Name: wet-spinning-based molding process of gelatin for tissue regeneration
Uploaded: 2019-03-07T14:15:00
Description: Watch this Scientific Journal Video about Wet-spinning-based Molding Process of Gelatin for Tissue Regeneration at JoVE.com

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>
	...

<ol>
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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 ...

This protocol improves a basic concept of wet-spinning to develop a material from a single fiber strip into various strips that can be used for any preferred medical application. This technique is simple, inexpensive, and does not require the addition of synthetic polymers for customization of the natural polymers into a resource of protein for materials of interest. This technique can be extended to biomaterial development for killing cold therapy such as central or peripheral nervous system treatment, blood vessel replacement, or direct urinary tract reconstruction.This method is intended for medical application including as a strategy for tissue neuralization or regenerative medicine. This technique can be easily implemented by beginners. Note that, after several molding repetitions, the quality of the solution should always be checked to maintain an optimal formation.To form the wet spinning tube, first submerge a 24 gauge by three quarter inch peripheral venous catheter into freshly prepared gelatin solution for three seconds, followed by immersion in freshly prepared acetone solution for one minute. Repeat these steps 20 times. After the last acetone immersion, let the molded tube dry for five minutes at room temperature, and use a hemostat to carefully transfer the gelatin tube from the catheter into a glass Pasteur pipette with 2.5%polycaprolactone dichloromethane solution for an overnight under-hood incubation at room temperature.The next morning, sterilize the gelatin for two hours under ultraviolet light before briefly submerging the tube in 75%ethanol. Then wash the tube two times with stem cell medium to remove the residual ethanol. To cultivate the gelatin tube with the cell culture, replace the supinate end of human adipose stem cells from a culture flask with one milliliter of 0.25%Trypsin-EDTA for a 3.5 minute incubation at 37 degrees Celsius and 5%carbon dioxide.When the cells have detached, arrest the reaction with one milliliter of fetal bovine serum and transfer the cell suspension into a microcentrifuge tube. Collect the cells by centrifugation and resuspend the pellet in one milliliter of stem cell medium. Then place the gelatin tube in one well of a six-well plate containing three milliliters of stem cell medium.After counting the cells, seed four times 10 to the fourth of human adipose stem cells into the tube for a two week incubation in the cell culture incubator. After confirming the appropriate level of sedation by lack of response to toe pinch in a female eight week old Sprague Dawley rat, shave the hair from the trapezius area and sterilize the exposed skin with povidone-iodine solution. Cover the rat with sterile surgical drapes to reduce the bacterial contamination of the surgical site and use surgical scissors to make a shallow, two centimeter long skin incision.Place the gelatin tube directly into the layer between the fascia and the muscle and use a nylon suture to close the wound. Then sterilize the incision with povidone-iodine solution. Induce the rat via an intramuscular injection of ketoprofen and cefazolin and maintain the animal under aseptic conditions for seven days.This user friendly wet spinning concept can be used to develop gelatin into fibers and tubes. Morphological observations of the gelatin tube by scanning electron microscopy reveals a not very smooth surface with a greater than 200 micrometer inner diameter and a thickness of approximately 20 micrometers. Staining of a human adipose stem cell culture gelatin tube for neural cell marker expression demonstrates a positive staining for the marker along with multiple detected nuclei suggesting that the cells can penetrate and adhere to the gelatin tube and differentiate into neural progenitor cells.Implantation of the gelatin tube into the fascia layer confirms its safety and biocompatibility, as no indication of redness, swelling, or other inflammation symptoms are observed within the local tissue and the tube is surrounded by well-developed connective tissue. While attempting this procedure of two point one, ensuring immersion of the mold in the specific coating time in each solution are very important to building up a precise tube form. Following this procedure, our experiments use animal model of human disease can be performed to accurately investigate the outcome of the biomaterials for tissue regeneration strategy.After its development, this technique paved the way for researchers in developing simple correlative nature polymers into any type of material for a decided medical application. Under normal use condition, the polycaprolactone dichloromethane solution is considered to present minimal environmental hazard, but don't forget to perform any steps with this chemical in an appropriate biosafety cabinet.

Research

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