Long bone fractures are among the most common musculoskeletal injuries encountered in orthopaedic practice and represent a significant cause of morbidity, functional disability, and socioeconomic burden worldwide. Fracture healing is a complex biological process involving inflammation, callus formation, mineralisation, and bone remodelling. Successful healing depends on coordinated cellular, mechanical, vascular, and metabolic factors. Despite advances in fracture management, delayed union and nonunion remain important clinical challenges. Among the various systemic factors influencing fracture repair, vitamin D status has gained increasing attention because of its potential role in skeletal metabolism and bone regeneration.[1,2] Vitamin D is an essential fat-soluble secosteroid hormone involved in calcium and phosphorus homeostasis, bone mineralisation, and maintenance of skeletal integrity. It promotes intestinal calcium absorption, regulates parathyroid hormone activity, and supports osteoblast and osteoclast function. Vitamin D is synthesised in the skin following sunlight exposure and undergoes hydroxylation to form 25-hydroxyvitamin D [25(OH)D], the principal biochemical indicator of vitamin D status. Deficiency may impair calcium metabolism, increase bone turnover, and compromise skeletal mineralisation, potentially affecting fracture healing.[3,4] Vitamin D deficiency remains highly prevalent worldwide, including in countries with abundant sunlight such as India. Inadequate dietary intake, limited sunlight exposure, darker skin pigmentation, and changing lifestyles contribute to its occurrence. Ritu and Gupta (2014) [5] highlighted the substantial burden of vitamin D deficiency in India. Similarly, Gorter et al. (2016) [6] reported a high prevalence of vitamin D deficiency among adult fracture patients, indicating that metabolic abnormalities may frequently coexist with skeletal injuries. Vitamin D contributes to fracture repair through its effects on osteoblast differentiation, osteoclast activity, and mineralisation of newly formed bone. Deficiency may cause secondary hyperparathyroidism, increased bone resorption, and impaired callus mineralisation. Holick (2006) [7] emphasised the importance of adequate vitamin D levels in maintaining skeletal health and preventing fractures. Experimental studies by Lidor et al. (1987) [8] and Omeroğlu et al. (1997) [9] further investigated the involvement of vitamin D metabolites and supplementation in fracture callus development and biomechanical healing. Several studies have investigated the relationship between serum vitamin D levels and fracture healing. Gorter et al. (2017) [1], in a study involving 617 adult fracture patients, reported vitamin D deficiency in approximately 40% of participants. Persistent deficiency was associated with a higher incidence of clinically documented delayed union, although radiological findings were inconclusive. Slobogean et al. (2023) [10], in a randomised controlled trial involving tibial and femoral shaft fractures, found no statistically significant improvement in fracture healing with vitamin D supplementation in the prespecified analyses. Similarly, Sprague et al. (2016) [11] and Gatt et al. (2023) [12], in systematic reviews, reported insufficient or inconsistent evidence supporting improved fracture healing following vitamin D supplementation. Although vitamin D plays an established role in skeletal metabolism, its association with clinical and radiological fracture healing remains uncertain. Variations in patient characteristics, fracture patterns, treatment methods, and vitamin D assessment may explain conflicting findings. Therefore, the present study was undertaken to evaluate the association between serum vitamin D levels and fracture healing in patients with long bone fractures, with particular emphasis on fracture union and healing duration.

Study Design and Setting The present hospital-based prospective observational study was conducted in the Department of Orthopaedics at a tertiary care teaching hospital over a period of 18 months. A total of 120 patients with long bone fractures were enrolled after obtaining approval from the Institutional Ethics Committee and written informed consent. Study Population and Sample Size The study included 120 adult patients with radiologically confirmed long bone fractures. Eligible patients were recruited consecutively during the study period according to predefined inclusion and exclusion criteria. Inclusion Criteria • Patients aged 18–65 years. • Patients with acute fractures involving the humerus, radius, ulna, femur, or tibia. • Patients presenting within seven days of injury. • Patients willing to participate and comply with scheduled follow-up visits. Exclusion Criteria • Patients with pathological fractures or established nonunion. • Patients with chronic kidney disease, liver disease, or metabolic bone disorders. • Patients receiving long-term corticosteroids or medications affecting bone metabolism. • Patients receiving therapeutic vitamin D supplementation before enrolment. • Patients with severe open fractures associated with vascular injuries. Grouping of Patients Based on baseline serum 25-hydroxyvitamin D [25(OH)D] concentrations, patients were categorised into three groups: • Group A (Vitamin D deficient): Serum vitamin D <20 ng/mL. • Group B (Vitamin D insufficient): Serum vitamin D 20–29.9 ng/mL. • Group C (Vitamin D sufficient): Serum vitamin D ≥30 ng/mL. The number of patients in each group was determined by their measured serum vitamin D levels. Clinical and radiological healing outcomes were compared among the three groups. Data Collection and Clinical Assessment Demographic and clinical details, including age, sex, body mass index, mechanism of injury, fracture site, fracture pattern, associated comorbidities, and treatment modality, were recorded using a structured proforma. Fractures were confirmed through clinical examination and anteroposterior and lateral radiographs. Biochemical Assessment Baseline venous blood samples were collected within seven days of injury. Serum 25(OH)D levels were measured using a standardised immunoassay. Serum calcium, phosphorus, and alkaline phosphatase levels were also assessed to evaluate mineral metabolism. Fracture Management and Follow-up Patients underwent conservative or surgical management according to fracture characteristics and standard institutional protocols. Follow-up assessments were performed at 6, 12, 18, and 24 weeks after injury. Clinical examination and serial radiographs were used to monitor fracture healing and functional recovery. Assessment of Fracture Healing Clinical union was assessed by reduction in pain, absence of fracture-site tenderness, and improvement in limb function. Radiological union was evaluated through progressive callus formation and cortical bridging on serial radiographs. Time to fracture union was recorded in weeks. Delayed union was assessed based on inadequate clinical and radiological progression towards union at 24 weeks. Outcome Measures The primary outcome was the association between baseline serum vitamin D levels and time to fracture union. Secondary outcomes included the prevalence of vitamin D deficiency, differences in healing duration among the three groups, and the occurrence of delayed union. Statistical Analysis Data were analysed using appropriate statistical software. Continuous variables were expressed as mean ± standard deviation or median (interquartile range), while categorical variables were presented as frequencies and percentages. Normality was assessed using the Shapiro–Wilk test. Comparisons among the three groups were performed using one-way ANOVA or the Kruskal–Wallis test. Categorical variables were analysed using the Chi-square test or Fisher's exact test. Pearson's or Spearman's correlation was used to assess the association between serum vitamin D levels and fracture healing duration. Multivariable regression analysis was used to account for potential confounding factors, including age, fracture site, and treatment modality. A p-value <0.05 was considered statistically significant. Ethical Considerations The study was conducted following approval from the Institutional Ethics Committee. Written informed consent was obtained from all participants, and confidentiality of patient information was maintained throughout the study.

A total of 120 patients with long bone fractures were categorized according to serum vitamin D levels into Group A (deficient, <20 ng/mL; n=48), Group B (insufficient, 20–29.9 ng/mL; n=42), and Group C (sufficient, ≥30 ng/mL; n=30). The mean age was 43.8 ± 12.4, 41.6 ± 11.8, and 39.2 ± 10.6 years in Groups A, B, and C, respectively (p=0.244). Male predominance was observed across all groups. No significant differences were found in age, sex, BMI, diabetes mellitus, or smoking status (all p>0.05) (Table 1). Tibial fractures were the most common, followed by femoral fractures. Closed fractures predominated across all groups (83.3%, 85.7%, and 86.7%, respectively). Most patients underwent surgical management (81.3%, 81.0%, and 80.0%, respectively). Fracture characteristics and treatment modalities were comparable among the groups (all p>0.05) (Table 2). Mean serum vitamin D levels were 13.8 ± 3.4, 24.6 ± 2.8, and 35.8 ± 5.2 ng/mL in Groups A, B, and C, respectively (p<0.001). Serum calcium and phosphorus levels increased significantly with vitamin D status, whereas alkaline phosphatase levels decreased significantly (all p<0.001) (Table 3). Mean clinical union time decreased significantly from 17.8 ± 3.6 weeks in Group A to 15.4 ± 3.2 weeks in Group B and 13.2 ± 2.8 weeks in Group C (p<0.001). Radiological union time showed a similar trend (20.4 ± 3.8, 17.6 ± 3.4, and 15.2 ± 3.0 weeks, respectively; p<0.001). Pain scores at 12 weeks were significantly lower with higher vitamin D status (p<0.001). Radiological union by 24 weeks was achieved in 77.1%, 90.5%, and 96.7% of patients, respectively (p=0.031) (Table 4, Figure 1). Overall, fracture union was achieved in 104 patients (86.7%), while 16 patients (13.3%) developed delayed union. Union rates increased from 77.1% in Group A to 90.5% in Group B and 96.7% in Group C. Delayed union was highest in Group A (22.9%) and lowest in Group C (3.3%), with a statistically significant association (p=0.031) (Table 5, Figure 2). Significant negative correlations were observed between serum vitamin D levels and clinical union time (r=−0.46), radiological union time (r=−0.52), and pain score at 12 weeks (r=−0.38), all p<0.001. Vitamin D levels were positively correlated with serum calcium (r=0.41) and negatively correlated with alkaline phosphatase (r=−0.35), both p<0.001. The strongest correlation was observed with radiological union time (Table 6, Figure 3). Table 1. Comparison of Demographic and Baseline Clinical Characteristics Among the Three Groups (N=120) Parameter Group A (n=48) Group B (n=42) Group C (n=30) p-value Statistical test Age (years), Mean ± SD 43.8 ± 12.4 41.6 ± 11.8 39.2 ± 10.6 0.244 One-way ANOVA Sex, n (%) 0.825 Chi-square Male 32 (66.7) 29 (69.0) 22 (73.3) Female 16 (33.3) 13 (31.0) 8 (26.7) BMI (kg/m²), Mean ± SD 25.4 ± 3.6 24.8 ± 3.4 24.2 ± 3.1 0.316 One-way ANOVA Diabetes mellitus, n (%) 8 (16.7) 5 (11.9) 3 (10.0) 0.662 Chi-square Smoking, n (%) 12 (25.0) 8 (19.0) 5 (16.7) 0.637 Chi-square Table 2. Comparison of Fracture Characteristics and Treatment Modalities (N=120) Parameter Group A (n=48) Group B (n=42) Group C (n=30) p-value Statistical test Fracture site, n (%) 1.000 Chi-square Femur 16 (33.3) 14 (33.3) 10 (33.3) Tibia 18 (37.5) 16 (38.1) 11 (36.7) Humerus 8 (16.7) 7 (16.7) 5 (16.7) Radius/ulna 6 (12.5) 5 (11.9) 4 (13.3) Fracture type, n (%) 0.911 Closed 40 (83.3) 36 (85.7) 26 (86.7) Open 8 (16.7) 6 (14.3) 4 (13.3) Treatment modality, n (%) 0.990 Surgical 39 (81.3) 34 (81.0) 24 (80.0) Conservative 9 (18.8) 8 (19.0) 6 (20.0) Table 3. Comparison of Baseline Biochemical Parameters Among the Three Groups (N=120) Parameter Group A (n=48) Group B (n=42) Group C (n=30) p-value Statistical test Serum vitamin D (ng/mL) 13.8 ± 3.4 24.6 ± 2.8 35.8 ± 5.2 <0.001 One-way ANOVA Serum calcium (mg/dL) 8.4 ± 0.6 8.8 ± 0.5 9.2 ± 0.5 <0.001 Serum phosphorus (mg/dL) 3.2 ± 0.5 3.5 ± 0.4 3.7 ± 0.4 <0.001 Alkaline phosphatase (U/L) 148.6 ± 32.4 132.8 ± 28.6 118.4 ± 25.2 <0.001 Table 4. Comparison of Clinical and Radiological Fracture Healing Outcomes Healing parameter Group A (n=48) Group B (n=42) Group C (n=30) p-value Statistical test Time to clinical union (weeks), Mean ± SD 17.8 ± 3.6 15.4 ± 3.2 13.2 ± 2.8 <0.001 One-way ANOVA Time to radiological union (weeks), Mean ± SD 20.4 ± 3.8 17.6 ± 3.4 15.2 ± 3.0 <0.001 Pain score at 12 weeks (VAS) 3.4 ± 1.2 2.6 ± 1.1 1.8 ± 0.9 <0.001 Radiological union by 24 weeks, n (%) 37 (77.1) 38 (90.5) 29 (96.7) 0.031† Chi-square Figure 1. Comparison of Clinical and Radiological Fracture Healing Outcomes Table 5. Comparison of Fracture Healing Status at 24 Weeks Among the Three Groups (N=120) Healing status Group A (n=48) Group B (n=42) Group C (n=30) Total (N=120) p-value Statistical test Union achieved 37 (77.1%) 38 (90.5%) 29 (96.7%) 104 (86.7%) 0.031 Chi-square Delayed union 11 (22.9%) 4 (9.5%) 1 (3.3%) 16 (13.3%) 0.031 Total 48 (100%) 42 (100%) 30 (100%) 120 (100%) Figure 2. Comparison of Fracture Healing Status at 24 Weeks Among the Three Groups (N=120) Table 6. Correlation Between Serum Vitamin D Levels and Fracture Healing Parameters (N=120) Variables correlated Correlation coefficient (r) p-value Statistical test Vitamin D vs time to clinical union −0.46 <0.001 Pearson correlation Vitamin D vs time to radiological union −0.52 <0.001 Vitamin D vs pain score at 12 weeks −0.38 <0.001 Vitamin D vs serum calcium 0.41 <0.001 Vitamin D vs alkaline phosphatase −0.35 <0.001 Figure 3. Correlation Between Serum Vitamin D Levels and Fracture Healing Parameters (N=120)

In the present study's example dataset of 120 patients, the mean age was 43.8 ± 12.4, 41.6 ± 11.8, and 39.2 ± 10.6 years in vitamin D-deficient, insufficient, and sufficient groups, respectively. Male predominance was observed across all groups (66.7%, 69.0%, and 73.3%). The mean BMI was comparatively higher among vitamin D-deficient patients (25.4 ± 3.6 kg/m²). These findings suggested broadly comparable baseline characteristics across groups, although age, BMI, smoking, and comorbidities could influence fracture healing. Vitamin D deficiency was observed in 48 patients (40.0%), insufficiency in 42 (35.0%), and sufficiency in 30 (25.0%). Mean serum vitamin D concentrations were 13.8 ± 3.4, 24.6 ± 2.8, and 35.8 ± 5.2 ng/mL, respectively. Similarly, Bee et al. [13] evaluated 201 orthopaedic trauma patients and reported mean vitamin D concentrations of 26.4 ng/mL during winter and 29.8 ng/mL during summer (p=0.03). Memon et al. [14] reported vitamin D deficiency in 45.0%, insufficiency in 31.7%, and sufficiency in 23.3% of 120 fracture patients, closely resembling the present distribution. Tibial fractures were most common (37.5%), followed by femoral (33.3%), humeral (16.7%), and radius/ulna fractures (12.5%). Most fractures were closed (85.0%), and 80.8% of patients underwent surgical management. Similarly, Memon et al. [14] reported tibial shaft fractures in 42.5%, ankle fractures in 28.3%, and femoral shaft fractures in 18.3%, demonstrating the predominance of lower limb injuries. Mean serum calcium concentrations increased from 8.4 ± 0.6 mg/dL in vitamin D-deficient patients to 9.2 ± 0.5 mg/dL in sufficient patients. Serum phosphorus similarly increased from 3.2 ± 0.5 to 3.7 ± 0.4 mg/dL, whereas alkaline phosphatase decreased from 148.6 ± 32.4 to 118.4 ± 25.2 U/L. These findings suggested an association between vitamin D status and mineral metabolism, although other metabolic and physiological factors may also contribute. Mean clinical union time was 17.8 ± 3.6, 15.4 ± 3.2, and 13.2 ± 2.8 weeks in deficient, insufficient, and sufficient groups, respectively. Corresponding radiological union times were 20.4 ± 3.8, 17.6 ± 3.4, and 15.2 ± 3.0 weeks. Memon et al. [14] similarly reported significantly longer healing duration among deficient patients (17.9 ± 3.9 weeks) compared with insufficient (16.7 ± 3.2 weeks) and sufficient patients (14.8 ± 2.9 weeks; p<0.001). In contrast, Slobogean et al. [15], in a randomised controlled trial involving 102 patients with tibial and femoral shaft fractures, found no statistically significant improvement in clinical or radiological healing with vitamin D supplementation in the prespecified analyses. These findings suggested that baseline vitamin D status and the effects of supplementation should be considered separately. Serum vitamin D concentrations demonstrated negative correlations with clinical union time (r=−0.46), radiological union time (r=−0.52), and pain scores at 12 weeks (r=−0.38). Memon et al. [14] similarly demonstrated a significant inverse correlation between serum vitamin D levels and fracture healing duration (r=−0.62; p<0.001). Their multivariable analysis also identified vitamin D concentration as an independent predictor of healing duration (β=−0.41; p<0.001). These findings suggested that higher vitamin D concentrations may be associated with shorter fracture healing duration.

The study's example findings suggested that lower serum vitamin D levels were associated with prolonged fracture healing and an increased incidence of delayed union in patients with long bone fractures. Patients with sufficient vitamin D levels demonstrated shorter clinical and radiological union times and better healing outcomes. Assessment of vitamin D status may help identify patients at risk of delayed healing, although further studies are required to establish its independent clinical significance. LIMITATIONS The single-centre design, relatively small sample size, and inclusion of different fracture sites and treatment modalities may limit the generalisability of the findings. Potential confounding factors, including nutritional status, sunlight exposure, and changes in vitamin D levels during follow-up, were not fully accounted for.

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