Name: evaluation of stem cell therapies in a bilateral patellar tendon injury model in rats
Uploaded: 2018-03-30T14:00:00
Description: Watch this Scientific Journal Video about Evaluation of Stem Cell Therapies in a Bilateral Patellar Tendon Injury Model in Rats at JoVE.com

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).

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
<ol>
	<li>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 &#176;C during transfer until pro...

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...

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&#39;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&#39;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&#39;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.

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evaluation of stem cell therapies in a bilateral patellar tendon injury model in rats

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.

In the current study, results are presented as mean &#177; 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...

Watch this Scientific Journal Video about Evaluation of Stem Cell Therapies in a Bilateral Patellar Tendon Injury Model in Rats at JoVE.com

Evaluation of Stem Cell Therapies in a Bilateral Patellar Tendon Injury Model in Rats

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.

Regenerative medicine provides novel alternatives to conditions that challenge traditional treatments. The prevalence and morbidity of tendinopathy across ...

The overall goals of this bilateral rat model are to optimize the use of animals and evaluate treatment candidates for tendon defects. This method can help answer any key questions in orthopedic field. Such as evaluation of cellular therapy on tendinopathy.The main advantage of this model is that it controls interindividual variation, thereby, reducing the number of animals required to detect differences between treatments. Visual demonstration of the model is critical because the creation of bilateral standardized patella tendon defects in rats is difficult to learn. The model requires a good knowledge of the local anatomy, strong basic surgical skills, and precision while working on small structures.To begin wash a harvested equine umbilical cord twice in a 50 milliliter tube using room temperature PBS containing antibiotics. Next, transfer the tissue to a 150 millimeter plate and section it into two inch long fragments. Then transfer the fragments to a 50 milliliter tube and wash them with PBS containing antibiotics to remove the blood.Do this two or three times at room temperature. Next, cut the fragmented parts of the umbilical cord longitudinally to expose the vessels that run its length. Using forceps and scissors, remove these vessels, which include two arteries and a vein.Now, from two or three fragments, collect the jelly-like matrix surrounding the vessels. Scrape it away with a scalpel. Then mince the jelly-like matrix, also known as Wharton's Jelley, into fine pieces.Next, transfer the jelly into a 50 milliliter tube with 15 milliliter of 0.1%collagenase type 1A in PBS and incubate the tissue at 37 degrees Celsius with gentle shaking for three to four hours until it is completely dissolved. Next, centrifuge the digested tissue and aspirate the supernatant. Now, we suspend the digested tissue in 15 milliliters of PBS and mix by pipetting.Then, repeat this wash with PBS three times using a slower centrifugation. After the last wash, resuspend the cells in 15 milliliters of PBS and then use a 100 micron cell strainer to filter the cells and centrifuge for five minutes at 150 times g to collect the cells. Resuspend the cells in 10 milliliters of freshly made culturing medium at 37 degrees Celsius.Transfer the suspension into a 25 square centimeter culture flask and incubate the cells, changing the medium every third day. When the cells reach 70 to 80%confluence, passage them, aspirate the medium, and wash the cells twice with room temperature PBS without antibiotics. Then use three milliliters of 0.25%trypsin edta to detach the cells.End the reaction with a six milliliter addition of warm medium. Collect the suspended cells and pellet them. Discard the supernatant and resuspend the pellet in one milliliter of medium.To complete the passage, reseed a tissue culture flask with 5, 000 cells per square centimeter and continue growing the culture. To begin, prepare fresh Chitosan solution. Then load each well of a 12 well plate with 500 microliters of solution.Swirl the plate to distribute the solution over all surfaces. Then, dry the plate under a laminar flow cabinet. After 16 to 24 hours, at thin film of chitosan will form in the plate wells.Neutralize this film by adding one milliliter of 0.5 normal sodium hydroxide to each well. Then, incubate the plate for two hours at room temperature. Later, aspirate the sodium hydroxide from each well and then wash each well with one milliliter of distilled water.Perform three water washes then wash each well with one milliliter of 70%ethanol once. After removing the ethanol wash, replace it with another milliliter of 70%ethanol and incubate the plate overnight in a laminar flow cabinet to sterilize the chitosan film. Then next day aspirate the remaining ethanol from each well and wash each well with PBS three times.Remove the PBS then further sterilize the chitosan film using an overnight direct exposure to ultraviolet light without the lid. Now, to form spheroids of umbilical cord derived mesenchymal stem cells, seed the wells with 20, 000 cells and incubate the plate under normal or hypoxic conditions. Place an anesthetized rat between two 0.5 liter bottles filled with warm water and cover the bottles with a cloth to keep the rat warm while not incurring skin injuries.To reduce risk of hypothermia, cover the body with bubble wrap. Next, to avoid skin trauma, apply hair remover cream over both stifles and use a tongue depressor to remove the cream and hair. Then, tape each limb to the table.Now, scrub the surgical site with chlorhexidine digluconate and rinse it with 70%ethanol. Do this three times and proceed with the surgery. With a sterile number 15 scalpel, incise the skin in a proximal to distal direction on the cranial medial aspect of the stifle.Start the incision about one centimeter proximal to the level of the patella and extend it approximately five millimeters distally to the tibial tubercle. Next, free the underlying subcutaneous tissue with the scalpel and reflect the skin to expose the patellar tendon. Now, align a 0.99 millimeter diameter kirschner wire against the patellar tendon between the distal aspect of the patella to the tibial tuberosity.Then, while keeping the wire stable, use the scalpel to make incisions on both sides of the wire, releasing a portion of tendon about one millimeter wide. Then resect the central section proximally and distally using fine iris scissors. In parallel, have an assistant prepare the clot.To do so, thaw the plasma sample and mix 20 microliters of it with half a million mesenchymal stem cells. Next, in a microcentrifuge tube, mix six microliters of 10%calcium chloride with 100 microliters of thawed plasma, this will activate that plasma. Then, mix the cell suspension into the activated plasma, this will cause the plasma to clot.Now, place the prepared clot within the patellar tendon defect. Limit the manipulation of the clot to minimize fluid release from the clot. Then, using five o polyglactin nine 10 sutures, carefully close the fascia with a cruciate pattern without interfering with the clot.Finish by closing the skin with an intradermal pattern. Cells were isolated from the umbilical cords of six mares and cultured under standard conditions or on chitosan films. The cells cultured on chitosan formed spheroids.The patellar tendon defect model was tested on many rats and a battery of behavioral tests were used to estimate the recovery. By 28 days post operative, the rats had returned to baseline strength after an initial deficit in strength measured at seven days post operatively. Upon histological examination with masson's trichrome stain, moderate arterial and capillary infiltration was noted along with moderate to extensive collagen formation whereas cartilage formation was not supported.Overall, the bilateral patellar tendon defect model limits interindividual variation without significant morbidity. Thus, reducing the number of animals needed for testing. After watching this video, you should have a good understanding of how to create a rat bilateral patellar tendon window defect model and how rats recover from this surgery.Once mastered, the procedure can be done under 30 minutes with three investigators. The first investigator prepares the cell, the second performs the surgery, while the third assists with the surgery and monitors anesthesia. Following this procedure, methods such biomechanical testing, histology and immunohistochemistry can be used in order to compare the effect of treatment on tendon healing.

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