Mesenchymal Stem Cell Allograft Improved Pain Management in Dogs with Osteoarthritis ()
1. Background
Osteoarthritis is the most common degenerative joint disease characterized by articular cartilage damage, particularly affecting the hands and knees [1] . Its occurrence is age-related and is the main cause of disability in the elderly [2] . Obesity and joint overuse are important factors leading to the degeneration of articular cartilage [3] . Its incidence will continue to increase with the aging of the population and the increase in the proportion of the obese population [4] . The pathogenesis of osteoarthritis is complicated. During the pathogenesis, it is accompanied by the degeneration of articular cartilage and the formation of osteophytes. The resulting discomfort and pain severely affect the quality of life for patients. The current treatment of this disease includes early and mid-term treatments such as exercise, weight loss, anti-inflammatory and analgesic drugs, and finally joint replacement. These treatments have limitations, and there is no effective method to prevent the occurrence and progression of osteoarthritis [5] .
In recent years, stem cell therapy has been applied to the treatment of osteoarthritis. Due to the characteristics of easy acquisition and rapid proliferation of stem cells, the method of using stem cells to replace diseased tissues has been widely studied, such as degenerative diseases, tumors, trauma, etc., which is called regenerative therapy [6] . Mesenchymal stem cells (MSCs) are an important part of stem cell regeneration therapy, which have been used in clinical trials of various diseases such as ulcerative colitis, diabetes and its complications, and liver cirrhosis. In addition, MSCs have been clinically used in diseases such as Crohn’s disease [7] . In recent years, studies have shown that MSCs can be used for the treatment of osteoarthritis. The role of MSC in the treatment of osteoarthritis has been discovered for more than ten years. As early as 2003, after Murphy et al. induced goat knee OA, they injected MSCs into the knee joints and found that articular cartilage degeneration, osteophyte remodeling and subchondral sclerosis were reduced in the cell-treated joints [8] . Numerous studies and clinical trials since then have shown the great potential of MSC in the treatment of osteoarthritis. This study aims to evaluate whether such benefits can be reproduced in dogs, who as companion pets are suffering from similar diseases. In this study reported clinical data of using allograft MSCs to treat dogs with osteoarthritis.
2. Methods
2.1. Animals
Companion pet dogs (including females and males) with osteoarthritis, aged seven years and older, were enrolled in the study after the owners signed a consent form. Prior to the study, consulting veterinarians performed a standard physical exam. The diagnosis of osteoarthritis was based on veterinarians’ examination. Standard blood work was collected and added to the study file at the veterinarian offices. The dogs were offered the treatment of MSC cells: For this particular analysis, the treatment group (n = 10) that received the intra-articular injections of the stem cell product. A group of pet owners who declined the MSC infusion, untreated control group (n = 6) that received standard non-steroidal anti-inflammatory drugs (NSAIDs) treatment. The type and dosage of NSAIDs given to the control animals were determined individually by licensed veterinarians as part of their regular practice. Dogs with active infections were excluded from this study as well as those pet owners who choose not to be involved in this study and subsequent follow up. The study protocol was reviewed and approved by the institutional animal care and use committee (IACUC).
2.2. Preparation and Culture of Stem Cells
For this study, mesenchymal stem cells (MSCs) were derived from commercially available canine bone marrow and cultured in humidified 37˚C tissue culture incubators in canine MSC growth medium containing 10% fetal bovine serum (Corning 35-010-CV) and 1% antibiotic-antimycotic (Corning 30-004-CI). After a period of tissue culture, tissue culture flasks containing the expanding MSC were divided and subdivided into new tissue culture flasks. For subculturing, MSCs were removed from the flask by trypsin-EDTA (Gibco 15-400-C4) and neutralized by complete media. MSC passages 3-7 were used for subsequent experiment. For cell freezing, MSCs were removed from the flask and the media was replaced by DMSO based BamBanker freezing media (Bulldog Bio BB-01), aliquoted at 5 × 105 cells/vial, and frozen in liquid nitrogen. Vials were labelled with specific lot number, date and identification as Canine MSC cells. Lots were collected and quarantined and an aliquot was tested and maintained until the results were collected, analyzed and reviewed by our quality control individual. Approved lots were released from quarantine and place in the release. One vial from each lot was retained as a long term archived sample and placed in low temperature storage. Frozen MSC aliquots were shipped on dry ice to the vet office where they were stored in a low temperature liquid nitrogen dewar. The storage dewar was monitored daily and filled with liquid nitrogen weekly to ensure the samples remained frozen during the storage interval.
2.3. Treatment Protocol
Vials of MSC cells were collected from the storage dewar, Lot # recorded and the vial was rapidly thawed in a 37˚C sterile water bath. The vial was sprayed with 70% ethanol and the vial opened with the contents drawn into a 3 cc sterile syringe with a 18 gauge needle. The companion dogs were treated with 1 ml suspension product (5 × 105 cells/mL) via intra articular infusion after the site was shaved and cleaned with aseptic spray. Animals were monitored post implantation for a period of 2 - 3 hours post infusion before being released back to their owners. Pets were followed up at 1 month and 3 months after injection. Below parameters were measured at baseline and each follow-up visit: 1) Physical Exam by veterinarian with routine blood work. 2) Owner-reported pain and activity from questionnaire. 3) Imaging evaluations by MRI and/or X-ray. 4) Specific blood levels of serum inflammatory markers, such as C-reactive protein and erythrocyte sedimentation rate.
2.4. Assessment of Pain and Mobility
The dogs returned to their homes after their participation in the study. At one month post stem cell injection, the owners were asked to evaluate the degree of pain and mobility of their dogs through well-established questionnaire. Out of the dogs treated with MSCs, 10 were assessed using the Helsinki Chronic Pain Index (Figure 1) and using the Liverpool Osteoarthritis in Dogs (LOAD) Owner questionnaire (Figure 2). Control groups were also evaluated by Helsinki Chronic Pain Index and LOAD index after one month of initial vet visit. The dogs continued to live with their owners and received ongoing care in their familiar environment as part of regular pet visits as per their owners.
2.5. Statistical Analysis
Quantitative values were presented as mean ± standard deviation. Statistical analysis was conducted using SPSS Statistics software. A t-test was used to compare differences between the treatment and the control groups at specific time points. For categorical data, the Chi-Square test was employed to assess differences between groups. A value of p < 0.05 was considered statistically significant.
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Figure 2. Liverpool osteoarthritis in dogs (LOAD).
3. Results
The MSCs products were manufactured in bulk and labelled with lot # and manufacturing date. Samples from each lot was assessed for quality control parameters (Table 1) and shipped in dry ice (−140˚C) under strict quality assurance regulations and procedures; no aerobic, anaerobic, or fungal contamination was detected.
After MSC treatment, dogs exhibited an 81.2% ± 6.8% decrease in Helsinki Chronic Pain Index questionnaire scores. LOAD Owner questionnaire showed that their mobility increased by 77.9% ± 10.1%. However, the mobility of 6 untreated control dogs without MSCs treatment decreased by 6.2% ± 7.8% (Table 2). There were significant differences between the MSCs-treated group and control group (p < 0.001).
All dogs tolerate the MSCs injection well. There were no adverse reactions found in the MSCs treated dogs during the treatment or during follow-up. There were no acute or chronic complications or issues following the injections.
4. Discussion
Chondrocytes are considered difficult to regenerate, and none of the existing treatments can alter the outcome of osteoarthritis [9] . Clinical investigators have encountered numerous challenges in treating osteoarthritis, prompting the search for potential cures. Stem cell regeneration therapy has been extensively explored in various diseases and is thus regarded as a promising avenue for addressing osteoarthritis. This method holds the potential to facilitate the repair of degenerated chondrocytes and promote the generation of new chondrocytes [10] . Previous studies have demonstrated that MSCs and their derived exosomes may expedite meniscus regeneration, while also accelerating cartilage formation. Furthermore, their presence can hinder cartilage degradation and suppress osteoclast activity through paracrine signaling rather than direct differentiation [11] [12] [13] [14] . Similarly, our study showed positive results in safety and efficacy of MSCs therapy in pet dogs with osteoarthritis.
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Table 1. Sample quality control reports generated per shipment of MSCs.
Endotoxin <5 EU/mL 0.8 EU/mL Gram Stain Negative Pass
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Table 2. Chronic pain and osteoarthritis scores. MSC-treated dogs with osteoarthritis were evaluated before and after 1 month of treatment. Dogs with osteoarthritis receiving no MSC treatment were used as control, and evaluated for osteoarthritis score 1 month apart.
Pain and immobility constitute the primary factors underlying the adverse emotional experiences of individuals afflicted with osteoarthritis, severely impinging on their quality of life. In the absence of intervention, the likelihood of disability becomes significantly elevated. Khatab et al. demonstrated that the MSC secretome could effectively diminish joint pain and mitigate cartilage damage in an osteoarthritis mouse mode [15] . In a study by He et al., MSC-derived exosomes were found to downregulate the expression of IL-1β within cartilage tissue of osteoarthritic rats. This process attenuated inflammation within chondrocytes and alleviated neuropathic and inflammatory pain [16] . Stanley et al., in a canine osteoarthritis model, confirmed that umbilical cord-derived MSCs notably alleviated pain in comparison to the control group [17] .
Intravenous administration of MSCs has also exhibited beneficial effects in osteoarthritis. Carlien et al. revealed that equine peripheral blood-derived MSCs ameliorated pain and lameness in dogs afflicted with osteoarthritis [18] . In a prospective study, the application of adipose-derived MSCs via intra-articular injection to 329 osteoarthritis patients yielded symptomatic improvement in 87.9% of cases, with a remarkable 10.8% of patients achieving complete recovery [19] . In summary, both the intra-articular injection of MSCs and the utilization of their cellular constituents have demonstrated the potential to attenuate or even reverse osteoarthritis symptoms in animal models.
In 2022, $35.9 billion was spent on Veterinary services in the United States. Osteoarthritis in pet dogs brings not only mental but also financial stress to owners. In this study, we employed the questionnaire based on the Helsinki Chronic Pain Index and the LOAD Owner questionnaire to assess pain scores and the mobility of the pet dogs. We then measured changes in these two sets of data before and after MSCs treatment in dogs with osteoarthritis. The Helsinki Chronic Pain Index comprises an 11-question survey aimed at assessing the degree of chronic pain in dogs and monitoring its changes [20] . On the other hand, the LOAD questionnaire was utilized to investigate clinical symptoms such as joint pain and the range of motion reduction in dogs with osteoarthritis during various activities [21] . Our results demonstrated that, in comparison to the control group, dogs treated with MSCs exhibited reduced pain and significantly improved mobility, with their overall mobility being significantly enhanced (p < 0.001) without any adverse reactions or other complications. This safe and effective treatment may greatly relieve the mental and financial stress of pet dog owners.
While this study highlights the significant potential of mesenchymal stem cell therapy in alleviating osteoarthritis symptoms, our findings come with certain limitations. Firstly, widespread adoption of this therapy may require additional time. Secondly, the data collected through questionnaires is subject to a degree of subjectivity. Finally, further exploration is needed to fully understand the underlying mechanisms of this treatment. To gain deeper insights, future studies will involve a larger cohort of dogs.
5. Conclusion
Transplantation of stem cells is a promising technology that has garnered considerable scientific interest. This study in companion dogs has demonstrated both short-term safety and efficacy of MSC therapy in treating osteoarthritis in pet dogs. It holds significant promise, and further research in this area is anticipated. Future studies are planned to further examine and validate the technology, including the long-term safety especially potential for tumorigenicity, as well as long-term efficacy including whether the disease recurs and if repeated MSC injection will be necessary.