Use of Beta-tricalcium Phosphate Bone Graft with Collagen Membrane as Guided Bone Regeneration in Long Bone Fractures with Bone Loss in Dogs: A Clinical Study

Six (6) dogs were surgically treated with suitable bone plate as internal fixation with beta-tricalcium phosphate (β-TCP) bone graft along with collagen membrane placed at the fracture site. Out of six dogs treated, five were surgically treated with locking compression plate system and one with veterinary cuttable plate (VCP) system depending on the body weight of the patient involving three femoral fractures, two tibial fractures and one radius-ulna fracture. The dogs were examined for loss of function, abnormal motility, deformity or change in angulation of affected limb, pain and crepitation at the fracture site. Clinical signs and neurological status of the dogs was recorded. Following initial clinical assessment, the dogs were subjected to pre-operative radiographic examination in two orthogonal views. The owners were advised to withhold food for about 12 hours and water for about 6 hours to dogs prior to surgery. Atropine sulphate @ 0.04 mg/kg body weight was administered subcutaneously as pre-anaesthetic medication followed 10 minutes later by xylazine hydrochloride @ 1 mg/kg body weight intramuscularly. Ten minutes later, general anaesthesia was induced with intramuscular injection of ketamine hydrochloride @ 10 mg/kg body weight and maintained with intravenous infusion of propofol @ 4 mg/kg body weight. Standard surgical approaches were made for radius-ulna, femur and tibia fractures as recommended by Johnson (2013). Following the surgical exposure of the fracture site, the fracture fragments were aligned, reduced and held with bone holding forceps to restore the length and correct rotational orientation before securing the plate. Locking compression plate was then placed over the bone and the plate was held in position with plate holding forceps and secured to the bone with locking screws placed on either end with proximal most and distal most screws placed initially and bone plating was accomplished by insertion of additional screws in both proximal and distal fragments leaving the fracture line. Similarly required length of VCP was cut based on radiographic measurement and bone plating was accomplished. Following the fracture fixation with suitable bone plating, 0.5 gm of sterile  beta-tri calcium phosphate bone graft was placed in a sterile petridish and mixed with 1 ml of fresh blood collected from cephalic vein (Franch et al., 2006). The graft was placed with a small bone curette to fill the bone defect at the fracture site (Fig 1). The bone defect filled with the graft was surrounded snugly by the collagen membrane. The membrane with bone graft was secured at the fracture site with polyglactin 910 no 2.0 (Fig  2). Soft tissue closure was done immediately after graft placement. Ceftriaxone sodium  was administered @ 25 mg/kg body weight as intramuscular injection twice a day for 7 days postoperatively. Injection Meloxicam  was administered once a day @ 0.3 mg/kg body weight by intramuscular route for 3 days. Owners were advised to restrict the movement of the animal for the first 2 weeks of surgery and then to allow leash walking for the next few weeks.
 

 

       
A sterile β-TCP bone graft with nano crystalline granules of particle size 200-300 microns was used. The osseous ingrowth and the rate of degradation is determined by microporosity of the graft with ideal pore size between 300 and 500µm. This was also in correlation with Damron (2007) and Tanaka et al., (2017). None of the cases showed any adverse tissue reaction to β-TCP, thus confirming its complete biocompatibility. Sasaki et al., (2017) and Grado et al., (2018) observed that beta-tri calcium phosphate have an excellent record of biocompatibility with no reports of systemic toxicity or foreign body reactions. 
       
Radiographic evaluation was carried out immediately after the surgery and on 7^th, 15^th, 30^th, 45^th and 60^th post-operative day. In present study, all the fractures healed by primary bone healing with minimal callus formation. This may be due to the anatomical reduction of fractures along with neutralization plate fixation which lead to primary bone healing with minimal callus formation making it difficult to define the time of radiographic union  Similar observations were made by Gibert et al., (2015). However, in all cases the fracture line was initially visible on all postoperative radiographs, with disappearance of the fracture line over time indicative of bone healing. The β-TCP bone graft applied at the fracture site appeared indistinct in immediate post-operative radiographs in five dogs which could be mainly due to the smaller particle size of the graft, quantity of the graft used in relation to the defect size, fracture configuration which include type of the fracture and size of the defect. Anker et al., (2005) upheld the fact that the limitation involved in evaluating graft incorporation via radiograph review, as the radioopaque appearance of the graft was challenging to assesss at times. This difficulty had been noted in past studies involving use of β-TCP as construed by Bucholz (1987) and Fleming et al., (2000). In one dog graft appeared as radio opaque granular structure in immediate post-operative radiograph.
       
By 7^th post-operative day the radiolucent fracture line was discernible. In this study bone healing was seen as early as 15^th post-operative day in which the fracture line was filled with early smooth opaque callus. By 30^th post-operative day, evidence of callus formation and faint radiolucent fracture line was observed with diffused arrangement of the beta-tricalcium phosphate granules. By 45^th post-operative day the fracture gap gradually decreased and the appearance of progressive bridging callus with adequate radio-density was observed radio graphically in five dogs whereas in one dog plate exposure at incision site along with proximal screw pullout was noticed. By 60^th post-operative day the fracture line disappeared and the callus became radio-dense with distinct cortical shadow pointing to the restitution of corticomedullary continuity as shown in Fig 3. This could be elicited from this finding that the resorption of the graft with its osteoconductive nature was seen by 8-12weeks. This was in corroboration with the findings of Ogose et al., (2004), Shim et al., (2013), Campana et al., (2014) and Sasaki et al., (2017). The haematological and serum biochemical values fluctuated non significantly within the physiological limits through different post-operative days in all six dogs.
 

The β-TCP bone graft used along with collagen membrane proved to be effective in promoting early bone healing and rapid later phase bone healing and provided high osteoconductive  support  and early resorption. The results obtained do serve as basic clinical research outcome, however future controlled studies with larger sample size need to be undertaken in a clinical setting to validate the osteoconductive properties of β-TCP bone graft along with bioresorbable collagen membrane.