Osteoarthritis (OA) is a painful and progressive disease that involves the permanent, long term deterioration and destruction of the cartilage and components surrounding the joints, which results in chronic pain, inflammation and decreased mobility (Henrotin et al., 2014).

The hip  is one of the most common areas of OA in the body. Hip dysplasia is highly prevalent in dogs, and affects primarily large and giant breeds of dogs, never the less this disease can also occur  in medium-sized breeds and rarely in small breeds (Butler and Gambino, 2017). German Shepherds, Labrador Retrievers, Rottweiler’s, Great Danes, Golden Retrievers and Saint Bernards appear to have a higher incidence, however these are all very popular breeds and may be over represented because  of their popularity (Butler and Gambino, 2017). Dogs  of  all ages are  subject  to  hip  dysplasia  and  the resultant OA (Gail et al., 2001).

Hip dysplasia is associated with abnormal joint structure and a laxity of the muscles, connective tissue, and ligaments that would normally support the joint. As joint laxity develops, the articular surfaces of the two bones lose contact with each other (Kyriazis and Prassinos, 2016). This separation of the two bones within the joint is called a subluxation, and this causes a drastic change  in the size and shape of the articular surfaces (Butler and Gambino, 2017). OA of the hip is the result of the degeneration of the joint due to a laxity caused by hip dysplasia (Butler and Gambino, 2017). When a dog has hip dysplasia, the joint wears out abnormally and the protective cartilage on  the surface of the joint deteriorates and the resultant bone-to-bone contact creates pain (Butler and Gambino, 2017).

Moreover, the degenerative changes of OA appear frequently causing inflammation and pain (Henrotin et al., 2014). Pain is also related with the inflammation of the joint (synovitis) (Henrotin et al., 2014). This pain makes the dogs shift most of their body weight to the front end. As a result, dogs showing few clinical symptoms may develop increased shoulder musculature while hind limb musculature remains under-developed (Kyriazis and Prassinos, 2016). As damage to the joint progresses and secondary OA sets in, symptoms of stiffness and lameness may be present (Kyriazis and Prassinos, 2016).

Some forms of degenerative joint disease can be treated with surgery but drugs are the most common  therapy for OA treatment. Nevertheless, pharmacological treatment is limited to clinical signs alleviation (Comblain et al., 2016). In this way, non-steroidal anti-inflammatory (NSAIDs) are commonly prescribed to address anti-inflammatory mechanisms. Unfortunately, the use of NSAIDs may be associated with detrimental  effects, especially gastrointestinal side effects (Comblain et al., 2016). Some clinical studies have highlighted the beneficial effects of dietary supplement for the treatment of OA in dogs (Comblain et al., 2016).

Glucosamine and chondroitin are two compounds that have been widely used for the treatment of OA in animals. These aminosaccharides act as a preferred substrate for the biosynthesis of glycosaminoglycan(GAG) chains, and subsequently for the production of aggrecan, main proteoglycan used by chondrocytes to build the cartilage extracellular matrix (ECM). They also exert anti-inflammatory and anti-catabolic effects through the inhibition of nuclear factor κB (NF-κB) binding activity (Comblain et al., 2016).

Ovopet® is an innovative ingredient obtained from egg shell membranes at Eggnovo S.L. via a patented process. Eggshell membrane discovery as a natural source of glycosaminoglycan’s, such as chondroitin sulfate and hyaluronic acid among others, has led to the consideration of this  product as a potential approach for the treatment of OA as shown in a previous study (Blasco et al., 2016).

The goal of the present study is to assess the effectiveness of Ovopet® in the treatment of hip dysplasia and the consequent OA in dogs.

Dog selection

A group of adults (63% female), privately owned arthritic dogs were used in this study. Their age  was between 1 to 13 years old, with an average of 8±0.56 years. All the participants were medium-sized dogs. Their body mass ranged from 15 to 40 kg, with an average of 34±1.05kg. The body condition of the 66% of the dogs was ideal according to the body condition score published in the manufacturer’s site  (http://www.affinitypetcare.com/veterinary/obesity/obesity_dog/flash/bcs.html).The German Shepherd was the most common breed (40% of dogs) followed by Labrador and Golden (15% of dogs each). All dogs had radiographic evidence of hip dysplasia. They were recently diagnosed dogs in all cases , so they had not been previously treated with chondroprotective supplements or diets. Besides the afore mentioned stated inclusion criteria, the owner had to describe at least one of the following signs: difficulty to stand up, difficulty to jump, difficulty to climb stairs or clear lameness. The study protocol was performed in compliance with national guidelines for research onanimals . Throughout the study, dogs remained with their owners. The owner’s consent was obtained  at the beginning of the study.

Nutraceuticals

Ovopet® was obtained from Eggnovo S.L. (Navarra, Spain) in a sustainable and environmentally friendly manner without the use of chemicals. It consists of egg membranes separated from eggshells by a patented process. Compositional analysis of Ovopet® has identified a high content of protein (collagen types I-V-X, elastin, keratin) and moderate quantities of GAGs (chondroitin sulfate, HA) and glucosamine. Snacks were prepared by a Spanish pet food manufacturer.

Study protocol

The randomized and double-blind with placebo study was carried out in cooperation with Eudald Toralles  and Sentmenat Veterinary Clinics (Barcelona). The veterinary monitoring was performed at days 0, 10, 20, 30 and 40. Veterinary Clinics were in charge of recruiting the dogs with hip dysplasia, getting the owners signed consent and performing the standard clinical  tests according to the study protocol. If the inclusion criteria were met, the dogs could take part in the study. Firstly, the veterinary filled in the dogs’ data (breed, age, body condition, sex and diet), did a clinical examination of the dog before starting the treatment and evaluated the OA grade with the Kellgren-Lawrence scale (Kohn et al., 2016). Based on the weight of the dog the veterinary established, the amount of fodder and the number of snacks that dogs had to intake during the study. This amount was revised in every veterinary visit to adjust the dosage of fodder. The performed protocol in each assessment day was as follows:

(1) To assess the muscle atrophy by measuring the perimeter of the rear legs

(2) To evaluate the extension-flexion range of the hip

 (3) To fill in a hip functional scale questionnaire

(4) To fill in an evaluation questionnaire together with the owner

(5) To fill in the side effects questionnaire

(6) To draw blood for measuring inflammatory blood markers (at day 0 and 40)

(7) To make a sonography of the hip (at day 0 and 40)

Two groups of patients were established, one taking the chondroprotective supplement with Ovopet ® (N=30) and the other group taking the placebo supplement (the same recipe but without Ovopet®) (N=10). The treatment lasted 40 days and the recommended daily dose of Ovopet® was 15 mg Kg^-1 dog day^-1. The only authorised pharmacological treatment, when the lameness was intense or the quality of life decreased markedly was Metacam. The owners and the veterinary noted down if the dog needed rescue drugs.

Kellgren-Lawrence classification of OA degree

The OA degree (from 1 to 4) was classified using the Kellgren-Lawrence grading scale. The grades in the scale are described as follows:

Grade 1: Doubtful narrowing of joint space and possible osteophytic lipping.

Grade 2: Definite osteophytes, definite narrowing of joint space.

Grade 3: Moderate multiple osteophytes, definite narrowing of joint space, some sclerosis and  possible deformity of bone contour.

Grade 4: Large osteophytes, marked narrowing of joint space, severe sclerosis and definitedeformity of bone contour.

Assessment of muscular atrophy

The circumference of each thigh was measured at standard anatomical references based on the Bioarth assessment scale (Villaret al., 2016; Cuervo et al., 2014). The same investigator performed all measurements using a measure ribbon.

Assessment of flexion-extension range

To evaluate the hip mobility range, the veterinarian measured the flexion and extension degrees of the hip every ten days based on the Bioarth assessment scale (Villaret al., 2016; Cuervo et al., 2014). The range of movement was measured bilaterally for the hip using a goniometer. These measurements were taken for the maximum possible extensionand flexion values.

Hip functional scale

The hip functional scale is an adaptation of the Bioarth assessment scale (Villaret al., 2016) to quantify the degree of OA. This is the questionnaire that the supplement manufacturer R&D department trained personnel employs for researching. The questionnaire contains two different sections: functional limitation and joint mobility. The functional limitation assesses the lameness before starting to walk, the lameness during walk, the resistance to walk, the resistance to run and play, the difficulty to climb stairs and the difficulty in little jumps. The joint mobility assesses the pain at manual mobilisation of the hip, the pain during palpation and the pain during movement. These parameters were assessed by the veterinarian in the clinical visits every ten days. The scale for most of the questions goes from one (no functional limitation or no pain) to four (complete functional limitation orextreme pain). However, some  items use  a  three-point  scale,  such  as,  difficulty  in  little jumps,pain during palpation and pain during movement.

Evaluation questionnaire with the owner

The evaluation questionnaire that the veterinarian completed with the owner is based partially on the Canine Brief Pain Inventory (Brown et al., 2018) and is divided in three sections: description of function, description of positive behaviour and visual scale.The description of the function compiles different questions in relation to the general activity of the dog, the ability to standing up from lying down, the ability to walk, the ability to run and the ability to climb stairs. The description of positive behaviour includes two questions: mood and feel like playing. The visual scale refers to the severity of the pain that the owner thinks that his/her dog suffers. Each section has different items and each item has a numerical rating scale from 0 to 10. Zero means no pain (or that pain does not interfere) and ten means extreme pain (or that pain interferes completely). Besides individual measurements for each parameter we obtained an average value for all the parameters included in the description of the function and another for the description of positive behaviour. The improvement with respect to the beginning of the study was calculated for each treatment (mean day 40 – mean day 0) x100 / mean day 0). We also calculated the difference in improvement between therapy and placebo (mean treatment – mean placebo).

Blood analysis

Blood was obtained from the cephalic vein for biochemical analyses of inflammation markers. It was collected under aseptic conditions in 5 ml tubes, and then centrifuged for 5 min at 2000 rpm to collect serum. Serum samples were analysed for Tumour necrosis factor-α (TNF-α) and nitric oxide (nitrite/nitrate) (NO). TNFα assay was performed using canine TNFα commercial ELISA set from R&D (Ref. CATA00, R&D systems, La Joya, CA), following the manufacturer’s instructions. No assay was performed as previously described (Miranda, Espeyand Wink, 2001; García-Robledo, Corzo and Papaspyrou, 2014).

Sonography

Sonographic evaluations were performed under routine sedation in all dogs using the Vivid I (GE Healthcare, Wauwatosa, WI, USA) ultrasound system at day 0 and 40 after treatment. Linear transducer 12L-RS of high frequency (5-13 MHz) was used for the ultra sound examination. The animals were placed in dorsal decubit, with posterior members in neutralposition (between 10° flexion and 30° extension, 10° and 30° abduction and 0° and 10°external rotation), the knees forming an angle of 90° between the femur and the tibia/fibula. The images of the coxofemoral joints (both hips) were obtained in three sagittal views parallel to the longitudinal axis of the lateral side of the hip, dorsal to the greater trochanter of the femur. The measurement of the distance between the femur head and the acetabulum was taken for each view and the major value was considered for statistical analysis.

Statistical analysis

Data were analysed using the GraphPad (GraphPad Prism version 6.0 for Windows, GraphPad Software, Inc) program and the Epi Info TM (Epi Info version 7 for Windows, CDC). Data were assessed for normality with the D’Agostino and Pearson normality test, and for homoscedasticity with the Bartlett’s test. Parametric analyses were performed with one-way ANOVA with repeated measures followed by  Holm  Sidak’s  multiple  comparison   post-test.  Non-parametric samples were compared with Friedman test for related samples.

All participants completed the study. The percentage of dogs taking part in the supplement study was similarly distributed among grade 1, grade 2, grade 3 and grade 4 of OA. Most of the dogs fulfilled two of the following inclusion criteria (48%): difficulty to stand up, difficulty to jump, difficulty to climb stairs or clear lameness, where the difficulty to stand up was the most common sign. Dogs were not administered rescue pain medication. The possible side effects related to the treatment such as: changes in appetite, vomits, diarrhoea and skin reactions were assessed in each veterinary visit. No significant side effects were seen during the treatment with Ovopet® and the veterinary did not relate the observed symptoms with Ovopet® as similar reactions were seen in the placebo group. Moreover, an initial test was carried out in 10 dogs giving them ten times the recommended daily dosage for 50 days to assess that Ovopet® was safe.

Assessment of muscular atrophy

A gradual improvement in the muscular perimeter in the group treated with Ovopet® was observed along the study reaching a significant improvement of 7.3% in the right (p<0.0001) and 8.9% in the left (p<0.0001) rear legs after 40 days of treatment with Ovopet® (Figure 1A and B). The placebo group showed only a slight increase (2.8%, p=0.3155 and 3.2%, p=0.0011) which differs from the continuous improvement showed in both legs of treated dogs since day 20 of treatment.

Assessment of flexion-extension range

To evaluate the hip mobility range, the veterinarian measured the flexion and extension degrees of the hip every ten days. Two patterns were observed depending on the treatment; the control group showed a significant increase in the flexion range (right rear leg, p=0.0266and left rear leg, p=0.0243) during the study while in the dogs treated with Ovopet® this parameter was significantly reduced (p=0.0001) in the right rear leg and it showed a tendencyto  decrease in the left rear leg (Figure 1C and D). The opposite occurred with the extension angles, which augmented significantly only in the treated group (right rear leg, p<0.0001 and left rearleg, p=0.0011) (Figure 1E and F).

Hip functional scale

The functional limitation assesses the lameness before starting to walk, the lameness during walk, the resistance to walk, the resistance to run and play, the difficulty to climb stairsand the difficulty in little jumps (Figure 2A, B, C, D, E and F, respectively). In general, the pattern of each treatment was different. In the treated group of dogs, for the lameness before starting to walk (p=0.005), the lameness during walk (p=0.0389), the resistance to run and play (p=0.0038) and the difficulty in little jumps (p=0.01), there was a significant  decrease while the placebo group remained stable or even augmented, although not significantly (Figure 2A, B, D and F). The assessment of the resistance to walk (p=0.0015) and the difficulty to climb stairs (p=0.0024) showed a progressive decrease after  treatment which failed to reach statistical significance in the ANOVA post hoc tests. The opposite  occurred with the placebo group, which showed a tendency to increase progressively (resistance to walk) or remained stable (difficulty to climb stairs) (Figure 2C and E).

The joint mobility assesses the pain at manual mobilisation of the hip, the pain during palpation  and the pain during movement, parameters that the veterinarian measured in the clinical visits every ten days (Figure 3A, B and C respectively). In the group treated with Ovopet® there was a gradual decrease in pain since day 10 until the end of the study. There were statistically significant differences in the pain at manual mobilization  of the hip (p=0.0008) and in the pain during palpation (p=0.0193) while the pain during  movement only showed a tendency to decrease. Evaluation questionnaire with the owner compiles different questions in relation to the general activity of the dog (Figure 4A), the ability to stand up from lying down (Figure 4B), the ability to walk (Figure 4C), the ability to run (Figure 4D) and the ability to climb stairs (Figure 4E).

Here, the placebo effect associated with the owners of the dogs regarding the function description was not observed in this study population which is in concordance with the statement of other researchers saying that when looking at the group average change in animal OA, there is no a placebo effect (Gagnon et al., 2017). Similarly, to assess canine behavioural changes affected by pain, an owner-reported instrument was employed. The detailed behaviour-based assessment performed by the ownershave been extensively employed (Belshaw et al ., 2014; Essner et al., 2017) and offer the advantage of an extended assessment of dogs in their typical environmentand routine (Sharkey, 2013). These results are in good concordance with the data obtained in the hip functional scale for functional limitation that was assessed by the veterinarian. They are also in concordance with data of mobility range, which revealed an improvement of the flexion and extension angles, and with muscular atrophy, which showed an increase of the rear legs perimeter.

In OA and in synovitis there is an increase in the production of pro-inflammatory factors (Mobasheriet al., 2017; Bhattaram and Chandrasekharan, 2017). The anti-inflammatory properties attributed to eggshell membrane that are potentially responsible for reducing inflammatory cytokines IL-1β and TFN-α are shown in rats (Ruff and De Vore, 2014). There is a need to prove these effects in clinical studies with eggshell membrane products used as nutraceutical compounds for dogs. Our finding of decreased NO blood concentration in treated dogs, although failed to besignificantly different, may indicate that a similar anti-inflammatory mechanism was conferred by Ovopet®. Moreover, hip joint space was analysed by ultrasounds, revealing a significant narrowing of the hip joint space. The results obtained for blood measurements of NO and the sonographic evaluation of hip joint space indicates that Ovopet® was capable of reducing the inflammation.

The goal of dietary supplements intended to treat OA is to provide relief to the major clinical signs of OA. Ovopet® arises as an effective supplement to decline inflammatory   pain,  functional  disability,  lameness  and inflammation associated to hip dysplasia and OA, and therefore, to improve the quality of life of dogs.   The data obtained in the present study support the use of this dietary supplement as an effective treatment for hip dysplasia and OA. Further research is needed to ascertain and better understand the mechanisms underlying the mode of action of eggshell membrane in osteoarthritic joints. For instance, the measurement of some biomarkers such as fragments of collagen type II (de Sousa et al., 2017) could be useful to gain insights in this complex mechanism.

The authors have not declared any conflict of interests.

The authors wishes to express their gratitudes to the Eco-innovation Initiative of the European Union for co-founding the research.