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

Summary

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.

Abstract

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&E) and Masson'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.

Introduction

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 developed^2,3.

TE involves the use of stem cells, scaffolds, and growth factors⁴ to promote the restoration of inflamed, fibrotic, and edematous organs and tissues^1,5,6. 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 pericardium^8,9. Furthermore, collagen can be obtained as a biopolymer to produce two- and three-dimensional scaffolds through bioprinting or electrospinning^10,11. 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 potential^13,14. 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 horses^3,14. 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.

Protocol

The present research was approved by the ethics committee of the School of Medicine (DI/115/2015) at the Universidad Nacional Autónoma de México (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 Medicine, UNAM, for this study.

1. Obtaining collagen matrix scaffolds from bovine femur

1. Obtain the condyle from bovine femur from a slaughterhouse certified by health and agricultural authorities of Mexico.
   1. Carefully dissect the condyle fat, muscle, and cartilage with a surgical instrument. Cut the condyle fragments into 3 cm x3 cm fragments using a saw cutter and clean the fat and blood with a towel. Wash the condyle fragments with water.
   2. Boil (92 °C) the fragments with 1 L of an anionic detergent (10 g/L) for 30 min. Wash the condyle fragments twice to remove any remnants of the anionic detergent.
   3. Dry the condyle fragments for 3 h using filter paper (0.5 mm).
2. Prepare a triangular (1 cm x1 cm x1 cm) CMS of thickness 0.5 cm from the fragments mentioned in step 1.1 (Figure 1A).
   1. Demineralize the fragments in 100 mL of 0.5 M HCl for 10 min with constant agitation. Remove the HCl.
      NOTE: Neutralize the HCl with sodium hydroxide (10 M).
   2. Rinse the fragments three times with 100 mL of distilled water, 15 min each time, with constant agitation. Dry the CMS with filter paper (0.5 mm) for 1 h.
   3. Use a stereo-microscope¹⁵ to analyze the structural properties of the CMS (size of pores, pore formation, and porous interconnection) (Figure 1B).
   4. Use a scanning electron microscope¹⁶ to analyze the rough surface of the CMS trabeculae (Figure 1C).
   5. Use the dissection instrument for blending and stretching to evaluate the mechanical changes (plasticity and flexibility) of the CMS (Figure 1D).
   6. Pack the CMS into the sterilization pouch and sterilize it with hydrogen peroxide plasma for 38 min. Store the sterile CMS in the original pack in a dry area at 20-25 °C until use.

2. Preparation of the surgical area and handling and preparation of the animal model

1. Sanitize the surgical area, worktable, microsurgery microscope, and seat with 2% chlorhexidine solution. Sterilize all surgical instruments, surgical sponge, swabs, and disposable surgical drape through heat sterilization (121 °C/30 min/100 kPa)
2. Assign the rats (n=5) into three groups of five rats per group: 1. sham, 2. hepatectomy, and 3. hepatectomy plus CMS, and follow all the groups on days 3, 14, and 21 days.
   1. Administer ketamine (35 mg/kg) and xylazine (2.5 mg/kg) intramuscularly in the hind limb.
      NOTE: The sedative period typically lasts for 30-40 min.
   2. Shave the abdominal skin (5 cm x 2 cm) using surgical soap and a double-edged blade, and disinfect the skin using topical 10% povidone-iodine solution in three rounds¹⁷.
   3. Place the animal on a warm plate in the decubitus dorsal position, with the neck hyperextended to maintain a permeable airway (Figure 2).
   4. Evaluate the depth of the anesthesia through the respiratory pattern and loss of the withdrawal reflex in the limbs.
   5. Place a disposable surgical drape around the shaved skin and make an incision (2.5 cm) on the albous line with a scalpel, using the xiphoid process as a reference point. Avoid the abdominal wall blood vessel to prevent bleeding.
   6. Put the abdominal retractor in place and observe the abdominal cavity. Using the dissection forceps, extract the left liver lobe and place it on the metal plate (Figure 3A).
      NOTE: In the sham group, only extract the left liver lobe and then return the liver to the abdominal cavity. Suture the abdominal wall and skin with a 3-0 nylon suture.
   7. In the experimental groups with and without CMS, use a scalpel blade and sterile scalpel blade (#15) to perform hepatectomy (approximately 40%) with two cuts. Use a triangular metallic template (1 cm x 1 cm x 1 cm) to perform the hepatectomy (Figure 3B).
   8. To prevent bleeding of the liver, maintain surgical compression with a swab on the edge of the liver for 5 min.
   9. Hydrate the CMS in sterile saline solution for 20 min before the surgical procedure. Implant the CMS in the hepatectomy site with four stitches sutures between the liver tissue and the CMS to prevent displacement of the biomaterial. Use 7-0 non-absorbable polypropylene sutures (Figure 3C).
      NOTE: Do not remove the sutures in a second surgery; sutures can be used as a reference to identify the site of CMS implantation.
   10. Return the liver to the abdominal cavity and suture the abdominal wall and skin with a 3-0 nylon suture. Clean the surgical incision with a surgical iodine-soaked sponge in two rounds. Observe and monitor the vital signs of the animals.

3. Postoperative care

1. Administer meglumine flunixin (2.5 mg/kg) intramuscularly in the hind limb. Administer analgesics as approved by the institutional animal care and use committee.
2. For anesthesia recovery, place the animals in individual polycarbonate boxes with laboratory animal bedding in a noise-free area with temperature control (23 °C).
3. Observe the recovery of the animals and monitor their water and food consumption for 2 h. Monitor animals post operatively as approved by the institutional animal care and use committee.

4. Evaluation of liver function in serum

1. Collect blood samples (500 µL) from the lateral tail veins of the anesthetized animals before the surgical procedure (baseline values) and at evaluation days 3, 14, and 21.
2. Centrifuge the blood samples at 850 × g/10 min at room temperature (23 °C); separate the serum and store it at -80°C until use.
3. Perform a panel of liver function tests: serum albumin (ALB), alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin (TB), and direct bilirubin (DB) (Table 1).

5. Euthanasia and tissue management

1. Anesthetize the animals for each evaluation (days 3, 14, and 21) by following the protocol described above.
2. Euthanize via methods approve by the institutional animal care and use committee.
3. Perform an incision (5-6 cm) on the albous line with a scalpel to observe the organs of the abdominal cavity and take photographs of the hepatectomy area of the sham and the experimental groups, with and without the CMS.
4. Take liver samples (2 cm x 2 cm; 0.40-0.45 g) from all the study group animals and place them in a 4% formaldehyde solution for 24 h for subsequent histological evaluation.

6. Histological analysis

1. Preserve the liver tissues in 4% formaldehyde and dehydrate the tissue using a series of alcohol concentrations (60%, 70%, 80%, 90%, 100%); place them in xylene (1h) and embedded them in paraffin¹⁶.
2. Cut the paraffin blocks with a microtome into 4 µm-thick sections for the preparation of eight slides.
3. Perform H&E and Masson's trichrome staining¹⁶.
4. Observe the stained sections under a light microscope to select the representative areas of the liver, with and without CMS. Obtain photomicrographs at 4x, 10x, and 40x magnification and process the images using appropriate software¹⁸ (see the Table of Materials).

Results

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...

Discussion

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 potential^2,4,13. The removal of a portion of the liver is a critical step in this procedure because of the ...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anionic detergent	Alconox	Z273228
Biopsy cassettes	Leica	3802453
Camera DMX	Nikon	DXM1200F
Centrifuge	Eppendorf	5424
Chlorhexidine gluconate 4%	BD	372412
Cover glasses 25 mm x 40 mm	Corning	2980-224
Eosin	Sigma-Aldrich	200-M	CAS 17372-87-1
Ethyl alcohol, pure	Sigma-Aldrich	459836	CAS 64-17-5
Flunixine meglumide	MSD	Q-0273-035
Glass slides 75 mm x 25 mm	Corning	101081022
Hematoxylin	Merck	H9627	CAS 571-28-2
Hydrochloric acid 37%	Merck	339253	CAS 7647-01-0
Ketamine	Pisa agropecuaria	Q-7833-028
Light microscope	Nikon	Microphoto-FXA
Microsurgery stereomicroscope	Zeiss	OPMI F170
Microtainer yellow cape	Beckton Dickinson	365967
Microtome	Leica	RM2125
Model animal: Wistar rats	Universidad Nacional Autónoma de México
Nylon 3-0 (Dermalon)	Covidien	1750-41
Polypropylene 7-0	Atramat	SE867/2-60
Povidone-iodine10% cutaneous solution	Diafra SA de CV	1.37E+86
Scanning electronic microscope	Zeiss	DSM-950
Sodium hydroxide, pellets	J. T. Baker	3722-01	CAS 1310-73-2
Software ACT-1	Nikon	Ver 2.70
Stereomicroscope	Leica	EZ4Stereo 8X-35X
Sterrad 100S	Johnson and Johnson	99970
Surgipath paraplast	Leica	39601006
Synringe of 1 mL with needle (27G x 13 mm)	SensiMedical	LAN-078-077
Tissue Processor (Histokinette)	Leica	TP1020
Tissue-Tek TEC 5 (Tissue embedder)	Sakura Finetek USA	5229
Trichrome stain kit	Sigma-Aldrich	HT15
Unicell DxC600 Analyzer	Beckman Coulter	BC 200-10
Xylazine	Pisa agropecuaria	Q-7833-099
Xylene	Sigma-Aldrich	534056	CAS 1330-20-7