Gereç ve Yöntem: Çalışmada toplam 48 adet erişkin Sprague-Dawley rat altı gruba ayrıldı: sağlıklı kontrol, sham kontrol, apiksaban, rivaroksaban, edoksaban ve dabigatran (n=8 her grupta). Tüm deney gruplarında sağ tibianın metafiziyel bölgesinde 4 mm çapında ve 4 mm derinliğinde standart kemik defekti oluşturuldu. Kontrol grubuna herhangi bir farmakolojik ajan uygulanmazken, deney gruplarına 4 hafta boyunca belirlenen dozlarda (apiksaban 5 mg/kg, rivaroksaban 3 mg/kg, edoksaban 3 mg/kg, dabigatran 10 mg/kg) oral gavaj ile ilaç verildi. Dört haftanın sonunda tüm ratlar ötenazi edildi, tibialar dekalsifiye edilerek hematoksilen-eozin ile boyandı ve defekt alanları longitudinal ve vertikal olarak ölçüldü.

Bulgular: Longitudinal defekt alanı açısından gruplar arasında anlamlı fark bulundu (p=0.019). Rivaroksaban grubunda ortalama longitudinal defekt alanı kontrol grubuna kıyasla anlamlı derecede düşük bulundu (p=0.011). Diğer gruplarda azalma olmasına rağmen istatistiksel anlamlılık sağlanmadı. Vertikal defekt alanında gruplar arası genel fark anlamlı değildi (p=0.06), ancak dabigatran (p=0.019) ve rivaroksaban (p=0.005) gruplarında kontrol grubuna göre anlamlı azalma tespit edildi. Özellikle rivaroksabanın hem longitudinal hem de vertikal ölçümlerde kemik iyileşmesini en belirgin şekilde artırdığını, dabigatranın ise vertikal yönde anlamlı iyileşme sağladığını göstermektedir.

Sonuç: Elde edilen veriler, bazı oral antikoagülanların doğrudan kemik iyileşmesi üzerine olumlu etkiler gösterebileceğini ortaya koymaktadır.

Oral anticoagulants are among agents for the prophylaxis of non-valvular atrial fibrillation, venous therapeutic the preferred thromboembolism, and cardiovascular diseases⁴. Their clinical use began with warfarin, a vitamin K antagonist, and later expanded to include a new generation of drugs known as direct oral anticoagulants (DOACs), such as apixaban, rivaroxaban, edoxaban, and dabigatran⁵. Atrial fibrillation has been identified as an independent and strong risk factor for stroke, and consequently, anticoagulant therapy has become increasingly common, particularly in elderly populations⁶.

In recent years, research has focused not only on the effects of anticoagulant drugs on coagulation mechanisms but also on their influence on the skeletal system. In particular, warfarin, a member of the vitamin K antagonist class, has been reported to impair bone mineralization by inhibiting osteoblast function, thereby increasing the risk of calcification in both bone and vascular tissues⁷. Systematic reviews and meta-analyses have shown a significant association between warfarin use and cardiovascular as well as valvular calcification⁸. This phenomenon is believed to be linked to warfarin’s inhibition of γ-carboxylation of vitamin K–dependent proteins, which play critical roles not only in the coagulation process but also in bone mineralization and the inhibition of vascular matrix calcification⁷. In particular, insufficient carboxylation of osteocalcin and matrix Gla protein (MGP), vitamin K dependent proteins found in bone and vascular tissues has been associated with the progression of vascular calcification and a reduction in bone mineral density. Accordingly, long-term use of vitamin K antagonists may have adverse effects not only on the cardiovascular system but also on the skeletal system⁹. Animal studies investigating the effects of anticoagulants on bone regeneration are limited. Preliminary findings suggest that DOACs, such as rivaroxaban, may exert significant effects on parameters such as osteoblastic proliferation, bone density, and callus formation¹⁰. However, the underlying mechanisms remain unclear, and it is thought that different DOACs may exert varying degrees of effect.

In this research, the effects of systemically delivered anticoagulants, including apixaban, rivaroxaban, edoxaban, and dabigatran, on bone repair were investigated using a rat tibial defect model and comparative histopathological assessment. The aim was to elucidate the potential effects of these widely prescribed clinical agents on bone regeneration and to contribute experimental evidence to the literature in this field.

Sprague Dawley rats, aged at least 3.5–4 months and weighing approximately 250–300 g each, were used in the study. In cases where female animals were preferred, vaginal smears were performed to synchronize the estrous cycle, and only those in the same stage were included in the experiment.

The animals were randomly divided into six groups:

Healthy control group (n = 8): Group with no defect created

Sham control defect group (n = 8): Surgical bone defect created; no treatment administered.

Apixaban group (n = 8): Bone defect + apixaban at 5 mg/kg, administered three times per week.

Rivaroxaban group (n = 8): Bone defect + rivaroxaban at 3 mg/kg, administered daily.

Edoxaban group (n = 8): Bone defect + edoxaban at 3 mg/kg, administered daily.

Dabigatran group (n = 8): Bone defect + dabigatran at 10 mg/kg, administered daily.

After induction of anesthesia through intraperitoneal administration of ketamine (50 mg/kg) and xylazine (10 mg/kg), the periosteum was elevated, and a 4 mm diameter by 4 mm depth defect was drilled in the right tibia’s metaphyseal portion, extending to the corticocancellous structure. The soft tissues were repositioned to their original anatomical position, and primary closure was achieved using 4-0 silk sutures. Postoperatively, tramadol hydrochloride (1 mg/kg) and penicillin (50 mg/kg) were administered intramuscularly for the first three days.

The designated anticoagulants were administered to the respective groups via oral gavage at the specified doses and frequencies for a duration of four weeks. No medication was given to the healthy control or sham groups. All animals were ethically sacrificed at the end of the four-week experimental period. The right tibias were harvested, fixed in 10% neutral formalin for 72 hours, and subsequently decalcified in 10% formic acid for approximately one week. Tissue processing was performed using an automated tissue processor (Leica TP1020, Germany), and samples were embedded in paraffin. Sections with a thickness of 3 μm were prepared using a rotary microtome (Leica RM2125 RTS, Germany) and stained with hematoxylin–eosin. Microscopic evaluations were conducted using a light microscope (Olympus BX42, Japan).

Histological evaluation was performed based on the percentage of new bone formation and the degree of fibrosis within the defect areas. For morphometric analysis, the widest (longitudinal) and deepest (vertical) dimensions of the defects were measured.

The obtained data were analyzed using parametric statistical methods. Comparisons between groups were performed using the one-way analysis of variance (ANOVA), and in cases where significant differences were detected, Tukey’s HSD post hoc test was applied. A p-value of <0.05 was considered statistically significant.

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Figure 1: Width measurements of the defect area and new bone formation (NBF) areas in the defect region of the healthy control and experimental groups

According to the vertical bone analysis, the largest defect area was measured in the sham control group (843.25±169.90 μm). A significant reduction was observed in the rivaroxaban (637.75±75.23 μm; p=0.002) and dabigatran (666.00±70.07 μm; p=0.012) groups compared to the sham control group, whereas the reductions observed in the apixaban (717.00±101.42 μm) and edoxaban (742.63±95.90 μm) groups did not reach statistical significance (Table 1).

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Table 1: Vertical bone analysis of the groups

Longitudinal bone analysis results also showed a statistically significant difference between the groups (p<0.001). The largest defect area was measured in the sham control group (906.00±115.63 μm). In the rivaroxaban group (605.38±92.86 μm), a significantly smaller defect area was detected compared to the sham control (p=0.006). Although the defect areas in the dabigatran (732.38±144.00 μm), apixaban (817.00±257.72 μm), and edoxaban (801.25±198.37 μm) groups were smaller than those in the sham control group, these differences did not reach statistical significance (Table 2).

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Table 2: Defect area longitudinal analysis of the groups

The findings indicate that systemic rivaroxaban administration significantly improved bone defect healing in both vertical and longitudinal directions, while dabigatran achieved a statistically significant improvement only in the vertical direction. Although apixaban and edoxaban groups showed a trend toward improvement, these changes did not reach statistical significance.

In the longitudinal analysis, the smallest defect area was observed in the rivaroxaban group (p = 0.011). In the vertical measurements, both rivaroxaban and dabigatran groups exhibited significantly smaller defect areas compared to the control group. This indicates that both agents had a positive effect on bone healing.

Although a reduction in defect areas was also observed in the apixaban and edoxaban groups, these decreases, while numerically lower than in the control group, did not reach statistical significance. This suggests that, although these agents may exert some effects on bone tissue, their efficacy might be more limited compared to other DOACs such as rivaroxaban and dabigatran. Pharmacologically, DOACs act as either factor Xa inhibitors (apixaban, edoxaban, rivaroxaban) or thrombin inhibitors (dabigatran), and their impact on the osteoblast/osteoclast balance may differ among agents¹¹.

Gigi et al.¹² reported that rivaroxaban exerts effects that support osteoblast activity and enhance bone mineralization. Duy Mai et al.¹¹ stated that dabigatran directly inhibits thrombin, thereby suppressing osteoclast activity, limiting bone resorption, and supporting healing. The positive effects of dabigatran have been confirmed in other studies as well^10,13. Rocha et al.¹⁴ suggested that dabigatran may inhibit RANKL-mediated osteoclastogenesis, thereby reducing inflammatory mediators and contributing to increased new bone formation. These studies are consistent with the present results regarding the effects of dabigatran and rivaroxaban, supporting our findings. The effects of edoxaban on bone tissue have been addressed in only a limited number of studies, and its role in osteogenesis remains unclear. Zhang et al.¹⁵ have reported similar observations for edoxaban.

The sham group consisted of subjects in which only a surgical defect was created without systemic drug administration. This group represented the natural healing process of bone tissue in response to surgical trauma. The significantly larger defect areas observed in the sham group compared to the rivaroxaban and dabigatran groups suggest that these anticoagulants may accelerate the regenerative process. Similarly, Siltari et al.¹⁶ reported that suppression of surgery-induced local inflammation by systemic anti-inflammatory and anticoagulant effects may positively influence osteogenic cell activity; this finding has also been supported by Shahbazi et al.¹⁷ and other studies.

The present findings indicate that, unlike traditional anticoagulants such as warfarin, DOACs may have the potential to avoid negative effects on bone. Wu et al.¹⁸ reported that warfarin, through vitamin K antagonism, inhibits the carboxylation of bone proteins such as osteocalcin and matrix Gla protein, leading to adverse effects on both the vascular and skeletal systems. Liu et al.¹⁹ stated that DOACs, by not interfering with these mechanisms, may represent a safer option for bone health. Based on the present data and these reports, DOACs may be considered as an alternative to warfarin in order to avoid such negative effects, especially when their beneficial effects on bone are also taken into account.

In this context, the study findings provide important contributions at both the experimental and clinical levels. Our results confirm previously reported positive effects of rivaroxaban and dabigatran on bone regeneration^10,12,14. In patients receiving DOAC therapy, the potential contribution of these agents to bone healing should be considered during preoperative planning for surgical interventions.

In conclusion, the findings of this study indicate that certain systemically administered direct oral anticoagulants may positively influence the healing process of bone defects created in rat tibias. In particular, measurements in the rivaroxaban and dabigatran groups revealed significantly smaller defect areas compared to the control group, suggesting that both agents may accelerate bone regeneration and enhance the healing process. Although reductions in defect area were also observed in the apixaban and edoxaban groups, these changes were not statistically significant.

The obtained data suggest that rivaroxaban and dabigatran may have the potential to promote osteogenic processes and accelerate bone regeneration, whereas the effects of edoxaban and apixaban in this regard may be more limited. These results emphasize the importance of considering the potential influence of anticoagulant selection on the healing process in clinical situations where postoperative bone healing is critical. Further prospective and controlled human studies are required to translate the findings obtained from this experimental animal model into clinical practice.

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