Effects of soluble guanylate cyclase activator vericiguat on fracture healing in rats

Effects of soluble guanylate cyclase activator vericiguat on fracture healing in rats | Authorea try { document.documentElement.classList.add('js'); } catch (e) { } var _gaq = _gaq || []; _gaq.push(['_setAccount', 'G-8VDV14Y67G']); _gaq.push(['_trackPageview']); (function() { var ga = document.createElement('script'); ga.type = 'text/javascript'; ga.async = true; ga.src = ('https:' == document.location.protocol ? 'https://ssl' : 'http://www') + '.google-analytics.com/ga.js'; var s = document.getElementsByTagName('script')[0]; s.parentNode.insertBefore(ga, s); })(); Skip to main content Preprints Collections Wiley Open Research IET Open Research Ecological Society of Japan All Collections About About Authorea FAQs Contact Us Quick Search anywhere Search for preprint articles, keywords, etc. Search Search ADVANCED SEARCH SCROLL This is a preprint and has not been peer reviewed. Data may be preliminary. 2 September 2025 V1 Latest version Share on Effects of soluble guanylate cyclase activator vericiguat on fracture healing in rats Authors : Azat Dzhumukov [email protected] , Bilal Karabak , Aziz Demirci , Ferhat Yıldırım Hoyur , and Muhammed Çağatay Engin 0000-0002-9302-9587 Authors Info & Affiliations https://doi.org/10.22541/au.175678772.28215855/v1 Published Joint Diseases and Related Surgery Version of record Peer review timeline 131 views 98 downloads Contents Abstract Supplementary Material Information & Authors Metrics & Citations View Options References Figures Tables Media Share Abstract Objective: The primary purpose of this study was to evaluate the direct effects of vericiguat on the healing of fractures. The study investigated how different doses of vericiguat affect femoral shaft fracture healing through radiological, biomechanical and histological methods. Methods: In this study, 60 female Wistar-Albino rats were used. The study comprised of six mouse groups with ten rats in each group: Group 1 (normal controls), Group 2 (positive controls - only broken ones), Group 3 (low-dose vericiguat - 3 mg/kg), Group 4 (high-dose vericiguat - 6 mg/kg), Group 5 (fracture + low-dose vericiguat), Group 6 (fracture + high-dose vericiguat). Under general anaesthesia, standard closed fractures were created in the right femurs of rats in the fracture groups. Radiological examinations were performed on days 7, 14, and 28. At the end of day 28, the rats were sacrificed, and the fracture healing tissues were examined biomechanically and histologically. Results: The 28th day biomechanical assessment showed significant differences in maximum load values between the fractured groups (Group 2: 88.75±20.34 N, Group 5: 83.54±17.89 N, Group 6: 39.07±15.21 N; p=0.003). The same results were observed when looking at the stiffness values (Group 2: 64.71±18.43 N/mm, Group 5: 99.20±25.67 N/mm, Group 6: 40.47±12.85 N/mm). In the histological evaluation according to the Huo scale, Group 5 showed the highest quality of healing (8.6±1.35) and a significant difference was found between Group 2 and Group 5 (p=0.014). In the radiological evaluation at 7, 14, and 28 days, no statistically significant differences were detected between the groups according to the Lane-Sandhu scoring system. Conclusion: The experimental research on vericiguat effects on rat fracture healing demonstrated that low doses of 3 mg/kg produced positive outcomes in histopathology and biomechanics. The high dose of 6 mg/kg produced negative biomechanical effects together with tissue necrosis and inflammation. INTRODUCTION Bone restoration to its original form and operational state without scar tissue formation defines the biological process of fracture healing. The healing process of fractures starts immediately after injury and requires the coordinated action of multiple molecular elements and cellular components and biomechanical factors [1]. It is reported that 5–10% of fractures occurring annually in the United States experience healing problems of varying degrees [2]. The failure of fractures to heal properly requires additional medical procedures and repeated surgeries and extended work disability and substantial financial expenses. The underlying causes of these complications include multifactorial aetiology such as age, nutritional status, hormonal changes, inadequate reduction, concomitant diseases, infections, medications used, and the characteristics of the trauma [3]. Research into bone metabolism signalling pathways during recent years uncovered fresh therapeutic targets. The research community now focuses on guanylate cyclase because this enzyme produces cyclic guanosine monophosphate (cGMP) while controlling vital cellular processes including signal transduction and gene expression and cell movement [4, 5]. Research shows that guanylate cyclase activation produces dual effects on osteoclast differentiation at the bone tissue level [6]. The increase of guanylate cyclase activity through mechanical stimulation leads to osteoblast proliferation [7]. Vericiguat is a new-generation oral activator of soluble guanylate cyclase that has established itself in cardiovascular medicine. The clinical evidence from 2020 demonstrated its ability to decrease cardiovascular death rates and hospital admissions among heart failure patients [8]. The mechanism of action of vericiguat is based on the activation of the nitric oxide-soluble guanylate cyclase-cGMP signaling pathway through direct cGMP production. This signaling pathway is known to play critical roles in bone metabolism and remodeling processes [4]. There are limited studies in the literature on the effects of soluble guanylate cyclase activators on bone tissue, and there are no experimental studies specifically investigating their direct effects on fracture healing processes. The current understanding suggests that vericiguat may influence fracture healing because it affects signaling pathways which control bone metabolism. The main objective of this research is to assess the healing effects of vericiguat at various concentrations on rat femur fractures through clinical and radiological and biomechanical and histopathological assessments. The study includes secondary objectives to evaluate how vericiguat affects normal bone tissue. The Local Ethics Committee for Animal Experiments at approved this experimental study through Decision No: 92 on 25.05.2023. This research constitutes the first experimental study in the literature to investigate the effects of vericiguat on fracture healing. MATERIALS AND METHODS Study Population and Sample The research obtained approval from the Animal Experiments Local Ethics Committee for the experimental study through decision number E-42190979-000-2300144669 on 05.05.2023. The research implemented a randomised controlled experimental study which took place from May 2023 to October 2023. The study used 80% power and 5% alpha error for sample size calculation with 60 Wistar-Albino female rats distributed across 10 subjects per group. The study included female rats aged 2.7–2.9 months weighing between 150–280 grams who maintained good health status. The research excluded rats that were pregnant or had previous surgical procedures or showed signs of infection or abnormal movement disorders or had general health conditions that made anesthesia unsafe. The research defined Vericiguat (Bayer AG, Leverkusen, Germany) administration as an oral gavage administration of 3 mg/kg/day (low dose) and 6 mg/kg/day (high dose) based on the animal body weights. Study Procedures Standardized methods enabled the research team to conduct radiological assessments at days 7, 14 and 28 and to perform biomechanical and histopathological assessments on day 28. Laboratory animals needed to stay at 22°C with 50±5% humidity levels and a 12-hour light/dark cycle under standard laboratory conditions according to the laboratory protocol. The anaesthesia protocol required intraperitoneal administration of Ketamine (Ketasol 10% Richter Pharma AG, Wels, Austria) at 52 mg/kg combined with Xylazine (Rompun 2%, Bayer AG, Leverkusen, Germany) at 8 mg/kg. The follow-up protocol included both daily clinical observations and weekly weight measurements together with radiological examinations at specific time points. Radiological evaluations maintained their reliability through blind assessment by three orthopaedic specialists who achieved an inter-observer reliability coefficient of 0.85. Standardised test parameters along with INSTRON 5982 (Instron Corporation, Norwood, MA, USA) device calibration validated the biomechanical tests. Intervention Protocol and Surgical Method The research subjects received random assignment to six groups consisting of Group 1 (NK) with healthy controls (n=10) and Group 2 (PK) with fracture+K-wire controls (n=10) and Group 3 (DVN) with low-dose vericiguat (n=10) and Group 4 (YVN) with high-dose vericiguat (n=10) and Group 5 (DVP) with fracture + K-wire + low-dose vericiguat (n=10) and Group 6 (YVP) with fracture + K-wire + high-dose vericiguat (n=10). The researchers used the sealed envelope method for randomisation and remained unaware about the group distribution. A 1 cm incision was made in the anterior region of the right knee to expose the femoral condyles (Figure 1A), an intramedullary entry hole was prepared (Figure 1B), and a Kirschner wire (1.0 mm diameter, Zimmer Biomet, Warsaw, IN, USA) was inserted (Figure 1C). The forceps for fracture creation used a three-point bending principle (Figure 1D) to perform the procedure while the K-wire tip was cut (Figure 1E) and the incision was closed with 4.0 silk suture (Ethicon, Johnson & Johnson, New Brunswick, NJ, USA) (Figure 1F). The first oral gavage administration of vericiguat started on the day of surgery followed by continuous administration until day 28. Statistical Analysis IBM SPSS 20.0 (IBM Corp., Armonk, NY, USA) software performed the data analysis. The Shapiro-Wilk test, Kolmogorov-Smirnov test together with Q-Q plot analysis were used to evaluate the normal distribution of continuous variables. The one-way ANOVA test was used for normally distributed data but the Kruskal-Wallis test was used for data that did not follow normal distribution. The Tukey test was employed for post-hoc analyses when variances were homogeneous yet the Tamhane’s T2 test was used when variances were not homogeneous. The study had no missing data since all participants finished the research. The Lane-Sandhu criteria evaluated radiological scoring and the Huo scale was used for histopathological assessment. The researchers performed subgroup analyses independently for both fracture and non-fracture groups. Units of Measurement The biomechanical test results were presented as maximum load capacity in Newton (N) and stiffness values in Newton/millimetre (N/mm). The weights of animals were expressed in grams (g), and drug doses in milligram/kilogram/day (mg/kg/day). The numerical scoring system of 1–10 was used for histopathological evaluation and a 0–4 scoring system was used for radiological assessment. Follow-up periods were reported in days (7th, 14th, 28th day). Statistical Analysis Criteria The study reports P values alongside the analyzed groups and the statistical methods used for comparison. The results contain two decimal points for P≥0.01 (e.g., P=0.03) and three decimal points for P<0.01 (e.g., P=0.007). P values below 0.001 appear as P<0.001 rather than their specific numerical values. The data appears in mean (SD) format where SD stands for standard deviation. The study set P<0.05 as the statistical significance level and applied a 95% confidence interval for all calculations. RESULTS In this experimental study conducted using a total of sixty female Wistar-Albino rats over the course of the study period, six different groups were formed, and the effects of vericiguat at various doses on fracture healing were comprehensively investigated. The experimental protocol resulted in no animal losses and no serious complications were observed (Table 1). The Lane-Sandhu scoring system was used to evaluate fracture healing through radiographic assessments. Serial anteroposterior radiographs were used to evaluate the radiological healing process of all groups at days 7, 14 and 28 (Figure 2). The negative control group maintained normal bone architecture during the first week but the fracture-induced groups showed no callus formation. The second week radiographic evaluations showed callus formation in the positive control group and similar healing patterns in the vericiguat-treated groups. The mean values were 1.6 in the positive control group, 1.6 in the low-dose positive group, and 1.8 in the high-dose positive group. The fourth week radiographic images showed complete bone union in the positive control group and significant callus maturation in the low- and high-dose positive groups. All fracture groups showed significant improvement at the end of the fourth week but no statistically significant differences were detected between the groups (Table 2). The most striking findings of the study were revealed through the biomechanical test results. No adverse effects of vericiguat administration on bone structure were observed in the groups without fractures. The negative control group showed an average load value of 115.32 N, the low-dose negative group 106.87 N, and the high-dose negative group 98.05 N. No statistically significant differences were found between these groups. However, significantly different results were obtained in the fracture-induced groups. The high-dose vericiguat positive group showed the lowest mechanical resistance at 39.07 N compared to all other groups. This value was approximately half that of the positive control group (88.75 N) and the low-dose positive group (83.54 N) (Table 3; Table 4). The hardness values followed the same pattern as the other parameters. The high-dose positive group had the lowest hardness value of 40.47 N/mm while the low-dose positive group had higher values of 99.20 N/mm compared to the positive control (64.71 N/mm). The results showed that vericiguat has dose-dependent effects (Table 4). Histopathological examinations provided the most comprehensive evaluations of the study. In the callus formation assessment according to the Huo scale, the low-dose positive group showed the highest value with an average score of 8.6. The group displayed sophisticated bone development through advanced ossification processes and mature bone tissue formation. The positive control group maintained their tissue development at fibrous and cartilage stages with an average score of 2.6 while the high-dose positive group achieved an intermediate level of improvement with a score of 4.4. The statistical analysis revealed significant differences between the groups (Table 5). The complication assessments produced the most remarkable findings. When evaluated for necrosis, no necrosis infiltration was observed in any of the groups without fractures or in the positive control group. The high-dose positive group showed necrosis in six out of ten animals. Three of these animals showed mild necrosis and one had severe necrosis. The distribution of inflammation findings was similar to the necrosis findings. The high-dose positive group showed severe inflammation in eight animals while the other groups had much milder findings (Table 5). The histopathological imaging results supported these findings. The control groups maintained their normal bone architecture but the positive control group showed fibrous tissue formation and early cartilage callus development. The low-dose positive group showed advanced callus formation and mature bone tissue and widespread ossification areas. The high-dose positive group showed a disrupted healing process with fibrous tissue infiltration and necrotic areas and inflammatory cell accumulation (Figure 3; Figure 4). The detailed results show that vericiguat produces a dose-dependent dual effect on bone fracture healing. The drug promotes bone healing at low doses yet produces severe adverse effects and interferes with healing at high doses. DISCUSSION The research shows that vericiguat produces dose-dependent effects which either promote or hinder fracture healing processes. The histopathological results from low-dose vericiguat (3 mg/kg) administration indicated bone healing support but high-dose (6 mg/kg) administration resulted in tissue-level necrosis and inflammation while decreasing biomechanical performance. The study provides essential clinical and scientific value because it represents the first investigation of vericiguat effects on fracture healing in existing literature. The closed fracture model used in our study was based on protocols developed by Jackson and colleagues in 1970 and improved by Bonnarens and Einborn in 1984 [9]. The closed fracture model was selected because it reduces the occurrence of delayed union and infection risks and non-union complications that open osteotomy techniques present. The literature shows that using an intramedullary K-wire improves surgical stability [10] and our study adopted this method to increase methodological reliability. The selection of female Wistar-Albino rats as experimental animals is consistent with similar studies in the literature [11]. The choice of this approach stems from its high availability and rapid adaptation capabilities and resistance to infections and low cost. The selection of female animals who have not mated before remains essential because it reduces experimental variability and prevents hormonal influences on study outcomes. Our study achieved complete animal survival because of strict sterilization protocols and professional team collaboration. Sun and colleagues performed an in vitro study which showed vericiguat affects osteoclast differentiation through dose-dependent mechanisms [6]. The effects of vericiguat on osteoclast differentiation follow an inverse pattern according to concentration: low doses between 100 nM and 1 μM enhance differentiation but high doses between 4 μM and 8 μM block this process. This effect occurs via the VASP/IκB-α/NF-κB signaling pathway. The positive histopathological findings observed in the low-dose group and the necrosis and inflammation findings in the high-dose group in our study appear consistent with these molecular mechanisms. The low-dose administration of vericiguat led to faster callus development and produced superior histopathological scores (mean score 8.6) which indicated optimal osteoclast activity for bone remodeling. The high-dose group experienced negative effects on fracture healing because excessive osteoclast suppression led to bone resorption inhibition and delayed bone remodeling. The high-dose vericiguat group (39.07 Newton) showed mechanical resistance values that were approximately half of the positive control group (88.75 Newton) and the low-dose group (83.54 Newton). The significant drop in bone quality became evident through this substantial reduction. The hardness measurements followed the same pattern as the mechanical resistance results because the high-dose group produced the lowest values at 40.47 N/mm. Homer and colleagues demonstrated in their study that sGC agonists at toxic doses between 25 mg/kg and 500 mg/kg enhance osteoclastic bone resorption [12]. The study results confirm the negative effects of high-dose vericiguat administration in our research. The necrosis and inflammation found in callus tissue indicates that excessive bone resorption weakens the biomechanical properties of the tissue. The lack of statistically significant differences between groups in radiological evaluations using the Lane-Sandhu scoring system suggests that this method may be insufficient for detecting subtle changes in bone quality. The main study limitation arises from the absence of advanced imaging tools including micro-computed tomography (micro-CT). Future research needs to study callus development through more precise imaging techniques. Tao and Shen demonstrated in their research that vericiguat combined with β-tricalcium phosphate enhanced osteogenic activity in ovariectomised rats [13]. The current study shows that a 5 mg dose produced better results than the high-dose group in our study even though the other study produced negative results. The main factors behind this disagreement stem from β-tricalcium phosphate supplementation synergy and the different study durations (12 weeks vs. 4 weeks) and natural differences between osteoporotic bone defects and healthy bone fractures. The positive findings from Tao and Shen’s study using 10 mg/kg vericiguat on elderly (24-month-old) rats [14] indicate that both age and oxidative stress levels affect how vericiguat works. The age difference between our 8-week-old rats and the study subjects and the varying follow-up periods in our research explain the observed discrepancies in results. Our research reveals a major inconsistency between the results obtained through histopathological examination and radiological imaging. Despite the histopathological evaluation according to the Huo scale showing a significantly higher quality of improvement (average score of 8.6) in the low-dose vericiguat group compared to the positive control group (score of 2.6), no significant difference was detected between the groups in the Lane-Sandhu radiological scoring. The literature supports this inconsistency because Zhu and colleagues demonstrated that the Lane-Sandhu X-ray scoring system failed to detect treatment group differences in rabbit radius models especially during early bone quality changes [15]. Menger and colleagues found that radiographic scores failed to match histopathological results in elderly mice while conventional radiography proved insufficient for detecting small bone tissue quality changes [16]. The biomechanical stiffness values (99.20 N/mm) in the low-dose group exceeded those of the positive control group (64.71 N/mm) by 53%. The superior results demonstrated both successful healing and the creation of a callus structure that exceeded normal bone strength. Menger and colleagues also reported that bending stiffness values paralleled histopathological findings and serve as the most reliable indicator of bone quality [16]. The mechanical properties of collagen-based hydrogels directly influence the formation of advanced callus and mature bone tissue according to De Pace and colleagues [17]. The histopathological examination revealed advanced callus formation and mature bone tissue formation and widespread ossification areas which establish the morphological basis for this mechanical superiority. The findings observed in the high-dose vericiguat group indicate that changes at the micro and macro levels in bone healing may develop with different kinetics and that overdose can cause serious toxicity. The histopathological score (4.4) in this group exceeded the positive control group (2.6) yet the biomechanical test results (39.07 Newton) were extremely low which shows that callus formation does not always lead to functional improvement. The literature supports this paradox as Karasu and colleagues found histopathological improvement alongside substantial wound healing impairment in patients receiving high-dose methotrexate treatment [18]. In the same study, the severe (+++) necrotic area reported on day 7 in the high-dose group is consistent with our findings. The quality of callus tissue appears to be substantially impaired by necrosis (found in 6 out of 10 animals) and widespread inflammation (severe in 8 animals). The results of Kyllönen and colleagues in their studies with BMP-7 were similar, showing that excessive doses of BMP-7 are detrimental to bone healing, with no fusion observed at doses of 10–30 µg, while bone fusion was only observed at a dose of 90 µg (three times the normal dose) [19]. These results highlight the need for an appropriate dose range for bone healing. The performance in healthy bones showed a dose-dependent decline from NK: 115.32 N to DVN: 106.87 N to YVN: 98.05 N. Although this gradual decline pattern did not reach statistical significance, it indicates potential risks associated with long-term use of vericiguat. Razavi and colleagues also emphasised that callus hardness should reflect physiological callus growth and that optimal mechanical performance is directly related to bone quality [20]. The research indicates that vericiguat treatment for fracture healing needs precise dose adjustments because its therapeutic benefits exist within a very limited range. The research by Singh and colleagues demonstrated that nitroglycerin as a nitric oxide donor produces effects that depend on the frequency of administration [21]. The research showed that daily administration of low-dose nitroglycerin stops bone mineral density decline but multiple daily doses of the same amount become ineffective. The research demonstrates that the NO-sGC-cGMP pathway requires precise control because optimal dosing represents a critical factor. In an in vitro study by Abnos and colleagues, it was noted that NO supports osteoblast proliferation at low concentrations but has the opposite effect at high concentrations [22]. The biphasic effect pattern matches the dose-dependent results from our study which demonstrates why dose optimization is needed to understand vericiguat’s role in bone healing. In a study by Joshua and colleagues, another sGC agonist, cinaciguat, was shown to increase bone mineral density and support osteoblast function [4]. The research results indicate that sGC activation could have beneficial effects on bone health. The positive results of low-dose vericiguat in our study suggest that this drug may support fracture healing at optimised doses. Our research benefits from a controlled experimental setting and a multi-group comparative design and a multidisciplinary evaluation approach (radiological, biomechanical, histopathological). As the first study to investigate the effects of vericiguat on fracture healing, it makes an important contribution to the scientific literature. The study lacks biochemical analysis results and plasma vericiguat concentration measurements and sensitive imaging methods including micro-CT. The 4-week follow-up duration might not be enough to assess long-term effects of the treatment. Conclusion The experimental research showed that vericiguat produces dose-dependent bidirectional effects when used in rat femur fracture models. The administration of 3 mg/kg vericiguat resulted in enhanced bone healing yet 6 mg/kg vericiguat produced severe adverse effects. The low-dose group showed a threefold increase in histopathological scores (8.6 vs 2.6) and their biomechanical stiffness values exceeded the control group by 53% while their callus organisation was superior. The high-dose group experienced a 50% decrease in biomechanical performance together with necrosis and severe inflammation. The medication did not produce any pathological effects on normal bones but radiological tests failed to show changes in bone quality. The research represents the first investigation of sGC activator effects on bone healing processes in scientific literature. Future research needs to include clinical trials together with sensitive imaging techniques and biochemical parameter assessments. Acknowledgment: N/A Conflict of interest: The authors declare that they have no conflict of interest to disclose. Funding: This study did not receive financial support. Data availability: Data used in this study can be provided on reasonable request. References: 1. Steppe L, Megafu M, Tschaffon-Müller MEA, Ignatius A, Haffner-Luntzer M. Fracture healing research: recent insights. Bone Rep 2023;19:101686. doi:10.1016/j.bonr.2023.101686. 2. Emeli A, İnanmaz ME, Işık C, Bentli R, Karakurt L, Belhan O. Serum ghrelin levels during fracture healing and immunohistochemical investigation of ghrelin in bone tissue. Acta Med Mediterr 2014;30:137-42. 3. Gogna S, Maxwell J, Policastro AJ, Latifi R. Nutrition support in elderly patients undergoing surgery. In: Latifi R, editor. Surgical decision making in geriatrics. 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Negative pressure wound therapy enhances bone regeneration compared with conventional therapy in a rabbit radius gap-healing model. Exp Ther Med 2021;21:474. doi:10.3892/etm.2021.9905. 16. Menger MM, Manuschewski R, Ehnert S, Rollmann MF, Maisenbacher TC, Tobias AL, et al. Radiographic, biomechanical and histological characterization of femoral fracture healing in aged CD-1 mice. Bioengineering 2023;10:275. doi:10.3390/bioengineering10020275. 17. De Pace R, Molinari S, Mazzoni E, Perale G. Bone regeneration: a review of current treatment strategies. J Clin Med 2025;14:1838. doi:10.3390/jcm14061838. 18. Karasu A, Kuşcu Y, Kayıkcı C, Yıldırım S, Kuşcu O, Kiliçlioğlu M. Effect of low- and high-dose methotrexate on wound healing in rats. Acta Cir Bras 2025;40:e403225. doi:10.1590/acb403225. 19. Kyllönen L, D’Este M, Alini M, Eglin D. Local drug delivery for enhancing fracture healing in osteoporotic bone. Acta Biomater 2015;11:412-34. doi:10.1016/j.actbio.2014.09.005. 20. Razavi AH, Nafisi N, Ghiasi MS, Oftadeh R, Hanna P, Lechtig A, et al. A computational model that integrates unrestricted callus growth, mechanobiology, and angiogenesis can predict bone healing in rodents. Sci Rep 2024;14:29826. doi:10.1038/s41598-024-80502-2. 21. Singh S, Sharma P, Dixit D, Mandal MB. Role of nitric oxide in determination of large intestinal contractility in neonatal rats. Indian J Physiol Pharmacol 2024;68:9-17. doi:10.25259/IJPP_374_2023. 22. Abnos MH, Sargolzaei J, Maleklou M. Exogenous nitric oxide up-regulates the Runx2 via Bmp7 overexpression to increase the osteoblast matrix production in vitro. Avicenna J Med Biochem 2022;10:58-64. doi:10.34172/ajmb.2022.08. Figures: Figure 1. Surgical procedure for experimental femoral fracture model in rats: (A) Anterior approach with exposure of femoral condyles through skin incision, (B) Preparation of intramedullary entry point in the femur, (C) Kirschner wire (K-wire) insertion for internal stabilization, (D) Closed fracture creation using custom-designed three-point bending forceps, (E) K-wire trimming to prevent postoperative skin irritation, (F) Surgical site closure with sutures. Figure 2. Radiological assessment of fracture healing progression across all experimental groups. Serial anteroposterior radiographs taken at 7, 14, and 28 days post-surgery (left to right). (A) NK: Negative control with intact femurs showing normal bone structure. (B) PK: Positive control demonstrating fracture healing with K-wire fixation alone, showing callus formation progression. (C) DVN: Low-dose vericiguat (3 mg/kg/day) in intact femurs. (D) YVN: High-dose vericiguat (6 mg/kg/day) in intact femurs. (E) DVP: Fracture healing with low-dose vericiguat treatment showing callus development. (F) YVP: Fracture healing with high-dose vericiguat treatment. Figure 3. Histopathological evaluation of control and healthy groups at 28 days (H&E staining, ×100 magnification). (A) NK group showing normal bone tissue architecture with healthy trabecular structure. (B) PK group demonstrating fibrous tissue formation and early cartilaginous callus development at fracture site. (C) DVN group exhibiting preserved normal bone morphology with intact cortical and trabecular bone. Scale bar = 100 μm. Figure 4. Histopathological evaluation of vericiguat treatment groups at 28 days (H&E staining, ×100 magnification). (D) YVN group showing preserved normal bone architecture with minimal structural changes. (E) DVP group demonstrating advanced callus formation with mature bone tissue and extensive ossification at fracture site. (F) YVP group exhibiting fibrous tissue infiltration, areas of necrosis, and inflammatory cell accumulation with impaired healing. Scale bar = 100 μm. Tables: Table 1. Experimental groups and study protocol Table 2. Radiographic evaluation using Lane-Sandhu scoring system Table 3. Individual biomechanical test results at day 28 Table 4. Biomechanical test results - Group means and statistical comparisons Table 5. Histopathological evaluation and complications at day 28 Supplementary Material File (table 1.docx) Download 14.84 KB File (table 2.docx) Download 14.86 KB File (table 3.docx) Download 17.48 KB File (table 4.docx) Download 17.92 KB File (table 5.docx) Download 15.87 KB Information & Authors Information Version history V1 Version 1 02 September 2025 Peer review timeline Published Joint Diseases and Related Surgery Version of Record 16 Mar 2026 Published Copyright This work is licensed under a Non Exclusive No Reuse License. Authors Affiliations Azat Dzhumukov [email protected] TC Saglik Bakanligi Torbali Devlet Hastanesi View all articles by this author Bilal Karabak Erzurum Bolge Egitim ve Arastirma Hastanesi View all articles by this author Aziz Demirci Erzurum Bolge Egitim ve Arastirma Hastanesi View all articles by this author Ferhat Yıldırım Hoyur TC Saglik Bakanligi Trabzon Yavuz Selim Kemik Hastaliklari ve Rehabilitasyon Hastanesi View all articles by this author Muhammed Çağatay Engin 0000-0002-9302-9587 Ataturk Universitesi Arastirma Hastanesi View all articles by this author Metrics & Citations Metrics Article Usage 131 views 98 downloads .FvxKWukQNSOunydq8rnd { width: 100px; } Citations Download citation Azat Dzhumukov, Bilal Karabak, Aziz Demirci, et al. Effects of soluble guanylate cyclase activator vericiguat on fracture healing in rats. Authorea . 02 September 2025. 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