Evaluation of the Therapeutic Potential of Canine Yolk Sac Cells in Dogs with Hips Dysplasia

Currently, stem cell therapy has been a useful tool in regenerative medicine with high therapeutic potential due to the capacity for self-renewal and differentiation of these cells. In recent years, orthopedics has been seeking new methods of treatment to obtain the effect of repair of articular defects more effectively and without invasive procedures. Therefore, many studies involving cell therapy in order to improve the articular repair are being conducted. The aim of this study was to evaluate the therapy with yolk sac cells in dogs with hip dysplasia. For the evaluation of the treated animals, we rated the lameness score, muscular atrophy score, a questionnaire about quality of life, radiographic evaluation, synovial fluid analysis and blood count. Bilateral intra-articular applications of yolk sac cells were performed on days 0, 30 and 60, being held the last valuation on day 90. The results showed no significant statistical difference when observed the treated animals compared to the control group. However, observing the clinical improvement of these animals throughout the treatment was evident that cell therapy is beneficial to the animals.


Introduction
In the veterinary medicine, the Hip Dysplasia (HD) has been presented as one of the main osteo-articular pathologies that affect the dogs, being responsible for about 50% of specialists' visits [1]. The prevalence varying from 0.0 to 71,8%. There is a strong association between the clinical diagnosis of hip status and veterinary care and mortality related to HD [2]. Since the earliest reports of HD, there are more papers written about this disease than about any other disease related to small animal orthopaedics [3]. HD is a change in the development of the femur that presents varying degrees of hip laxity, progressive remodeling of the hip structures and subsequent development of Osteoarthritis (OA) [4].
Hip laxity allows subluxation of the joint during growth, resulting in abnormal development of the acetabulum, neck and femoral head.
Repetitive subluxation and reduction lead to excessive cartilage wear and damage to the dorsal acetabular ridge [5]. Although HD has a recessive, intermittent and polygenic genetic basis [6], multiple environmental factors modify the expression of this predisposition, affecting the way it manifests itself and its severity [7][8][9][10]. Many studies have reported heritabilities of HD (0.01-1.75) [11] and some have observed favourable genetic progress in reducing expression [11,12]. It can be observed in several breeds of dogs, however, it is prevalent in large dogs and fast growing, for example, Labrador Retriever, New found lands, Rottweilers, St.
The history and clinical signs of affected animals include gait abnormalities such as lameness, short steps, rabbit leap, as well as exercise intolerance and difficulty in getting up and climbing stairs [16]. Among the treatment modalities, surgical and conservative ones are described. This second one is restricted to weight control, exercise restriction, physical rehabilitation, pain management and administration of nutraceutical supplements [17][18][19][20][21]. The surgical procedures described in animals are juvenile pubic sinfisiodesis [19,22], colocephalectomy [16], triple pelvic osteotomy [23], total hip replacement [19,24] and capsular denervation [3]. Currently, cell therapy has been a useful tool in regenerative medicine with high therapeutic potential due to the capacity for self-renewal and differentiation of these cells. In recent years orthopedics has been looking for new methods for a treatment that obtains the effect of repairing joint defects more effectively and without invasive procedures. Therefore, many studies involving stem cells aimed at improving joint repair are being performed [25].

Material and Methods
This study was submitted and approved by the Committee of

Dogs
Eight animals of the canine species were selected, with no preference of race, sex and age, coming from the private clinics of São Paulo, with bilateral coxofemoral dysplasia confirmed by a recent radiographic examination. These animals should not present any other orthopedic disease and have the ability to wander normally.
After the selection, the animals were randomized in control group and treated group (YS).

Clinical Evolution
In order to evaluate the efficacy of the cellular therapy, the clinical evolution of the animals was analyzed by means of a comparison between the pre-and post-treatment evaluations, the latter being on days 30, 60 and 90 days, as well as the simple x-ray of the hip joint to evaluate the evolution of the arthrosis, complete blood count to evaluate possible hematological alterations resulting from the treatment, and analysis of the synovial fluid to evaluate the changes present directly in the fluid of this joint.

Surgery, Cell Therapy and Tests
All animals were submitted to 8 hour water and food fasting for the sedation and anesthesia procedures, in which Meperidine (0.4 mg / kg, intramuscular, União Química, Brazil) and Propofol (4.0 mg / kg, intravenous, Propovan®, Cristália, Brazil) for subsequent radiographic examination with cranio-caudal projection of the coxofemoral joint. After this examination, with the animals in lateral decubitus, tricotomy and asepsis of the pelvic region were performed near the hip joint. A Thuoy 19G needle (Terumo Corporation, Japan) was used percutaneously to reach the hip joint to collect synovial fluid, which was filled into EDTA (anticoagulant) -containing tubes. Sterile saline solution (0.5ml) was then applied to the joint in the animals of group C, and canine Yolk Sac Cells (YS) (0.5ml / 1x107 cells). The same procedure was performed on the contralateral limb. A blood sample was collected by venous puncture of the jugular and packed in tubes containing EDTA.
After the procedures, all animals received analgesic and antibiotic medication, Dipirone (1ml / animal, subcutaneous route, Algivet®, Vetnil, Brazil) and Enrofloxacin (5mg / kg, single dose subcutaneous use, Baytril 10% ®, Bayer Laboratory SA, Brazil). Three applications (day 0, 30 and 60 after application) and four evaluations (day 0, 30, 60 and 90 after application) of each patient were performed on each item. For this study the animals were followed during the 90 days, the data were evaluated by t-test and two-tailed test, being adopted as value of statistical significance p <0.05.
To evaluate the scores of muscle atrophy, claudication and Ortolani test were assigned values from 0 to 4, 0 being the absence of the problem and 4 the maximum degree observed, according to Hudson [26].

Results
No radiographic changes were observed between the groups before and after treatment. There were also no changes in hematological examinations after the procedures. There were also no reports of worsening of claudication of the animals, remaining

Discussion
Hip dysplasia was the joint disease of choice because it had the highest prevalence of joint affections causing degenerative joint disease in dogs worldwide. Its main feature is the abnormal formation of the coxofemoral joint with different degrees of dislocation and subluxation, and its prevalence is higher in animals of large and giant races, although it has been observed in the terrier breeds, as well as Bulldogs and Pugs [13][14][15]. The cause of the hip dysplasia is multifactorial and the treatments are diverse and controversial [27]. The only surgery that treats hip dysplasia per se is the total hip replacement [19,24]. Surgery is often used to ameliorate the discomfort of hip dysplasia. The surgical procedure often depends on the age of the animal at the time of admission, the preference and recommendation of the surgeon, the financial investment, the potential risks and the necessary care in the postoperative period. However, it is still unclear whether this will result in a better outcome and hence whether it is the most recommended [28]. Dogs as experimental models of stem cell treatments have provided preclinical information relevant to treatments in both human medicine and veterinary medicine [29].
Another important aspect is the great growth of cell therapy in the veterinary clinic for the treatment of several canine diseases [30], since Mesenchymal Stem Cells (MSCs) have shown promising results in preclinical studies and in the central nervous system and in cartilaginous bone defects [31].
Autologous MSCs demonstrated therapeutic effects in experimental studies, including improved tendon healing in horses and increased regeneration of meniscal tissue and delayed progression of OA in goat model [32,33]. However, stem cells of fetal origin are more accessible because they are obtained from extra embryonic tissue, such as the yolk sac [34], placenta [35], amnion [36] and chorion [37], which are usually discarded after birth. Fetal stem cells have low immune responses, since they are at the maternal-fetal interface [38]. In the present study we could observe the low immune response of this cell type, since none of the treated animals showed signs compatible with this problem.
Fetal stem cells also show rapid growth and good plasticity [39] and can replace adult and embryonic stem cells in cell therapy [40].
Moreover, they are able to differentiate into specific cells such as cardiomyocytes, as well as to present the differentiation properties in already established osteogenic, adipogenic and chondrogenic lines [41], making these cells an alternative source of multipotent cells [42]. For these reasons we choose for this study the therapy with yolk sac cells, which has properties of mesenchymal progenitor cells, plasticity and potential do differentiation [34,43] for the treatment of joint diseases in the canine model, aiming to validate this therapy in articular diseases, such as, for example, the hip dysplasia, enabling a new clinical management in these cases .
In order to isolate progenitor cells capable of maintaining the hematopoietic lineage in vitro, great interest has been gained in obtaining stem cells from the yolk sac. When these cells were cultured in specific culture medium, the yolk sac progenitor cells were able to differentiate into bone, cartilaginous and muscular tissue [34]. In view of these results, we believe that these cells

Conclusion
Positive changes were observed in the animals treated with yolk sac cells when performing the Ortolani test and quality of life questionnaire. No evolution of osteoarthrosis was observed in the animals submitted to cell therapy. There were no adverse effects on the use of yolk sac cells in this treatment. We conclude that although more confirmatory studies are needed, therapy with yolk sac cells is a promising tool for treating hip dysplasia in dogs.