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. 2025 May 6;9(1):31–40. doi: 10.1002/ame2.70022

Biocompatibility and healing patterns in experimentally induced canine tibial fractures using Pedicle screw‐Rod external fixation

Mohammad Mahdi Gooran 1, Ramin Mazaheri‐Khameneh 1,✉, Seyed Mohammad Hashemi‐Asl 1, Rahim Hobbenaghi 2
PMCID: PMC12907973  PMID: 40326210

Abstract

Background

Biological osteosynthesis preserves blood supply and promotes rapid healing by aligning fracture fragments without direct surgical exposure. Pedicle screws are primarily designed for internal fixation in spinal procedures. A key objective of many orthopedic studies is to assess the biocompatibility of implants with bone and adjacent soft tissue. This study aims to evaluate the biocompatibility and effects of the Pedicle screw‐Rod configuration as a novel external fixation method in canine tibial osteotomy.

Methods

With ethics approval, eight healthy, intact male dogs, aged 10–12 months and weighing between 20 and 22 kg, underwent a minimally invasive medial tibial approach for surgical fixation of tibial osteotomy using a Pedicle screw‐Rod configuration. Postoperative evaluations included ultrasound assessments at the osteotomy site and histological evaluations at the bone‐screw interface.

Results

B‐mode ultrasound evaluation indicated healing progress at all osteotomy sites. The color Doppler examination revealed an initial increase in signals in the surrounding soft tissue during the first 4 weeks post‐operation, followed by a decrease in signals within the adjacent soft tissue between the 5th and 8th weeks. During this latter period, the signals were primarily concentrated on the bone surface and the callus. The bone‐screw interface at various screw sites exhibited similar histological changes, indicating effective integration of the newly formed woven bone into the screw threads.

Conclusions

Fixation of non‐articular tibial osteotomy with Pedicle screw‐Rod configuration resulted in secondary bone healing, characterized by abundant callus formation and neovascularization. This implant demonstrated favorable biocompatibility with bone and surrounding soft tissue, without significant complications.

Keywords: dog, fracture, neovascularization, osteointegration, Pedicle screw‐Rod, tibia

This study demonstrates that using a Pedicle screw‐Rod configuration for the external fixation of non‐articular tibial osteotomy aligns well with the principles of biological osteosynthesis, resulting in secondary bone healing characterized by callus formation and neovascularization. The implant exhibited favorable biocompatibility with the bone and surrounding soft tissue, without significant complications.

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1. INTRODUCTION

A minimally invasive bone fixation method that supports early, painless limb movement is vital for veterinary surgeons, as it enhances bone healing and preserves surrounding soft tissues. 1 Biological osteosynthesis prioritizes maintaining the bone's blood supply during fracture fixation, facilitating early healing and minimizing complications. 2 , 3 , 4

Unilateral external skeletal fixation (ESF) is widely used for treating open fractures, especially in challenging soft‐tissue conditions, as it aligns with minimally invasive surgical principles by placing fixation pins away from the fracture site to preserve the biological environment. 5 ESF allows easy manipulation and adjustment, but common complications include pin loosening and pin tract infections. 6 , 7 The pin‐bone interface is critical for transferring forces and supporting skeletal loads, with mechanical degradation being the primary issue. Osteointegration at this interface is essential to prevent pin loosening. 5

Pedicle screws (PS) are primarily used for internal spinal fixation to correct deformities and stabilize the spine until union is achieved. 8 Magerl introduced the first percutaneous PS insertion method for external spinal fixation, 9 and in 1995, Matthews and Long developed a fully percutaneous PS system (PPS). 10 Advances in screw design and fixation apparatus have enabled the PPS system's use in minimally invasive surgery (MIS) for spinal stabilization across regions from the thorax to the pelvis. 11 This approach addresses conditions such as vertebral fractures, scoliosis, metastases, osteoporotic fractures, and discitis/pyogenic spondylosis. 12 , 13 , 14 , 15 , 16 , 17 Biomechanical studies show that PS exhibit strong performance under various mechanical loads, regardless of bone density, focusing on design and thread shape. 18

Various imaging methods, including radiography, cross‐sectional imaging (ultrasonography, CT, MRI), and scintigraphy, can assess bone healing. 19 , 20 , 21 , 22 , 23 , 24 While radiography is the primary method, it is insufficient for evaluating surrounding soft tissue, crucial for soft tissue callus formation in secondary bone healing. 25 Previous research has shown that ultrasonography is a sensitive and accurate method for detecting early fracture healing in diaphyseal long bone fractures. 26 , 27 Monitoring neovascularization can help assess healing progress and identify delayed healing or nonunion. 28 Neovascularization occurs during fracture healing and subsequently diminishes. 28 While scintigraphy and intraosseous venography have been used to track neovascularization, their dependence on sedation or general anesthesia limits their practicality in veterinary medicine. Alternatives like laser Doppler and Doppler ultrasound, which use color coding for velocity measurement, have been utilized. 29 , 30

A primary objective of orthopedic research is to evaluate the biocompatibility of implants with bone and surrounding soft tissue. 31 This study aims to complement existing findings by assessing the efficacy and biocompatibility of a novel minimally invasive fixation technique for non‐articular tibial osteotomy using the Pedicle screw‐Rod (PS‐R) configuration. The investigation focuses on ultrasonographic evaluations, neovascularization rates, bone healing patterns, and histological analysis of the bone‐screw interface.

2. METHODS

2.1. Animal management and study design

The present study included eight healthy, intact male mixed‐breed dogs, aged 10 to 12 months (10.75 ± 0.88), with a body weight of 20–22 kg (20.81 ± 0.79). Standardization was conducted to minimize the influence of bone structure on outcomes, focusing on bone length, cortical bone thickness, and body condition score relative to hind limb muscle bulk. Each dog was housed individually in a kennel under standardized sanitary conditions, provided with uniform dry commercial food, and had unrestricted access to water. Prior to the study, clinical and paraclinical evaluations, and biochemical analyses, were conducted to rule out underlying diseases. All selected dogs were dewormed and vaccinated at least 30 days before the study. This study aimed to evaluate the biocompatibility of the PS‐R configuration as a novel, minimally invasive external fixation device for tibial osteotomy, with a focus on its interaction with bone and adjacent soft tissue. Because this was a descriptive study, all dogs were placed in a single group to assess the efficacy and biocompatibility of the PS‐R configuration throughout the healing process. This study was reviewed and approved by the Animal Ethics Committee at the Urmia University Veterinary Faculty (Urmia, Iran; Ref: IR‐UU‐AEC‐3/79).

2.2. Anesthesia procedures

In all dogs, pre‐anesthesia involved a combination of Acepromazine maleate 1% (0.01 mg/kg, IM; Alfasan, Woerden, Netherlands) and Midazolam (0.2 mg/kg, IM; Rotexmedica, Trittau, Germany). For anesthesia induction, a mixture of Propofol 1% (4 mg/kg, IV; B. Braun, Melsungen AG, Germany) and Midazolam (0.2 mg/kg, IV) was used. Anesthesia maintenance was performed using Isoflurane (Baxter Healthcare Corporation, Deerfield, USA) in oxygen within a semi‐closed circuit via a tracheal tube. Cefazolin (22 mg/kg, IV; Tum Ekip Pharmaceuticals, Istanbul, Turkey) was administered during anesthesia induction and repeated every 90 minutes during the surgical procedure to prevent infections.

2.3. Surgical procedure

All surgical procedures were performed under aseptic conditions by the same surgeons (the first two authors of this article). Following standard surgical preparation, a 2‐cm medial incision was made at the mid‐shaft of the right tibia. The subcutaneous tissue and periosteum were dissected to expose the tibial diaphysis, and a complete transverse osteotomy of the tibia and fibula was performed using a sagittal blade (Stryker, Kalamazoo, USA), based on previous research on canine tibial osteotomy. 32 , 33 Unilateral fixation was achieved from the medial aspect of the bone. To place the monoaxial Pedicle screws, a small skin incision was made, and the soft tissue was dissected to expose the tibial diaphysis. Screw holes were drilled perpendicular to the longitudinal axis of the tibia using a 3.2 mm drill bit (70% of the screw's outer diameter) with an orthopedic drill (Stryker, Kalamazoo, USA). The fixation was performed using four 4.5 mm titanium monoaxial PS (Atra Orthoped, Urmia, Iran) and a 6.0 mm titanium Rod (Atra Orthoped, Urmia, Iran). 34 Two screws were inserted away from the osteotomy gap for axial support, while the other two were placed closer to the gap to reduce stress at the bone‐screw interface. All screws were positioned parallel to each other and perpendicular to the longitudinal axis of the tibia in the same plane (Figure 1A). After accurate realignment of the tibia, a 1.0 mm metal spacer was inserted into the osteotomy gap, and the titanium Rod was secured through the screw heads with specialized Pedicle screw nuts (Atra Orthoped, Urmia, Iran) (Figure 1B). Sterile saline irrigation was continuously applied during the osteotomy and screw insertion to prevent thermal bone damage. A temporary 1.0 mm metal spacer was placed in the osteotomy gap to maintain uniform spacing. The periosteum and subcutaneous tissue were sutured using a simple continuous pattern with 2–0 Dexon (Covidien, Mansfield, USA), while the skin was closed using a simple interrupted pattern with 2–0 nylon (Ethicon Inc., Somerville, USA). An aseptic bandage was applied immediately postoperatively. Following recovery from anesthesia, each dog was housed in an individual private kennel for the recovery period. 34

FIGURE 1.

FIGURE 1

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Intraoperative photographs (A, B). A complete transverse osteotomy was performed at the mid‐shaft of the tibia and fibula, followed by the unilateral placement of monoaxial Pedicle screws at the medial aspect of the bone (A). After accurately realigning the tibia's longitudinal and torsional axes, precisely apposing the bone fragments, and inserting a 1.0 mm temporary metal spacer in the osteotomy gap, a 6.0 mm titanium Rod was inserted through the Pedicle screw heads. The PS‐R configuration was secured with specialized Pedicle screw nuts (B).

2.4. Postoperative care

Postoperative care included daily dressing changes over the incision and screws for the first 2 weeks, followed by dressing replacement every 48 h until complete bone union and implant removal. The dogs were confined to cage rest for five days post‐operation, after which they were allowed supervised leash walks twice daily. Pain management consisted of tramadol hydrochloride (4 mg/kg, PO, q8h; Aburaihan Pharmaceutical Co, Tehran, Iran) and meloxicam (0.1 mg/kg, PO, q24h; Metacam, Boehringer Ingelheim Vetmedica, USA) for 5 days. Antibiotic prophylaxis with cefazolin (25 mg/kg, IV, q12h) was administered for 5 consecutive days.

2.5. Ultrasonographic evaluations

Bone healing progression and neovascularization at the tibial osteotomy site were evaluated using B‐mode and color Doppler ultrasound (P40, SonoScape Medical Corp, Guangdong, China). A linear transducer (3–17 MHz) with spatial compounding was used for ultrasound evaluations. Color Doppler ultrasound settings included a Doppler frequency of 6.7 MHz, a pulse repetition frequency of 1.3 kHz, and a wall filter of 102 Hz. Evaluations began preoperatively and were conducted weekly until bone union was achieved and the fixation device was removed. The examination area was prepared by clipping the hair and applying ultrasound coupling gel to ensure accuracy. Sedation was not required for any patient. The probe was positioned longitudinally (sagittally), capturing three standard directions (cranial, lateral, and medial) of the osteotomy site. All ultrasound images were stored in JPG format and analyzed in a blinded manner by an expert radiologist. ImageJ 1.44p software (Wayne Rasband, National Institutes of Health, USA) was used for all measurements. B‐mode ultrasound images were used to assess healing stages, with a scoring system ranging from 1 to 5 based on echogenicity and tissue organization (Table 1). 25 , 26 , 27 Color Doppler ultrasound was employed to evaluate neovascularization at the osteotomy site using a quantitative scoring system (0–3), determined by the number and area of Doppler signals across all examined directions: grade 0 (no Doppler signals), grade 1 (fewer than 5 signals with a total area <5 mm2), grade 2 (5–10 signals with a total area of 5–10 mm2), and grade 3 (more than 10 signals with an area >10 mm2). 25 , 28 Scoring was limited to the vascularization associated with the callus at the osteotomy site, excluding nearby blood vessels and tissue reactions related to the orthopedic implant.

TABLE 1.

Stages of fracture healing based on characteristics of B‐mode ultrasonographic images 25 , 27 used to monitor dogs that underwent Pedicle screw‐Rod configuration for the fixation of tibial osteotomy.

Stage of fracture healing Characteristics
1 Tissue within the fracture gap appears anechoic to hypoechoic, with possible hematomas and fragments; no vascularization is visible by use of Doppler ultrasonography
2 Tissue appears hypoechoic (soft tissue callus) but is now heterogeneous; vascularization (determined by use of Doppler ultrasonography) is clearly present in the soft tissue
3 Evidence of bridging of the fracture gap with inhomogeneous tissue (mix of hypoechoic and hyperechoic areas); vascularization (determined by use of Doppler ultrasonography) is present but less abundant than in stage 2
4 Increasingly homogeneous, hyperechoic image of the tissue at the fracture site (acoustic shadow returns); vascularization is still present and appears to be located on the bone surface rather than in the soft tissue
5 Mature callus is present; homogeneous, hyperechoic tissue bridging the facture gap; the surface of this bridge will progressively appear smoother, compared with previous interrogations; vascularization is not detectable by use of Doppler ultrasonography
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2.6. Bone biopsies

Following complete recovery of the tibial osteotomy, the fixation apparatus was removed, and bone samples were aseptically collected under general anesthesia. Screw insertion sites were numbered sequentially from proximal to distal (Figure 1B), with biopsies obtained from at least two randomly selected locations: one near the osteotomy site and another farther away to minimize trauma (n = 20). Limited surgical access was employed to expose the tibial diaphysis, where core bone biopsies (5.5 mm in diameter) were extracted using a low‐rotation pen trephine and subsequently fixed in 10% buffered formalin for histological analysis. Following sample collection, soft bandages were applied, and the animals received appropriate postoperative care. Upon full recovery, they were sterilized and either returned to the shelter or placed for adoption.

2.7. Histological evaluation

The decalcification process and sample preparation were conducted following the method outlined by Jackson et al. 31 Histological sectioning was performed using an automated microtome set to 4.0 μm, followed by standard staining with Verhoeff–Van Gieson stain. Histological evaluation of the healing response and biocompatibility at the bone‐screw interfaces in all study animals was performed in a blinded manner by an expert pathologist, using the semi‐quantitative scoring scheme developed by Jackson et al. 31 This scoring scheme offers a comprehensive assessment of the bone‐screw interfaces, evaluating various parameters, including cortical and medullary bone growth along the implant, bone quality at the interfaces, inflammation, neovascularization, and fibrosis at the bone‐screw interface (Table 2).

TABLE 2.

Histological scoring system at the bone‐screw interfaces for evaluating implant biocompatibility with bone. This system assesses parameters such as cortical and medullary bone growth along the implant, bone quality at the interfaces, inflammation, neovascularization, fibrosis, and additional relevant features. 31

Parameter Score
0 1 2 3 4
Cortical and medullary bone growth along implant Absent Minimal, 1%–25% of implant site contains osseointegration Mild, 26%–50% of implant site contains osseointegration Moderate, 51%–75% of implant site contains osseointegration Marked, 76%–100% of implant site contains osseointegration
Bone quality at interface No bone or osteogenic islands Osteoid, osteogenic/chondroid islands Mostly woven bone Mixture of woven and lamellar bone Mostly lamellar bone
Inflammation (polymorphonuclear cells, lymphocytes, plasma cells,…) Absent Rare, 1–5/hpf (giant cells ¼ 1–2/hpf) 6–10/hpf (giant cells ¼ 3–5/hpf) Moderate, heavy infiltrates Packed (giant cells ¼ sheets)
Neovascularizatin Absent Minimal capillary proliferation, focal, 1–3 buds Groups of 4–7 capillaries with supporting fibroblastic structures Broad band of capillaries with supporting fibroblastic structures Extensive band of capillaries with supporting fibroblastic structures
Fibrosis Absent Narrow band Moderately thick band Thick band Extensive band
Other features (necrosis, mineralization, granulomas,… [other than implant]) Absent Minimal Mild Moderate Marked
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2.8. Statistical analysis

Initially, the normality of the data was assessed using the Shapiro–Wilk test. All data from this study were non‐parametric, and Friedman's two‐way test was employed for analysis. For the post‐hoc analyses, the Wilcoxon signed‐rank test was performed on significant data. Data are presented as median and interquartile range. All statistical analyses were conducted using SPSS software (Version 27 for Windows, SPSS Inc., Chicago, IL, USA) by an expert statistician, with p < 0.05 considered significant.

3. RESULTS

3.1. Ultrasonographic evaluations

B‐mode ultrasound assessments confirmed progressive healing at all osteotomy sites (Figure 2). In the first week post‐operation, the median healing stage was recorded as 2.00 (2.00, 2.00). By the 10th week, a mature callus with homogeneous, hyperechoic tissue filling the osteotomy gap was observed, along with a progressively smoother bone bridge surface, reaching a final healing stage of 5.00 (5.00, 5.00) (Table 3 and Figure 2). Statistical analysis indicated a significant trend in bone healing over time (Table 3 and Figure 3A).

FIGURE 2.

FIGURE 2

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Sagittal ultrasound images (B‐mode and color Doppler) of the tibial osteotomy site at four time points (pre‐operative, and weeks 4, 6, and 10 post‐operative) from three directions (C = Cranial, L = Lateral, M = Medial). The images illustrate the progressive healing of the tibial osteotomy, accompanied by fluctuations in Doppler signals. Prior to surgery, no osteotomy gap or Doppler signal was present in any direction. At 4 weeks post‐operation, bridging of the osteotomy gap was observed, with heterogeneous tissue displaying both hypoechoic and hyperechoic regions. Doppler imaging revealed abundant vascularization extending from surrounding soft tissues into the callus. The healing stage of the osteotomy (rated from 1 to 5, with 1 indicating no healing and 5 indicating complete healing) was assessed through ultrasound by analyzing echogenicity and tissue organization at the osteotomy site, as well as neovascularization patterns. By week 4, healing was evaluated as stage 3, with a peak color Doppler ultrasound score of 2, indicating heightened vascularization. At 6 weeks post‐operation, a more homogeneous and hyperechoic tissue image was seen at the osteotomy site. Doppler imaging showed persistent vascularization, now primarily localized to the bone surface rather than surrounding soft tissue. Healing at this stage was assessed as stage 4 with a color Doppler score of 1. By 10 weeks post‐operation, a mature callus was evident, with homogeneous hyperechoic tissue completely filling the osteotomy gap. The surface of this bridge appeared smoother compared to earlier assessments. No Doppler signals were detected at this stage. Healing was assessed as stage 5 by B‐mode ultrasonography, with a color Doppler score of 0.

TABLE 3.

Median and interquartile range of post‐operative B‐mode and color Doppler ultrasonographic scores in all studied animals. Statistical analysis yielded a p value of 0.001, indicating a significant trend over time.

Weeks post‐operation B‐mode ultrasound Color Doppler ultrasound
Week 1 2 (2, 2) 1 (1, 0.50)
Week 2 2 (2, 2) 2 (2, 1)
Week 3 2 (2.50, 2) 2 (2, 2)
Week 4 3 (3, 3) 2 (2.50, 2)
Week 5 3 (3, 3) 1 (2, 1)
Week 6 3 (3.50, 3) 1 (1, 1)
Week 7 4 (4, 3) 1 (1, 0)
Week 8 4 (4, 4) 0 (1, 0)
Week 9 4 (5, 4) 0 (0, 0)
Week 10 5 (5, 5) 0 (0, 0)
p value 0.001 0.001
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FIGURE 3.

FIGURE 3

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Post‐operative ultrasound scoring evaluation for all studied animals, featuring both B‐mode ultrasound (A) and color Doppler ultrasound (B). Statistical analysis of both parameters yielded a p value of 0.001, indicating a significant trend over time.

Color Doppler ultrasound evaluations demonstrated an increase in the average number and area of Doppler signals until the 4th week post‐operation, followed by a decline from the 5th to the 8th week. No Doppler signals were detected from the 9th week onward (Table 3 and Figure 2). The cranial osteotomy surface exhibited the fewest signals, while the lateral surface displayed the highest. Until the 4th week, signal presence in the surrounding soft tissue increased, but from the 5th to the 8th week, signals became concentrated on the bone surface and within the bridging callus (Figure 2). Statistical analysis confirmed a significant trend in neovascularization at the osteotomy site over time (Table 3 and Figure 3B).

Ultrasound assessments continued until complete osteotomy healing and fixation device removal. The optimal healing score of 5, as determined by B‐mode ultrasound, was achieved at 10 weeks post‐operation, with consistent and reproducible results thereafter. Similarly, color Doppler ultrasound detected no significant signals beyond 8 weeks, eliminating the need for further evaluation. Consequently, ultrasonographic results beyond 10 weeks are not reported to avoid redundancy (Table 3).

3.2. Histological evaluation

Histological analysis of samples from the bone‐screw interface revealed minimal differences in tissue response to the implant materials and screw anchorage. All screw insertion sites exhibited good bone apposition, close contact with the implant, and active bone remodeling. None of the samples showed loss of bone‐screw contact, osteolysis in the cortex, or necrosis at the bone‐screw interface (Figure 4). This positive outcome may result from enhanced remodeling in the callus and cortex around the screws, likely due to a rigorous screw care routine and a low infection rate. A narrow band of fibrous tissue was observed in some screw threads between the bone and the implant in a few samples. Most evaluated samples did not contain inflammatory cells, although a few samples occasionally showed the presence of polymorphonuclear cells. Statistical analysis of the histological results revealed no significant differences in the bone‐screw interface locations among the four screws implanted from proximal to distal. This analysis was based on the semi‐quantitative scoring scheme proposed by Jackson et al. 31 (Figure 5A–E).

FIGURE 4.

FIGURE 4

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Histological observation of the bone‐screw interface with Verhoeff‐Van Gieson staining at 12 weeks post‐operation. Histological evaluation showed minimal tissue response to the implant material and screw anchoring, with good bone deposition, close contact with the implant, and active bone regeneration. Newly formed woven bone was effectively integrated into the screw threads, with no signs of bone‐screw contact loss, cortical osteolysis, or necrosis at the interface.

FIGURE 5.

FIGURE 5

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Evaluation of the histological scoring system in samples taken from the bone‐screw interfaces (BSI) in the studied animals (A–E) included assessments of cortical and medullary bone growth along the implant (A), bone quality at the interface (B), inflammation (C), neovascularization (D), and fibrosis (E). Statistical analysis yielded a p value ≥0.05 for all five parameters, indicating no significant difference for each parameter.

4. DISCUSSION

Neovascularization, a key indicator of bone healing, was assessed based on the number and area of Doppler signals at the osteotomy site. 25 Previous studies have demonstrated a strong correlation between Doppler‐detected vascularization and histological findings. 35 In a study of 108 human patients with simple fractures, color Doppler ultrasound detected neovascularization within the first week post‐operation, followed by vessel maturation, characterized by fewer but larger and more branched signals as healing progressed. 36 The present study yielded similar results to Bottinelli et al. 36 with neovascularization observed within the first postoperative week and vascular maturation becoming evident over time. Risselada et al. 28 also investigated neovascularization in dogs with long bone fractures, reporting a time‐dependent expansion and regression of vessels. Their study found that ESF led to a greater number of Doppler signals and larger signal areas in the early healing phase, peaking 11–20 days post‐fixation before decreasing and localizing to the bone surface and callus. Consistent with previous research, our study demonstrated a time‐dependent increase in Doppler signals, peaking at 28 days post‐operation and declining until signals were undetectable by the 9th week. Similar to Risselada et al. 28 fractures treated with ESF exhibited greater neovascularization, initially in the surrounding soft tissues and later extending to the callus bridge. Our study confirmed previous findings, demonstrating that in the early stages of healing, Doppler signals were primarily detected in the surrounding soft tissues and gradually became localized to the bone surface and callus bridge as the healing process progressed. Pozzi et al. 25 further highlighted that the localization of neovascularization is indicative of fracture healing type, distinguishing secondary from direct healing. All fractures treated with the minimally invasive osteosynthesis (MIO) technique in the referenced study achieved bone union through secondary healing, with Doppler ultrasound signals initially observed in the soft tissue and later concentrated on the bone surface as healing progressed. Similarly, our study found that all fixed osteotomies exhibited callus formation indicative of secondary healing, with Doppler ultrasound evaluations showing a comparable pattern to that reported by Pozzi et al. 25

Previous research demonstrated that B‐mode ultrasound is more effective than radiography for early detection of fracture healing in dogs, enabling quicker diagnosis and intervention to prevent complications such as delayed or nonunion. 26 , 27 Consistent with these findings, our study confirmed that B‐mode ultrasound detected bone healing earlier and with greater sensitivity than radiography. 37 Similarly, Pozzi et al. 25 reported faster healing in the minimally invasive osteosynthesis (MIO) group compared to the open reduction and internal fixation (ORIF) group. However, no significant differences between radiographic and ultrasound evaluations were observed at 4‐week intervals. The authors recommended 2‐week follow‐up intervals for better detection of differences in healing assessments, a suggestion supported by our study. In our research, we conducted weekly ultrasound examinations post‐operation, allowing us to observe all stages of bone fracture healing (Table 1) as documented in prior studies using B‐mode ultrasound. 26 , 27 The ultrasonographic evaluations in this study provided critical insights into the temporal progression of bone healing. B‐mode ultrasound assessments indicated that optimal healing, characterized by a score of 5, was achieved by 10 weeks postoperatively. This milestone corresponded with improved echogenicity, tissue organization at the osteotomy gap, and the formation of a well‐structured callus, signifying sufficient mechanical stability to support weight‐bearing. Furthermore, the absence of detectable Doppler signals beyond 8 weeks postoperatively suggests that vascularization had stabilized, marking the transition from active bone healing to the remodeling phase. Clinically, these findings correlate with the progressive restoration of normal limb function, as the dogs gradually resumed weight‐bearing without evidence of discomfort or instability.

Histological analysis is highly sensitive for evaluating the implant‐bone interface but is unsuitable for clinical monitoring due to its invasiveness. 38 The stability of the pin‐bone interface is critical for ESF and fracture healing, and its dynamics change over time. 39 The inflammatory response at the bone‐screw interface plays a crucial role in implant stability and postoperative pain. 31 Histological assessments in this study revealed minimal inflammation, indicating a controlled biological reaction without adverse effects. While a certain level of inflammation is a normal physiological response, excessive or prolonged inflammation can hinder osteointegration, increase postoperative pain, and compromise implant stability. 31 Additionally, excessive inflammation can stimulate osteoclastic activity, leading to localized bone resorption, micromotion, and further instability. 40 Persistent inflammation may also contribute to fibrotic tissue formation around the screws, impairing bone‐implant contact and increasing the risk of loosening. Clinically, this may result in prolonged discomfort, delayed recovery, and reduced weight‐bearing capacity. 37 , 40 The minimal inflammatory response observed in this study underscores the biocompatibility of the PS‐R configuration, suggesting its potential to reduce postoperative pain, enhance healing, and improve functional outcomes. 34 Histological analysis also demonstrated minimal fibrosis, further highlighting the favorable integration of the PS‐R configuration with bone and soft tissues. While fibrosis is a natural response to tissue injury and may provide mechanical cushioning, excessive fibrosis can form a non‐osseous barrier, preventing direct bone‐implant integration. 40 , 41 This lack of osteointegration can compromise implant stability, increasing the risk of micromotion, loosening, and potential failure. 37 The limited fibrotic response observed in this study suggests that the PS‐R configuration promotes a more natural bone healing environment, reducing the risk of implant‐related complications. The combination of minimal fibrosis, limited inflammation, adequate neovascularization, and robust bone formation at the bone‐screw interface indicates that the PS‐R configuration provides stable fixation while minimizing adverse biological responses. This is particularly beneficial for long‐term implant performance, as excessive fibrosis has been associated with an increased risk of infection, reduced biomechanical strength, and compromised functional outcomes. 40 Schell et al. 38 analyzed the pin‐bone interface following tibial osteotomy stabilization, reporting improved pin‐bone contact within three weeks but noting increased fibrous tissue and osteolysis with prolonged implantation. A limitation of the present study was the timing of sampling, as it was designed to evaluate both the bone‐screw interface and osteotomy site healing. Consequently, sampling was conducted only after complete osteotomy healing and device removal. Histological findings in our study differed from those of Schell et al. 38 Unlike their observations, no fibrotic tissue, bone lysis, or screw loosening was detected at any bone‐screw interface sites. Instead, screw threads were filled with woven bone, indicating strong integration. While Schell et al. 38 reported that middle pins in external skeletal fixation experienced the least loosening due to their mechanically protected position, Pommer et al. 42 found no correlation between pin loosening and factors such as position or treatment duration. The present study aligns with Pommer et al. 42 showing no significant histological differences at the bone‐screw interface or along the screw trajectory, regardless of location. Furthermore, no association was found between screw position and loosening, with all interface sites exhibiting similar histological characteristics.

This study had several limitations, primarily a small sample size and the absence of a control group or alternative treatment for comparison, a decision influenced by ethical considerations. As the initial phase of an experimental investigation, this study aimed to assess the feasibility, validation, and in vivo characteristics of a novel tibial fixation method in dogs. If the results proved promising, future research was planned to incorporate comparative analyses with conventional tibial fixation methods under identical conditions to strengthen the findings and further develop this technique. The PS‐R configuration for non‐articular tibial osteotomy fixation demonstrated favorable biocompatibility with bone and surrounding soft tissue, without significant complications. However, minor adverse reactions, including limb swelling and mild soft tissue responses at the proximal Pedicle screw insertion site, were observed. Additionally, as with other external fixation methods, maintaining the dressing at the surgical and screw insertion sites posed challenges and required additional attention during postoperative care. Nonetheless, these issues were minimal and did not affect the bone fixation process during healing. Moreover, our study did not include an ex vivo biomechanical evaluation of the PS‐R configuration under simulated physiological loads. ESF systems are widely used for fracture stabilization; however, their effectiveness is influenced by factors such as the clinical application site, fixation components, and mechanical properties. Despite their versatility, ESF systems have limitations in terms of stiffness and rigidity. 43 An optimal fixation system should be easy to apply, biocompatible, lightweight, cost‐effective, and sufficiently rigid to support fracture healing without secondary displacement. 44 Therefore, biomechanical testing under standardized loading conditions is essential to assess the stiffness and overall performance of external fixation systems. 43 Future research should address these limitations by including larger sample sizes, comparative treatment groups, and biomechanical assessments to better evaluate the technique's efficacy.

5. CONCLUSION

This study provides complementary insights into the efficacy and biocompatibility of a novel surgical fixation technique, emphasizing ultrasonographic and histological evaluations for non‐articular tibial osteotomy using the PS‐R configuration. Findings indicate that this fixation method promotes secondary healing through biological osteosynthesis, evidenced by significant callus formation and neovascularization. Histological analysis revealed consistent bone integration at the bone‐screw interface, with newly formed woven bone effectively incorporating into the screw threads. The PS‐R configuration demonstrated favorable biocompatibility with bone and surrounding soft tissue, without notable complications. Further research with a larger sample size, application to different long bones, and comparative studies against standard tibial fixation methods is recommended before clinical implementation.

AUTHOR CONTRIBUTIONS

Mohammad Mahdi Gooran: Conceptualization; data curation; formal analysis; investigation; methodology; software; validation; writing – original draft. Ramin Mazaheri‐Khameneh: Conceptualization; data curation; funding acquisition; methodology; project administration; supervision; validation; writing – review and editing. Seyed Mohammad Hashemi‐Asl: Data curation; investigation; validation; writing – review and editing. Rahim Hobbenaghi: Data curation; investigation; validation; writing – review and editing.

FUNDING INFORMATION

This study was supported by the Vice Chancellor of Research and Technology at Urmia University, Urmia, Iran.

CONFLICT OF INTEREST STATEMENT

The authors declare no competing interests.

ETHICS STATEMENT

This study was reviewed and approved by the Animal Ethics Committee at the Urmia University Veterinary Faculty (Urmia, Iran; Ref: IR‐UU‐AEC‐3/79). All methods were conducted in accordance with the relevant guidelines and regulations. The current study adhered to the ARRIVE guidelines (Animals in Research: Reporting In Vivo Experiments). The dogs were used for research purposes with the informed consent and full knowledge of the animal shelter administration. After the study was completed, all of them were neutered and either adopted or returned to the shelter.

DECLARATION OF GENERATIVE AI AND AI‐ASSISTED TECHNOLOGIES IN THE WRITING PROCESS

During the preparation of this study, the authors used ChatGPT to rewrite some parts of the manuscript. After using this tool/service, the authors reviewed and edited the content if needed and take full responsibility for the content of the publication.

CONSENT TO PARTICIPATE STATEMENT

Informed consent for the publication of identifying images or other personal or clinical details was obtained from all participants.

CONSENT TO PUBLISH STATEMENT

The authors gave the publisher the permission of to publish the work.

ACKNOWLEDGMENTS

This study was derived from the research project conducted as part of the first author's (Mohammad Mahdi Gooran) dissertation, submitted in partial fulfillment of the requirements for the Doctor of Veterinary Science (DVSc) degree at Urmia University, Urmia, Iran. The authors would like to thank the Vice Chancellor of Research at Urmia University, Urmia, Iran, for their support. Special thanks are extended to Dr. Hadi Keshipour for assistance with statistical analysis.

Gooran MM, Mazaheri‐Khameneh R, Hashemi‐Asl SM, Hobbenaghi R. Biocompatibility and healing patterns in experimentally induced canine tibial fractures using Pedicle screw‐Rod external fixation. Anim Models Exp Med. 2026;9:31‐40. doi: 10.1002/ame2.70022

DATA AVAILABILITY STATEMENT

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Data Availability Statement

The data that support the findings of this study are available from the corresponding author upon reasonable request.

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