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  • Podsumowanie
  • Streszczenie
  • Wprowadzenie
  • Protokół
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  • Dyskusje
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Podsumowanie

This paper describes the preparation and evaluation of umbilical cord matrix-derived mesenchymal stem cells spheroids with a bilateral patellar tendon defect model in a rat. This model was associated with an acceptable morbidity and was found to detect differences between untreated and treated tendons, and between the two treatments tested.

Streszczenie

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across species, combined with the limited healing properties of this tissue, have prompted the search for cellular therapies and propelled the development of experimental models to study their efficacy. Umbilical cord matrix-derived mesenchymal stem cells (UCM-MSC) are appealing candidates because they are abundant, easy to collect, circumvent the ethical concerns and risk of teratoma formation, yet resemble primitive embryonic stem cells more closely than adult tissue-derived MSCs. Significant interest has focused on chitosan as a strategy to enhance the properties of MSCs through spheroid formation. This paper details techniques to isolate UCM-MSCs, prepare spheroids on chitosan film, and analyze the effect of spheroid formation on surface marker expression. Consequently, creation of a bilateral patellar tendon injury model in rats is described for in vivo implantation of UCM-MSC spheroids formed on chitosan film. No complication was observed in the study with respect to morbidity, stress rising effects, or tissue infection. The total functional score of the operated rats at 7 days was lower than that of normal rats, but returned to normal within 28 days after surgery. Histological scores of tissue-healing confirmed the presence of a clot in treated defects evaluated at 7 days, absence of foreign body reaction, and progressing healing at 28 days. This bilateral patella tendon defect model controls inter-individual variation via creation of an internal control in each rat, was associated with acceptable morbidity, and allowed detection of differences between untreated tendons and treatments.

Wprowadzenie

Tendon injury is one of the most common causes of significant pain and muscle atrophy across species1. In veterinary medicine, tendon and ligament injuries are of special interest in horses, as 82% of all injuries in race horses involve the musculoskeletal system, and 46% of those affect tendons and ligaments2,3. Scar tissue formation affects the biomechanical properties of healed tendons and explains the guarded prognosis for return to athletic use after flexor tendon injuries; re-injury occurs within 2 years in up to 67% of horses treated conservatively4. Regenerative medicine provides novel alternatives to a condition that challenges traditional treatments. Autologous stem cell therapy has produced some encouraging results5,6 but is limited by the morbidity associated with tissue collection, delayed administration due to processing/reprogramming of cells, and the influence of the patient's health status (such as age) on the properties of stem cells7,8. These limitations provide a rationale for investigating allogeneic stem cells as an off-the-shelf alternative. Fetal adnexa-derived cells are appealing candidates because they circumvent the ethical concerns and risk of teratoma formation associated with embryonic stem cells. Among fetal adnexa, umbilical cord matrix (UCM), also named Wharton's Jelly, is abundant and easy to collect.

Regardless of cell source, enhancing stemness is essential to establish a cell bank for allogenic regenerative medicine. From a functional standpoint, stemness can be defined as the potential for self-renewal and multi-lineage differentiation9. Evidence of stemness relies on proliferation and differentiation assays, along with expression of gene markers Oct4, Sox2, and Nanog9. One strategy to enhance stemness relies on the use of biomaterials to serve as void fillers and carriers enhancing proliferation and differentiation of UCM-MSCs. This approach eliminates concerns regarding manipulation of transcriptional factors to reprogram mature cells into induced pluripotent cells. Among biomaterials considered as potential carriers for stem cells, chitosan is appealing for its biocompatibility and degradability10. This natural aminopolysaccharide is formed by alkaline deacetylation of chitin, the second most abundant natural polysaccharide, primarily obtained as a subproduct of shellfish10. We have previously investigated interactions between MSCs and chitosan scaffolds, and observed the formation of spheroids11,12,13,14,15,16. We also reported on the superiority of chondrogenesis on chitosan matrices12,13,14,15,16,17,18. More recently, two independent studies described spheroids formation by adipose tissue and placenta tissue derived MSCs cultured on a chitosan film19,20. This formation of spheroids not only enhanced stemness, but also improved the retention of stem cells after in vivo implantation20.

The prevalence and morbidity of tendinopathy across species have prompted the development of experimental models to study the pathophysiology of tendinopathies and test new therapies such as stem cell injections. In horses, collagenase-induced tendonitis is a common model to demonstrate efficacy using MSCs in tendon repair21. The relevance of this approach is limited, as injections cause acute inflammatory changes, whereas clinical tendinopathies usually result from chronic overstrain22,23. In addition, chemical induction of tendon disease induces a healing response and does not replicate the impaired healing process present in clinical cases22,23. Excision of a segment of the superficial digital flexor tendon has been described as a surgical model of tendonitis in horses24. More recently, a minimally invasive approach was used to restrict the traumatic damage to the central core of the superficial digital flexor tendon25. Surgical models do not simulate the fatigue mechanism that may lead to natural tendon disease, and tend to lack reproducibility in the extent of damage created25. Regardless of the model, the morbidity and cost associated with equine models of tendon diseases are additional limitations, which justify an interest in rodent models as a first step for in vivo evaluation of novel therapies.

One of the main advantages of experimental models in rodents consists of the cost and ability to control inter-individual variability. Rodents can be standardized with respect to various physiological factors due to their rapid growth rates and relatively short life spans, limiting sources of variation and therefore reducing the number of animals required to detect differences. Strategies to induce tendon diseases in rodents have relied on chemical induction, but also on surgical creation of partial tendon defects21. Surgical models may simulate natural tendinopathies better than chemical models, but can lead to higher morbidity and catastrophic failure of the damaged tendon. In that respect, rats seem better candidates than mice for these models, as their size allows creation of larger defects, thereby facilitating evaluation of tissue healing. Sprague-Dawley rats have been used in experimental studies of tendinopathies in four major tendon groups: rotator cuff, flexor, Achilles, and patellar tendons26. Among these, models involving the patellar tendon are especially appealing because of the larger size of this tendon and the ease of accessing it27. The patellar tendon attaches the quadriceps muscle to the tibial tuberosity. Within this extensor mechanism, the patella is a sesamoid bone that directs the action of the quadriceps and delineates the proximal extent of the patellar tendon. The presence of bony anchors at the proximal and distal extents of the patellar tendon facilitates biomechanical tests. Models involving the patellar tendon typically rely on unilateral surgical defects, with a contralateral intact tendon serving as a control28,29. The most common patellar tendon defect model involves excising the central portion (1 mm in width) of the patellar tendon from the distal apex of the patella to the insertion of the tibial tuberosity, while the contralateral patellar tendon is left intact. Measures of outcomes have included histology, non-destructive biomechanical testing or biomechanical testing to failure, ultrasound imaging, ex vivo fluorescence imaging, gross observation, and functional tests28,30,31. Unilateral models do not allow comparison of a proposed treatment with conservative management of a similar injury within the same animal. Similarly, comparison between several treatments requires separate animals. A bilateral model would eliminate inter-individual variations and reduce the number of animals required for a study32. However, bilateral injuries may increase morbidity, and bilateral lameness could impede treatment evaluation. A few studies briefly report the use of bilateral patellar tendon defects in rats but focus on the effects of treatments rather than peri-operative management and morbidity of the model33,34.

This study's long-term goal is to develop a strategy to improve stemness and in vivo survival of UCM-MSCs destined to allogenic transplantation. To achieve this goal, we have recently reported improved stemness of UCM-MSCs by formation of spheroids on chitosan film and incubation under hypoxic environment35. These in vitro properties were associated with improved biomechanical properties of patellar tendon defects treated with conditioned UCM-MSCs. Based on these results, the rat bilateral patellar tendon defect model seems suitable to test candidate treatments for tendon injuries36. The purpose of the study reported here is to provide detailed protocols for isolation and characterization of UCM-MSCs, preparation of a biologic delivery system for stem cells, creation and treatment of bilateral patella tendon defects, and post-operative recovery and evaluation of tissue healing within the defects.

Protokół

All methods described here have been approved by the Institutional Animal Care and Use Committee (IACUC) of Western University of Health Sciences.

1. Isolation and Expansion of MSCs from Equine Umbilical Cord Matrix

  1. Obtain the placenta from an adult mare (pregnant) after observed foaling and aseptically isolate the umbilical cord from the placenta. Keep the umbilical cord in phosphate buffered saline (PBS) with 1% penicillin-streptomycin (P/S) at 4 °C during transfer until processing.
  2. Wash the umbilical cord twice with room temperature PBS with 1% P/S in a 50 mL tube. Section the umbilical cord into 2-inch-long fragments on 150 mm plate and wash in room temperature PBS with 1% P/S in a 50 mL tube two or three times, until most of the blood is rinsed out.
  3. Cut the umbilical cord longitudinally to expose vessels. Remove vessels, including 2 arteries, a vein, and an allantoic stalk from the cord using forceps and scissors. Collect umbilical cord matrix (Wharton's Jelly), which is the jelly-like matrix surrounding vessels, onto a 150 mm plate by scraping with a scalpel, and mince the jelly into fine pieces.
  4. Place Wharton's Jelly from 2–3 umbilical cord fragments (approximately 12–15 g) in a 50-mL tube with 15 mL of 0.1% (w/v) collagenase type IA solution in PBS. Incubate at 37 °C with gentle shaking for 3-4 h until dissolved.
  5. Centrifuge digested tissue at 300 x g for 15 min and aspirate supernatant. Resuspend digested tissue in 15 mL of room temperature PBS with 1% P/S, mix by pipetting, centrifuge at 150 x g for 5 min, and aspirate supernatant (wash). Repeat washing twice more.
  6. Resuspend washed cells in 15 mL of room temperature PBS with 1% P/S, mix by pipetting, strain by with 100 µm cell strainer, centrifuge at 150 x g for 5 min, and aspirate supernatant.
  7. Prepare culture medium (CM) by mixing low glucose Dulbecco's Modified Eagle's Medium (LG-DMEM) with fetal bovine serum (FBS) and 1% P/S. Sterilize the medium by filtration.
  8. Resuspend strained cells in 10 mL of CM pre-warmed to 37 °C, mix by pipetting, transfer into a 25 cm2 tissue culture flask, and incubate in 5% CO2 at 90% humidity and 37.0 °C. Change CM every 3 days until the culture reaches 70–80% confluence, when the culture will be passaged.
  9. To passage the culture, aspirate CM from flask, wash with 5 mL of room temperature PBS twice, and detach with 3 mL of 0.25% trypsin/ethylenediaminetetraacetic acid (EDTA) at 37 °C for 5 min.
  10. Neutralize trypsin/EDTA with 6 mL of CM pre-warmed to 37.0 °C, mix by pipetting, transfer into a 15 mL tube, centrifuge at 150 x g for 5 min, and aspirate supernatant. Resuspend detached cells in 1 mL of CM, mix by pipetting. Count the viable cells using trypan blue and hemocytometer. Re-seed into a 25 cm2 tissue culture flask at 5,000 cells/cm2, and incubate in 5% CO2 at 90% humidity and 37.0 °C.

2. Preparation of Spheroids with UCM-MSCs Cultured on Chitosan Films

  1. To prepare 100 mL of 1% (w/v) chitosan solution, add 1 g of chitosan in 99 mL of distilled water (dH2O), and mix well with a magnetic stirrer.
  2. Add 670 µL of glacial acetic acid and continue mixing until chitosan dissolves and becomes viscous. This usually takes 3–4 h.
  3. Add 500 µL of chitosan solution into each well of the 12-well tissue culture plate, and swirl the plate to distribute chitosan solution evenly and cover all of the bottom surface.
  4. Dry the chitosan solution coated plate under laminar flow cabinet without a cover overnight for 24 h. Once dried, thin film will be formed and adhered to plate.
  5. Neutralize chitosan film by adding 1 mL of 0.5 N sodium hydroxide (NaOH) solution into each well and incubate for 2 h at room temperature. Aspirate NaOH from each well and wash each well with 1 mL of dH2O three times. Wash each well with 1 mL of 70% ethanol (EtOH) once.
  6. To sterilize chitosan film, add 1 mL of 70% EtOH into each well and incubate overnight in laminar flow cabinet. Aspirate remaining EtOH from each well in the following day. Wash each well with 1 mL of sterile PBS three times. Sterilize chitosan film with ultraviolet light under laminar flow cabinet overnight without a cover.
  7. To form spheroids of UCM-MSCs, seed expanded and passaged cells into each well at 5,000 cells/cm2, and incubate in 5% CO2 at 90% humidity and 37.0 °C.
    NOTE: Cells can be incubated under hypoxia or normoxia, depending on the investigator's interest.

3. Expression of Surface Markers Analyzed via Flow Cytometry

  1. Preparation of a single cell suspension
    1. Standard plate
      1. Remove medium from each well and wash twice with room temperature PBS.
      2. Detach cells with 500 µL of a cell dissociation reagent into each well of a 12-well plate for 5–10 min at room temperature.
      3. After incubation, add 1 ml of staining buffer (PBS containing 0.5% BSA) into each well and mix by pipetting to detach cells.
      4. Transfer cell suspension into 15 mL conical tube and centrifuge at 150 x g for 5 min.
    2. Chitosan plate
      1. Collect all spheroids by aspirating medium using a pipette with a 1,000 µL tip. Transfer collected medium into a 15-mL conical tube. After collecting medium, wash well by adding 1 mL of PBS, and transfer washed PBS into the same 15 mL conical tube.
      2. Centrifuge spheroids at 150 x g for 5 min and remove supernatant.
      3. Add 500 µL of the cell dissociation reagent (e.g., accutase) and incubate for 5–10 min at room temperature. Mix by pipetting using a 1 mL tip until spheroids dissociate and are no longer visible.
      4. Add 1 mL of staining buffer into the single cell suspension and centrifuge at 150 x g for 5 min.
  2. Washing cells
    1. Remove the supernatant from the conical tube and re-suspend the cells in 3 mL of cold staining buffer. Keep cells on ice throughout experiment from this step.
    2. Centrifuge at 300 x g for 5 min (washing).
    3. Wash twice.
  3. Staining cells
    1. Centrifuge at 300 x g for 5 min and remove supernatant.
    2. Count viable cells using a trypan blue and hemocytometer. Re-suspend 1 x 106 cells in 50 µL blocking buffer (PBS containing 10% horse serum) and incubate for 30 min on ice.
    3. Add 10 µL fluorescein isothiocyanate (FITC) conjugated antibodies (CD44, CD90, CD105, CD34, major histocompatibility complex (MHC) class II, or isotype control for each antibody) and 40 µL of staining buffer, then incubate protected from light for 1 h on ice.
    4. Add 3 mL of cold staining buffer and mix, centrifuge at 300 x g for 5 min, and aspirate supernatant (washing).
    5. Repeat washing twice.
    6. Re-suspend the cell pellet in 0.5 mL of staining buffer
    7. Add 5 µL of 7-AAD (viability dye) and incubate for 30 min on ice
    8. Analyze stained cell samples by flow cytometer. Exclude debris by their smaller SSC and FSC, and identify viable cells with lower uptake of 7-AAD. Plot FL1 and FL2 on the y- and x- axes, respectively. Use isotype control to create a gate above the diagonal line. Measure the percentage of positively stained cells in the area. Count at least 20,000 events/sample (Supplementary Figure 1).
    9. Measure the percentage of cells stained with antibodies by gating viable cells and auto-fluorescence, and subtract percentage of cells stained with isotype control.

4. Bilateral Patellar Tendon Defect Model in the Rat

  1. Select Sprague-Dawley rats (adult male, 4–5 months old, body weight 350–375 g). Note the relatively large size of the rats used for this model.
  2. Anesthetize and apply artificial eye lubricant the rat with 8% sevoflurane in 2 L/min 100% oxygen delivered via mask, until disappearance of pinch-toe reflex in the induction chamber.
  3. Administer an intramuscular injection of Meloxicam (1 mg/kg) as preemptive analgesia.
  4. Place the rat between two 0.5 L water bottles filled with warm water and covered with a cloth to maintain body temperature and position, while preventing skin injury. Tape each extremity to the table. To reduce risk of hypothermia, cover the body with bubble wrap (Figure 1).
  5. Maintain anesthesia with continuous flow of 5% sevoflurane in 1 L 100% oxygen mixture via nose cone, with the animal on dorsal recumbency on water heating pad.
  6. To avoid skin trauma, do not clip the surgical sites. Instead, apply hair remover cream over both stifles. Use a tongue depressor to remove the cream and hair.
  7. Scrub the surgical site with chlorhexidine digluconate scrub, and rinse with 70% ethanol 3 times.
  8. Incise the skin with a sterile #15 scalpel blade in a proximal to distal direction, on the craniomedial aspect of the stifle. Start the incision about 1 cm proximal to the level of the patella, and extend it approximately 5 mm distal to the tibial tubercle.
  9. Reflect the skin to expose the patellar tendon by freeing the underlying subcutaneous tissue with a #15 scalpel blade.
  10. Using the #15 scalpel blade, excise the central third of each patellar tendon (1 mm) from the distal aspect of the patella to the tibial tuberosity.
    1. Align a 0.99 mm-diameter Kirschner wire against the tendon as a template to standardize the size of the defect in each limb (Figure 2).
    2. Make 2 full-thickness incisions on each side of the Kirschner wire with a #15 scalpel blade to isolate the central portion of the tendon (Figure 3). Resect the central section proximally and distally with fine Iris scissors (Figure 4).
    3. Before closing the fascia of the stifle, insert the clot (mixed cell suspension and ACP) for the appropriate treatment groups as described in 5.5. Close the fascia with a cruciate pattern and skin with an intradermal pattern using 5-0 polyglactin 910 sutures (Figure 5).
  11. Repeat the procedure on the contralateral stifle.
    NOTE: One defect is randomly assigned to a treatment (stem cells conditioned on chitosan or cultured on standard plates). The contralateral defect is left empty, to serve as internal control.
  12. After surgery, administer 4 tablets of enrofloxacin (2 mg/tablet, orally, once daily) and a tablet of meloxicam (2 mg/tablet, orally, once daily) for 7 days. These doses were recommended by a laboratory animal veterinarian and approved in the IACUC protocol.

5. Delivery of MSCs within the Patellar Tendon Defect

  1. Anesthetize a healthy rat which is not used for patellar tendon defect creation with 8% sevoflurane in 2 L/min 100 % oxygen delivered via mask, until disappearance of pinch-toe reflex in the induction chamber.
  2. Collect 5 mL blood by cardiac puncture from the anesthetized Sprague-Dawley rat (adult male, 4–5 months old, body weight 350–375 g), in a 5 mL syringe with a 20 G needle containing 1 mL of acid-citrate-dextrose (5:1 v/v).
  3. Euthanize the rat after cardiac puncture and blood collection, by intracardial injection of pentobarbital (100 mg/kg), while under the same anesthesia.
  4. Centrifuge the sample at 350 x g for 15 min at room temperature. Transfer supernatant and store aliquots (120 µL each) at -20 °C until use.
  5. Immediately before in vivo implantation, thaw the above aliquot and mix 20 µL with 0.5 x 106 MSCs (from either 1.9 or 3.1.2.1) detached from standard plates using trypsin/EDTA, or collect from chitosan plates by flushing (with PBS/medium).
  6. Add 6 µL of 10% calcium chloride (CaCl2) to the remaining 100 µL of thawed plasma in a well of 96-well plate to activate the plasma and induce formation of a clot (activated conditioned plasma: ACP).
  7. Mix cell suspension (20 µL) and ACP (100 µL) to form a clot (Figure 6). Place the clot within the patellar tendon defect created before closing the fascia of the stifle (see step 4.10.3).

6. Functional Outcome

  1. Monitor rats twice a day for signs of pain, based on the rat grimace scale (RGS)37,38 and swelling over the implantation sites.
    NOTE: A scoring system (0–6) was developed to evaluate ambulatory function based on 3 activities, including timed hind limb standing with forelimbs supported (0-3), timed unassisted hind limb standing (0–2), and the ability to climb a 17 cm plastic cage wall (0–1) (Table 1).
  2. Evaluate rats for the two hind limb standing activities in the morning and in the evening of each time point to calculate an average score.
  3. Evaluate rats for the ability to successfully climb a 17 cm plastic cage wall with both hind limbs reaching the top.

7. Gross Appearance and Histopathology of the Patellar Tendon

  1. Euthanize rats at 7 days (to evaluate inflammation) or 28 days (to evaluate tissue healing) post treatment by intracardial injection of pentobarbital (100 mg/kg) under anesthesia with 8 % sevoflurane and 2 L 100% oxygen delivered via mask.
  2. Harvest patella-tendon-tibia tuberosity units by scalpel and scissors after euthanasia. Remove soft tissues and ligaments around the stifle, except for the patellar tendon.
  3. Examine each specimen for gross appearance and thickening of the tendon (Figure 7).
  4. Orient the specimen by placing a surgeon's knot of 5.0 Polydioxanone on the proximal and lateral aspect of the tendon. Fix each specimen in 10% neutral buffered formalin solution and obtain transverse sections (5 µm) from the mid-portion of each tendon.
  5. Stain the sections using hematoxylin and eosin, and Masson's Trichrome staining following standard protocols.
  6. Examine section to detect the presence of hematoma within the defect, as well as pathological changes such as inflammation.
  7. Evaluate the histological score of sections using the previously published scoring system, based on collagen grade, degree of angiogenesis, and cartilage formation (Table 2)39.

Wyniki

In the current study, results are presented as mean ± SD (standard deviation). Cells were isolated from the umbilical cords of 6 mares, and percentage of isolated cell lines expressing each cell surface marker under standard or chitosan conditioning were compared with a Friedman test, as a non-parametric analysis of variance with repeated measures. For tendon defect model creation, 8 rats were used for 7 days post-surgery assessment and 12 rats were used for 28 days assessment. Resul...

Dyskusje

Equine cells were selected for this project because we eventually intend to test candidate approaches in the management of natural tendinopathies in horses. Indeed, tendon injuries in horses are appealing as natural models of tendinopathy in man because of the biological similarity between the equine superficial digital flexor and Achilles tendon in humans41. The cell surface markers CD44, CD90, CD105, CD34, and MHC II were selected for immunophenotyping of cells, in accordance with the criteria r...

Ujawnienia

The authors have no conflict of interest to disclose.

Podziękowania

The authors would like to acknowledge Dr. Su, PhD, for her statistical analysis of the data. The authors also thank Dr. McClure, DVM, PhD DACLAM, for her advice on the anesthesia and pain management protocols used in the study. This project was supported by grants from Western University of Health Sciences Office of the Vice President for Research (12678v) and USDA Section 1433 funds (2090).

Materiały

NameCompanyCatalog NumberComments
PBS 10xHycloneSH30258.01Consumable
Collagenase type IAWorthingtonLS004197Consumable
DMEM low glucoseHycloneSH30021.FSConsumable
Fetal Bovine SerumHycloneSH30910.03Consumable
Penicillin/Streptomycin 100xHycloneSV30010Consumable
Trypsin 0.25%HycloneSH30042.01Consumable
AccutaseInnovative Cell TechnologiesAT104Consumable
Trypan blueHycloneSV30084.01Consumable
Dimethyl SulfoxideSigmaD2650Consumable
ChitosanSigmaC3646Consumable
Sodium HydroxideSigmaS8045Consumable
Bovine Serum AlbuminHycloneSH30574.01Consumable
Round bottom polystyrene tubeCorning149591AConsumable
Mouse anti-horse CD44 (FITC)AbD serotecMCA1082FConsumable
Mouse anti-rat CD90 (FITC)AbD serotecMCA47FTConsumable
Mouse anti-horse MHC-II (FITC)AbD serotecMCA1085FConsumable
Mouse IgG1 (FITC) - Isotype ControlAbD serotecMCA928FConsumable
Mouse monoclonal [SN6] to CD105 (FITC)abcamab11415Consumable
Mouse IgG1 (FITC) - Isotype Controlabcamab91356Consumable
Mouse anti-human CD34 (FITC)BDBDB560942Consumable
Mouse IdG1 kappa (FITC)BDBDB555748Consumable
7-AADBDBDB559925Consumable
BD Accuri C6 Flow CytometerBDEquipment
Vacutainer 5 mLMed Vet InternationalRED5.0Consumable
Acid-citrate-dextroseSigmaC3821Consumable
Calcium ChlorideSigmaC5670Consumable
SevofluraneJD Medical60307-320-25Consumable
RatsCharles RiverStrain code: 400Experimental animal
Rat surgical kitHarvard apparatus728942Equipment
Surgical Blade #15MEDLINEMDS15115Consumable
Rat MD's Baytril (2 mg/Tablet),
Rimadyl (2 mg/Tablet)		Bio ServF06801Consumable
Polyglactin 910, 5-0EthiconJ436GConsumable
Eosin alchol shandonThermo scientific6766007Consumable
Harris HematoxylinThermo scientific143907Consumable

Odniesienia

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