Name: three-dimensional collagen matrix scaffold implantation as a liver regeneration strategy
Uploaded: 2021-06-29T14:15:00
Description: Watch this Scientific Journal Video about Three-Dimensional Collagen Matrix Scaffold Implantation as a Liver Regeneration Strategy at JoVE.com

The authors wish to thank the personnel of the Laboratory Animal Facility of the Experimental Medicine Unit, Nurse Carolina Ba&#241;os G. for technical and surgical support, Marco E. Gudi&#241;o Z. for support in microphotographs, and Erick Apo for support in liver histology. The National Council supported this research for Science and Technology (CONACyT), grant number SALUD-2016-272579 and the PAPIIT-UNAM TA200515.

The present research was approved by the ethics committee of the School of Medicine (DI/115/2015) at the Universidad Nacional Aut&#243;noma de M&#233;xico (UNAM) and the ethics committee of the Hospital General de Mexico (CI/314/15). The institution fulfills all technical specifications for the production, care, and use of laboratory animals and is legally certified by national law (NOM-062-ZOO-1999). Male Wistar rats weighing 150-250 g (6-8 weeks old) were obtained from the Laboratory Animal Facility of the School of Me...

<ol>
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Organ transplantation is the mainstay of treatment in patients with liver fibrosis or cirrhosis. A few patients benefit from this procedure, making it necessary to provide therapeutic alternatives for patients on the waiting list. Tissue engineering is a promising strategy that employs scaffolds and cells with regenerative potential2,4,13. The removal of a portion of the liver is a critical step in this procedure because of the ...

The liver is one of the most important organs involved in maintaining homeostasis and protein production1. Unfortunately, liver disease is the leading cause of death worldwide. In advanced stages of liver damage, which include cirrhosis and hepatocellular carcinoma, liver transplantation is the clinically recommended procedure. However, due to the scarcity of donors and the low rate of successful transplants, new techniques in tissue engineering (TE) and regenerative medicine (RM) have been developed2,3.
TE involves the use of stem cells, scaffolds, and growth factors4 to promote the restoration of inflamed, fibrotic, and edematous organs and tissues1,5,6. The biomaterials used in scaffolds mimic the native ECM, providing the physical, chemical, and biological cues for guided cellular remodeling7. Collagen is one of the most abundant proteins obtained from the dermis, tendon, intestine, and pericardium8,9. Furthermore, collagen can be obtained as a biopolymer to produce two- and three-dimensional scaffolds through bioprinting or electrospinning10,11. This group is the first to report the use of collagen from a bone source for the regeneration of liver tissue. Another study reports the use of scaffolds synthesized from bovine collagen, which was obtained from skin, with homogeneous and closely situated pores, without any communication between them12.
Decellularization preserves the native ECM, allowing the subsequent incorporation of cells with stem cell potential13,14. However, this procedure is still in the experimental phase in the liver, heart, kidney, small intestine, and urinary bladder from mice, rats, rabbits, pigs, sheep, cattle, and horses3,14. Currently, the resected liver mass volume is not replaced in any of the animal hepatectomy models. However, the use of additional support or network (biomaterials) that enables cell proliferation and angiogenesis could be essential for the prompt restoration of liver parenchymal functions. Thus, scaffolds could be employed as alternative approaches to regenerate or repair tissue in chronic liver diseases, in turn, eliminating limitations due to donation and the clinical complications of liver transplantation.

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			<td>Anionic detergent</td>
			<td>Alconox</td>
			<td>Z273228</td>
			<td></td>
		</tr>
		<tr>
			<td>Biopsy cassettes</td>
			<td>Leica</td>
			<td>3802453</td>
			<td></td>
		</tr>
		<tr>
			<td>Camera DMX</td>
			<td>Nikon</td>
			<td>DXM1200F</td>
			<td></td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5424</td>
			<td></td>
		</tr>
		<tr>
			<td>Chlorhexidine gluconate 4%</td>
			<td>BD</td>
			<td>372412</td>
			<td></td>
		</tr>
		<tr>
			<td>Cover glasses 25 mm x 40 mm</td>
			<td>Corning</td>
			<td>2980-224</td>
			<td></td>
		</tr>
		<tr>
			<td>Eosin</td>
			<td>Sigma-Aldrich</td>
			<td>200-M</td>
			<td>CAS 17372-87-1</td>
		</tr>
		<tr>
			<td>Ethyl alcohol, pure</td>
			<td>Sigma-Aldrich</td>
			<td>459836</td>
			<td>CAS 64-17-5</td>
		</tr>
		<tr>
			<td>Flunixine meglumide</td>
			<td>MSD</td>
			<td>Q-0273-035</td>
			<td></td>
		</tr>
		<tr>
			<td>Glass slides 75 mm x 25 mm</td>
			<td>Corning</td>
			<td>101081022</td>
			<td></td>
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			<td>Hematoxylin</td>
			<td>Merck</td>
			<td>H9627</td>
			<td>CAS 571-28-2</td>
		</tr>
		<tr>
			<td>Hydrochloric acid 37%</td>
			<td>Merck</td>
			<td>339253</td>
			<td>CAS 7647-01-0</td>
		</tr>
		<tr>
			<td>Ketamine</td>
			<td>Pisa agropecuaria</td>
			<td>Q-7833-028</td>
			<td></td>
		</tr>
		<tr>
			<td>Light microscopy</td>
			<td>Nikon</td>
			<td>Microphoto-FXA</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtainer yellow cape</td>
			<td>Beckton Dickinson</td>
			<td>365967</td>
			<td></td>
		</tr>
		<tr>
			<td>Microtome</td>
			<td>Leica</td>
			<td>RM2125</td>
			<td></td>
		</tr>
		<tr>
			<td>Model animal: Wistar rats</td>
			<td>Universidad Nacional Aut&#243;noma de M&#233;xico</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Nylon 3-0 (Dermalon)</td>
			<td>Covidien</td>
			<td>1750-41</td>
			<td></td>
		</tr>
		<tr>
			<td>Polypropylene 7-0</td>
			<td>Atramat</td>
			<td>SE867/2-60</td>
			<td></td>
		</tr>
		<tr>
			<td>Povidone-iodine10% cutaneous solution</td>
			<td>Diafra SA de CV</td>
			<td>1.37E+86</td>
			<td></td>
		</tr>
		<tr>
			<td>Scaning electronic microscopy</td>
			<td>Zeiss</td>
			<td>DSM-950</td>
			<td></td>
		</tr>
		<tr>
			<td>Sodium hydroxide, pellets</td>
			<td>J. T. Baker</td>
			<td>3722-01</td>
			<td>CAS 1310-73-2</td>
		</tr>
		<tr>
			<td>Software ACT-1</td>
			<td>Nikon</td>
			<td>Ver 2.70</td>
			<td></td>
		</tr>
		<tr>
			<td>Stereoscopy macroscopy</td>
			<td>Leica</td>
			<td>EZ4Stereo 8X-35X</td>
			<td></td>
		</tr>
		<tr>
			<td>Sterrad 100S</td>
			<td>Johnson and Johnson</td>
			<td>99970</td>
			<td></td>
		</tr>
		<tr>
			<td>Surgipath paraplast</td>
			<td>Leica</td>
			<td>39601006</td>
			<td></td>
		</tr>
		<tr>
			<td>Synringe of 1 mL with needle (27G x 13 mm)</td>
			<td>SensiMedical</td>
			<td>LAN-078-077</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue Processor (Histokinette)</td>
			<td>Leica</td>
			<td>TP1020</td>
			<td></td>
		</tr>
		<tr>
			<td>Tissue-Tek TEC 5 (Tissue embedder)</td>
			<td>Sakura Finetek USA</td>
			<td>5229</td>
			<td></td>
		</tr>
		<tr>
			<td>Trichrome stain kit</td>
			<td>Sigma-Aldrich</td>
			<td>HT15</td>
			<td></td>
		</tr>
		<tr>
			<td>Unicell DxC600 Analyzer</td>
			<td>Beckman Coulter</td>
			<td>BC 200-10</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylazine</td>
			<td>Pisa agropecuaria</td>
			<td>Q-7833-099</td>
			<td></td>
		</tr>
		<tr>
			<td>Xylene</td>
			<td>Sigma-Aldrich</td>
			<td>534056</td>
			<td>CAS 1330-20-7</td>
		</tr>
	</tbody>
</table>

three-dimensional collagen matrix scaffold implantation as a liver regeneration strategy

Liver diseases are the leading cause of death worldwide. Excessive alcohol consumption, a high-fat diet, and hepatitis C virus infection promote fibrosis, cirrhosis, and/or hepatocellular carcinoma. Liver transplantation is the clinically recommended procedure to improve and extend the life span of patients in advanced disease stages. However, only 10% of transplants are successful, with organ availability, presurgical and postsurgical procedures, and elevated costs directly correlated with that result. Extracellular matrix (ECM) scaffolds have emerged as an alternative for tissue restoration. Biocompatibility and graft acceptance are the main beneficial characteristics of those biomaterials. Although the capacity to restore the size and correct function of the liver has been evaluated in liver hepatectomy models, the use of scaffolds or some kind of support to replace the volume of the extirpated liver mass has not been assessed.
Partial hepatectomy was performed in a rat liver with the xenoimplantation of a collagen matrix scaffold (CMS) from a bovine condyle. Left liver lobe tissue was removed (approximately 40%), and an equal proportion of CMS was surgically implanted. Liver function tests were evaluated before and after the surgical procedure. After days 3, 14, and 21, the animals were euthanized, and macroscopic and histologic evaluations were performed. On days 3 and 14, adipose tissue was observed surrounding the CMS, with no clinical evidence of rejection or infection, as was vessel neoformation and CMS reabsorption at day 21. There was histologic evidence of an insignificant inflammation process and migration of adjacent cells to the CMS, observed with the hematoxylin and eosin (H&#38;E) and Masson&#39;s trichrome staining. The CMS was shown to perform well in liver tissue and could be a useful alternative for studying tissue regeneration and repair in chronic liver diseases.

Bone demineralization affects the mechanical properties of CMS without altering the original shape or interconnection of its pores. CMS can have any shape, and therefore, can be adjusted to the size and shape of the selected organ or tissue19. In the present protocol, we used a triangular CMS (Figure 1A-D). A rat model was used to evaluate the regenerative capacity of the CMS xenoimplant in the liver. Although the liv...

Watch this Scientific Journal Video about Three-Dimensional Collagen Matrix Scaffold Implantation as a Liver Regeneration Strategy at JoVE.com

Three-Dimensional Collagen Matrix Scaffold Implantation as a Liver Regeneration Strategy

Liver diseases are induced by many causes that promote fibrosis or cirrhosis. Transplantation is the only option for recovering health. However, given the scarcity of transplantable organs, alternatives must be explored. Our research proposes the implantation of collagen scaffolds in liver tissue from an animal model.

Liver diseases are the leading cause of death worldwide. Excessive alcohol consumption, a high-fat diet, and hepatitis C virus infection promote fibrosis, ...

This method involves placing an implant of collagen matrix as a scaffold in the liver to promote liver regeneration. This implantation method has opened the door for a strategist exploring the restoration of the shape and balloon of the resected tissues, thereby reducing anesthesia, surgical duration, and recovery time. This method can be used to promote regeneration involving models of liver fibrosis, as well as other mammalian species.This method is based on tissue engineering and regenerative medicine to promote the restoration of injured tissue. Begin by sanitizing the surgical area work table, microsurgery microscope, and seat with a 2%chlorhexidine solution, then hydrate the collagen matrix scaffold obtained from a bovine condyle in sterile saline solution for 20 minutes. Next, using surgical soap and a double-edged blade, shave the abdominal skin of an anesthetized six to eight-week-old male Wistar rat, then disinfect the skin three times using a 10%povidone iodine solution.Place the animal on a warm plate in the decubitus dorsal position with the neck hyperextended to maintain a permeable airway. Evaluate the depth of anesthesia through the respiratory pattern and loss of withdrawal reflex in the limbs. Then place a disposable surgical drape around the shaved skin and using the xiphoid process as a reference point, make a 2.5 centimeter incision on the alpis line with dissection forceps, taking care to avoid the abdominal wall blood vessel to prevent bleeding.After placing the abdominal retractor, observe the abdominal cavity. Using the dissection forceps, extract the left liver lobe and place it on the metal plate. Using a sterile one by one by one centimeter triangular metallic template, perform a hepatectomy.To prevent bleeding of the liver, maintain surgical compression with a swab on the edge of the liver for five minutes. Next, using 7-O non-absorbable polypropylene sutures, implant the hydrated collagen matrix scaffold in the hepatectomy site with four stitches between the liver tissue and the scaffold to prevent displacement of the biomaterial. Return the liver to the abdominal cavity and suture the abdominal wall and skin with a 3-O nylon suture.Clean the surgical incision twice with a surgical iodine-soaked sponge and monitor the vital signs of the animal. Then place the animal in lateral decubitus position. After administering an analgesic, place the animal in an individual polycarbonate box with laboratory animal bedding in a noise-free area with temperature control.Observe the recovery from anesthesia and monitor water and food consumption for two hours. There was no difference in the liver function between the SHAM group and the experimental groups with and without the collagen matrix scaffold at three, 14, and 21 days, indicating that the collagen matrix scaffold does not disrupt liver function. Histological analysis on day three shows that the presence of the collagen matrix scaffold in the liver did not promote a foreign body reaction and the conspicuous presence of lax connective tissue is observed.On days 14 and 21, the lax connective tissue is more abundant with the collagen matrix scaffold trabeculae surrounded by hepatocytes suggesting that hepatocytes have migrated to the collagen matrix scaffold. Areas of hepatocytes irrigated by a vessel can be seen and the hepatocytes adhering to the collagen matrix scaffold trabeculae are sometimes surrounded by lax connective tissue. While performing the hepatectomy, use aseptic technique to avoid infection and apply compression afterwards to prevent bleeding from the liver.Also, use 7-O sutures for implantation of collagen matrix scaffold to avoid tearing. After implantation of the collagen matrix scaffold in a fibrotic animal model, the advantages of sinogenic implantation for liver regeneration can be evaluated. The use of collagen matrix scaffold to replace the liver extubated mass has not been evaluated until now.These genogenic materials paves the way as another therapeutic option in the field of the chronic liver diseases.

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Invasion of surrounding normal tissues is generally considered to be a key hallmark of malignant (as opposed to benign) tumors. For some cancers in ...

Method of Direct Segmental Intra-hepatic Delivery Using a Rat Liver Hilar Clamp Model

Major hepatic surgery with inflow occlusion, and liver transplantation, necessitate a period of warm ischemia, and a period of reperfusion leading to ...

Three-Dimensional Printing of a Complex Aortic Anomaly

Complex congenital aortic anomalies include diverse types of malformations that may be clinically asymptomatic or present with respiratory or esophageal ...

Murine Precision-Cut Liver Slices as an Ex Vivo Model of Liver Biology

Understanding the mechanisms of liver injury, hepatic fibrosis, and cirrhosis that underlie chronic liver diseases (i.e., viral hepatitis, non-alcoholic ...

3D Imaging of the Liver Extracellular Matrix in a Mouse Model of Non-Alcoholic Steatohepatitis

Non-alcoholic steatohepatitis (NASH) is the most common chronic liver disease in the United States, affecting more than 70 million Americans. NASH can ...