​Efficacy of Multi-ion Doped Plasma Spray Nano-hydroxyapatite Coated Titanium Implants in Long Bone Fracture Repair in Dogs- Radiographical and Bone Markers Evaluation

Numerous biochemical assays are available for measuring serum concentrations of markers of bone metabolism to evaluate healing process in fracture bones. Bone markers have been studied in general with regard to age and breed related differences in dogs (Breur et al., 2004), their clinical applications in osteomyelitis (Southwood et al., 2003) in rabbits, osteosarcomas and osteoarthritis (Garzoto et al., 2000 and Lucas et al., 2008), delayed bone union and non-union in tubular bones (Komnenou et al., 2005) and experimental bone fracture healing (Paskalev et al., 2005). Bone markers have been used in veterinary orthopaedics to assess skeletal maturation, effects of hormones on bone metabolism and in the diagnosis of bone pathology (Allen et al., 2000 and Allen, 2003). On the other hand, biochemical markers of bone cell activity are widely used in the research on metabolic diseases like osteoporosis (Greenblatt et al., 2017) and in diseases like Paget’s disease (Alvarez et al., 2001) in humans.
       
Bone alkaline phosphatase (BALP) and carboxy-terminal telopeptide of type 1 collagen (CTX) are the emerging bone markers in orthopaedics that can aid in the earlier assessment of treatment effects in fracture repairs compared to measurement of bone density (Allen et al., 2000). Fracture complications can be avoided by an early measurement of bone turnover markers like BALP and CTX that assess accurately the bone healing stages. BALP is an extracellular enzyme produced by osteoblasts. Serum BALP concentration is a specific and sensitive indicator of osteoblast activity and bone formation and a high serum concentration of BALP is indicative of high level of osteoblastic activity (Lin et al., 2016). CTX contains important inter molecular cross-linking agents of type 1 collagen and residues of cross-linking agents. The serum CTX concentration is an ideal marker of bone resorption (Seibel et al., 2001 and Lin et al., 2016). The current study was undertaken to clinically evaluate the efficacy of multi-ion doped plasma spray nano-hydroxyapatite coated titanium implants in long bone fracture repair in dogs by radiographical evaluation supported by concurrent evaluation of BALP and CTX.

After a detailed clinical, orthopaedic, neurological and radiological examination, twenty-four dogs with long bone fractures were selected from 78 dogs presented to the Department of Surgery and Radiology and Veterinary Clinical Complex of Sri Venkateswara Veterinary University, Tirupati, for a period of two years i.e., from November 2017 to November 2019. The selected dogs were free from concurrent fracture of flat bones, pelvis fractures, spinal fractures, neurological, metabolic or infectious diseases. The data regarding the incidence of long bone fractures in dogs were collected and variables like age, breed, sex, bone, limb involved, weight of the animal, etiology of fracture, location, type of fracture and clinical symptoms were recorded and analysed. The 24 dogs were divided into four groups with six dogs in each group and fracture repair was carried out in all the dogs. Open reduction and internal immobilization were done with uncoated titanium intramedullary pins (group I), or with plasma spray nano-hydroxyapatite coated titanium intramedullary pins (group II), or with plasma spray multi-ion (5% zinc, 2.5% strontium and 2.5% fluorine) doped nano-hydroxyapatite coated titanium intramedullary pins (group III) or with plasma spray multi-ion (5% zinc, 5% strontium and 2.5% silver) doped nano-hydroxyapatite coated titanium intramedullary pins.
       
Lateral and cranio-caudal radiographs of the fractured bone were taken at 90^o angle (Shales, 2008) for confirmatory diagnosis, classification of fractures, selection of implants and approach. In all the dogs, fractures were classified according to the fracture configuration using Winquist-Hansen (W-H) and AO (ASIF) classification. Preparation, characterization and standardization of multi-ion doped plasma spray nano-hydroxyapatite titanium intramedullary pins were done at Central Glass and Ceramic Research Institute (CGCRI), Kolkata. Steinmann pins of thickness sufficient to fill up 70 - 80 per cent of the marrow cavity at the isthmus were used. Cerclage wire of appropriate size was used for ancillary fixation in necessary cases.
       
Atropine-Xylazine @ 0.02 mg and 1 mg/kg body weight, IM as pre-anaesthetic medication and after an interval of 30 minutes, Ketamine-Diazepam at the dose rate of 8 mg/kg body weight and 0.3 mg /kg body weight respectively were administered IV for induction. Following induction of anaesthesia, dogs were intubated with endotracheal tubes of suitable size. General anaesthesia was maintained with 1.5 to 2 per cent Isoflurane.
       
Lameness grade was assigned to all the dogs during the pre and post-treatment period based on the weight bearing nature during stance and while walking (Vasseur et al., 1995). Radiographs of repaired fractures were taken post-operatively on 0^th day, immediately after 3^rd, 6^th and 9^th week to assess the fracture healing by radiographic scoring system (Hayashi et al., 2008) (Table 1). Blood samples were collected in vacutainer tubes post-operatively on 0^th day, immediately after 3^rd, 6^th and 9^th week to evaluate the levels of serum calcium, serum phosphorus and serum alkaline phosphatase, BALP and CTX. Serum concentrations of BALP and CTX were estimated using ELISA kits developed by Bioassay Technology Laboratory. Functional limb outcome during post-operative period was assessed in all the dogs by periodical clinical examinations. The data collected were statistically analysed using SPSS-ANOVA Post Hoc tests. Implants were removed on 9th post-operative week after evaluating the radiographs for fracture healing.
 

Out of 78 long bone fractures in dogs, femoral fractures accounted for 69.23% followed by radius (14.10%), humerus (10.25%) and tibia (6.41%). Singh et al., (2017) and Uddin et al., (2017) also documented similar observations. Femur was found to be the most commonly affected long bone which might be due to the counteracting pulls of flexor and extensor muscles and also due to more exposure of hind quarters directly to the major force of impact. Similar findings were recorded by Dvorak et al., (2000). In the present study, left limb was most commonly involved in fracture, as observed by Rani et al., (2004) and Raghunath et al., (2007).
       
Fractures were classified based on AO/ASIF classification and Winquist - Hansen (W-H) classification. In the present study majority of long bone fractures were of grade I (66.66%) followed by grade II, IV, V and III. Gondalia and Siddiqui (2015) reported higher incidence of grade II fractures in dogs. AO/ASIF classification of fractures revealed that majority of long bone fractures were simple (75.64%) followed by compound (17.95%) and complex type (6.41%). Diaphyseal fracture was the most observed (71.80%) fracture type. Dvorak et al., 2000 and Kumar et al., 2007 also documented similar observation.
         
Lameness was graded on the basis of gait and weight bearing in all the animals at 0^th day, 3^rd, 7^th, 14^th, 21^st, 30^th, 45^th and 60^th post-operative days. Lameness was not observed in all the dogs on 60^th post-operative day. Dogs subjected to open reduction and internal immobilization with group-IV pins had the earliest limb usage without lameness by 21^st post-operative day.
       
Uncoated titanium implants were stable in all the dogs throughout the study in group I dogs. Stable reduction and stable implants were observed in all the animals by 3^rd week. Radiographic evaluation score immediately after 6th and 9th post-operative week was 4 and 5 in all the dogs (Fig 1).
 

       
Fracture reduction was stable in all the group II dogs during the entire period of study. After 3^rd post-operative week, most of the dogs had radiographic evaluation score of 3 and on 6^th post-operative week, the score was 4. By 9th post-operative week, the score was 5 in 3 dogs and the remaining 3 dogs had a score of 6.
       
Fracture reduction was stable in all the group III dogs during the entire period of study. Radiographic evaluation of all the dogs in the group after 3^rd, 6^th and 9^th post-operative week revealed score of 4, 4 and 6 respectively.  
       
Fracture reduction was stable in all the group IV dogs during the entire period of study. Radiographic evaluation of dogs on 3^rd post-operative week revealed score 5 in most of the dogs and the score was 5 and 6 on 6^th and 9^th post-operative week in all the dogs.
       
BALP values increased significantly up to 6^th post-operative week in group I dogs followed by a significant decrease by 9^th week. In group II, III and IV dogs, BALP values increased up to 3^rd post-operative week and decreased later on (Table 2). The increase in BALP values up to 3^rd post-operative week and subsequent decrease, after 3^rd to 9^th post-operative week was rapid in dogs of group III and group IV and this might be due to the use of multi-ion doped nano-hydroxyapatite coated titanium intramedullary pins for fracture repair in these group of dogs. The rapid increase in BALP values by 3^rd post-operative week caused due to the intramedullary pins in group III and group IV dogs was suggestive of a rapid bone turnover at the fracture site facilitating faster fracture healing and early limb usage which on observation was also corroborated by the radiographic scores, lameness grading and serum biochemical studies. This was in agreement with the findings of Sousa et al., (2015) who reported higher BALP concentrations during early fracture healing in dogs. Southwood et al., (2003) also reported increased serum BALP concentration at 8 weeks in femur defects in a rabbit model.
 


The mean±SE values of CTX in group I and IV dogs were significantly different on 0^th day, after 3^rd, 6^th and 9^th week post operatively (Table 2). In group II dogs, the CTX values remained unchanged up to 3^rd post-operative week but were significantly different after 6^th and 9^th post-operative weeks. The CTX values of group III dogs were not significantly different up to 6^th post-operative week but were significantly different on 9^th post-operative week. The CTX values were at peak levels on 9^th post-operative week in all the groups and this was rapid and higher in group III and highest in group IV dogs indicating bone reabsorption and bone tissue remodelling was rapid due to osteoclastic activity at the fracture site in these group of dogs. The highest CTX values in group IV followed by group III dogs from 3^rd week to 9^th week is indicating the completion of fracture healing and near completion of bone tissue remodelling facilitated by the multi-ion doped nano-hydroxyapatite coated titanium intramedullary implants. Completion of fracture healing was evidenced by 3^rd week in the radiographic scores and serum biochemical studies in these three groups. Lucas et al., (2008) observed significant increase in serum CTX concentration in dogs with osteosarcoma. However, Herrman et al., (2002) reported no significant difference in concentrations of CTX in normal and delayed healing of fractures in human. Moghaddam et al., (2011) reported lower serum concentration of CTX after surgery in delayed fracture healing in humans. However, Belic et al., (2010) reported higher CTX values in heavier dogs. 

Long bone fracture repair using multi-ion doped nano-hydroxyapatite coated titanium implants in dogs was evaluated by radiographical studies and by bone markers evaluation. Majority of dogs subjected to open reduction and internal immobilization with multi-ion doped nano hydroxyapatite coated titanium intramedullary pinning, showed early limb usage with no post-operative complications like pin migration and pin track infection. However, using multi-ion doped i.e., either 5% zinc, 5% strontium and 2.5% silver ion doped (group IV) or 5% zinc, 2.5% strontium and 2.5% fluorine ion doped (group III) nano hydroxyapatite coated titanium intramedullary implants for long bone fracture repair in dogs resulted in excellent functional limb out come with the earliest limb usage by 3^rd post-operative week as evidenced by radiographical evaluation scores and serum concentration levels of BALP and CTX values. Radiographical evaluation scores improved from 5 in 3^rd / 6^th post-operative week to a score of 6 by 9^th post-operative week.  BALP values increased rapidly up to 3^rd post-operative week in group III and group IV dogs suggesting rapid bone turnover at the fracture site facilitating faster fracture healing and early limb usage. CTX values were at peak levels on 9^th post-operative week in all the groups and this was rapid and higher in group III and highest in group IV dogs indicating osteoclastic activity at the fracture site resulting in completion of fracture healing and near completion of bone tissue remodelling facilitated by the multi-ion doped nano-hydroxyapatite coated titanium intramedullary implants with no post-operative complications. Radiographical evaluation clubbed with concurrent evaluation of serum bone marker concentrations could aid in the earlier assessment of treatment effects in fracture repairs and hence fracture complications could be avoided as BALP and CTX values can precisely assess the bone healing stage during fracture repair.