impractical to use liquid injection molding for low-cost, rapid prototyping applications. We have devised a method for rapid and low-cost production of liquid elastomer injection molded devices that utilizes fused deposition modeling 3D printers for mold design and a modified desiccator as an injection system. Low costs and rapid turnaround time in this technique lower the barrier to iteratively designing and prototyping complex elastomer devices. Furthermore, CAD models developed in this process can be later adapted for metal mold tooling design, enabling an easy transition to a traditional injection molding process. We have used this technique to manufacture intravaginal probes involving complex geometries, as well as overmolding over metal parts, using tools commonly available within an academic research laboratory. However, this technique can be easily adapted to create liquid injection molded devices for many other applications.

We have devised a method for low-cost and rapid prototyping of liquid elastomer rubber injection molded devices by using fused deposition modeling 3D printers for mold design and a modified desiccator as a liquid injection system.

Biologically inert elastomers such as silicone are favorable materials for medical device fabrication, but forming and curing these elastomers using ...

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. ...

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to intrinsic parameters within the process allow a high degree of control over scaffold characteristics including fiber diameter, alignment and porosity. By developing scaffolds with similar dimensions and topographies to organ- or tissue-specific extracellular matrices (ECM), micro-environments representative to those that cells are exposed to in situ can be created. 
The airway bronchiole wall, comprised of three main micro-environments, was selected as a model tissue. Using decellularized airway ECM as a guide, we electrospun the non-degradable polymer, polyethylene terephthalate (PET), by three different protocols to produce three individual electrospun scaffolds optimized for epithelial, fibroblast or smooth muscle cell-culture. Using a commercially available bioreactor system, we stably co-cultured the three cell-types to provide an in vitro model of the airway wall over an extended time period.
This model highlights the potential for such methods being employed in in vitro diagnostic studies investigating important inter-cellular cross-talk mechanisms or assessing novel pharmaceutical targets, by providing a relevant platform to allow the culture of fully differentiated adult cells within 3D, tissue-specific environments.

Adapting the Electrospinning Process to Provide Three Unique Environments for a Tri-layered In Vitro Model of the Airway Wall

Advancements in biomaterial technologies enable the development of three-dimensional multi-cell-type constructs. We have developed electrospinning protocols to produce three individual scaffolds to culture the main structural cells of the airway to provide a 3D in vitro model of the airway bronchiole wall.

Electrospinning is a highly adaptable method producing porous 3D fibrous scaffolds that can be exploited in in vitro cell culture. Alterations to ...

Qualitative Characterization of the Aqueous Fraction from Hydrothermal Liquefaction of Algae Using 2D Gas Chromatography with Time-of-flight Mass Spectrometry

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in complex mixtures. It is often used to analyze gasoline, jet fuel, diesel, bio-diesel and the organic fraction of bio-crude/bio-oil. In most of those analyses, the first dimension of separation is non-polar, followed by a polar separation. The aqueous fractions of bio-crude and other aqueous samples from biofuels production have been examined with similar column combinations. However, sample preparation techniques such as derivatization, solvent extraction, and solid-phase extraction were necessary prior to analysis. In this study, aqueous fractions obtained from the hydrothermal liquefaction of algae were characterized by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry without prior sample preparation techniques using a polar separation in the first dimension followed by a non-polar separation in the second. Two-dimensional plots from this analysis were compared with those obtained from the more traditional column configuration. Results from qualitative characterization of the aqueous fractions of algal bio-crude are discussed in detail. The advantages of using a polar separation followed by a non-polar separation for characterization of organics in aqueous samples by two-dimensional gas chromatography coupled with time-of-flight mass spectrometry are highlighted.

A two-dimensional gas chromatography-time-of-flight mass spectrometry method is described for characterization of the aqueous fraction of bio-crude produced from hydrothermal liquefaction of algae. This protocol can also be employed to analyze the aqueous fraction of liquid products from fast pyrolysis, catalytic fast pyrolysis, catalytic deoxygenation and hydro-treating.

Two-dimensional gas chromatography coupled with time-of-flight mass spectrometry is a powerful tool for identifying and quantifying chemical components in ...

Alternating Magnetic Field-Responsive Hybrid Gelatin Microgels for Controlled Drug Release

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of smart soft devices for biomedical applications. Although a number of magnetically-responsive drug delivery systems have demonstrated efficacies through either in vitro proof of concept studies or in vivo preclinical applications, their use in clinical settings is still limited by their insufficient biocompatibility or biodegradability. Additionally, many of the existing platforms rely on sophisticated techniques for their fabrications. We recently demonstrated the fabrication of biodegradable, gelatin-based thermo-responsive microgel by physically entrapping poly(N-isopropylacrylamide-co-acrylamide) chains as a minor component within a three-dimensional gelatin network. In this study, we present a facile method to fabricate a biodegradable drug release platform that enables a magneto-thermally triggered drug release. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and thermo-responsive polymers within gelatin-based colloidal microgels, in conjunction with an alternating magnetic field application system.

We present a facile method to fabricate a biodegradable gelatin-based drug release platform that is magneto-thermally responsive. This was achieved by incorporating superparamagnetic iron oxide nanoparticles and poly(N-isopropylacrylamide-co-acrylamide) within a spherical gelatin micro-network crosslinked by genipin, in conjunction with an alternating magnetic field application system.

Magnetically-responsive nano/micro-engineered biomaterials that enable a tightly controlled, on-demand drug delivery have been developed as new types of ...

A Paired Bead and Magnet Array for Molding Microwells with Variable Concave Geometries

A spheroid culture is a useful tool for understanding cellular behavior in that it provides an in vivo-like three-dimensional environment. Various spheroid production methods such as non-adhesive surfaces, spinner flasks, hanging drops, and microwells have been used in studies of cell-to-cell interaction, immune-activation, drug screening, stem cell differentiation, and organoid generation. Among these methods, microwells with a three-dimensional concave geometry have gained the attention of scientists and engineers, given their advantages of uniform-sized spheroid generation and the ease with which the responses of individual spheroids can be monitored. Even though cost-effective methods such as the use of flexible membranes and ice lithography have been proposed, these techniques incur serious drawbacks such as difficulty in controlling the pattern sizes, achievement of high aspect ratios, and production of larger areas of microwells. To overcome these problems, we propose a robust method for fabricating concave microwells without the need for complex high-cost facilities. This method utilizes a 30 x 30 through-hole array, several hundred micrometer-order steel beads, and magnetic force to fabricate 900 microwells in a 3 cm x 3 cm polydimethylsiloxane (PDMS) substrate. To demonstrate the applicability of our method to cell biological applications, we cultured adipose stem cells for 3 days and successfully produced spheroids using our microwell platform. In addition, we performed a magnetostatic simulation to investigate the mechanism, whereby magnetic force was used to trap the steel beads in the through-holes. We believe that the proposed microwell fabrication method could be applied to many spheroid-based cellular studies such as drug screening, tissue regeneration, stem cell differentiation, and cancer metastasis.

This manuscript introduces a robust method of fabricating concave microwells without the need for complex high-cost facilities. Using magnetic force, steel beads, and a through-hole array, several hundred microwells were formed in a 3 cm x 3 cm polydimethylsiloxane (PDMS) substrate.

A spheroid culture is a useful tool for understanding cellular behavior in that it provides an in vivo-like three-dimensional environment. Various ...

Three-dimensional Tissue Engineered Aligned Astrocyte Networks to Recapitulate Developmental Mechanisms and Facilitate Nervous System Regeneration

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to replace lost neurons and regenerate axonal pathways. However, during nervous system development, neuronal migration and axonal extension often occur along pathways formed by other cells, referred to as &#34;living scaffolds&#34;. Seeking to emulate these mechanisms and to design a strategy that circumvents the inhibitory environment of the CNS, this manuscript presents a protocol to fabricate tissue engineered astrocyte-based &#34;living scaffolds&#34;. To create these constructs, we employed a novel biomaterial encasement scheme to induce astrocytes to self-assemble into dense three-dimensional bundles of bipolar longitudinally-aligned somata and processes. First, hollow hydrogel micro-columns were assembled, and the inner lumen was coated with collagen extracellular-matrix. Dissociated cerebral cortical astrocytes were then delivered into the lumen of the cylindrical micro-column and, at a critical inner diameter of &#60;350 &#181;m, spontaneously self-aligned and contracted to produce long fiber-like cables consisting of dense bundles of astrocyte processes and collagen fibrils measuring &#60;150 &#181;m in diameter yet extending several cm in length. These engineered living scaffolds exhibited &#62;97% cell viability and were virtually exclusively comprised of astrocytes expressing a combination of the intermediate filament proteins glial-fibrillary acidic protein (GFAP), vimentin, and nestin. These aligned astrocyte networks were found to provide a permissive substrate for neuronal attachment and aligned neurite extension. Moreover, these constructs maintain integrity and alignment when extracted from the hydrogel encasement, making them suitable for CNS implantation. These preformed constructs structurally emulate key cytoarchitectural elements of naturally occurring glial-based &#34;living scaffolds&#34; in vivo. As such, these engineered living scaffolds may serve as test-beds to study neurodevelopmental mechanisms in vitro or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding following CNS degeneration in vivo.

We showcase the development of self-assembled, three-dimensional bundles of longitudinally aligned astrocytic somata and processes within a novel biomaterial encasement. These engineered &#34;living scaffolds&#34;, exhibiting micron-scale diameter yet extending centimeters in length, may serve as test-beds to study neurodevelopmental mechanisms or facilitate neuroregeneration by directing neuronal migration and/or axonal pathfinding.

Neurotrauma and neurodegenerative disease often result in lasting neurological deficits due to the limited capacity of the central nervous system (CNS) to ...

Patterning the Geometry of Human Embryonic Stem Cell Colonies on Compliant Substrates to Control Tissue-Level Mechanics

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that correspond to primary germ layer formation during embryogenesis. Thus, these cells represent a powerful tool with which to examine the mechanisms that drive early human development. We have developed a method to culture human embryonic stem cells in confined colonies on compliant substrates that provides control over both the geometry of the colonies and their mechanical environment in order to recapitulate the physical parameters that underlie embryogenesis. The key feature of this method is the ability to generate polyacrylamide hydrogels with defined patterns of extracellular matrix ligand at the surface to promote cell attachment. This is achieved by fabricating stencils with the desired geometric patterns, using these stencils to create patterns of extracellular matrix ligand on glass coverslips, and transferring these patterns to polyacrylamide hydrogels during polymerization. This method is also compatible with traction force microscopy, allowing the user to measure and map the distribution of cell-generated forces within the confined colonies. In combination with standard biochemical assays, these measurements can be used to examine the role mechanical cues play in fate specification and morphogenesis during early human development.

Extracellular matrix ligands can be patterned onto polyacrylamide hydrogels to enable the culture of human embryonic stem cells in confined colonies on compliant substrates. This method can be combined with traction force microscopy and biochemical assays to examine the interplay between tissue geometry, cell-generated forces, and fate specification.

Human embryonic stem cells demonstrate a unique ability to respond to morphogens in vitro by self-organizing patterns of cell fate specification that ...

Protocols of 3D Bioprinting of Gelatin Methacryloyl Hydrogel Based Bioinks

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed from mammal collagen. Thus, the arginine-glycine-aspartic acid (RGD) sequences and target motifs of matrix metalloproteinase (MMP) remain on the molecular chains, which help achieve cell attachment and degradation. Furthermore, formation properties of GelMA are versatile. The methacrylamide groups allow a material to become rapidly crosslinked under light irradiation in the presence of a photoinitiator. Therefore, it makes great sense to establish suitable methods for synthesizing three-dimensional (3D) structures with this promising material. However, its low viscosity restricts GelMA's printability. Presented here are methods to carry out 3D bioprinting of GelMA hydrogels, namely the fabrication of GelMA microspheres, GelMA fibers, GelMA complex structures, and GelMA-based microfluidic chips. The resulting structures and biocompatibility of the materials as well as the printing methods are discussed. It is believed that this protocol may serve as a bridge between previously applied biomaterials and GelMA as well as contribute to the establishment of GelMA-based 3D architectures for biomedical applications.

Presented here is a method for the 3D bioprinting of gelatin methacryloyl.

Gelatin methacryloyl (GelMA) has become a popular biomaterial in the field of bioprinting. The derivation of this material is gelatin, which is hydrolyzed ...