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

Podsumowanie

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.

Streszczenie

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.

Wprowadzenie

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 surgery^1,2. The tissue at the damaged area is lost and replaced with hypertrophically induced astrocytes³, eventually forming a dense glial scar^4,5. This matrix acts as a barrier that blocks the recovery of nerve function^6,7 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 — 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 invertebrates^13,14, 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.

Protokół

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

1. Solution preparation
   1. Dissolve 5 g of gelatin powder in 100 mL of double-distilled water to obtain 5% (w/v) solution concentration.
   2. Stir the mixture slowly at 60 - 70 °C overnight to achieve a completely homogeneous dispersion without any bubbles.
2. Wet spinning
   1. 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.
      1. Plug a 26 G x 1/2 inch (0.45 mm x 12 mm) syringe as the solution injector into clear vinyl tubing.
      2. Prepare 100 mL of 99.5% acetone solution in a beaker glass to be used as the coagulation bath.
      3. 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.
      4. 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).
      5. Let the gelatin fibers in the PCL/DCM solution dry overnight, under the hood at room temperature.
   2. Tube formation
      NOTE: A schematic of the method is shown in Figure 1B.
      1. Use a peripheral venous catheter of 24 G x 3/4 inch (0.7 mm x 19 mm) as tube mold.
      2. Load the catheter into the gelatin solution and hold it there for 3 s.
      3. Load the catheter into the acetone solution and hold for 1 min.
      4. Load the catheter again into the gelatin solution and hold it there for 3 s.
      5. Repeat this alternate procedure 20x.
      6. Take out the catheter from the acetone solution and let the molded tube dry at room temperature for 5 min.
      7. 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.
      8. Let the Pasteur pipette containing the gelatin tube and PCL/DCM solution dry overnight, under the hood at room temperature.

2. Morphology of the Gelatin Tube

1. Mount the piece of dried gelatin tube on a carbon stub.
2. 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).

3. Culture of Human Adipose Stem Cells

1. 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.
2. Cut the tissues into small pieces (less than 1 mm²) with a scalpel blade.
3. Transfer the tissues into a 15 mL plastic tube and centrifuge at 500 x g for 5 min.
4. Remove the supernatant and incubate the sediment in a plastic tube containing 10 mL of Dulbecco's modified Eagle'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 °C, in a humidified atmosphere containing 95% air and 5% CO₂, for 1 day.
5. 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 °C, in a humidified atmosphere containing 95% air and 5% CO₂.
6. 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 °C, in a humidified atmosphere containing 95% air and 5% CO₂.
7. Discard the supernatant, add 1 mL of 0.25% trypsin-ethylenediaminetetraacetic acid (EDTA), and incubate it at 37 °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.
8. Add 1 mL of FBS, suspend the mixture, and transfer it to a microcentrifuge tube.
9. 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 °C in a humidified atmosphere containing 95% air and 5% CO₂.
10. Maintain the subculture by changing the stem cell medium every 3 days.

4. Cultivation of Cells on the Gelatin Tube

1. 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.
2. Collect the human adipose stem cells (hASCs) from the culture flask.
   1. Remove the medium from the culture flask.
   2. Add 1 mL of 0.25% trypsin-EDTA and incubate at 37 °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.
   3. Add 1 mL of FBS, suspend the mixture, and transfer it to a microcentrifuge tube.
   4. 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.
3. 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 °C, in a humidified atmosphere containing 95% air and 5% CO₂.
4. Change the stem cell medium every 3 days to provide enough nutrition for cell growth.

5. Immunocytochemistry

1. Remove the stem cell medium and wash the well with PBS.
2. Fix the tube with cells with 0.1% acetic acid glacial for 30 min at room temperature.
3. Wash the well 2x with PBS.
4. Add a surfactant (NP-40, 0.05%) and incubate for 10 min at room temperature.
5. Wash the well 3x with PBS.
6. Add 2% normal goat serum and incubate at room temperature for 30 min.
7. Decant the solution, add primary antibody nestin (neural progenitor cell marker), and incubate overnight at 4 °C.
8. Wash the well 3x with PBS.
9. Add secondary antibody donkey anti-mouse-fluorescein isothiocyanate (FITC) and keep the sample in the dark for 2 h.
10. Wash the well 3x with PBS.
11. Add 1 mL of Hoechst 33342 to stain the nuclei and incubate for 10 min at room temperature.
12. Wash the well gently and observe it under a fluorescence microscope.

6. In Vivo Biocompatibility Test

NOTE: Rats with a weight between 201 - 225 g have been successfully tested using this protocol.

1. Anesthesia
   1. 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's fingers to confirm the anesthetization.
2. Sterilization of the surgical site
   1. Shave and clean the skin of rat’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).
   2. Cover the rat with sterile surgical drapes to reduce bacterial transfer and subsequent contamination of the surgical site.
3. Implantation of the gelatin tube
   1. Gently cut the skin of rat’s trapezius area with a surgical scissor.
   2. Create a wound of 2 cm and place the gelatin tube directly on the layer between the fascia and the muscle.
4. Postimplantation surgery
   1. Close the wound with nylon suture and wipe it with povidone-iodine solution (100 mg/mL).
   2. Induce the rat via an intramuscular injection of ketoprofen (2.5 mg/kg) and cefazolin (15 mg/kg).
   3. Keep the rat under aseptic conditions for 7 days.
5. Euthanasia
   1. 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.
   2. Cut the rat gently with a scalpel blade, remove the implanted tissue, and take photos for observation.

Wyniki

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...

Dyskusje

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 °C). A gentle solution preparation is a critical step wit...

Materiały

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