Haemato-biochemical evaluation of long bone fracture healing in dogs treated with stainless-steel and titanium dynamic compression plates

Introduction

Fracture healing is a dynamic and complex process involving coordinated cellular, molecular, and biochemical interactions that restore bone integrity. In veterinary orthopaedics, long bone fractures particularly those of the radius and ulna are among the most common musculoskeletal injuries in dogs, typically resulting from vehicular trauma or falls (Gadallah et al., 2009; Rudd and Whitehair, 1992) [10,24] . Successful management depends on achieving anatomical alignment, rigid stabilization, and early restoration of limb function. Dynamic compression plates (DCPs) are widely used for diaphyseal fracture fixation, providing stable reduction and mechanical support (Reddy et al., 2020; Hayashi et al., 2019) [22,12] . However, the choice of implant material significantly influences both mechanical and biological outcomes. Stainless-steel plates, though effective, may induce stress shielding and corrosion (Uhthoff et al., 1981) [29] , whereas titanium alloys such as Ti-6Al-4V offer superior biocompatibility, corrosion resistance, and osseointegration due to an elastic modulus closer to that of cortical bone (Niinomi, 2002; Golish and Mihalko, 2011) [18,11] . Fracture healing is also accompanied by systemic physiological changes that can be assessed through haematological and biochemical parameters. Haematological indices such as haemoglobin concentration, packed cell volume, erythrocyte and leukocyte counts, and differential leukocyte profiles reflect the body's inflammatory and immune responses. Similarly, biochemical markers including serum calcium, phosphorus, and alkaline phosphatase (ALP) levels provide insights into bone metabolism and osteoblastic activity (Beale, 2004; Arens et al., 1996) [5,4] .

Despite their diagnostic importance, limited data are available on the comparative haematobiochemical responses to different implant materials during fracture healing in dogs. Hence, the present study was designed to evaluate the haematological and biochemical alterations associated with the use of stainless-steel and titanium DCPs in the treatment of canine radius-ulna fractures. The primary objective was to elucidate the systemic and metabolic responses during the healing process and to compare the biocompatibility and clinical performance of these two implant materials in canine orthopaedic practice.

Materials and Methods

Study Design and Case Selection

The present study aimed to evaluate and compare the haemato-biochemical responses during fracture healing in dogs treated with dynamic compression plates (DCPs) made of stainless steel and titanium alloy. Clinical cases of long bone fractures presenting with lameness of the thoracic or pelvic limbs were selected from the clinical wards. Twelve dogs with diaphyseal radius-ulna fractures, irrespective of age and sex, were included after thorough clinical, orthopaedic, neurological, and radiographic evaluation. Cases with compound fractures or neurological deficits were excluded. The animals were randomly allocated into two groups (N=6 each):  Group I (Control): Open reduction and internal fixation using stainless steel DCPs.

 Group II: Open reduction and internal fixation using titanium alloy DCPs.

Haemato-biochemical parameters were assessed on the preoperative day and on the immediate postoperative, 7th, 15 th , 30 th , 60 th , and 90 th postoperative days. Whole blood was collected aseptically in EDTA-coated vials for haematological analysis, including haemoglobin concentration, packed cell volume (PCV), erythrocyte sedimentation rate (ESR), total erythrocyte count (TEC), total leukocyte count (TLC), and differential leukocyte count (DLC). Serum separated from clot activator tubes was analyzed for calcium, inorganic phosphorus, and alkaline phosphatase (ALP) concentrations. Data were statistically analyzed using SPSS v20, employing independent t-tests for between-group comparisons and repeated-measures ANOVA with Turkey's HSD for periodic variations.

Results and Discussion

Haematological Parameters

The mean haemoglobin (Hb), packed cell volume (PCV), and total erythrocyte count (TEC) values (Tables 123) showed a non-significant decline from the preoperative to immediate postoperative periods in both groups, followed by a gradual rise from the 7th postoperative day onwards. This transient decline can be attributed to intraoperative haemorrhage and haemodilution due to intravenous fluid administration.

Restoration of these. [27] and Chaurasia et al. (2019) [8] , whereas Reddy et al. (2021) [23] observed delayed normalization by the 60th postoperative day. The stability of these parameters in the present study suggests minimal systemic blood loss and satisfactory postoperative recovery in both implant groups. The erythrocyte sedimentation rate (ESR), (Table 4) was significantly (p<0.05) elevated during the preoperative, immediate postoperative, and 7th postoperative days in both groups, followed by a gradual decline to baseline by the 90th postoperative day. The initial elevation represents the acute inflammatory phase triggered by trauma and surgical intervention, consistent with the observations of Phaneendra et al. (2018) [20] . The normalization of ESR by day 90 indicates resolution of inflammation and establishment of bone union. Ajadi et al. (2018) [2] also noted a positive correlation between ESR and the neutrophil-to-lymphocyte ratio during osteoarthritic inflammation, corroborating the present findings. Total leukocyte count (TLC), (Table 5) values increased significantly (p<0.05) from the preoperative to 7 th postoperative day and subsequently declined to normal levels by day 15. This biphasic trend aligns with the findings of Aiello and Mays (1998) [1] , Mahendra et al. (2007) [16] , and Chaurasia et al. (2019) [8] . The early leukocytosis can be attributed to the inflammatory response and stress-induced corticosteroid release following surgical trauma (Maiti et al., 1999; Toth et al., 2014) [17,28] . The subsequent normalization reflects resolution of inflammation and progression of reparative bone formation. Differential leukocyte counts (Table 6) revealed neutrophilia with relative lymphopenia during the preoperative and immediate postoperative periods, indicative of an acute inflammatory response (Schalm et al., 1975; Sastry, 1989) [26,25] . Neutrophil counts declined significantly (p<0.05) up to the 30th postoperative day, while lymphocyte counts rose correspondingly from the 30 th . .08 *Normal physiological range of neutrophil count in dogs is 58-85%, lymphocyte (8-21%), eosinophil (0-9%) and monocyte (0-9%) G-1: Group-I, G-2: Group-II Means bearing different superscripts within a row (A, B) differ significantly (p<0.05). To 90 th postoperative days. This reciprocal pattern signifies the transition from the acute inflammatory to the chronic reparative phase (Patil et al., 2017; Chaurasia et al., 2019) [19,8] . Eosinophil and monocyte counts fluctuated within physiological limits, confirming the absence of hypersensitivity or implant rejection. The normal eosinophil trends in both groups further support the biocompatibility of titanium and stainless-steel implants.

Biochemical Parameters

Although clinical and radiographic evaluations are routinely used for fracture assessment, biochemical markers such as serum alkaline phosphatase (ALP), calcium, and phosphorus provide valuable insights into the metabolic activities associated with bone regeneration (Breur et al., 2004; Allen et al., 1998) [7,3] . Serum ALP levels (Table 7) were significantly elevated (p<0.05) during the early postoperative period (up to day 7) in both groups, followed by a gradual decline to near-normal levels by day 90. This pattern corresponds to the period of active osteoblastic proliferation and matrix mineralization (Komnenou et al., 2005; Bhavani et al., 2022) [14,6] . Elevated ALP levels in the early healing phase reflect the osteogenic response at the fracture site, while the subsequent decline indicates callus maturation and cortical bridging (Rani and Ganesh, 2003; Chaurasia et al., 2019) [21,8] . Interestingly, the titanium DCP group (Group II) recorded comparatively lower ALP activity during the 30 th and 60 th postoperative days than the stainless-steel DCP group (Group I), suggesting faster osteoblastic stabilization and superior osseointegration with titanium implants. Serum phosphorus levels (Table 8) increased up to the 7 th postoperative day, followed by a gradual, non-significant decrease toward baseline values by the 90 th day. This early rise corresponds to active mineralization during callus formation, while the later decline indicates transition to bone remodelling (Chaurasia et [8,6,9] . The lower phosphorus levels observed in Group II during the late healing phase suggest faster attainment of metabolic equilibrium, reflecting efficient osteoblastic activity associated with titanium implants. Serum calcium levels (Table 9) demonstrated a biphasic trend, showing a mild decline from the preoperative to 7th postoperative day, followed by a steady rise up to the 90th day. This decrease may be due to calcium deposition in the developing callus and temporary hypocalcaemia associated with postoperative bone metabolism (Hegde et al., 2007; Patil et al., 2017) [13,19] . The subsequent rise represents bone remodelling and mineral consolidation. The calcium values in both groups remained within the physiological range throughout, indicating balanced calcium metabolism and absence of metabolic complications. While both implant materials exhibited similar haematological and biochemical trends, the titanium DCP group consistently showed earlier normalization of leukocyte, ALP, and phosphorus values, indicating a reduced inflammatory response and faster metabolic stabilization. These findings reinforce titanium's superior biocompatibility, corrosion resistance, and enhanced osseointegration compared to stainless steel. No significant deviations in physiological parameters were observed, confirming that both materials are safe and effective for internal fixation in canine long bone fractures. Means bearing different superscripts within a row (A, B) differ significantly (p<0.05). Intergroup differences were statistically non-significant, titanium DCPs exhibited faster biochemical normalization, earlier limb function, and fewer postoperative complications. Owing to their superior biocompatibility, corrosion resistance, and mechanical compatibility with bone, titanium DCPs are recommended over stainless-steel plates for internal fixation of canine long bone fractures, despite their higher cost.

Conclusion

Both stainless-steel and titanium dynamic compression plates (DCPs) effectively stabilized long bone fractures in dogs, supporting normal haematological and biochemical recovery throughout healing. Haematological parameters remained within physiological limits, indicating minimal systemic stress, while serum alkaline phosphatase and phosphorus increased during early osteogenesis and declined as healing progressed. Serum calcium showed an initial decrease followed by normalization, reflecting callus mineralization. Although.

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