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 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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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
- Obtain the condyle from bovine femur from a slaughterhouse certified by health and agricultural authorities of Mexico.
- 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.
- 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.
- Dry the condyle fragments for 3 h using filter paper (0.5 mm).
- 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).
- 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). - 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.
- Use a stereo-microscope15 to analyze the structural properties of the CMS (size of pores, pore formation, and porous interconnection) (Figure 1B).
- Use a scanning electron microscope16 to analyze the rough surface of the CMS trabeculae (Figure 1C).
- Use the dissection instrument for blending and stretching to evaluate the mechanical changes (plasticity and flexibility) of the CMS (Figure 1D).
- 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.
- Demineralize the fragments in 100 mL of 0.5 M HCl for 10 min with constant agitation. Remove the HCl.
2. Preparation of the surgical area and handling and preparation of the animal model
- 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)
- 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.
- 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. - 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.
- Place the animal on a warm plate in the decubitus dorsal position, with the neck hyperextended to maintain a permeable airway (Figure 2).
- Evaluate the depth of the anesthesia through the respiratory pattern and loss of the withdrawal reflex in the limbs.
- 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.
- 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. - 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).
- To prevent bleeding of the liver, maintain surgical compression with a swab on the edge of the liver for 5 min.
- 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. - 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.
- Administer ketamine (35 mg/kg) and xylazine (2.5 mg/kg) intramuscularly in the hind limb.
3. Postoperative care
- Administer meglumine flunixin (2.5 mg/kg) intramuscularly in the hind limb. Administer analgesics as approved by the institutional animal care and use committee.
- For anesthesia recovery, place the animals in individual polycarbonate boxes with laboratory animal bedding in a noise-free area with temperature control (23 °C).
- 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
- 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.
- 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.
- 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
- Anesthetize the animals for each evaluation (days 3, 14, and 21) by following the protocol described above.
- Euthanize via methods approve by the institutional animal care and use committee.
- 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.
- 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
- 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.
- Cut the paraffin blocks with a microtome into 4 µm-thick sections for the preparation of eight slides.
- Perform H&E and Masson's trichrome staining16.
- 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).
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Representative 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 liver is a friable and soft organ, the surgical procedure performed in this protocol ensured that the CMS remained in place (Figure 2 and Figure 3A-C). The partial hepatectomy of 40% of the left lobe enabled a portion of the organ to be assessed, keeping the rest of the liver intact, so that the changes in the implant and the native parenchyma could be compared. Additionally, we obtained blood samples to evaluate liver function before and after CMS implantation.
There were no differences in baseline concentrations of the biochemical parameters between the sham group and the experimental group with and without CMS implantation at 3 and 14 days; they remained within the reference values (ALB: 0.43-2.41 g/dL; ALP: 134-357.3 U/L; ALT: 41-83.1 UI/L; AST: 61.4-276.2 UI/L; TB: 0.01-0.43 mg/dL; DB: 0.1 mg/dL)20. In addition, there were no differences in the albumin, ALP, ALT, AST, TB, and DB levels between the sham group and the experimental groups at 21 days, indicating that the CMS does not disrupt liver function (Table 1).
During euthanasia, exploratory laparotomy revealed the typical color and correct size and shape of the liver. No inflammation or infection was observed at the implantation site in any of the cases. On day 3, there were no changes in the organ in relation to the abdominal cavity (Figure 4A). In a portion that contained liver tissue and CMS, blood was observed to have infiltrated the CMS, with incipient adherent omental fat (Figure 4B). On day 14, the amount of fat increased; there was blood vessel neoformation and the integration of the CMS into recipient liver tissue but no changes in the small intestine or organs of the abdominal cavity (Figure 5A). Omental fat was higher in the anterior zone (Figure 5B) compared with the visceral zone (Figure 5C) in the CMS implantation site. On evaluation day 21, CMS incorporation into the liver was more evident (Figure 6A). The increase in omental fat could be used as an indicator of the progress of regeneration, as it is a source of mesenchymal stem cells21. Moreover, at 21 days, the liver presented dense areas that corresponded to degradation and absorption, changing the size and original shape (Figure 6B).
To analyze the microstructure of the liver with the CMS implant, we performed histological analysis of a representative fragment of tissue from the implantation site, which was compared with tissue from the right liver lobe (control). Biopsies performed on days 3, 14, and 21 were processed and subjected to H&E and Masson's trichrome staining. The normal structure of the liver parenchyma was observed in the sham group at day 21 (Figure 7A and Figure 7E). On day 3, the histological analysis showed that the presence of the CMS in the liver did not promote a foreign body reaction, and the conspicuous presence of lax connective tissue was observed (Figure 7B and Figure 7F). On days 14 and 21, the lax connective tissue was more abundant, with the CMS trabeculae surrounded by hepatocytes, suggesting that hepatocytes had migrated to the CMS (Figure 7C and Figure 7G; Figure 7D and Figure 7H). The results also showed areas of hepatocytes that were irrigated by a vessel (Figure 8A). A close inspection revealed that the hepatocytes that had adhered to the CMS trabeculae were sometimes surrounded by lax connective tissue (Figure 8B-D). Two double-blinded independent pathologists performed the histopathological analysis. Immunohistochemistry and polymerase chain reaction-based assays are underway to evaluate the different proteins and genes related to the hepatic regeneration process.
Thus, the macroscopic and microscopic observations and the biochemical values support our proposition that the CMS is an ideal biomaterial for supporting and promoting liver regeneration. Nevertheless, CMS implantation must be evaluated for more than 21 days to determine its absorption time and complete liver tissue restoration. In addition, studies that evaluate the different proteins and genes related to hepatic regeneration in the presence of the CMS should be conducted.
Figure 1: Bone specimen and the CMS. (A) A triangular sample of the bone specimen was utilized to prepare the CMS. (B) A detailed view of the pores and interconnection or trabecular connection of the CMS, as observed by electron microscopy. (C) The CMS, as seen under a stereo-microscope. (D) CMS manipulation with an instrument was carried out to verify the modification of mechanical properties. Abbreviation: CMS = collagen matrix scaffold. Please click here to view a larger version of this figure.
Figure 2: Preparing the animal for surgery. The animal is in the decubitus dorsal position, showing the shaved abdominal area, prepared for hepatectomy and CMS implantation. Abbreviation: CMS = collagen matrix scaffold. Please click here to view a larger version of this figure.
Figure 3: Hepatectomy of the left lobe of the liver. (A) The left lobe was extracted and placed on the metallic plate to perform the hepatectomy. (B) Hepatectomy of 40% of the left lobe was carried out in a triangular shape, like the CMS. (C)The area of the hepatectomy is replaced by the CMS. Abbreviation: CMS = collagen matrix scaffold. Please click here to view a larger version of this figure.
Figure 4: Day 3 of evaluation. (A) Macroscopic observation of the implantation site on evaluation day 3. The liver implanted with (filled star) the CMS (dotted line) did not change in color or size. The small intestine (empty star) and the rest of the abdominal structures showed no alterations. (B) Sample of the liver (filled star) and the CMS (white arrow) covered with omental fat (red arrow). Abbreviation: CMS = collagen matrix scaffold. Please click here to view a larger version of this figure.
Figure 5: Day 14 of evaluation. (A) Macroscopic observation of the implantation site on evaluation day 14. The liver tissue (filled star) with the CMS did not change in color or size. The small intestine (empty star) and the rest of the structures and organs of the abdominal cavity showed no alterations. Sample of the liver with the implanted CMS: (B) anterior view; (C) posterior/visceral view. Omental fat (red arrow) covering the area. The CMS was integrated into the liver tissue. The omental fat (red arrow) mainly covers the area in the anterior view. The dotted line indicates the site of CMS implantation; sutures (blue thread). Abbreviation: CMS = collagen matrix scaffold. Please click here to view a larger version of this figure.
Figure 6: Day 21 of evaluation. (A) Macroscopic observation of the implantation site at evaluation day 21. The recipient liver (filled star) implanted with the CMS (dotted line) did not change color or size. The small intestine (empty star) and the rest of the abdominal structures showed no alterations. (B) Sample of the liver with the implanted CMS (dotted line) showing changes in size and shape. Abbreviation: CMS = collagen matrix scaffold. Please click here to view a larger version of this figure.
Figure 7: Histology of the liver and the liver with CMS. (A) Normal liver (SHAM) showing the characteristic tissue architecture. (B) Day 3 of CMS implantation showing lax connective tissue in the trabeculae of the CMS (dashed ovals). (C) Transition zone between the native liver (right side) and the CMS (left side), with the trabeculae of the CMS (arrow). (D) Native liver in contact with the CMS (dotted lines) at day 21. (E) Normal liver with Masson's trichrome stain. (F) The CMS implanted in the liver; invasion of lax connective tissue at day 3 (dashed oval). (G) On day 14, native liver with the CMS trabeculae, showing an area of hepatocytes (filled star) outside the native liver, indicating that the native hepatocytes had migrated through the CMS. (H) On day 21, the native hepatocytes have migrated toward the CMS (arrow). A and E: Scale bars = 200 µm: 4x, B-D and F-H: Scale bars = 100 µm: 10x. A-D: H&E staining; E-H: Masson's trichrome staining. Abbreviations: CMS = collagen matrix scaffold; H&E = hematoxylin and eosin. Please click here to view a larger version of this figure.
Figure 8: Histology of the liver and CMS. (A) Pool of hepatocytes (white arrow) present in the trabeculae of the CMS (black arrow). The hepatocytes are irrigated by a vessel (filled star). (B) Hepatocytes (white arrow) have migrated and adhered to the trabeculae of the CMS (black arrow). (C) A group of hepatocytes (dashed circle) are observed between two trabeculae (black arrows) of the CMS. (D) Hepatocytes have adhered to trabeculae (black arrows) of CMS in the presence of lax connective tissue (dashed oval). A and C: Scale bars = 100 µm: 10x. B and D: Scale bars = 20 µm: 40x. Abbreviation: CMS = collagen matrix scaffold. Please click here to view a larger version of this figure.
| Days | SHAM | Hepatectomy | Implantation CMS | |
| ALB (g/dL) | 3 | 1.4±0 | 1.25±0.07 | 1.4±0 |
| 14 | 1.3±0.5 | 1.85±0.07 | 1.8±0.14 | |
| 21 | 1.3±05 | 1.6±0.1 | 1.7± 0 | |
| ALT (IU/L) | 3 | 73±1.4 | 3.5±0.7 | 54.6±6.4 |
| 14 | 84±0 | 59.5±4.9 | 57±4.2 | |
| 21 | 57±0 | 73.5±14.8 | 73.5±14.8 | |
| AST (IU/L) | 3 | 106±1.4 | 80±6.6 | 90±1.4 |
| 14 | 127±5.5 | 94±21.2 | 83.5±17.6 | |
| 21 | 123.7±27.3 | 92.5±24.7 | 101±31.1 | |
| TB (mg/dL) | 3 | 0.33±0.05 | 0.25±0.07 | 0.3±0 |
| 14 | 0.5±0 | 0.2±0 | 0.15±0 | |
| 21 | 0.3±0 | 0.4±0 | 0.4±0.14 | |
| DB (mg/dL) | 3 | 0.1±0 | 0.05±0.07 | 0.1±0 |
| 14 | 0.1±0 | 0.1±0 | 0.1±0 | |
| 21 | 0.1±0 | 0.1±0 | 0.1±0 |
Table 1: Liver function. Liver function tests were performed for the sham and experimental groups with and without CMS at 3, 14, and 21 days. Abbreviations: CMS = collagen matrix scaffold; ALB = Albumin; ALP = Alkaline phosphatase; ALT= Alanine aminotransferase; AST = Aspartate aminotransferase; TB = Total bilirubin; DB = Direct bilirubin.
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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 potential2,4,13. The removal of a portion of the liver is a critical step in this procedure because of the profuse bleeding of this vascularized organ. Therefore, hemostasis of the surgical bed must be performed to prevent this complication. Furthermore, the binding of the liver tissue to the CMS, which is essential for ensuring tissue-biomaterial interaction, is facilitated through the use of sutures. However, it must be done carefully to avoid tearing the liver and causing later bleeding.
Although the biomaterial proposed herein is of bovine origin (xenogenic), there are no data of biomaterial reaction in rats, making selective hepatectomy possible. In contrast, the 2/3 hepatectomy model has been shown to cause the death of the animals because of the removal of a large amount of liver tissue14. Moreover, we developed a stainless steel metallic template because we had difficulty standardizing the size in the hepatectomy and the CMS. Sterilization of the CMS was challenging because the existing techniques modified the structure of collagen and its biochemical properties. Hence, sterilization by heat, gamma radiation, and ethylene oxide was explored as sterilization methods before determining that the plasma of hydrogen peroxide was the optimal technique13.
It is essential to determine the maximum size of the CMS that could be implanted in the liver and evaluate the biological response at a systemic level. Further, it is necessary to optimize and investigate the efficacy of this technique in a larger animal species. Furthermore, it is important to investigate the effects of the CMS implantation in the liver for a longer period (>30 days), which will allow the assessment of the extent of the biosorption of the CMS and the regeneration of the hepatic tissue. Further, the effects of the CMS implantation must be examined in animal models with liver damage. This implantation method has opened the door to strategies exploring the restoration of the shape and volume of resected tissue, thereby reducing anesthesia, surgical duration, and recovery time4,13.
The CMS was obtained from a natural source and preserved its physical and chemical properties compared to synthetic biomaterials made using complex methodologies, such as bioprinting or electrospinning, which are not biocompatible or bioabsorbable2,3. The primary component of this CMS is collagen type I, which is the main protein of the ECM that enables cell adhesion and proliferation22. Additionally, the trabeculae of the CMS pores enable cell migration and the continuous flow of growth factors, blood, and other mediators of the regeneration process. According to the tissue to be repaired, this research group has experience designing the CMS in different shapes, preserving its 3D structure. For example, cylindrical CMS were implanted in dog urethra and bile duct in pigs and yielded promising results in tissue regeneration23,24.
The implantation of this CMS could be an alternative treatment to stimulate tissue regeneration and restore the fibrogenesis-fibrolysis balance in liver cirrhosis due to different etiologies (e.g., virus, alcohol, metabolic factors). Proliferation, migration, and inflammation assays must be performed to identify the molecular and cellular mechanisms triggered by the CMS. In conclusion, this paper describes a reproducible procedure for hepatectomy as well as the examination of the regeneration process through the xenoimplantation of biomaterials.
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Disclosures
The authors declare that they have no competing financial interests.Benjamín León-Mancilla is a doctoral student from the Programa de Doctorado en Ciencias Biomédicas, Universidad Nacional Autónoma de México (UNAM) and he received DGAPA-UNAM fellowship.
Acknowledgments
The authors wish to thank the personnel of the Laboratory Animal Facility of the Experimental Medicine Unit, Nurse Carolina Baños G. for technical and surgical support, Marco E. Gudiño 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.
Materials
| 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 |
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