Dose-dependent effects of topical vancomycin and teicoplanin on fusion mass formation in a rat posterolateral lumbar arthrodesis model

Dose-dependent effects of topical vancomycin and teicoplanin on fusion mass formation in a rat posterolateral lumbar arthrodesis model | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Dose-dependent effects of topical vancomycin and teicoplanin on fusion mass formation in a rat posterolateral lumbar arthrodesis model Cemil Burak Demirkiran, Deniz Kara, Anil Pulatkan, Arzu Gunes, and 3 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8358911/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Purpose Topical glycopeptide antibiotics are widely used during spinal fusion procedures to reduce the risk of surgical site infection; however, their dose-dependent effects on fusion biology remain unclear. This experimental study aimed to compare the effects of locally applied vancomycin and teicoplanin at different doses on fusion mass formation in a rat posterolateral lumbar arthrodesis model. Methods Forty-five female Wistar rats underwent unilateral L4–L5 posterolateral fusion and were randomized into five groups (n = 9 each): control (no topical antibiotic), low-dose vancomycin (14.3 mg/kg), high-dose vancomycin (71.5 mg/kg), low-dose teicoplanin (15 mg/kg), and high-dose teicoplanin (50 mg/kg). Autologous iliac crest bone graft was combined with the assigned antibiotic solution and placed onto the decorticated fusion bed. Fusion was evaluated at 8 weeks using micro-computed tomography (fusion mass volume), manual palpation (fusion score), and qualitative histological assessment (hematoxylin–eosin and Masson’s trichrome staining). Results Micro-CT fusion scores did not differ significantly among groups (p = 0.106). In contrast, fusion mass volume showed a significant intergroup difference (p = 0.018). The high-dose vancomycin group demonstrated the lowest fusion mass volume (median 7.3 mm³ [4.4–14.9]), which was significantly reduced compared with the control group (13.6 mm³ [10.3–19.7]) and both teicoplanin groups (median range 13.1–13.7 mm³; post hoc p ≤ 0.007). Manual palpation scores showed a non-significant trend toward lower values in the high-dose vancomycin group (p = 0.073). Histologically, fusion masses in the high-dose vancomycin group appeared less organized, whereas both teicoplanin groups demonstrated bone morphology comparable to controls. Conclusion In this rat posterolateral fusion model, high-dose topical vancomycin was associated with a reduction in fusion mass volume, despite similar fusion scores. In contrast, topical teicoplanin at both low and high doses did not adversely affect fusion mass formation. These findings highlight the importance of dose selection when using topical vancomycin and indicate that teicoplanin may be considered a bone-compatible alternative for local application during spinal fusion. Spinal Fusion Micro-computed tomography Vancomycin Teicoplanin Bone Development Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Introduction Topical antibiotics are widely employed as adjunctive agents for the prevention and management of surgical site infections, particularly in clinical settings where the effectiveness of systemic antibiotics may be limited ( 1 , 2 ). This limitation is commonly attributed to suboptimal tissue penetration and the presence of implantable materials, both of which are well-recognized risk factors for infection. Spinal fusion surgery represents a prototypical high-risk procedure in this regard, as it frequently involves the placement of bone grafts or foreign materials within relatively poorly vascularized environments, thereby creating local conditions that predispose to infection. Accordingly, numerous experimental and clinical studies have evaluated the use of topical antibiotics—most notably vancomycin ( 3 – 8 ), as well as tobramycin ( 7 ), and gentamicin ( 9 ) to reduce infection rates associated with spinal procedures. Teicoplanin, a glycopeptide antibiotic with a molecular structure similar to that of vancomycin, demonstrates antibacterial activity that is comparable to or, in some settings, superior to vancomycin ( 10 ). Despite favorable pharmacological characteristics, including good bone penetration, evidence regarding the use of teicoplanin in orthopedic surgery remains limited ( 1 , 11 , 12 ). A clinical study has suggested that teicoplanin may serve as a viable alternative for the prevention of surgical site infections following spinal fusion surgery without adversely affecting fusion outcomes ( 1 ). Nevertheless, data regarding the direct effects of teicoplanin on spinal fusion biology remain scarce. Although vancomycin is widely applied intraoperatively for infection prophylaxis in spinal surgery and has been reported in several studies not to impair fusion, ongoing discussion persists as to whether alternative local antibiotics might provide comparable infection control while preserving—or potentially improving—fusion integrity ( 7 , 13 ). The relationship between local antibiotic dose and bone healing, particularly in the setting of spinal arthrodesis, remains controversial ( 6 , 14 ). Although several studies have reported that commonly used topical antibiotics, such as vancomycin, do not adversely affect spinal fusion, concerns persist regarding the potential negative effects of supraphysiological local concentrations on bone regeneration and cellular viability ( 7 ). Experimental evidence suggests that excessively high antibiotic concentrations may impair the function of fibroblasts and osteoblasts, which play a critical role in the fusion process. Consequently, determining a topical antibiotic strategy that balances effective infection prophylaxis with preservation of fusion capacity remains an important consideration in spinal surgery ( 6 , 7 ). While a previous experimental study compared local and systemic administration of glycopeptides, including vancomycin and teicoplanin, in the prevention of vascular graft infections in a rat model, direct comparative data regarding the local application of vancomycin and teicoplanin in the context of spinal fusion are lacking ( 15 ). The aim of this study was to evaluate the effects of two different doses of locally applied vancomycin and teicoplanin on spinal fusion in a rat posterolateral arthrodesis model. Accordingly, the investigation focused on fusion-related outcomes rather than antimicrobial efficacy. We hypothesized that high-dose topical vancomycin would adversely affect fusion outcomes, whereas teicoplanin would better preserve fusion mass quality and structural integrity. Materials and Methods Study Design The protocol of this animal study was approved by the Local Ethics Committee for Animal Research (Date: 25.05.2022; Approval No: E.62255). All experimental procedures were conducted in accordance with the Animal Welfare Act and its implementing regulations, as well as the principles outlined in the Guide for the Care and Use of Laboratory Animals . A total sample size of 45 animals (nine per group) was calculated to detect a mean difference of 5 units in radiological fusion scores, assuming a standard deviation of 4, with 80% statistical power and a 95% confidence level in a five-group comparison model. This estimation was based on previous literature and pilot observations ( 7 ). Accordingly, forty-five female Wistar rats (12 weeks old; body weight 250–300 g) were obtained from the obtained from an accredited institutional animal laboratory (Istanbul, Turkey). Animals were housed in standard cages under controlled environmental conditions (temperature 22–24°C; constant humidity) with a 12-hour light/dark cycle and provided ad libitum access to food and water. Throughout the study period, animal welfare was closely monitored by a veterinarian. Postoperative pain and distress were assessed daily based on general activity, posture, grooming behavior, and food intake. Supportive care was provided when necessary, and all procedures were performed in accordance with institutional animal welfare protocols to minimize discomfort and stress. Administration of Antibiotics Low- and high-dose regimens of water-soluble vancomycin and teicoplanin were selected based on previously published experimental studies evaluating local antibiotic application in spinal fusion and orthopedic models ( 6 , 7 , 12 , 16 , 17 ). These dose ranges were chosen to represent clinically relevant concentrations and supraphysiological exposure levels commonly investigated in experimental settings. Antibiotic solutions were prepared by dilution in 100 µL of sterile saline, as described by Ishida et al. ( 7 ). The vancomycin and teicoplanin doses were derived from standard adult human dosing regimens (vancomycin: 1 g corresponding to 14.3 mg/kg; teicoplanin: 15 mg/kg) and proportionally adjusted for local administration in the rat model, consistent with previously reported experimental protocols ( 7 ). Animals were randomly assigned to five groups (n = 9 per group): control (Group C, no antibiotic), low-dose vancomycin (Group LD-Van, 14.3 mg/kg), high-dose vancomycin (Group HD-Van, 71.5 mg/kg), low-dose teicoplanin (Group LD-Tei, 15 mg/kg), and high-dose teicoplanin (Group HD-Tei, 50 mg/kg) (Fig. 1 ). Posterolateral Arthrodesis Procedure Preoperative prophylaxis was provided via intraperitoneal administration of cefazolin (20 mg/kg). Anesthesia was induced using intraperitoneal ketamine (36 mg/kg) and xylazine (4 mg/kg). Pain responses were monitored intraoperatively by a veterinarian, and maintenance doses were administered as required. Autologous bone graft material was harvested intraoperatively by collecting approximately one-fourth of each rat’s iliac bone using a bone scraper and rongeur. All grafts were obtained from the recipient animals themselves; therefore, no additional donor animals were used. The harvested bone grafts were mixed with 100–200 µL of the preprepared antibiotic solutions immediately prior to implantation (Fig. 2 ) . Unilateral posterolateral lumbar fusion was performed in accordance with previously described techniques ( 7 ). Following placement of the animals in the prone position, the L4 and L5 vertebrae were identified by palpation. A 4-cm midline skin incision was made, followed by a single paramedian fascial incision approximately 0.5 cm lateral to the spinous processes on the left side. The transverse processes of L4 and L5 were exposed using an intermuscular approach, and the fusion bed was decorticated using an electric burr. The bone–antibiotic mixture was then placed unilaterally over the left posterolateral fusion site ( 7 , 17 ). Follow-up Procedure During the eight-week follow-up period, one rat required resuturing due to superficial skin dehiscence on the third postoperative day. No other complications, including infection, nerve palsy, hemiparesis, or mortality, were observed in the animals. At the end of the study, all animals were euthanized using an overdose of intraperitoneally administered pentobarbital sodium (≥ 200 mg/kg), in accordance with institutional ethical guidelines. Radiological Analysis Micro-computed tomography (micro-CT) imaging of the harvested lumbar spines was performed using a U-CT system (MILabs, MicroCT-OI). Specimens were positioned with the fusion mass centered on the scanning bed. Scans were acquired at 50 kVp and 45 µA, with an exposure time of 40 ms per projection, followed by reconstruction of axial, coronal, sagittal, and three-dimensional (3D) images. Coronal micro-CT sections were independently evaluated by two investigators blinded to the study groups. Fusion was graded according to established criteria as complete fusion (continuous fusion tissue spanning the operated segments, 2 points), partial fusion (narrowing within the fusion mass, 1 point), or no fusion (discontinuity of the fusion mass, 0 points), as previously described ( 7 , 13 ). A modified micro-CT score was then assigned to each harvested spine by averaging the unilateral scores of each fusion mass ( 7 ) (Fig. 3 ). Fusion mass volume, defined as the primary radiological outcome measure, was quantified on axial micro-CT sections using the Volume Calculator plugin of ImageJ software (National Institutes of Health, USA). The region of interest (ROI) was delineated as newly formed bone lateral to the transverse processes at the operated levels. Palpation Analysis Fusion was assessed by manual palpation after removal of surrounding soft tissues from the harvested vertebral segments. Two investigators, blinded to group allocation, independently evaluated each specimen and graded fusion as complete fusion (absence of motion across the operated segment, 2 points), partial fusion (limited motion, 1 point), or no fusion (motion comparable to adjacent segments, 0 points), according to previously described methods ( 6 , 18 ). A modified palpation score was calculated for each specimen by averaging the unilateral fusion scores ( 7 ). Histopathological Analysis The L4–L5 vertebral segments were fixed in 10% neutral buffered formalin for 72 hours and subsequently decalcified in 10% ethylenediaminetetraacetic acid (EDTA) solution (pH 7.4), with the decalcifying solution changed every 48 hours. Decalcification was completed on day 28, after which the specimens were re-fixed for 24 hours, rinsed under running tap water for 30 minutes, dehydrated through a graded alcohol series, cleared in xylene, and embedded in paraffin wax at 60°C overnight. Serial coronal sections of the L4–L5 vertebral levels were obtained at a thickness of 5 µm, with particular focus on the fusion mass region on the operated (unilateral) side. Sections from the contralateral, non-operated side in the control group were also examined to serve as internal controls for bone integrity comparison. The sections were mounted on glass slides, air-dried, and incubated overnight at 60°C. Hematoxylin and eosin (H&E) and Masson’s trichrome staining were performed to evaluate bone integrity and collagen fiber density. Histological assessments were conducted under light microscopy at 10× and 40× magnifications ( 6 , 7 ). Statistical Analysis Statistical analyses were performed using Jamovi (version 2.3.28.0; Jamovi Project, 2023) and JASP (version 0.19.1; Jeffreys’ Amazing Statistics Program, 2024). Continuous variables, including fusion mass volume, were summarized as median (minimum–maximum) values. Given the non-normal distribution of the data, intergroup comparisons were conducted using the Kruskal–Wallis test, followed by Dunn’s post hoc test for pairwise comparisons. A p value ≤ 0.05 was considered statistically significant. Results Although the micro-CT–based fusion scores were lower in the high-dose vancomycin (HD-Van) group (median: 0.5 [0.0–0.5]) compared with the other experimental groups, this difference did not reach statistical significance (p = 0.106). The corresponding median fusion scores were 1.0 [1.0–2.0] in the control group (C), 1.0 [0.5–1.5] in the low-dose vancomycin (LD-Van) group, 1.0 [1.0–1.5] in the low-dose teicoplanin (LD-Tei) group, and 1.0 [1.0–1.0] in the high-dose teicoplanin (HD-Tei) group (Table 1 ). Overall, micro-CT evaluation demonstrated comparable fusion scores across the study groups despite the observed numerical differences (Table 1 ). In contrast, micro-CT–based fusion mass volume differed significantly among the groups (p = 0.018). The lowest median fusion mass volume was observed in the HD-Van group (7.3 mm³ [4.4–14.9]). Median fusion mass volumes were 13.6 mm³ [10.3–19.7] in the control group, 12.2 mm³ [6.2–16.5] in the LD-Van group, 13.1 mm³ [7.2–18.3] in the LD-Tei group, and 13.7 mm³ [7.7–18.3] in the HD-Tei group. Post-hoc analysis demonstrated that the fusion mass volume in the HD-Van group was significantly lower than that in the control (p = 0.043), LD-Tei (p = 0.006), and HD-Tei (p = 0.007) groups, whereas no statistically significant difference was observed between the HD-Van and LD-Van groups (p = 0.318). Manual palpation scores did not differ significantly among the groups (p = 0.073). The median palpation score was 0.0 [0.0–1.0] in the HD-Van group, compared with 1.5 [1.0–2.0] in the control group, 1.0 [1.0–1.5] in the LD-Van group, 1.0 [0.5–1.5] in the LD-Tei group, and 1.5 [1.0–2.0] in the HD-Tei group. Table 1 Comparison of the groups regarding micro-CT fusion score, fusion mass volume, and palpation scores. Group C (n = 9) Group LD-Van (n = 9) Group HD-Van (n = 9) Group LD-Tei (n = 9) Group HD-Tei (n = 9) p-value Micro-CT fusion score 1.0 [1.0, 2.0] 1.0 [0.5, 1.5] 0.5 [0.0, 0.5] 1.0 [1.0, 1.5] 1.0 [1.0, 1.0] 0.106 Micro-CT fusion mass volume ( mm 3 ) 13.6 [10.3–19.7] b 12.2 [6.2–16.5] a, b 7.3 [4.4–14.9] a 13.1 [7.2–18.3] b 13.7 [7.7–18.3] b 0.018 Palpation score 1.5 [1.0, 2.0] 1.0 [1.0, 1.5] 0.0 [0.0, 1.0] 1.0 [0.5, 1.5] 1.5 [1.0, 2.0] 0.073 a, b: Different superscript letters indicate statistically significant differences between groups (Dunn post hoc test, p < 0.05). Micro-CT: Micro computed tomography. Note Bold p-values indicate statistical significance (p ≤ 0.05). Histopathological Analysis In rats in Group C, microfractures within the trabecular bone were frequently observed on the operated side, where the surgical bone graft model was created, whereas trabecular structures and bone marrow areas on the non-operated contralateral side appeared unaffected (Fig. 4 a, b). Histopathological examination of bone tissues from the low-dose teicoplanin (LD-Tei) group demonstrated the presence of trabecular microfractures, while overall trabecular architecture remained preserved compared with the operated side of Group C. Similarly, in the high-dose teicoplanin (HD-Tei) group, preservation of trabecular morphology was observed (Fig. 5 a, b). In the low-dose vancomycin (LD-Van) group, trabecular bone morphology appeared preserved, whereas in the high-dose vancomycin (HD-Van) group, trabecular microfractures were more frequently observed, indicating reduced preservation of trabecular architecture (Fig. 6 a, b). In the control groups, collagen fibres in the trabeculae appeared reduced on the operated side, where the surgical bone graft model was created. On the non-operated contralateral side, the density of collagen fibres and bone marrow were observed to be physiological (Fig. 7 a, b). In all treated groups (Group LD-Tei, Group HD-Tei, Group LD-Van, and Group HD-Van), collagen fibre density was preserved in the trabeculae and the bone marrow. No difference in trabecular collagen density was observed among treated groups (Fig. 8 a, b, c, d). Discussion This study comparatively evaluated the effects of low- and high-dose vancomycin and teicoplanin on fusion-related outcomes and bone morphology in an experimental posterolateral arthrodesis model. To the best of our knowledge, this is among the first in vivo studies to assess and directly compare the impact of topical vancomycin and teicoplanin on spinal fusion outcomes in a rat posterolateral fusion model using autologous iliac crest graft. Although micro-CT–based fusion scores were lower in the high-dose vancomycin group, this difference did not reach statistical significance. In contrast, fusion mass volume was significantly reduced in the high-dose vancomycin group compared with the control, low-dose teicoplanin, and high-dose teicoplanin groups. On histological assessment, the high-dose vancomycin group demonstrated less organized trabecular architecture with trabecular microfractures. These findings were qualitative and descriptive in nature and, in the absence of quantitative histomorphometric measures, should be interpreted accordingly. Several studies investigated the impact of topical vancomycin use on the rates of intervertebral fusion and surgical site infections in patients who underwent lumbar fusion procedures ( 1 , 2 , 8 , 19 , 20 ). These studies have reported that topical vancomycin had a protective effect against surgical site infections and that varying vancomycin doses did not negatively impact intervertebral fusion rates ( 8 ). Despite its reported benefits, concern remains that local vancomycin exposure may adversely affect bone healing. Experimental studies have reported vancomycin-related toxicity to osteoblasts, although its cytotoxicity has been described as lower than that of some other antibiotics, including aminoglycosides, quinolones, and gentamicin ( 6 , 21 , 22 ). Guimbard-Pérez et al. ( 3 ) reported that vancomycin powder (200 mg, five times the dose used in humans) mixed with graft material resulted in a 30% reduction in graft fusion rates in rabbits. In a study featuring a rat model of posterolateral fusion with rhBMP-2-induced osteogenesis, Mendoza et al. ( 6 ) did not find a significant difference in fusion scores, rates, and new bone formation between groups administered low-dose (14.3 mg/kg) and high-dose (143 mg/kg) vancomycin. They concluded that the powder form of vancomycin at doses equivalent to those routinely used by surgeons for fusion procedures may not reduce fusion rates ( 6 ). However, the fact that they did not use more commonly used bone-graft materials prevents generalizing their findings to clinical practice. Additionally, there is no consensus on what constitutes a high dose of vancomycin. In some studies, doses ranging from 71 mg/kg to 200 mg/kg, which were stated to be 5 times higher than the vancomycin dose used by spine surgeons, have been defined as high doses ( 3 , 7 ).On the other hand, Ishida et al. ( 7 ), as in this study, defined 71.5 mg/kg vancomycin as a high dose, contrary to Mendoza et al.( 6 ). They reported that intraoperative local application of vancomycin, particularly at a supraphysiological dosage, led to an impairment in fusion-mass formation, and also detected reduced fusion-mass volume based on micro-CT analysis and less osseous fusion mass based on histological analysis and obtained lower manual palpation scores, in line with our findings that high-dose vancomycin may be associated with impaired fusion-mass formation ( 7 ). Vancomycin is extensively utilized in clinical practice for infection control; nevertheless, its potential deleterious effects on bone regeneration, particularly when applied locally at high concentrations, remain a subject of ongoing debate. The heterogeneity in dosing protocols, the mode of application—either as a powder or in solution—and the diversity of graft materials employed across studies significantly hinder the consistent interpretation of its safety profile in the context of spinal fusion. In the present study, vancomycin in solution form was administered directly to the surgical site at two distinct dosages (71.5 mg/kg as high dose and 14.3 mg/kg as low dose), using a rat posterolateral fusion model with a standardized autograft substitute. Post-hoc analysis using the Dunn test revealed that fusion mass volume was significantly reduced in the high-dose vancomycin group compared to the control group (p = 0.043), whereas no statistically significant difference was observed between the high-dose and low-dose vancomycin groups (p = 0.318). In contrast, micro-CT–based fusion scores did not differ significantly among the groups. Taken together, these findings indicate that higher local vancomycin exposure may be associated with reduced fusion-mass formation in this model, highlighting the importance of careful dose selection when topical antibiotics are used during spinal fusion procedures. Teicoplanin has been studied less extensively than vancomycin, with prior work focusing mainly on osteomyelitis, implant-associated infections, vascular graft infections, and wound healing ( 11 , 12 , 15 , 17 , 23 , 24 ). For instance,Yasim et al.( 15 ) reported that prophylactic intraperitoneal administration of teicoplanin was less effective than antibiotic-soaked grafts in preventing infection in a vascular graft model. In contrast, another study suggested that intraperitoneal teicoplanin administration may support new bone formation following intramedullary tibial fixation in rats ( 12 ). Similarly, Cai et al. ( 24 ) demonstrated that teicoplanin-loaded wound dressings offered promising local protection against implant-related infections. Şener et al. ( 17 ), investigating intramuscular teicoplanin application in autogenous bone grafts, reported favorable outcomes with teicoplanin-loaded bone cement compared with other approaches. Overall, these findings are model-dependent and may be influenced by factors such as teicoplanin dose, local pH, and graft material characteristics. Evidence regarding the direct effects of topical teicoplanin on bone regeneration remains limited. In the present study, teicoplanin was applied topically at two doses (15 mg/kg and 50 mg/kg). Both teicoplanin groups demonstrated significantly greater fusion mass volumes compared with the high-dose vancomycin group (p = 0.006 and p = 0.007, respectively). No statistically significant difference was observed between either teicoplanin group and the control group, suggesting that topical teicoplanin did not show a detrimental association with fusion mass formation in this model. Based on the micro-CT findings, teicoplanin may warrant consideration as an alternative topical agent when considering potential effects on fusion alongside infection prophylaxis, although confirmatory studies incorporating infection models and quantitative histologic measures are warranted. The Limitations of the Study This study has several limitations that should be considered when interpreting the findings. First, the lack of a standardized definition for “high” and “low” topical doses of vancomycin and teicoplanin complicates extrapolation to clinical practice, where dosing regimens vary. Although doses were selected to reflect common clinical use, their pharmacokinetic and biological relevance in a rat model remains uncertain. Second, this was not an infection model; therefore, conclusions regarding prophylactic efficacy against spinal fusion–related infections cannot be drawn. Third, histological findings were based on qualitative assessment rather than quantitative histomorphometric analysis; the absence of objective metrics (e.g., trabecular thickness, bone volume fraction, or new bone area) limits reproducibility and interpretive strength. Finally, although the rat model provides a controlled platform to evaluate local antibiotic effects on fusion, it does not replicate the anatomical, biomechanical, and physiological complexity of the human spine. Future studies should incorporate clinically relevant infection models, quantitative bone evaluation methods, and longer-term functional outcomes. Declarations Author Contribution Conceptualization:Deniz Kara, Cemil Burak Demirkiran; methodology: Cemil Burak Demirkiran, Anil Pulatkan, formal analysis and investigation: Bilal Sulak, Mehmet Anil Pulatkan; writing — original draft preparation ;Arzu Gunes, Ilknur Keskin- histological analysis, Cemil Burak Demirkiran writing — review and editing:Deniz Kara, Cemil Burak Demirkiran; supervision: Cemil Burak Demirkiran, Nuh Mehmet Elmadag. All authors read and approved the final manuscript. References Elmadag NM, Kara D, Pulatkan A, Ucan V, Cesme DH, Aliyev O et al (2024) Local Prophylactic Teicoplanin Effect on Spinal Fusion Surgery: A Comparative Retrospective Study. 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Spine Res Soc 25(4):1021–1028 Shiels SM, Raut VP, Patterson PB, Barnes BR, Wenke JC (2017) Antibiotic-loaded bone graft for reduction of surgical site infection in spinal fusion. spine journal: official J North Am Spine Soc 17(12):1917–1925 Egawa S, Hirai K, Matsumoto R, Yoshii T, Yuasa M, Okawa A et al (2020) Efficacy of Antibiotic-Loaded Hydroxyapatite/Collagen Composites Is Dependent on Adsorbability for Treating Staphylococcus aureus Osteomyelitis in Rats. J Orthop Res 38(4):843–851 Cai XZ, Jin AD, Yan SG (2010) Did local teicoplanin delivery systems inhibit or aggravate implant-related infection? Int Orthop 34(3):453–454 author reply 5 Additional Declarations No competing interests reported. Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. 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1","display":"","copyAsset":false,"role":"figure","size":199149,"visible":true,"origin":"","legend":"\u003cp\u003eFlowchart of the study\u003c/p\u003e","description":"","filename":"Figure1.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8358911/v1/800542aef9bc06823e9e465d.jpg"},{"id":100006528,"identity":"a1daf27a-4658-49fe-8fc5-74872385463e","added_by":"auto","created_at":"2026-01-12 05:40:48","extension":"jpg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":366243,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e Surgical exposure of the iliac and facet joint regions in the donor rat prior to bone harvesting. \u003cstrong\u003eb.\u003c/strong\u003e Bone grafts harvested from the iliac region and prepared for use in the spinal fusion site.\u003c/p\u003e","description":"","filename":"Figure2.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8358911/v1/797d729a27efef04fba00276.jpg"},{"id":100006535,"identity":"14eeb74c-f9ab-4fc7-a675-46eaec179fa3","added_by":"auto","created_at":"2026-01-12 05:40:48","extension":"jpg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":316777,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003ea.\u003c/strong\u003e Coronal micro-CT image. \u003cstrong\u003eb.\u003c/strong\u003e3D micro-CT reconstruction\u003c/p\u003e","description":"","filename":"Figure3.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8358911/v1/0e4848776460205496eb732f.jpg"},{"id":100006533,"identity":"b5287003-ffce-4821-bdc0-4ea27bbd57d1","added_by":"auto","created_at":"2026-01-12 05:40:48","extension":"jpg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":553694,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative microscopic images from the control groups. \u003cstrong\u003ea. \u003c/strong\u003eThe operated side, where the surgical bone graft model was created. \u003cstrong\u003eb.\u003c/strong\u003e The non-operated contralateral side. The star: intact trabecular structures. The dashed circle: areas of microfractures within the trabecular bone. (Hematoxylin \u0026amp; Eosin, 10X).\u003c/p\u003e","description":"","filename":"Figure4.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8358911/v1/72cd2e5780ae3615e7b0cd8b.jpg"},{"id":100361408,"identity":"ba9dc5b2-9fb5-44d2-899a-74148c089eea","added_by":"auto","created_at":"2026-01-16 07:45:05","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":3451562,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative microscopic images from the teicoplanin-treated groups. \u003cstrong\u003ea.\u003c/strong\u003e Group LD-Tei. \u003cstrong\u003eb.\u003c/strong\u003e Group HD-Tei. The star: intact trabecular structures, the dashed circle: areas of microfractures within the trabeculae (Hematoxylin \u0026amp; Eosin, 10X).\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8358911/v1/9242f3c6565d8ceccec4c9f5.png"},{"id":100006538,"identity":"86b2986f-927d-415e-86ff-3f75343bd343","added_by":"auto","created_at":"2026-01-12 05:40:49","extension":"jpg","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":450389,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative microscopic images from the vancomycin-treated groups\u003cstrong\u003e a. \u003c/strong\u003eEffective preservation of bone morphology in Group LD-Van.\u003cstrong\u003e b.\u003c/strong\u003e Inadequate preservation of bone morphology in Group HD-Van. The star: intact trabecular structures, the dashed circle: areas of microfractures within the trabeculae. (Hematoxylin \u0026amp; Eosin, 10X).\u003c/p\u003e","description":"","filename":"Figure6.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8358911/v1/e46f72387ada726348eb0f17.jpg"},{"id":100006543,"identity":"22007ed4-d801-4540-a9be-90c863e32660","added_by":"auto","created_at":"2026-01-12 05:40:49","extension":"jpg","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":555856,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative microscopic images from the control groups. \u003cstrong\u003ea. \u003c/strong\u003eThe operated side, where the surgical bone graft model was created. \u003cstrong\u003eb. \u003c/strong\u003eThe non-operated contralateral side. Blue colour: collagen fibres in the trabeculae, red colour: nuclei, yellow colour: erythrocytes, triangle: trabeculae\u003cstrong\u003e \u003c/strong\u003e(Masson's trichrome staining, 40X).\u003c/p\u003e","description":"","filename":"Figure7.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8358911/v1/bbd5fcd8216e82a9f0b547e4.jpg"},{"id":100006541,"identity":"65c20ce9-9a3f-409e-8ed7-51809532248e","added_by":"auto","created_at":"2026-01-12 05:40:49","extension":"jpg","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":806367,"visible":true,"origin":"","legend":"\u003cp\u003eRepresentative microscopic images of trabeculae and bone marrow from the treated groups. \u003cstrong\u003ea.\u003c/strong\u003e Group LD-Tei \u003cstrong\u003eb.\u003c/strong\u003e Group HD-Tei \u003cstrong\u003ec. \u003c/strong\u003eGroup LD-Van\u003cstrong\u003e d.\u003c/strong\u003e Group HD-Van. Blue colour: collagen fibres in the trabeculae, red colour: nuclei, yellow colour: erythrocytes, triangle: trabeculae\u003cstrong\u003e, \u003c/strong\u003ecross: bone marrow\u003cstrong\u003e \u003c/strong\u003e(Masson's trichrome staining, 40X).\u003c/p\u003e","description":"","filename":"Figure8.jpg","url":"https://assets-eu.researchsquare.com/files/rs-8358911/v1/885c7a5f744538a232efa54f.jpg"},{"id":100803969,"identity":"20ec8284-10fb-42f1-889e-3e6c25e95778","added_by":"auto","created_at":"2026-01-21 14:32:29","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":8014193,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8358911/v1/606d8791-5475-4981-b766-3ba9d091385b.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Dose-dependent effects of topical vancomycin and teicoplanin on fusion mass formation in a rat posterolateral lumbar arthrodesis model","fulltext":[{"header":"Introduction","content":"\u003cp\u003eTopical antibiotics are widely employed as adjunctive agents for the prevention and management of surgical site infections, particularly in clinical settings where the effectiveness of systemic antibiotics may be limited (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e). This limitation is commonly attributed to suboptimal tissue penetration and the presence of implantable materials, both of which are well-recognized risk factors for infection. Spinal fusion surgery represents a prototypical high-risk procedure in this regard, as it frequently involves the placement of bone grafts or foreign materials within relatively poorly vascularized environments, thereby creating local conditions that predispose to infection. Accordingly, numerous experimental and clinical studies have evaluated the use of topical antibiotics\u0026mdash;most notably vancomycin (\u003cspan additionalcitationids=\"CR4 CR5 CR6 CR7\" citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e), as well as tobramycin (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), and gentamicin (\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e) to reduce infection rates associated with spinal procedures.\u003c/p\u003e \u003cp\u003eTeicoplanin, a glycopeptide antibiotic with a molecular structure similar to that of vancomycin, demonstrates antibacterial activity that is comparable to or, in some settings, superior to vancomycin (\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e). Despite favorable pharmacological characteristics, including good bone penetration, evidence regarding the use of teicoplanin in orthopedic surgery remains limited (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). A clinical study has suggested that teicoplanin may serve as a viable alternative for the prevention of surgical site infections following spinal fusion surgery without adversely affecting fusion outcomes (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e). Nevertheless, data regarding the direct effects of teicoplanin on spinal fusion biology remain scarce. Although vancomycin is widely applied intraoperatively for infection prophylaxis in spinal surgery and has been reported in several studies not to impair fusion, ongoing discussion persists as to whether alternative local antibiotics might provide comparable infection control while preserving\u0026mdash;or potentially improving\u0026mdash;fusion integrity (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe relationship between local antibiotic dose and bone healing, particularly in the setting of spinal arthrodesis, remains controversial (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e). Although several studies have reported that commonly used topical antibiotics, such as vancomycin, do not adversely affect spinal fusion, concerns persist regarding the potential negative effects of supraphysiological local concentrations on bone regeneration and cellular viability (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Experimental evidence suggests that excessively high antibiotic concentrations may impair the function of fibroblasts and osteoblasts, which play a critical role in the fusion process. Consequently, determining a topical antibiotic strategy that balances effective infection prophylaxis with preservation of fusion capacity remains an important consideration in spinal surgery (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). While a previous experimental study compared local and systemic administration of glycopeptides, including vancomycin and teicoplanin, in the prevention of vascular graft infections in a rat model, direct comparative data regarding the local application of vancomycin and teicoplanin in the context of spinal fusion are lacking (\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eThe aim of this study was to evaluate the effects of two different doses of locally applied vancomycin and teicoplanin on spinal fusion in a rat posterolateral arthrodesis model. Accordingly, the investigation focused on fusion-related outcomes rather than antimicrobial efficacy. We hypothesized that high-dose topical vancomycin would adversely affect fusion outcomes, whereas teicoplanin would better preserve fusion mass quality and structural integrity.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003eStudy Design\u003c/h2\u003e \u003cp\u003e The protocol of this animal study was approved by the Local Ethics Committee for Animal Research (Date: 25.05.2022; Approval No: E.62255). All experimental procedures were conducted in accordance with the Animal Welfare Act and its implementing regulations, as well as the principles outlined in the \u003cem\u003eGuide for the Care and Use of Laboratory Animals\u003c/em\u003e.\u003c/p\u003e \u003cp\u003eA total sample size of 45 animals (nine per group) was calculated to detect a mean difference of 5 units in radiological fusion scores, assuming a standard deviation of 4, with 80% statistical power and a 95% confidence level in a five-group comparison model. This estimation was based on previous literature and pilot observations (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Accordingly, forty-five female Wistar rats (12 weeks old; body weight 250\u0026ndash;300 g) were obtained from the obtained from an accredited institutional animal laboratory (Istanbul, Turkey). Animals were housed in standard cages under controlled environmental conditions (temperature 22\u0026ndash;24\u0026deg;C; constant humidity) with a 12-hour light/dark cycle and provided ad libitum access to food and water.\u003c/p\u003e \u003cp\u003eThroughout the study period, animal welfare was closely monitored by a veterinarian. Postoperative pain and distress were assessed daily based on general activity, posture, grooming behavior, and food intake. Supportive care was provided when necessary, and all procedures were performed in accordance with institutional animal welfare protocols to minimize discomfort and stress.\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eAdministration of Antibiotics\u003c/h3\u003e\n\u003cp\u003eLow- and high-dose regimens of water-soluble vancomycin and teicoplanin were selected based on previously published experimental studies evaluating local antibiotic application in spinal fusion and orthopedic models (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e). These dose ranges were chosen to represent clinically relevant concentrations and supraphysiological exposure levels commonly investigated in experimental settings. Antibiotic solutions were prepared by dilution in 100 \u0026micro;L of sterile saline, as described by Ishida et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). The vancomycin and teicoplanin doses were derived from standard adult human dosing regimens (vancomycin: 1 g corresponding to 14.3 mg/kg; teicoplanin: 15 mg/kg) and proportionally adjusted for local administration in the rat model, consistent with previously reported experimental protocols (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Animals were randomly assigned to five groups (n\u0026thinsp;=\u0026thinsp;9 per group): control (Group C, no antibiotic), low-dose vancomycin (Group LD-Van, 14.3 mg/kg), high-dose vancomycin (Group HD-Van, 71.5 mg/kg), low-dose teicoplanin (Group LD-Tei, 15 mg/kg), and high-dose teicoplanin (Group HD-Tei, 50 mg/kg) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e\n\u003ch3\u003ePosterolateral Arthrodesis Procedure\u003c/h3\u003e\n\u003cp\u003ePreoperative prophylaxis was provided via intraperitoneal administration of cefazolin (20 mg/kg). Anesthesia was induced using intraperitoneal ketamine (36 mg/kg) and xylazine (4 mg/kg). Pain responses were monitored intraoperatively by a veterinarian, and maintenance doses were administered as required.\u003c/p\u003e \u003cp\u003eAutologous bone graft material was harvested intraoperatively by collecting approximately one-fourth of each rat\u0026rsquo;s iliac bone using a bone scraper and rongeur. All grafts were obtained from the recipient animals themselves; therefore, no additional donor animals were used. The harvested bone grafts were mixed with 100\u0026ndash;200 \u0026micro;L of the preprepared antibiotic solutions immediately prior to implantation (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e\u003cb\u003e)\u003c/b\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eUnilateral posterolateral lumbar fusion was performed in accordance with previously described techniques (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Following placement of the animals in the prone position, the L4 and L5 vertebrae were identified by palpation. A 4-cm midline skin incision was made, followed by a single paramedian fascial incision approximately 0.5 cm lateral to the spinous processes on the left side. The transverse processes of L4 and L5 were exposed using an intermuscular approach, and the fusion bed was decorticated using an electric burr. The bone\u0026ndash;antibiotic mixture was then placed unilaterally over the left posterolateral fusion site (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e).\u003c/p\u003e\n\u003ch3\u003eFollow-up Procedure\u003c/h3\u003e\n\u003cp\u003eDuring the eight-week follow-up period, one rat required resuturing due to superficial skin dehiscence on the third postoperative day. No other complications, including infection, nerve palsy, hemiparesis, or mortality, were observed in the animals. At the end of the study, all animals were euthanized using an overdose of intraperitoneally administered pentobarbital sodium (\u0026ge;\u0026thinsp;200 mg/kg), in accordance with institutional ethical guidelines.\u003c/p\u003e\n\u003ch3\u003eRadiological Analysis\u003c/h3\u003e\n\u003cp\u003eMicro-computed tomography (micro-CT) imaging of the harvested lumbar spines was performed using a U-CT system (MILabs, MicroCT-OI). Specimens were positioned with the fusion mass centered on the scanning bed. Scans were acquired at 50 kVp and 45 \u0026micro;A, with an exposure time of 40 ms per projection, followed by reconstruction of axial, coronal, sagittal, and three-dimensional (3D) images.\u003c/p\u003e \u003cp\u003eCoronal micro-CT sections were independently evaluated by two investigators blinded to the study groups. Fusion was graded according to established criteria as complete fusion (continuous fusion tissue spanning the operated segments, 2 points), partial fusion (narrowing within the fusion mass, 1 point), or no fusion (discontinuity of the fusion mass, 0 points), as previously described (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e, \u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e). A modified micro-CT score was then assigned to each harvested spine by averaging the unilateral scores of each fusion mass (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e) (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eFusion mass volume, defined as the primary radiological outcome measure, was quantified on axial micro-CT sections using the Volume Calculator plugin of ImageJ software (National Institutes of Health, USA). The region of interest (ROI) was delineated as newly formed bone lateral to the transverse processes at the operated levels.\u003c/p\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003ePalpation Analysis\u003c/h2\u003e \u003cp\u003eFusion was assessed by manual palpation after removal of surrounding soft tissues from the harvested vertebral segments. Two investigators, blinded to group allocation, independently evaluated each specimen and graded fusion as complete fusion (absence of motion across the operated segment, 2 points), partial fusion (limited motion, 1 point), or no fusion (motion comparable to adjacent segments, 0 points), according to previously described methods (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e). A modified palpation score was calculated for each specimen by averaging the unilateral fusion scores (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003c/div\u003e\n\u003ch3\u003eHistopathological Analysis\u003c/h3\u003e\n\u003cp\u003eThe L4\u0026ndash;L5 vertebral segments were fixed in 10% neutral buffered formalin for 72 hours and subsequently decalcified in 10% ethylenediaminetetraacetic acid (EDTA) solution (pH 7.4), with the decalcifying solution changed every 48 hours. Decalcification was completed on day 28, after which the specimens were re-fixed for 24 hours, rinsed under running tap water for 30 minutes, dehydrated through a graded alcohol series, cleared in xylene, and embedded in paraffin wax at 60\u0026deg;C overnight.\u003c/p\u003e \u003cp\u003eSerial coronal sections of the L4\u0026ndash;L5 vertebral levels were obtained at a thickness of 5 \u0026micro;m, with particular focus on the fusion mass region on the operated (unilateral) side. Sections from the contralateral, non-operated side in the control group were also examined to serve as internal controls for bone integrity comparison. The sections were mounted on glass slides, air-dried, and incubated overnight at 60\u0026deg;C.\u003c/p\u003e \u003cp\u003eHematoxylin and eosin (H\u0026amp;E) and Masson\u0026rsquo;s trichrome staining were performed to evaluate bone integrity and collagen fiber density. Histological assessments were conducted under light microscopy at 10\u0026times; and 40\u0026times; magnifications (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003eStatistical Analysis\u003c/h2\u003e \u003cp\u003eStatistical analyses were performed using Jamovi (version 2.3.28.0; Jamovi Project, 2023) and JASP (version 0.19.1; Jeffreys\u0026rsquo; Amazing Statistics Program, 2024). Continuous variables, including fusion mass volume, were summarized as median (minimum\u0026ndash;maximum) values. Given the non-normal distribution of the data, intergroup comparisons were conducted using the Kruskal\u0026ndash;Wallis test, followed by Dunn\u0026rsquo;s post hoc test for pairwise comparisons. A p value\u0026thinsp;\u0026le;\u0026thinsp;0.05 was considered statistically significant.\u003c/p\u003e \u003c/div\u003e"},{"header":"Results","content":"\u003cp\u003eAlthough the micro-CT\u0026ndash;based fusion scores were lower in the high-dose vancomycin (HD-Van) group (median: 0.5 [0.0\u0026ndash;0.5]) compared with the other experimental groups, this difference did not reach statistical significance (p\u0026thinsp;=\u0026thinsp;0.106). The corresponding median fusion scores were 1.0 [1.0\u0026ndash;2.0] in the control group (C), 1.0 [0.5\u0026ndash;1.5] in the low-dose vancomycin (LD-Van) group, 1.0 [1.0\u0026ndash;1.5] in the low-dose teicoplanin (LD-Tei) group, and 1.0 [1.0\u0026ndash;1.0] in the high-dose teicoplanin (HD-Tei) group (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Overall, micro-CT evaluation demonstrated comparable fusion scores across the study groups despite the observed numerical differences (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eIn contrast, micro-CT\u0026ndash;based fusion mass volume differed significantly among the groups (p\u0026thinsp;=\u0026thinsp;0.018). The lowest median fusion mass volume was observed in the HD-Van group (7.3 mm\u0026sup3; [4.4\u0026ndash;14.9]). Median fusion mass volumes were 13.6 mm\u0026sup3; [10.3\u0026ndash;19.7] in the control group, 12.2 mm\u0026sup3; [6.2\u0026ndash;16.5] in the LD-Van group, 13.1 mm\u0026sup3; [7.2\u0026ndash;18.3] in the LD-Tei group, and 13.7 mm\u0026sup3; [7.7\u0026ndash;18.3] in the HD-Tei group. Post-hoc analysis demonstrated that the fusion mass volume in the HD-Van group was significantly lower than that in the control (p\u0026thinsp;=\u0026thinsp;0.043), LD-Tei (p\u0026thinsp;=\u0026thinsp;0.006), and HD-Tei (p\u0026thinsp;=\u0026thinsp;0.007) groups, whereas no statistically significant difference was observed between the HD-Van and LD-Van groups (p\u0026thinsp;=\u0026thinsp;0.318).\u003c/p\u003e \u003cp\u003eManual palpation scores did not differ significantly among the groups (p\u0026thinsp;=\u0026thinsp;0.073). The median palpation score was 0.0 [0.0\u0026ndash;1.0] in the HD-Van group, compared with 1.5 [1.0\u0026ndash;2.0] in the control group, 1.0 [1.0\u0026ndash;1.5] in the LD-Van group, 1.0 [0.5\u0026ndash;1.5] in the LD-Tei group, and 1.5 [1.0\u0026ndash;2.0] in the HD-Tei group.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eComparison of the groups regarding micro-CT fusion score, fusion mass volume, and palpation scores.\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"7\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"char\" char=\".\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e\u0026nbsp;\u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eGroup C\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;9)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003cp\u003eLD-Van\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;9)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003cp\u003eHD-Van\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;9)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003cp\u003eLD-Tei\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;9)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c6\"\u003e \u003cp\u003eGroup\u003c/p\u003e \u003cp\u003eHD-Tei\u003c/p\u003e \u003cp\u003e(n\u0026thinsp;=\u0026thinsp;9)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c7\"\u003e \u003cp\u003ep-value\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMicro-CT fusion score\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003cp\u003e[1.0, 2.0]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003cp\u003e[0.5, 1.5]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.5\u003c/p\u003e \u003cp\u003e[0.0, 0.5]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003cp\u003e[1.0, 1.5]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003cp\u003e[1.0, 1.0]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.106\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003eMicro-CT fusion mass volume\u003c/b\u003e (\u003cem\u003emm\u003c/em\u003e\u003csup\u003e\u003cem\u003e3\u003c/em\u003e\u003c/sup\u003e)\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e13.6\u003c/p\u003e \u003cp\u003e[10.3\u0026ndash;19.7] \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e12.2\u003c/p\u003e \u003cp\u003e[6.2\u0026ndash;16.5] \u003csup\u003ea, b\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e7.3\u003c/p\u003e \u003cp\u003e[4.4\u0026ndash;14.9] \u003csup\u003ea\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e13.1\u003c/p\u003e \u003cp\u003e[7.2\u0026ndash;18.3] \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13.7\u003c/p\u003e \u003cp\u003e[7.7\u0026ndash;18.3] \u003csup\u003eb\u003c/sup\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e\u003cb\u003e0.018\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003e\u003cb\u003ePalpation score\u003c/b\u003e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003cp\u003e[1.0, 2.0]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003cp\u003e[1.0, 1.5]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e0.0\u003c/p\u003e \u003cp\u003e[0.0, 1.0]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e1.0\u003c/p\u003e \u003cp\u003e[0.5, 1.5]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.5\u003c/p\u003e \u003cp\u003e[1.0, 2.0]\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"char\" char=\".\" colname=\"c7\"\u003e \u003cp\u003e0.073\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003ea, b: Different superscript letters indicate statistically significant differences between groups (Dunn post hoc test, p\u0026thinsp;\u0026lt;\u0026thinsp;0.05).\u003c/p\u003e \u003cp\u003eMicro-CT: Micro computed tomography.\u003c/p\u003e \u003cp\u003e \u003cstrong\u003eNote\u003c/strong\u003e \u003cp\u003eBold p-values indicate statistical significance (p\u0026thinsp;\u0026le;\u0026thinsp;0.05).\u003c/p\u003e \u003c/p\u003e \u003cp\u003eHistopathological Analysis\u003c/p\u003e \u003cp\u003eIn rats in Group C, microfractures within the trabecular bone were frequently observed on the operated side, where the surgical bone graft model was created, whereas trabecular structures and bone marrow areas on the non-operated contralateral side appeared unaffected (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003ea, b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eHistopathological examination of bone tissues from the low-dose teicoplanin (LD-Tei) group demonstrated the presence of trabecular microfractures, while overall trabecular architecture remained preserved compared with the operated side of Group C. Similarly, in the high-dose teicoplanin (HD-Tei) group, preservation of trabecular morphology was observed (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003ea, b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the low-dose vancomycin (LD-Van) group, trabecular bone morphology appeared preserved, whereas in the high-dose vancomycin (HD-Van) group, trabecular microfractures were more frequently observed, indicating reduced preservation of trabecular architecture (Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003ea, b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn the control groups, collagen fibres in the trabeculae appeared reduced on the operated side, where the surgical bone graft model was created. On the non-operated contralateral side, the density of collagen fibres and bone marrow were observed to be physiological (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003ea, b).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn all treated groups (Group LD-Tei, Group HD-Tei, Group LD-Van, and Group HD-Van), collagen fibre density was preserved in the trabeculae and the bone marrow. No difference in trabecular collagen density was observed among treated groups (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003ea, b, c, d).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e"},{"header":"Discussion","content":"\u003cp\u003eThis study comparatively evaluated the effects of low- and high-dose vancomycin and teicoplanin on fusion-related outcomes and bone morphology in an experimental posterolateral arthrodesis model. To the best of our knowledge, this is among the first in vivo studies to assess and directly compare the impact of topical vancomycin and teicoplanin on spinal fusion outcomes in a rat posterolateral fusion model using autologous iliac crest graft. Although micro-CT\u0026ndash;based fusion scores were lower in the high-dose vancomycin group, this difference did not reach statistical significance. In contrast, fusion mass volume was significantly reduced in the high-dose vancomycin group compared with the control, low-dose teicoplanin, and high-dose teicoplanin groups. On histological assessment, the high-dose vancomycin group demonstrated less organized trabecular architecture with trabecular microfractures. These findings were qualitative and descriptive in nature and, in the absence of quantitative histomorphometric measures, should be interpreted accordingly.\u003c/p\u003e \u003cp\u003eSeveral studies investigated the impact of topical vancomycin use on the rates of intervertebral fusion and surgical site infections in patients who underwent lumbar fusion procedures (\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e, \u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e, \u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e, \u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e, \u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e). These studies have reported that topical vancomycin had a protective effect against surgical site infections and that varying vancomycin doses did not negatively impact intervertebral fusion rates (\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e).\u003c/p\u003e \u003cp\u003eDespite its reported benefits, concern remains that local vancomycin exposure may adversely affect bone healing. Experimental studies have reported vancomycin-related toxicity to osteoblasts, although its cytotoxicity has been described as lower than that of some other antibiotics, including aminoglycosides, quinolones, and gentamicin (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e, \u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e, \u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e). Guimbard-P\u0026eacute;rez et al. (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e) reported that vancomycin powder (200 mg, five times the dose used in humans) mixed with graft material resulted in a 30% reduction in graft fusion rates in rabbits. In a study featuring a rat model of posterolateral fusion with rhBMP-2-induced osteogenesis, Mendoza et al. (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e) did not find a significant difference in fusion scores, rates, and new bone formation between groups administered low-dose (14.3 mg/kg) and high-dose (143 mg/kg) vancomycin. They concluded that the powder form of vancomycin at doses equivalent to those routinely used by surgeons for fusion procedures may not reduce fusion rates (\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). However, the fact that they did not use more commonly used bone-graft materials prevents generalizing their findings to clinical practice. Additionally, there is no consensus on what constitutes a high dose of vancomycin. In some studies, doses ranging from 71 mg/kg to 200 mg/kg, which were stated to be 5 times higher than the vancomycin dose used by spine surgeons, have been defined as high doses (\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e, \u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e).On the other hand, Ishida et al. (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e), as in this study, defined 71.5 mg/kg vancomycin as a high dose, contrary to Mendoza et al.(\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e). They reported that intraoperative local application of vancomycin, particularly at a supraphysiological dosage, led to an impairment in fusion-mass formation, and also detected reduced fusion-mass volume based on micro-CT analysis and less osseous fusion mass based on histological analysis and obtained lower manual palpation scores, in line with our findings that high-dose vancomycin may be associated with impaired fusion-mass formation (\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e). Vancomycin is extensively utilized in clinical practice for infection control; nevertheless, its potential deleterious effects on bone regeneration, particularly when applied locally at high concentrations, remain a subject of ongoing debate. The heterogeneity in dosing protocols, the mode of application\u0026mdash;either as a powder or in solution\u0026mdash;and the diversity of graft materials employed across studies significantly hinder the consistent interpretation of its safety profile in the context of spinal fusion. In the present study, vancomycin in solution form was administered directly to the surgical site at two distinct dosages (71.5 mg/kg as high dose and 14.3 mg/kg as low dose), using a rat posterolateral fusion model with a standardized autograft substitute. \u003cb\u003ePost-hoc analysis using the Dunn test revealed that fusion mass volume was significantly reduced in the high-dose vancomycin group compared to the control group (p\u0026thinsp;=\u0026thinsp;0.043), whereas no statistically significant difference was observed between the high-dose and low-dose vancomycin groups (p\u0026thinsp;=\u0026thinsp;0.318).\u003c/b\u003e In contrast, micro-CT\u0026ndash;based fusion scores did not differ significantly among the groups. Taken together, these findings indicate that higher local vancomycin exposure may be associated with reduced fusion-mass formation in this model, highlighting the importance of careful dose selection when topical antibiotics are used during spinal fusion procedures.\u003c/p\u003e \u003cp\u003eTeicoplanin has been studied less extensively than vancomycin, with prior work focusing mainly on osteomyelitis, implant-associated infections, vascular graft infections, and wound healing (\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e, \u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e, \u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e). For instance,Yasim et al.(\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e) reported that prophylactic intraperitoneal administration of teicoplanin was less effective than antibiotic-soaked grafts in preventing infection in a vascular graft model. In contrast, another study suggested that intraperitoneal teicoplanin administration may support new bone formation following intramedullary tibial fixation in rats (\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e). Similarly, Cai et al. (\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e) demonstrated that teicoplanin-loaded wound dressings offered promising local protection against implant-related infections. Şener et al. (\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e), investigating intramuscular teicoplanin application in autogenous bone grafts, reported favorable outcomes with teicoplanin-loaded bone cement compared with other approaches. Overall, these findings are model-dependent and may be influenced by factors such as teicoplanin dose, local pH, and graft material characteristics.\u003c/p\u003e \u003cp\u003eEvidence regarding the direct effects of topical teicoplanin on bone regeneration remains limited. In the present study, teicoplanin was applied topically at two doses (15 mg/kg and 50 mg/kg). Both teicoplanin groups demonstrated significantly greater fusion mass volumes compared with the high-dose vancomycin group (p\u0026thinsp;=\u0026thinsp;0.006 and p\u0026thinsp;=\u0026thinsp;0.007, respectively). No statistically significant difference was observed between either teicoplanin group and the control group, suggesting that topical teicoplanin did not show a detrimental association with fusion mass formation in this model. Based on the micro-CT findings, teicoplanin may warrant consideration as an alternative topical agent when considering potential effects on fusion alongside infection prophylaxis, although confirmatory studies incorporating infection models and quantitative histologic measures are warranted.\u003c/p\u003e \u003cdiv id=\"Sec13\" class=\"Section2\"\u003e \u003ch2\u003eThe Limitations of the Study\u003c/h2\u003e \u003cp\u003eThis study has several limitations that should be considered when interpreting the findings. First, the lack of a standardized definition for \u0026ldquo;high\u0026rdquo; and \u0026ldquo;low\u0026rdquo; topical doses of vancomycin and teicoplanin complicates extrapolation to clinical practice, where dosing regimens vary. Although doses were selected to reflect common clinical use, their pharmacokinetic and biological relevance in a rat model remains uncertain. Second, this was not an infection model; therefore, conclusions regarding prophylactic efficacy against spinal fusion\u0026ndash;related infections cannot be drawn. Third, histological findings were based on qualitative assessment rather than quantitative histomorphometric analysis; the absence of objective metrics (e.g., trabecular thickness, bone volume fraction, or new bone area) limits reproducibility and interpretive strength. Finally, although the rat model provides a controlled platform to evaluate local antibiotic effects on fusion, it does not replicate the anatomical, biomechanical, and physiological complexity of the human spine. Future studies should incorporate clinically relevant infection models, quantitative bone evaluation methods, and longer-term functional outcomes.\u003c/p\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003ch2\u003eAuthor Contribution\u003c/h2\u003e\u003cp\u003eConceptualization:Deniz Kara, Cemil Burak Demirkiran; methodology: Cemil Burak Demirkiran, Anil Pulatkan, formal analysis and investigation: Bilal Sulak, Mehmet Anil Pulatkan; writing \u0026mdash; original draft preparation ;Arzu Gunes, Ilknur Keskin- histological analysis, Cemil Burak Demirkiran writing \u0026mdash; review and editing:Deniz Kara, Cemil Burak Demirkiran; supervision: Cemil Burak Demirkiran, Nuh Mehmet Elmadag. All authors read and approved the final manuscript.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eElmadag NM, Kara D, Pulatkan A, Ucan V, Cesme DH, Aliyev O et al (2024) Local Prophylactic Teicoplanin Effect on Spinal Fusion Surgery: A Comparative Retrospective Study. J Neurol Surg Cent Eur Neurosurg 85(6):539\u0026ndash;548\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKhan NR, Thompson CJ, DeCuypere M, Angotti JM, Kalobwe E, Muhlbauer MS et al (2014) A meta-analysis of spinal surgical site infection and vancomycin powder. J Neurosurg Spine 21(6):974\u0026ndash;983\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGuimbard-P\u0026eacute;rez JH, Nicol\u0026aacute;s-Ortiz P, Cristiani-Winer M, Orosco-Falcone D, Guti\u0026eacute;rrez N, Pomba M (2020) Aplicaci\u0026oacute;n de vancomicina en polvo sobre el injerto. \u0026iquest;Afecta la fusi\u0026oacute;n de columna en conejos? Acta Ortop\u0026eacute;dica Mexicana 34(5):276\u0026ndash;281\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWei J, Gu H, Tong K (2023) Intra-wound versus systemic vancomycin for preventing surgical site infection induced by methicillin-resistant S. aureus after spinal implant surgery in a rat model. J Orthop Surg Res 18(1):299\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKarau MJ, Zhang C, Mandrekar JN, Kohrs NJ, Puleo DA, van Wijnen AJ et al (2020) Topical vancomycin for treatment of methicillin-resistant Staphylococcus epidermidis infection in a rat spinal implant model. Spine Deform 8(4):553\u0026ndash;559\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eMendoza M, Sonn KA, Kannan A, Bellary SS, Mitchell SM, Park C et al (2015) The Effect of Vancomycin Powder on Bone Healing in a Rat Spinal Arthrodesis Model. Spine J 15(10):S158\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eIshida W, Perdomo-Pantoja A, Elder BD, Locke J, Holmes C, Witham TF et al (2019) Effects of Intraoperative Intrawound Antibiotic Administration on Spinal Fusion: A Comparison of Vancomycin and Tobramycin in a Rat Model. J Bone Joint Surg Am 101(19):1741\u0026ndash;1749\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eXu SJ, Liu XL, Shi JP, Shi JX (2024) The Effect of Topical Vancomycin Powder Application on the Rate of Intervertebral Fusion Following Lumbar Fusion: A Retrospective Study. World Neurosurg 185:e1216\u0026ndash;e23\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eJanssen DMC, Kramer M, Geurts J, Rhijn LV, Walenkamp G, Willems PC (2018) A Retrospective Analysis of Deep Surgical Site Infection Treatment after Instrumented Spinal Fusion with the Use of Supplementary Local Antibiotic Carriers. J Bone Jt Infect 3(2):94\u0026ndash;103\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYashiyama YYTKMBD (2000) Nephrotoxicity of teicoplanin in rats. Japan J Antibiot 53:660\u0026ndash;666\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYucel MO, Turhan Y, Arican M, Karaduman ZO, Saglam S, Tekce Y et al (2022) Rifaximine spacer application is not superior to local teicoplanin treatment in a rat model of osteomyelitis. North Clin Istanb 9(5):505\u0026ndash;513\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGocer H, Onger ME, Kuyubasi N, Cirakli A, Kir MC (2016) The effect of teicoplanin on fracture healing: an experimental study. Eklem Hastalik Cerrahisi 27(1):16\u0026ndash;21\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eRajkovic CJ, Tracz JA, DeMordaunt T, Davidar AD, Perdomo-Pantoja A, Judy BF et al (2024) Synthesis and evaluation of a novel vancomycin-infused, biomimetic bone graft using a rat model of spinal implant-associated infection. N Am Spine Soc J 18:100323\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKang DG, Holekamp TF, Wagner SC, Lehman RA (2015) Jr. Intrasite vancomycin powder for the prevention of surgical site infection in spine surgery: a systematic literature review. spine journal: official J North Am Spine Soc 15(4):762\u0026ndash;770\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYasim A, Gul M, Atahan E, Ciragil P, Aral M, Ergun Y (2006) Efficacy of vancomycin, teicoplanin and fusidic acid as prophylactic agents in prevention of vascular graft infection: an experimental study in rat. Eur J Vasc Endovasc Surg 31(3):274\u0026ndash;279\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eHaimoto S, Schar RT, Nishimura Y, Hara M, Wakabayashi T, Ginsberg HJ (2018) Reduction in surgical site infection with suprafascial intrawound application of vancomycin powder in instrumented posterior spinal fusion: a retrospective case-control study. J Neurosurg Spine 29(2):193\u0026ndash;198\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eSener M, Kazimoglu C, Karapinar H, Gunal I, Afsar I, Karatas Sener AG (2010) Comparison of various surgical methods in the treatment of implant-related infection. Int Orthop 34(3):419\u0026ndash;423\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eYee AJ, Bae HW, Friess D, Robbin M, Johnstone B, Yoo JU (2004) Accuracy and interobserver agreement for determinations of rabbit posterolateral spinal fusion. 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Spine Res Soc 25(4):1021\u0026ndash;1028\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShiels SM, Raut VP, Patterson PB, Barnes BR, Wenke JC (2017) Antibiotic-loaded bone graft for reduction of surgical site infection in spinal fusion. spine journal: official J North Am Spine Soc 17(12):1917\u0026ndash;1925\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eEgawa S, Hirai K, Matsumoto R, Yoshii T, Yuasa M, Okawa A et al (2020) Efficacy of Antibiotic-Loaded Hydroxyapatite/Collagen Composites Is Dependent on Adsorbability for Treating Staphylococcus aureus Osteomyelitis in Rats. J Orthop Res 38(4):843\u0026ndash;851\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eCai XZ, Jin AD, Yan SG (2010) Did local teicoplanin delivery systems inhibit or aggravate implant-related infection? Int Orthop 34(3):453\u0026ndash;454 author reply 5\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Spinal Fusion, Micro-computed tomography, Vancomycin, Teicoplanin, Bone Development","lastPublishedDoi":"10.21203/rs.3.rs-8358911/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8358911/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003ePurpose\u003c/h2\u003e \u003cp\u003eTopical glycopeptide antibiotics are widely used during spinal fusion procedures to reduce the risk of surgical site infection; however, their dose-dependent effects on fusion biology remain unclear. This experimental study aimed to compare the effects of locally applied vancomycin and teicoplanin at different doses on fusion mass formation in a rat posterolateral lumbar arthrodesis model.\u003c/p\u003e\u003ch2\u003eMethods\u003c/h2\u003e \u003cp\u003eForty-five female Wistar rats underwent unilateral L4\u0026ndash;L5 posterolateral fusion and were randomized into five groups (n\u0026thinsp;=\u0026thinsp;9 each): control (no topical antibiotic), low-dose vancomycin (14.3 mg/kg), high-dose vancomycin (71.5 mg/kg), low-dose teicoplanin (15 mg/kg), and high-dose teicoplanin (50 mg/kg). Autologous iliac crest bone graft was combined with the assigned antibiotic solution and placed onto the decorticated fusion bed. Fusion was evaluated at 8 weeks using micro-computed tomography (fusion mass volume), manual palpation (fusion score), and qualitative histological assessment (hematoxylin\u0026ndash;eosin and Masson\u0026rsquo;s trichrome staining).\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eMicro-CT fusion scores did not differ significantly among groups (p\u0026thinsp;=\u0026thinsp;0.106). In contrast, fusion mass volume showed a significant intergroup difference (p\u0026thinsp;=\u0026thinsp;0.018). The high-dose vancomycin group demonstrated the lowest fusion mass volume (median 7.3 mm\u0026sup3; [4.4\u0026ndash;14.9]), which was significantly reduced compared with the control group (13.6 mm\u0026sup3; [10.3\u0026ndash;19.7]) and both teicoplanin groups (median range 13.1\u0026ndash;13.7 mm\u0026sup3;; post hoc p\u0026thinsp;\u0026le;\u0026thinsp;0.007). Manual palpation scores showed a non-significant trend toward lower values in the high-dose vancomycin group (p\u0026thinsp;=\u0026thinsp;0.073). Histologically, fusion masses in the high-dose vancomycin group appeared less organized, whereas both teicoplanin groups demonstrated bone morphology comparable to controls.\u003c/p\u003e\u003ch2\u003eConclusion\u003c/h2\u003e \u003cp\u003eIn this rat posterolateral fusion model, high-dose topical vancomycin was associated with a reduction in fusion mass volume, despite similar fusion scores. In contrast, topical teicoplanin at both low and high doses did not adversely affect fusion mass formation. These findings highlight the importance of dose selection when using topical vancomycin and indicate that teicoplanin may be considered a bone-compatible alternative for local application during spinal fusion.\u003c/p\u003e","manuscriptTitle":"Dose-dependent effects of topical vancomycin and teicoplanin on fusion mass formation in a rat posterolateral lumbar arthrodesis model","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-12 05:40:43","doi":"10.21203/rs.3.rs-8358911/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"3e0115e4-597b-4212-9afc-0419a36d57cb","owner":[],"postedDate":"January 12th, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2026-01-19T09:54:33+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-12 05:40:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8358911","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8358911","identity":"rs-8358911","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}