Clinical and Radiological Evaluation of Modified DARthroplasty Using Rib Allograft Impregnated with the Mesenchymal Cells & PRP in Dogs

Hip dysplasia is an impaired development of the hip joint characterized by subluxation or complete luxation of joint with subsequent mild to severe degenerative changes mani- fested by pain and lameness (Tomlinson and Cook, 2002). The incidence of hip dysplasia is highest in large and giant breeds and has increased in the past 50 years which is a con- cern for dog owners, dog breeders, and vet- erinarians (Comhire and Snaps, 2008; Smith, 1998). While the definitive diagnosis of hip dysplasia must be based on the radiograph, other screening methods include clinical signs, palpation, ultrasound, computed to- mography, and magnetic resonance imaging (Flückiger, 2008; Ginja et al., 2009; Butler and Gambino, 2017). Treatment options of hip dysplasia range from conservative (ad- ministration of nonsteroidal anti-inflamma- tory drugs, chondroprotective drugs, weight maintenance, exercise moderation, complete rest, dietary changes, physical therapy, con- fining to restricted cages, nutraceuticals, acupuncture, and stem-cell therapy) to sur- gery (Marx et al., 2014; Vilar et al., 2014; Dycus et al., 2017; Corral, 2018). The surgi- cal options can be divided into two groups, depending on the animal’s age: (1) therapies aimed at alleviating pain consist of total hip replacement, femoral head, and neck os- tectomy, pectineal myectomy, coxofemoral denervation and (2) therapies aimed at pre- venting or lessening the amount of future degenerative joint disease(DJD) include ju- venile pubic symphysiodesis, triple pelvic osteotomy, double pelvic osteotomy, fem- oral neck lengthening, intertrochanteric os- teotomy and DARthroplasty (Moses, 2000; Denny et al., 2018; Witte, 2019). DARthro- plasty directly increases the size of the ac- etabulum by promoting the coverage of the

femoral head through implanting graft bone to the lateral rim (Slocum, 1998; Jacobs et al.; 2004, Grzegorzewski et al., 2013). DAR- throplasty is a relatively new center of atten- tion procedure for the prevention of early detrimental effects of luxation on joint el- ements (Yaprakci et al., 2014). Also, it has not been performed long enough to collect results from a large number of patients and there are  no  short-  or  long-term  scientif- ic studies documenting the effectiveness of DARthroplasty in the treatment of canine hip dysplasia (Luck, 2007). Mesenchymal stem cells (MSCs) are multipotential cells that can differentiate into osteoblasts, chondrocytes, stromal cells, adipocytes, fibrous tissue, myoblasts, tenocytes and become more com- monly utilized to promote bone graft fusion rate (Anderson et al., 2013, Lee et al., 2011). Platelet-rich plasma (PRP) containing many growth factors such as transforming growth factor-β, platelet-derived growth factor and insulin-like growth factor that  are involved in bone healing and tissue repair and also as an augmentation procedure to improve im- plant healing (Roffi et al., 2013, Jensen et al., 2005). This study aims to report the use of al- lograft bone impregnated with mesenchymal stem cells derived from bone marrow and platelet-rich plasma in canine DARtroplasty and to retrospectively evaluate the clinical and radiographic outcome after a 6-month follow-up.

Materials and Methods

In this study, twelve neutered adult male mongrel dogs, weighing ~ 22.5 ± 2.5 kg, were provided at 12 months of age by the University of Tehran (Tehran, Tehran Prov- ince, Iran). All experimental procedures involving animals were conducted accord-

ing to the Ethical Principles in Animal Ex- periments adopted by Faculty of Veterinary Medicine Research Committee and were ap- proved by the Experts Research Committee in Faculty of Veterinary Medicine Universi- ty of Tehran, Protocol 2345/95/2016. First- ly, animals received vaccination and they were wormed and castrated. After, the dogs underwent a radiographic and physical and orthopedic examination to obtain baseline information. Secondly, while coxofemoral luxation and hip osteoarthritis models have been previously described (Ozaydin et al, 2003, Little et al., 2016), a modified model of coxofemoral subluxation to induction of hip dysplasia was provided by capsulotomy, ligament capitis osis femoris transaction and capsulorrhaphy with craniolateral approach (Figure 1, A). Dogs were assessed to be at-risk for CHD by evidence of hip laxity, which was defined as orthopedic examina- tion and radiographs.

All dogs were subsequently divided into 3 groups of four dogs each: Control (acetab- ular coverage with allograft), PRP (acetab- ulum was covered by allograft impregnated with PRP), and MSCs group (covered ace- tabular with allograft and MSCs).

Surgical technique

Modified DARthroplasty was carried out on all dogs. Animals were taken off-feed for 12 h and water was withheld for 2 h. All re- search dogs were premedicated with a Ket- amine (10 mg/kg, IM, Alfasan, Netherlands) and Xylazine (0.5 mg/kg, IM, Alfasan, Neth- erlands) and afterward the left coxofemoral area was thoroughly clipped. After receiving a prophylactic dose of Cefazolin (22 mg/kg, IV, Exir, Iran), anesthesia was inducted by a combination of Ketamine (5 mg/kg, IV) and Diazepam (0.25 mg/kg, IV, Caspian Tamin, Iran). Following endotracheal intubation, in-

halation anesthesia was maintained with 2% Isoflurane in Oxygen via a rebreathing cir- cuit. Animals were positioned in left lateral recumbency, then thorax and left coxofem- oral region were aseptically prepared for the surgery with betadine and alcohol. After rou- tinely reaching the hip joint with a dorsal ap- proach, an arch-shaped  slot,  approximately 7 mm broad and 12.5 mm high, was created close dorsally to the joint capsule origin into the subchondral bone from the cranial end in anterior direction using a high speed round burr. An appropriate 2 mm cortical bone screw was selected based on measurement by depth gauge and radiograph. After ad- justment, the rib allograft was firmly packed into the slot and secured to the recipient bed with screw in a lag fashion in such a way as to cover the posteriolateral aspect of the fem- oral head and to rest on the capsule (Figure 1, B). The motion of the hip was checked to find out whether the graft did not restrict its full range, and where necessary the graft was made smaller. The next step included metic- ulous reinsertion of gluteal muscles and clo- sure of the wound routinely.

Allograft collection

Before the beginning of primary surgery, standard lateral approach to the right thorax on the area of greatest convexity was applied to harvest an approximately 25 mm segment of the 8th left rib, which was wrapped in sterile saline (0.9% NaCl) solution soaked gauze sponge to be transplanted in a recipi- ent dog which was prepared simultaneously. (Aranda et al., 2008; Boudrieau et al., 2004; Makridis et al., 2012).

PRP preparation

For obtaining autologous PRP, before each operation, peripheral venous blood was drawn into tubes containing an anticoagulant (ACD) under standard aseptic technique.

The four 10 ml ACD tubes were filled for each of the four dogs in the second group and immediately transferred to the laborato- ry. Blood samples were centrifuged (Smart- PReP2, Centrifuge, Harvest Plymouth .MA) for 5 min at 1000 g so that its three compo- nents (Erythrocytes, buffy coat, and plate- let-poor plasma) were formed based on the density of blood components. The buffy coat layer was closely collected by a pipette and pooled in a separate tube which was spun for a second time for 15 min at 1500 g so that the PRP portion was taken from the surface, 3 ml of PRP mixed with calcium chloride solu- tion was obtained. Four syringes  contain- ing PRP were delivered to operation room  so that a volume of 3 ml of PRP contain-  ing 882±199×103 plate‌let/μl was utilized around allograft site for each dog (Bearden et al., 2017; Lee et al., 2014; Messora et al., 2014; Shin et al., 2017; Dallari et al., 2016; Aldirawi et al., 2018).

Bone marrow MSCs isolation

Under strict aseptic condition bone marrow was obtained from the iliac wing of dogs three weeks before transplantation surgeries. Under anesthesia and using a Jamshidi bone marrow needle (15G), 9 mL bone marrow was aspirat- ed into a syringe containing 1mL heparinized saline solution. Immediately specimens were sent to the cell-culture facility. The BM-hep- arin blood was diluted at a 1:1 ratio with Dul- becco’s modified Eagle’s medium (DMEM; Gibco, USA) high glucose and carefully laid over the 3 ml Lymphosep density gradient medium (Biowest, France) in a 15 ml conical tube and then centrifuged for 30 min at 400 ×g. The mononuclear cell layer at the plasma-Fi- coll interface was removed and  resuspend-  ed in DMEM and finally centrifuged  at 400 gr for 10 min twice. The mononuclear cells were plated in tissue culture flasks at a den-

sity of 1×105 cells per 25 cm2 10% fetal bo- vine serum (FBS; Gibco, USA), 1000 IU/ml penicillin, and 100 μg/ml streptomycin were added to culture flasks and incubated at 37°C with 5% CO2 and 95% air at 100% humidity. Four days after the initial culture, plates were washed with Dulbecco’s Phosphate Buffered Saline (DPBS) to remove the nonadherent cell. From this point on, every 2nd day, we changed the medium until a confluence of 70-80% was achieved. The BMSCs, adherent cells, were harvested with 0.25% trypsin-ED- TA and subcultured until at passage 3 they were used in the third group. MSCs were in- jected into the gap around allograft in specif- ic dogs from which they had been collected. (Callegaro et al., 2018; Bearden et al., 2017; Jo et al., 2017; Long et al., 2013; Rafatpanah et al., 2018; Grabowski and Robertson, 2013; Mirghasemi et al., 2017; Thua et al., 2015).

Post–operative care

Radiographs were taken immediately af- ter the surgical procedure to be assessed as the baseline date for comparative purposes with follow-up radiograph  examinations  at 6 months. After recovery from general anes- thesia, the dogs were transferred to their re- spective cages. Cefazolin (22.5 mg/kg, IV), was administered every 12 h for five days and Tramadol (2 mg/kg q12h, Alborz Darou, Iran) was given for 3 days and changed to oral Tramadol (4 mg/kg q12h, Alborz Darou, Iran) for 5 days. Skin sutures were removed within 14 days. The orthopedic examination was conducted at 2 months and 6 months postoperatively also neurological examina- tions were performed to exclude conditions other than a hip disorder.

Evaluation

Ventrodorsal hip_extended radiographs were used to calculate Norberg angle and estimate femoral head coverage using the

scoring system developed by the Orthope- dic Foundation for Animals (OFA) via an MX-20 Cabinet X-ray System (35 kV, 300 μA and 240 s) under sedation (IM Ketamine (10 mg/kg) and Xylazine (0.5 mg/kg) and af- terward an IV cocktail of Ketamine (5 mg/ kg) and Diazepam (0.25 mg/kg)). The radio- graph image was used for diagnosis, DJD score (0-4), Norberg angle (NA), percent coverage (PG) of the femoral head by ace- tabulum, graft position, metallosis around screws, bone proliferation associated with the graft. The Clinical variables evaluated include Ortolani maneuver, gait observation for gross lameness score (an abnormality of gait or posture), muscle atrophy (decrease in mass of muscle), the hip extension to detect pain with passive movement and range of motion, presence of seroma (Off and Ma-

tis, 2010; Dueland et al.,  2010).  DJD also is known as  osteoarthritis,  was  defined  as a noninflammatory, noninfectious degener- ation of articular cartilage characterized by bone formation at the synovial margins and by fibrosis of periarticular soft tissue. Nor- berg angle (NA) was characterized as the re- lationship of the center of the femoral head to the craniolateral aspect of the dorsal ace- tabular rim, a NA of 105o or greater has been considered to indicate normal status. Percent Coverage is an indication of the support pro- vided by the acetabulum to oppose the force transmitted from the femur, a  PC  of  50% or greater was normal status (Figure 1, C). Ortolani sign was defined as the “clunk” or shift palpated as the femoral head enters the acetabulum during the abduction of the hip.

       
A

B                                                                       C

Figure 1. Radiographic images show (A) a hip subluxation following the modified induced coxofemoral subluxation proce- dure which is measured by NA and PC parameters in a standard ventrodorsal projection and (B) after a modified DARthro- plasty, the femoral head was completely covered with the rib allograft. (C) An illustration of periosteal reaction and allograft remodeling on the radiograph and indicates the amount of acetabular coverage.

Table 1. Clinical tests performed, time tested and percentage of the dogs tested.

Results

The authors have performed the DARthro- plasty with rib allograft on 12 hips. All dogs completed the study without major compli- cations. According to the same clinical re- sults that have been obtained at 3 months

and 6 months after surgery and the absence radiographs between post-op and 6 months, the comparison was mentioned between pre- operative and by next six months. In the control group, one graft was fixed using two screws that did not present a significant dif-

Table 2. NA and PC study showing means and standard errors in control, PRP , and MSC groups before and after 6 months of grafting.

ference in that final result compared with the other dogs. All dogs that showed positive Ortolani sign before surgery was converted to a negative after surgery. Eleven dogs had no apparent lameness at 6 months, whereas the other one had some lameness for approx- imately 2 weeks due to soft tissue injury in the control group. After the third week, all dogs had a subjectively normal gait. Post- operative re-evaluation indicated that  all dogs had normal range of motion without pain during passive movement of the limb. Muscle atrophy did not occur after surgery (Table 1). In the PRP group, one early patient had a little seroma, which was due to loose muscle closure. This responded well to aspi- ration without any sign of infection. In the MSCs group, one graft appeared slightly cra- nial to its expected location but fortunately, it was considered enough to cover the femoral head. None of the dogs revealed any DJD and metallosis findings radiographically. No dislocation of the allograft was observed in any of the dogs until to be remodeled (Fig. 1, C). The mean ± SD value of the preoperative NA was 101.25 ± 1.29 (range, 100o to 103o; median 101o), 101.75 ± 1.39  (range,  100o to 104o; median 101.5o) and 103.25 ± 0.82 (range, 102o to 104o; median 103.5o) for control, PRP and MSCs, respectively. After surgery, the mean ± SD of the same parame- ter increased to 107.75 ± 1.64 (range, 105o to 109o; median 108.50), 108.25 ± 1.82 (range,

107o to 109o; median 108.5o)  and 108.5 ±

1.12 (range, 107o to 110o; median 108.5o). In the control group, the PC values were 40.25% ± 2.04% (range, 37% to 42%; me- dian 41%) and 59.25% ± 3.27% (range, 55% to 64%; median 59) before and after surgery, respectively, and this increase was elicited in the PRP 40.25 ± 2.04% (range, 39% to 45%; median 41.5%) and 62.5% ± 2.28% (range, 60% to 62%; median 61) pre and post-op- eration,  respectively.  The  PC  surged   from

43.75 ± 1.47% (range, 42% to 46%; median 43.5%) to 66.25% ± 2.28% (range, 64% to 70%; median 68) postoperatively in MSCs group (Table 2). Bone proliferation was not- ed at the cranial aspect of the implant in all recipient dogs.

Discussion

DARthroplasy is a surgical intervention intended to improve the biomechanics of the hip, hip joint congruity, decrease abnormal hip joint laxity, normalize  articular  stress- es and reduce hip pain. It can be performed bilaterally and done with routine bone in- struments with a minimal fixation for a rea- sonable cost compared to other procedures (Witte, 2019). Dogs clinically affected by  hip dysplasia with palpable  or  radiograph- ic joint laxity before articular cartilage is badly damaged, are candidates for this pro- cedure (Hupp et al.,  2007).  DARthroplasy is also a viable surgical option for dogs in

which juvenile pubic symphysiodesis, triple pelvic osteotomy, and total hip replacement are precluded by cost, age, degree of laxity. This technique is an advanced procedure  and should be performed only by expert sur- geons and contraindicated when there are signs of advanced DJD, breakdown of the dorsal acetabular rim, or neurological dis- ease. Early reports indicated that the animals with significant DJD but without instability are not considered candidates for it, because the function of this procedure is to improve stability. This procedure results in an excel- lent return to normal function unless com- plications occur. Although temporary sciatic neuropraxia, screw breakage, infection and seroma have been reported as complications secondary to biocompatible osteoconductive polymer shelf arthroplasty, in our study none of them occurred postoperatively other than seroma in one case which responded to as- piration and pressure bandage. In this case, appropriate muscle suturing and dead space obliteration could have been useful to pre- vent seroma formation. To support our the- ory as to whether the modified procedure could lead to radiographic improvement of congruency and hip stability, NA and PC were analyzed before and after surgery, and the results of our analysis demonstrated a remarkable increase of NA and PC for hips and a subsequent decrease of subluxation and joint incongruency in all groups. Since both positive Ortolani incidence and NA  and PC in dogs had improved significantly at six months, our study demonstrated cox- ofemoral stability occurs following modi- fied DARthroplasty. Bone proliferation has been reported at the cranial of an implant on ilial wing and neck 17 weeks after surgery which was observed in our radiograph eval- uation indicating an active ossification site.

In Slocum’s series of cases, none of the pa- tients that have undergone DARthroplasty have demonstrated painful signs, which was already evident in our study. In our study,  the autologous bone graft was not applied since it is already known that it is the most effective bone substitute due to the presence of osteoprogenitor cells inside the graft that are reported to survive after transplantation (Kruyt et al., 2004) and it cannot be further enhanced by the addition of adjuvant factors, because of the presence of differentiated osteoblasts (Ronald et al., 2004). Although from a clinical point of view, a combination of bone allografts with ostegenetic factors is of great relevance, in the current study was radiologically observed the reliable bone healing at all groups, however, in PRP and MSCs groups the appreciable bone healing and remodeling were discerned. This result was probably because radiographs were taken only one time at 6 months postoper- atively. The absence of growth factors and multipotent MSCs from the periosteum and bone marrow causes bone allograft fracture and nonunion (Jensen et al., 2004, Long et al., 2013). The use of MSCs and PRP had  no adverse events and post-op complications clinically so MSCs or PRP impregnated al- lograft is a viable alternative if a dog needs bone grafting. In our experiment, the local injections of MSCs and PRP seem to have good clinical efficacy in distraction osteo- genesis, however, further investigations are needed. Shelf arthroplasty (or biocompati- ble orthopedic polymer shelf arthroplasty) was reported by Sertl and Jensen in the late 1980s as a new surgical treatment for the canine hip. In the procedure, for extending the dysplastic lateral rim on the acetabulum, a commercial polymer as a bony shelf was used (Sertl and Jensen, 1990). Although this