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Clinical outcomes following intra-articular injection of autologous adipose-derived mesenchymal stem cells for the treatment of osteoarthritis in dogs characterized by weight-bearing asymmetry

Aims This study investigates the effects of intra-articular injection of adipose-derived mesenchymal stem cells (AdMSCs) and platelet-rich plasma (PRP) on lameness, pain, and quality of life in osteoarthritic canine patients. Methods With informed owner consent, adipose tissue collected from adult dogs diagnosed with degenerative joint disease was enzymatically digested and cultured to passage 1. A small portion of cells (n = 4) surplus to clinical need were characterized using flow cytometry and tri-lineage differentiation. The impact and degree of osteoarthritis (OA) was assessed using the Liverpool Osteoarthritis in Dogs (LOAD) score, Modified Canine Osteoarthritis Staging Tool (mCOAST), kinetic gait analysis, and diagnostic imaging. Overall, 28 joints (25 dogs) were injected with autologous AdMSCs and PRP. The patients were followed up at two, four, eight, 12, and 24 weeks. Data were analyzed using two related-samples Wilcoxon signed-rank or Mann-Whitney U tests with statistical significance set at p < 0.05. Results AdMSCs demonstrated stem cell-like characteristics. LOAD scores were significantly lower at week 4 compared with preinjection (p = 0.021). The mCOAST improved significantly after three months (p = 0.001) and six months (p = 0.001). Asymmmetry indices decreased from four weeks post-injection and remained significantly lower at six months (p = 0.025). Conclusion These improvements in quality of life, reduction in pain on examination, and improved symmetry in dogs injected with AdMSCs and PRP support the effectiveness of this combined treatment for symptom modification in canine OA for six months. Cite this article: Bone Joint Res 2021;10(10):650–658.


Introduction
Osteoarthritis (OA) is characterized by degeneration of the articular cartilage, with loss of matrix, fibrillation, and formation of fissures causing loss of the cartilage surface. Associated changes include osseous metaplasia of intra-and extracapsular structures, abnormal periarticular calcification, and joint mechanics. OA is the most common cause of chronic pain in dogs, with an estimated 20% of dogs presenting with clinical signs. 1 It is a chronic degenerative condition commonly managed with analgesics and symptom-modifying medication, alone or in combination with supportive therapies such as exercise and physiotherapy. These management methods are, however, limited in their ability to attenuate disease progression, leaving arthroplasty as a common eventuality in OA patients. 1 Research has focused on using stem cell therapy for the management of OA. Not only do stem cells have differentiation and self-renewal properties, which may directly contribute to cartilage repair, but they also have anti-inflammatory and immunosuppressive abilities and secrete a variety of bioactive factors that allow them to be attracted to the site of injury, reducing pain and inflammation. [2][3][4][5] In the osteoarthritic joint, progressive cartilage degradation produces proinflammatory mediators which exacerbate the condition; consequently, the antiinflammatory properties of stem cells may be beneficial in reducing the inflammatory processes associated with OA. 6,7 Autologous adipose-derived mesenchymal stem cell (AdMSC) therapy requires isolating and expanding stem cells from the patient's fat using tissue culture techniques. The cells can then be administered into the osteoarthritic joint through intra-articular injection. The use of AdMSCs in preference to BMSCs is becoming increasingly common as adipose tissue is easy to obtain; the procedure for isolating the cells from the fat is simple, and adipose tissue has been shown to yield approximately 500 times more stem cells than bone marrow. [8][9][10] Studies reporting the application of MSCs in canine OA include autologous and allogeneic BMSCs and AdMSCs. 1,8,11,12 Human clinical trials assessing the effect of AdMSC in OA demonstrated preservation and increased cartilage volume compared to control patients as assessed by MRI, clinical exam, and cartilage scoring. [13][14][15] Platelet-rich plasma (PRP) is isolated from patient blood and consists of a mixture of growth factors such as hepatocyte growth factor (HGF), vascular endothelial growth factor (VEGF), platelet-derived growth factor (PDGF), and transforming growth factor-β (TGF-β). Intra-articular injection of PRP is associated with reduced inflammation, pain relief, improved limb function, and possible cartilage regeneration in human OA patients, [16][17][18][19] and has been shown to reduce lameness in dogs with OA. 20 The aim of this study was therefore to investigate the effect of stem cells and PRP injection on lameness and pain scores in osteoarthritic canine patients.

Methods
We do not believe the ARRIVE guidelines apply to our study because the dogs in this study were injected under the guidance of Recognised Veterinary Practice (RVP). All patients were receiving treatment at a veterinary practice, treatment for which the owners were paying and have given informed consent. Patient recruitment and radiological evaluation. A series of 25 skeletally mature dogs (28 joints) were recruited for this study (Table I). The mean age of the dogs included was 98.5 months (standard deviation (SD) 34.5); 18 to 138) and the mean weight was 32.8 kg (SD 13.0; 13 to 58.1). All patients included in this study were receiving their first stem cells and PRP injection as part of OA management.
Inclusion criteria included the presence of OA in one or more joints. Dogs were excluded if they had any other orthopaedic-related surgery to the injected and/ or the contralateral limb. A complete clinical evaluation was performed including haematology and serum biochemistry. Radiographs were reviewed to determine the degree of OA in the affected joint(s). The International Elbow Working Group Scoring Scheme was used to assess osteophyte size to grade the severity of OA. The scoring system used for all the joints including shoulder, hip, and stifle was: grade 0 = no osteophytes, grade 1 = osteophytes < 2 mm, grade 2 = osteophytes 2 mm to 5 mm, grade 3 = osteophytes > 5 mm. 21,22 Lameness and limb asymmetry were confirmed using pressure-sensitive walkway analysis (Strideway; Tekscan, USA) immediately prior to enrolment on the study. Adipose tissue collection. Adipose tissue was collected from each dog. Dogs were pre-medicated using intravenous (IV) administration of acepromazine (0.02 mg/kg; ACP; Elanco, UK) and methadone (0.3 mg/kg; Comfortan; Dechra, UK). General anaesthesia was induced with propofol 1 to 4 mg/kg (PropoFl; Zoetis, UK), the dog intubated, and then anaesthesia maintained with inhaled 2% isoflurane in oxygen. Adipose tissue was harvested from subcutaneous tissue in the inguinal region or falciform ligament. 23 Tissue and stem cell processing. The adipose tissue was washed with phosphate-buffered saline (PBS) (Thermo Fisher Scientific, UK) and 1% P/S solution. The fat was minced and digested with collagenase II enzyme (Sigma Aldrich, UK) for three hours at 37°C with agitation. Thereafter, the fat mixture was centrifuged at 644 ×g for five minutes. The resulting cell pellet was then resuspended in growth media composed of Dulbecco's Modified Eagle's Medium (DMEM) (Sigma Aldrich) supplemented with 10% (v/v) foetal calf serum (FCS) (First Link, UK) and 1% P/S solution, and cultured in a flask. Cells were maintained in the growth media until passage 1, after which they were trypsinized, counted, and mixed with PRP for injection into the joints. PRP collection. Using a syringe containing 2 ml of citrate anticoagulant (ACD-A, Anticoagulant Citrate Dextrose Solution, Zimmer Biomet, USA), 18 ml of blood was collected from the jugular vein. The blood was centrifuged in sediment PRP (sPRP) tubes (NTL Biologica, UK) at 1,900 ×g for three minutes, to separate the PRP from the blood. The leucocyte-rich platelets in the PRP fraction were then counted under a light microscope and mixed with the stem cells before injection. Platelets were added to AdMSCs at a ratio of ten platelets per AdMSC.
In vitro study for stem cell characterization: flow cytometry analysis of CD marker expression. Passage 1 cells surplus to clinical need were expanded further. Flow cytometry was used to investigate CD marker expression of cells at either passage 2 or 3 (n = 4). The cells were analyzed for expression of CD29, CD90 and CD105, CD34, and CD45.
Cells were fixed for 15 minutes with 4% formaldehyde solution and washed with 0.5% bovine serum albumin (BSA) solution. The cells were then incubated with the antibodies (Table II), and diluted as recommended by the company for one hour in the dark at room temperature. The excess antibody was washed off and the cells were analyzed using a flow cytometer (CytoFLEX; Beckman Coulter, USA). Analysis of CD105 required a fluorescent secondary antibody. After one-hour incubation in the primary antibody, the samples were incubated with the secondary antibody in the dark for 30 minutes. After the cells were washed in BSA, they were re-suspended in saline solution for flow cytometry analysis. The resultant data were analyzed with proprietary software (CytExpert; Beckman Coulter). Isotype antibody was used as the negative control.
Trilineage differentiation (performed on samples from four patients). Osteogenic differentiation -30,000 cells at passage 2 were seeded and treated with osteogenic  Chondrogenic differentiation -100,000 cells at passage 2 were seeded as a micro-mass and supplemented with chondrogenic media containing 2% FCS, 100 µM dexamethasone, 50 mM ascorbic acid, 0.05 M sodium pyruvate (Sigma Aldrich), 10 mg/ml transforming growth factor-β1 (Peprotech EC,UK), and insulin-transferrinselenium premix (Thermo Fisher Scientific). The samples were incubated at 37°C and 5% CO 2 . After 21 days, the samples were washed with distilled water and fixed for three hours using 100% methanol (Sigma Aldrich) at -20°C. The samples were then stained overnight for glycosaminoglycan production using Alcian Blue stain (Sigma Aldrich). Stem cell injection. Patients were sedated with an IV injection of medetomidine (12 µg/kg) plus methadone (0.02 mg/kg). The number of cells injected per joint was determined by the weight of the patient in approximately 1.5 ml of PRP (Table III). The site for intra-articular injection was clipped and cleaned using chlorohexidine followed by final alcohol prep. The injection was administered under aseptic conditions with a 5 ml syringe and one-inch 21-gauge needle. Elbows were injected by identifying the medial humeral condyle and olecranon and passing a needle caudomedial to craniolateral into the joint space. Coxofemoral joints were injected by identifying the great trochanter of the femur, externally rotating the femur, palpating a deficit between femoral head and pelvis, then passing a needle into the coxofemoral joint space. Stifles were injected by locating the distal aspect of the patella and tibial tuberosity. The needle was passed abaxial to the straight patella tendon angled underneath the patella. Joint fluid was aspirated to confirm intra-articular location. Post-procedural analgesia provided was by buprenorphine (0.01 mg/kg SC at six hours post-injection) and paracetamol (10 mg/kg by slow IV injection, then orally every 12 hours for the next 72 hours). Outcome measures. To assess mobility, a validated OA survey instrument, the Liverpool Osteoarthritis in Dogs (LOAD) questionnaire was given to owners at the time of study entry (18 dogs), two weeks (ten dogs), four weeks (13 dogs), eight weeks (13 dogs), 12 weeks (13 dogs), and 24 weeks (eight dogs) post-injection. 24 A reduction in LOAD scores reflects an improvement in mobility. 25 All Tekscan data were collected by the same person (LC) for this study. Tekscan data were collected on the day of injection to obtain the initial baseline data (25 dogs). Thereafter data were collected at two weeks (ten dogs), four weeks (15 dogs), eight weeks (18 dogs), 12 weeks (16 dogs), and 24 weeks (nine dogs) post-injection. Limb loading, corrected to percentage body weight (%BW) was used to calculate the asymmetry index, which is an indicator of weight distribution between the healthy and the osteoarthritic joint. Lameness was indicated by a high asymmetry value. When lameness occurs in a load-bearing limb, compensatory load adjustments are made in the contralateral limb, so a lower asymmetry index indicated equal weight distribution. 26,27 The %BW was measured by walking the dogs in a straight line four times across a pressure mat, at a consistent walk, approximately 100 cm/s. Prior to measurements, all dogs were acclimatized to the room and the pressure mat. Asymmetry index between contralateral limbs was calculated by: ((X r -X l )/(X r + X l ))*100, whereby X r and X l are the %BW for the right and left limb, respectively. 28 The Canine Osteoarthritis Staging Tool (COAST) is a standardized scoring system that uses observations from both the owner and veterinarian to monitor canine OA progression. 29 A modified version of the COAST (mCOAST) scoring system was used in the current study to assess patients at baseline (16 dogs), 12 weeks (16 dogs), and 24 weeks (13 dogs). The system was modified by excluding values assigned to follow-up radiological assessment. In this study, the final score was determined by adding scores together from each category, rather than taking the highest value as per the original COAST scoring system, and a reduction in the score would reflect an improvement in the progression of OA symptoms. 30 Statistical analysis. The data in this study were nonparametric, therefore statistical comparison was carried out using a Wilcoxon signed-rank or Mann-Whitney U test. All data were analyzed using SPSS version 25 (IBM, USA). Statistical significance was defined as p < 0.05.

Results
There were no adverse effects associated with either surgical harvest of fat tissue or after the injection of stem cells and PRP. Radiographs were available for 26 of 28 joints; three joints were assigned grade 0, three joints were assigned grade 1, eight joints were assigned grade 2, and 12 joints were assigned grade 3. Two joints were not radiographed. Of the 25 patients, three were injected in multiple joints. Altogether 28 joints were treated with AdMSCs and PRP (Table I).
In accordance with the International Society for Stem Cell Research, 31 AdMSCs differentiated to bone, fat, and chondrocytes (n = 4) after 21 days and stained positively for Alizarin Red, Oil Red O, and Alcian Blue stain, respectively ( Figure 1). The cells (n = 4) were also analyzed using flow cytometry and showed limited expression of CD34 and CD45 with positive expression of CD29, CD90, and CD105 (Figure 2). LOAD scores were significantly lower at week 4 (p = 0.021, Mann-Whitney U test) compared with preinjection. The mCOAST improved significantly after three months (p = 0.001, Mann-Whitney U test) and six months (p = 0.001, Mann-Whitney U test), implying that the injections made an improvement to the pain and mobility of the patients after four weeks, as assessed by the owner, and after three months (12 weeks) and  Flow cytometry results for adipose-derived mesenchymal stem cells. The cells were negative for CD34 and CD45 and highly expressed CD90 (n = 4). six months (24 weeks), as assessed by the veterinarian (Figure 3).
The asymmetry index decreased at week 2, possibly due to the physical effect of joint lavage from the injection procedure. The asymmetry index then increased at week 4 and then decreased consistently until 24 weeks (six months). Significant differences were recorded between weeks 8 and 24 (p = 0.021), weeks 4 and 24 (p = 0.008), weeks 12 and 24 (p = 0.011), and between weeks 24 and week 0/preinjection (p = 0.025, all Mann-Whitney U test) (Figure 4).

Discussion
Loss of articular cartilage with inflammation is a common feature of OA. It eventually results in impaired joint function. 32 Results from this study demonstrate that AdMSC and PRP therapy improved pain and lameness scores over time, with statistical and clinical significance seen at 12 weeks and six months compared with preinjection. Clinically, this is an important finding because although these results did not reflect the asymmetry index from gait analysis, the AdMSCs and PRP injections appear to have enabled patients to experience a better quality of life with less pain, delaying the need for more invasive surgical techniques. Similar results were reported in a study in osteoarthritic human patients, in which 18 patients were injected with AdMSCs in the knee joint. This study showed significant improvement in Western Ontario and McMaster Universities Osteoarthritis (WOMAC) score and size of the cartilage defect, with no adverse events. 33 Articular cartilage is susceptible to damage and has a poor potential for regeneration due to its lack of vascularity. As OA progresses, the load-bearing capacity and biomechanical properties of the thinning cartilage decreases and restoration of normal cartilage function is difficult to achieve. As well as the ability to differentiate into chondrocytes, stem cells also control the local microenvironment via anti-inflammatory and immunosuppressive factors, thereby protecting the cartilage from further tissue destruction and aiding regeneration of the remaining progenitor cells. 34 The role that injected stem cells play in cartilage regeneration is therefore unclear and it is difficult to predict how long the cells' stimulatory effect may last, and whether a reinjection of cells and PRP may be necessary. 13 From the asymmetry measurements in our study, the treatment appears to remain effective for months.
The growth factors in PRP have an anti-inflammatory and analgesic effect. 35,36 HGF has been shown to enhance the inhibition of nuclear factor kappa-light-chainenhancer of activated B cells (NF-κB), which decreases the production of the cytokine interleukin 6 (IL-6), leading to an increase in the anti-inflammatory cytokine IL-10. 37,38 Indeed, PRP injection has been shown to be effective after three and six months of injections in human patients with initial stage of knee OA, compared with patients injected with hyaluronic acid (HA). 39 Similar results have also been reported in canine patients injected with PRP, with an increase in PVF and improved pain and lameness scores at 12 weeks. 12,40 Additionally, it has been reported that when MSCs were treated with PRP, proliferation and chondrogenic differentiation in vitro were enhanced. 41,42 A study by Yun et al 12 demonstrated superior disease modification when AdMSCs were combined with PRP, as compared to stem cells or PRP alone, in an experimental model of canine OA, whereby the dogs were injected every week for a month. However, unlike the study by Yun et al, 12 dogs in our study had naturally occurring OA  and have shown a statistically significantly long-term positive response to a single injection of cells and PRP injection, until six months. This is therefore more viable in a clinical setting in veterinary and human medicine, because all patients would demonstrate variability in ages and weight, and their progress would be monitored using gait analysis and pain scores.
Unlike MRI, which can be very costly, radiological analysis can be used in clinical practice for diagnosis of OA in dogs. However, it mainly provides information on osseous changes such as sclerosis and osteophyte formation, but provides limited data on soft tissues. Osteophyte measurements can be graded and used to diagnose OA, but it has been shown that osteophyte size can vary between different breeds of dogs. 43 Therefore, radiological findings, taken in isolation, should be interpreted with caution, as they are not a reliable indicator of OA.
The dogs in this study were therefore assessed clinically and using gait analysis.
The dogs in this study were injected under the guidance of RVP. Therefore, the limitations of this study were as follows. Firstly, the lack of a placebo or control group that would receive an intra-articular injection of saline solution or PRP in a prospective blinded randomized control manner to conclusively assess the effect of the treatment arm in an unbiased manner. The authors acknowledge that it would have been preferable to compare the results from this case series with those injected with only PRP or HA, but there are constraints on randomization in many veterinary practices. Secondly, the restriction to collect invasive outcome measures in a clinical setting following treatments such as AdMSCs. MRI and arthroscopy could have been used to assess cartilage volume and infill. This would have identified whether the The asymmetry index calculated using the peak vertical force (PVF), calculated from the injected limb and the contralateral uninjected joint. Asymmetry index is a percentage, calculated by finding the difference in PVF between the treated and untreated limb and dividing by the sum of PVF for the same limbs. *p < 0.05, Mann-Whitney U test. Preinjection (n = 28), week 2 (n = 10), week 4 (n = 15), week 8 (n = 18), week 12 (n = 16), and six months (n = 9). improvement in lameness was due to cartilage regrowth or due to the anti-inflammatory effect of the cells. Determining the catabolic and anabolic biological markers in the synovial fluid would also have helped to establish the anti-inflammatory effect of the cells and PRP in OA. However, to subject these patients to additional anaesthesia for imaging and arthroscopy would have required an experimental licence. Finally, canine OA often affects joints bilaterally; it is difficult to report on the effects of stem cells in patients with asymmetrical OA. The effect of multiple joint OA may mask clinical outcomes following stem cell injection.
In conclusion, this study represents the first clinical series on the use of a combination of autologous cultureexpanded AdMSCs and PRP in the management of canine OA. We report a significant improvement in pain and functional scores and lameness. Although these findings support the idea of using AdMSCs in the management of OA, additional research needs to be performed in order to determine the optimal dose and timing, and to confirm whether combination therapy is better than monotherapy.