Indian Journal of Animal Research

  • Chief EditorK.M.L. Pathak

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Indian Journal of Animal Research, volume 55 issue 2 (february 2021) : 222-225

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

K. Preethi1,*, V. Gireesh Kumar1, K.B.P. Raghavender1, D. Pramod Kumar1, M. Lakshman1
1Department of Veterinary Surgery and Radiology, P.V. Narsimharao Telangana Veterinary University, College of Veterinary Science, Rajendranagar, Hyderabad-500 030, Telangana, India.
Cite article:- Preethi K., Kumar Gireesh V., Raghavender K.B.P., Kumar Pramod D., Lakshman M. (2020). 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 . Indian Journal of Animal Research. 55(2): 222-225. doi: 10.18805/IJAR.B-3930.
Background: Fractures associated with bone loss requires stabilization with suitable fixation devices, placement of appropriate bone grafts to fill up the bone defects and barrier membranes as space maintainers for enhanced bone regeneration. The aim of this study was to evaluate the use of beta-tricalcium phosphate (β-TCP) bone graft with collagen membrane as guided bone regeneration in long bone fractures with bone loss in dogs. 
Method: Six dogs with long bone fractures accompanied with bone loss in Radius-ulna, Femur and Tibia were surgically treated with suitable bone plate as internal fixation with β-TCP bone graft along with collagen membrane placed at the fracture site. 
Conclusion: The application of β-TCP along with collagen membrane for filling the bone defect is extremely simple, convenient and less time consuming and proved to be effective in promoting early bone healing with rapid later phase bone healing and provided osteoconductive support  and early resorption.
Treatment of long bone fractures with bone defects is the most intriguing and challenging aspect in veterinary orthopaedics. Therefore a variety of therapeutic modalities have been developed to enhance the bone healing response and fill the bone defects. The use of bone grafts is recommended in several surgical situations where the healing is difficult to achieve (Sen et al., 2018). Options for grafting include autograft, allograft, biomaterials and synthetic bone substitutes. The consensual ‘gold standard’ graft remains the autograft, which does not induce immunological reactions and has the ability to provide osteoinductive growth factors, osteogenic cells and acts as structural scaffold to new bone ingrowth (Bohner, 2010 and Rogers and Greene, 2012). However, this procedure is associated with prolonged anaesthetic times, limited availability, donor site morbidity (pain, intra-operative blood loss and risk of stress fracture), risk of local infection and predisposition to failure (Pinto et al., 2016). Sinibaldi (2014) vividly stated that inconvenience of allograft harvesting, processing, storage and quality assurance have limited their use. To address these problems, synthetic bone graft substitutes can be used to fill bone voided spaces which have led to extensive research in tissue engineering based on the development of osteoconductive scaffolds with osteoinductive growth factors either codelivered with or to aid in situ recruitment of osteogenic cell sources (Guda et al., 2013). Osteoconductive scaffolds are designed to provide a suitable substrate for the in-growth of bone tissue and supporting vasculature and intended to function as space maintainers for bony in-growth (Carloreis et al., 2011). To support the space maintenance function, barrier membranes are used as guided bone regeneration to prevent in-growth of faster growing fibrous tissues in bone defect spaces resulting in incomplete bone healing or nonunion (Queiroz et al., 2006, Hu et al., 2011 and Guda et al., 2013). Beta-tricalcium phosphate is the most common synthetic bone graft substitute used with collagen membrane in guided bone regeneration due to their osteoconductivity and ability to form a direct bond to host bone and to provide a local calcium source for bone regeneration (Jegoux et al., 2011 and Malhotra and Habibovic, 2016).
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.

Fig 1: Sterile â-TCP bone graft incorporated in the collagen membrane surrounding the fracture site with a small bone curette to fill the bone defect.


Fig 2: Collagen membrane along with â-TCP bone graft secured at the fracture site with polyglactin 910 no 2.0.

Out of six dogs selected for study four were males and two were females. The age of dogs ranged from 4 to 132 months with mean of 35.83 ± 20.33 and bodyweight ranged from 4 to 25 kgs with mean of 13.83 ± 2.78. Five dogs met with automobile accident and one dog had fall from height leading to long bone fractures. The collagen membrane used in this study is a bioresorbable high purity type-I cross linked membrane with porosity lesser than the penetrable size of an epithelial cell and found to be beneficial as it aided in bone regeneration in the defect space and maintained space as barrier membrane for fracture healing. This was in conformity with Oh et al., (2003) who stated that guided bone regeneration (GBR) treatment using collagen membrane enhanced bone regeneration, including bone height gain, new bone-to-implant contact and bone fill which is manifested at a later stage of healing and stated that space maintenance and prevention of membrane exposure during healing were the crucial factors for the success of GBR. The porous and compact layers of collagen membrane can not only enable osteogenic cell migration to make bone ingrowth possible, but also prevent the invasion of fibroblasts (Benque et al., 1999 and Jegoux et al., 2009). Securing the collagen membrane with polyglactin 910 immobilized the particulate bone graft in the desired position at the fracture site and thus prevented the migration of the graft into surrounding tissues. Similar procedure was adopted by Wessing et al., (2018).
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 7th, 15th, 30th, 45th and 60th 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 7th post-operative day the radiolucent fracture line was discernible. In this study bone healing was seen as early as 15th post-operative day in which the fracture line was filled with early smooth opaque callus. By 30th post-operative day, evidence of callus formation and faint radiolucent fracture line was observed with diffused arrangement of the beta-tricalcium phosphate granules. By 45th 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 60th 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.

Fig 3: Progressive radiographs in a dog with tibial fracture.

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.

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