Revista Cienfica, FCV-LUZ / Vol. XXXV Recibido:11/04/2025 Aceptado:09/07/2025 Publicado: 03/08/2025 hps://doi.org/10.52973/rcfcv-e35650 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico 1 of 6 Revista Cienfica, FCV-LUZ / Vol. XXXV hps://doi.org/10.52973/rcfcv-e35675 UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Biomechanical invesgaon of the effect of Vitamin C supplementaon on Osseointegraon of tanium implants in rat (Raus norvegicus) bia Invesgación biomecánica del efecto de la suplementación con vitamina C sobre la osteointegración de implantes de tanio en bias de rata (Raus norvegicus) ³Firat University, Faculty of Denstry, Department of Peridontology, Elazig, Turkiye. ⁴Firat University, Instute of Sciences, Department of Stascs, Doctorate Student ⁵Firat University, Faculty of Medicine, Department of Esthec, Plasc and Reconstrucve Surgery, Elazig, Turkiye. ⁶Turkish Jockey Club Elazig Racecourse Horse Hospital, Elazig, Turkiye. *Correspondence Author: mtanrisever@firat.edu.tr ABSTRACT In this study, it was aimed to evaluate some possible effects of vitamin C supplementaon on the osseointegraon process implants placed in rat bias. Thirty 3-month-old female Spraque Dawley rats (Raus norvegicus) were divided into 3 groups. In all groups, the corcocancellous part of the the metaphysis of the right bia bones were surgically reached. Monocorcal implant sockets were created by surgical methods under serum cooling, and then tanium implants with 2.5 mm diameter and 4 mm long tanium were integrated on these bone sockets. Group Sham (Control) (n=10): No addional applicaon was made during the experimental process. Group C1 (n=10): 5 mg/kg vitamin C was given to the rats with oral gavage applicaon in three days a week for four weeks of experimental process. Group C2 (n=10): 10 mg/kg vitamin C was given to the rats with oral gavage applicaon in three days a week for four weeks of experimental process. At the end of the four- week experimental setup, all rats were sacrificed and the experimental process was completed. The implants integrated in the bias of all rats were collected with the surrounding bone ssue aſter removing the soſt ssues for biomechanical analysis. Biomechanical reverse torque analysis was applied to all implants. Stascal analysis of the obtained data; Kruskall- Wallis test, no stascally significant difference was detected between the control and experimental groups (P>0,05). In conclusion, 5 and 10mg/kg vitamin C supplementaon has no posive or negave effects on osseointegraon. This situaiton can be explained in two ways; the first, the dosages of vitamin C used in this study is not sufficient to create an effect. Second, the endogenous synthesis of this vitamin is enough even though under these stress condions. Key words: Vitamin C; osseointegraon; rat bias RESUMEN Este estudio, tuvo como objevo evaluar algunos posibles efectos de la suplementación con vitamina C en el proceso de osteointegración en implantes de ratas. Treinta ratas Sprague Dawley, hembras, de 3 meses de edad, se dividieron aleatoriamente en tres grupos. Todos los ejemplares, fueron abordados quirúrgicamente en la porción corcoesponjosa de la metáfisis bial derecha. Se perforaron las corcales óseas y se integraron implantes de tanio de 2,5 mm de diámetro y 4 mm de longitud. Grupo control (n=10): no se realizó ninguna aplicación adicional. Grupo C1 (n=10): Se administraron 5 mg/ kg de vitamina C por sonda oral tres días a la semana durante cuatro semanas. Grupo C2 (n=10): recibiò 10 mg/kg de vitamina C por sonda oral tres días a la semana durante cuatro semanas. Al final de las cuatro semanas, se sacrificaron todas las rata, dando por finalizado el experimento. Los implantes integrados en las bias de todas las ratas se recogieron junto con el tejido óseo circundante tras la extracción de los tejidos blandos para su análisis biomecánico. Se aplicó un análisis biomecánico de torque inverso a todos los implantes. El análisis estadísco de los datos obtenidos se realizó mediante la prueba de Kruskall-Wallis. No se detectaron diferencias estadíscamente significavas entre los grupos control y experimental (p > 0,05).En conclusión, la suplementación con 5 y 10 mg/kg de vitamina C no ene efectos posivos ni negavos sobre la osteointegración. Esto se puede explicar de dos maneras: primero, las dosis de vitamina C ulizadas en este estudio no son suficientes para lograr un efecto; segundo, la síntesis endógena de esta vitamina es suficiente incluso en estas condiciones de estrés. Palabras clave: Vitamina C; osteointegración; bias de rata ¹Dicle University, Diyarbakir Agricultural Vocational School, Dairy and Breeding Program, Turkiye. ²Firat University, Faculty of Veterinary Medicine, Department of Surgery, Elazig, Turkiye. Can Ayhan Kaya 1 , Murat Tanrisever 2 * , Nurullah Duger 3 , Tuba Talo Yildirim 3 , Erhan Cahit Ozcan 5 , Umit Koray Can 6 , Turker Gelic 2 , Alihan Bozoglan 3 , Serkan Dundar 3 , 4

Vitamin C supplementaon on osseointegraon / Ayhan et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico INTRODUCTION Vitamin C, also known as ascorbic acid, is a glucose derivave. In addion to dissolving in water, it shows weak acid properes, it also has a white and crystalline structure. Vitamin C shows a stable structure against heat in acidic soluons, while alkaline can be separated under condions. When heated, the destrucon is increased with daylight [1]. Vitamin C is synthesized from glucose via the uronic pathway in the liver of most mammals (eg., pigs, dogs, cats, ruminants) and both in the liver and kidneys of poultry [2 , 3 ,[4]. However in primates, guiana pigs, some species of fish, birds, insects and invertebrates vitamin C cannot be syntesized from glucose due to the lack of the enzyme L-gulonolactone oxidase [5]. When vitamin C is examined chemically, it is seen that it has a strong reducing potenal with the ability to stabilize unpaired electrons. By this way vitamin C reduces compounds such as O 2 , NO 3 , cytochromes A and C. Helps some enzymes metal cofactor to maintain in their reduced state such as Cu + in monooxygenase, Fe 2+ in dioxygenase. Synthesis of hydroxyproline and hydroxylysine in collagen requires vitamin C. This vitamin acts also in oxidaon of tyrosine, synthesis of catecholamins from tyrosine, synthesis of bile acid and carnine. When the metabolic effects of vitamin C are examined, steroidogenesis in the adrenal cortex requires this vitamin. Vitamin C significantly increases iron absorpon by prevenng nitrosamine formaon during digeson. It has also been reported that vitamin C supports endothelial NO synthesis, which is necessary for the regulaon of cardiovascular funcon. Vitamin C provides its funcon in the synthesis of NO by increasing intracellular tetrahydrobiopterin (BH4) concentraons [6]. Therefore it scavenges free radicals, protect biomolecules from oxidaon and maintain metal ions in their reduced forms [1]. The most well-known symptom of vitamin C deficiency is scurvy. Scurvy is directly related to defecve collagen synthesis in the connecve ssue. Condions such as bleeding under the skin, soſt and swollen gums, excessive bleeding in the gums, loose teeth, cracked capillaries, impaired wound healing, muscle weakness, fague, depression, growth retardaon, decreased appete and low resistance to infecous diseases can be seen in both humans and animals due to vitamin C deficiency [7]. Although some organisms can syntesize and the others get vitamin C alimenterally, in some certain cases like stress of surgery, the demand for this vitamin may be greater than endogenous or exogenous sources. In a study on rats it is reported that; ACTH injecon caused a 30% decrease in adrenal ascorbic acid level within 20 minutes (min) and this decrease reached the maximum level of 60% within one hour (h) [8]. In the case of bone fractures which are also a big stress factors for the organism; the healing process begins with hypertrophy in chondrocytes and calcificaon in the matrix of carlages. A significant increase in alkaline phosphatase levels occurs with the mineralizaon mechanism. Due to this increase, there is a decrease in the major carlage protein collagen type II and an increase in type X. It is known that this mechanism in collagens works due to the presence of ascorbate. An in vitro study on chondrocytes indicates that, ascorbic acid smulated the alkaline phosphatase acvity [9]. In a study on rats (Raus norvegicus), Xu et al. [10], indicated that by the use of zymosan which increases the oxygen free radicals, the fracture healing process is impaired. It has been reported in the literature that vitamin C supplementaon increases bone healing in rats [11]. However Giordano et al. [12], claimed vitamin C has no posive effect on bone healing in fracture model in rats. The healing process of implant surgery and osseointegraon is the same with the same of fracture healing process. Bone implant fusion; osseointegraon is defined as the direct and ght bonding of an alloplasc material with the bone without any connecve ssue between it and the bone. Osseointegraon is extremely important for the implant material to funcon clinically and to remain in the mouth for a long me. Therefore we evaluated the osseointegraon process similar to fracture healing process. In osseointegraon, where direct contact between the implant and the bone is ensured, any connecve ssue infiltraon between the bone and the implant is not desired [11]. This study aimed to evaluate some possible effects of vitamin C supplementaon in rats, on the osseointegraon process of implants. MATERIAL AND METHODS Animals and study design Animal Ethics Commiee confirmed the design of the study with the approval number of (30.11.2020-426281). All experimental procedures were implemented in accordance with the Declaraon of Helsinki. 30 female Sprague Dawley rats, 3 months old and weighing 280-300 g (WL, Shimadzu, Japan), obtained from the Firat University Experimental Research Center were used in the study. In order to ensure standardizaon of the work, vaginal smears were performed and rats in the same estrus period were included in the study. The rats included in the study were kept in plasc cages in rooms with a standard temperature of 24±2°C, with a 12-hour light and 12-hour dark cycle. All of them were fed with a standard diet ad libitum and free acces to water. The nutrional values of the diet is shown in TABLE I. TABLE I The nutrional values of the diet (%). Methionine 0,43 Phosphorus 0,74 Crude Protein 24 Sodium 0,04 Crude fat 3,15 Mineral mix 0,1 Crude sellulose 4,96 Crude ash 4,91 The study groups were designed as; Group Sham (n=10): Aſter the reached the bial bones with the surgical applicaons the bone cavies were drilled and tanium implants with the 2,5 mm diameter and 4 mm length were integrated. Group C1 (n=10): 5 mg/kg of vitamin C was administered three days a week during the 4 weeks period by oral gavage was added to the same surgical procedure [11]. 2 of 6

Revista Cienfica, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico Group C2 (n=10) : 10 mg/kg vitamin C was administered three days a week during the 4 weeks period by oral gavage was added to the same surgical procedure [11]. At the end of the experimental setup; four weeks, all rats were euthanized and the experimental phase was terminated. The implants were gently removed from the soſt ssues along with the surrounding bone ssue. All samples were subjected to biomechanical reverse torque analysis in order to evaluate the osseointegraon levels. Surgical procedures All surgical procedures were performed under general anesthesia. Rats (Raus norvegicus) were fasted for 8 h before anesthesia. In order to provide anesthesia, 40 mg/kg Ketamine hydrochloride (Ketasol®, Richter Pharma, Austria) and 5 mg/ kg Xylazine hydrochloride (Rompun®, Bayer, Germany) were administered intramuscularly using appropriate syringes [11]. Mepivacaine hydrochloride (0,3 ml/kg, scandicaine epinephrine 1:100,000 to 2%; Septodont, France) was infiltrated into the surgical area to reduce bleeding [11]. The surgical area was shaved and washed with povidone iodine to aid sterilizaon. An approximately 1,5 cm long incision was made by using a number 15 scalpel, taking bone contact from the bial crest. Aſter the incision, the soſt ssues were dissected and the proximal part of the bial bone was reached using a periosteal elevator (FIG. 1) [11]. FIGURE 1. Skin incision made to reach the bia bone where the implant will be placed Implant cavies were formed in the right bial metaphyseal corcocancellous parts (FIG. 2) by a drill with a diameter of 2,5 mm and a length of 4 mm. FIGURE 2. The bone socket prepared in the bia corco cancellous region where the implant will be applied While this applicaon, first point drills and then 1,8 mm and 2,5 mm drills were used, respecvely. Titanium implants (Implance Dental Implant System, AGS Medical Corporaon, Istanbul, Turkiye) with a resorbable blast material (RBM) surface and a diameter of 2,5 mm and a length of 4 mm were placed in the bone sockets created by surgical methods (FIG. 3). FIGURE 3. Placing the implant into the prepared bone sockets The surgical procedures were ended by suturing the flaps to their original posions. To avoiding postoperave pain and infecon, anbiocs (50 mg/kg Penicillin) and analgesics (0,1 mg/ kg Tramadol hydrochloride) were administrated intramuscularly to all rats once a day for three days [11]. No addional procedures were performed during the four-week experimental setup. During the study period, all groups were monitored daily to avoiding the possible complicaons such as pain, separaon of wound edges, infecon, immobility and weight loss. At the end of the experiment, all rats were sacrificed aſter a four-week recovery period. The implants and surrounding bia bone were taken for biomechanical analysis of the samples (FIG. 4). 3 of 6

Vitamin C supplementaon on osseointegraon / Ayhan et al. UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico FIGURE 4. Removal of the implant along with the surrounding bone ssues The samples were stored in 10% buffered formalin soluon to preotect from dehydraon unl the biomechanical reverse torque analysis. Biomechanical analysis All samples were subjected to biomechanical analysis aſter being embedded in polymethyl methacrylate resin. In order to measure the torque of the implants, a ratchet and a hexagonal key were placed in the groove inside the implant. The ratchet was aached to the hexagonal key and manually applied slowly and gradually counterclockwise in a way that would be opposite to the direcon of placement of the implant in the socket. (Mark 10, NY, USA) (FIG. 5). When applying reverse torque analysis, the highest torque force (Ncm) measured during the first rotaon of the implant in the socket within the bone was automacally recorded. The data obtained for each implant was recorded as the biomechanical osseointegraon value of the implant. Stascal analysis For the stascal analysis, SPPS 22.00 soſtware was used. Shapiro Wilks and Kolmogorov-Smirnov tests was first applied to evaluate if the data was distributed normal or not. Aſter determining that the data show not normal distribuon, Kruskall Wallis test was applied. The Mann Whitney U test was applied in pairwise comparisons. Data is given as mean minimum- maximum. P-value < 0,05 was considered sufficient to indicate stascal significance. RESULTS AND DISCUSSION Five rats (two rats from sham and C2 groups, one rat from the C1 group) were excluded from the study because their implants did not fit properly and were not osteointegrated. The biomechanic bone implant connecon (BIC) data of the groups are shown in TABLE II. FIGURE 5. Performing the torque analysis with a digital torqmeter (Mark 10, NY, USA). TABLE II Biomechanic bone implant connecon (BIC) (Newton/cm) levels of the groups Groups N Mean Minimum Maximum P* Sham (Control) 8 5,74 3,7 7 0,12 C vit dosage 1 9 4,27 2,5 6 C vit dosage 2 8 4,93 2,8 9 * Kruskall Wallis test. Differences were not stascally significant (P>0,05). The Shapiro-Wilk and Kolmogorov-Smirnov tests were used to evaluate whether the data showed normal distribuon. Since it was understood that the data did not show normal distribuon, nonparametric tests were used in the analyses, and the data were presented with mean and minimum-maximum values. The mean BIC raos of groups sham (control animals), C1 and C2 are determined as 5,74N, 4,27N and 4,93N, respecvely. Although there is numarical difference between the groups, it is not found stascally significant (P>0.05). From these results, it was observed that the doses of vitamin C used in this study did not have any effect on the osseointegraon levels of the implants. In this experimental study, we invesgated the effects of vitamin C supplementaon with two different doses on implant 4 of 6

Revista Cienfica, FCV-LUZ / Vol. XXXV UNIVERSIDAD DEL ZULIA Serbiluz Sistema de Servicios Bibliotecarios y de Información Biblioteca Digital Repositorio Académico osteointegraon in rat bia by biomechanical method. Although some numerical variaons were found among the groups, the differences were not stascally significant which agrees with the results of Bozoglan et al and Giardiano et al [11 , 12]. Long et al. [13], claims that plasma ascorbic acid levels decreases to extremely low levels in the case of trauma or infecons due to increased catabolism and turnover rate. In the light of these data we supplemented our subjects with higher doses than the turnover amount. Researchers [9 , 14 , 15 , 16] worked on osteoblast cell cultures and established that ascorbic acid treatment induced matrix mineralisaon and collagen synthesis. Although we did not study on molecular detecons, the possibility of not observing any posive effect like them may be due to circadian rhtym difference between in vitro and in vivo studies. Yilmaz et al. [17], supplemented bone fractured rats with 0,5 mg/kg vitamin C. However, although they observed beneficial effects of vitamin C supplementaon, the difference may be due to the difference between the stascal evaluaon in this study and their light microscopic observaon results. The other possibility of the difference may be the way of supplementaon. In this study, subjects were given c vitamin supplement via oral gavage. Also farmacokinecs differences may occur between intraperitoneal and oral supplementaon. Similar to this study, in the study by Bozoglan et al., which was performed with oral gavage, it was reported that bone healing and osseointegraon values did not create a stascal difference [11]. Deyhim et al. [18], study on rats which developed osteoporosis aſter ovariectomy, oral vitamin C was administered 22 mg. While the researchers found an increase in bone density in the femoral bones of the rats aſter the experimental setup, they also found an increase in both bone density and bone mineral content in the lumbar bones. They reported that oral vitamin C supplementaon can treat the negave effects of osteoporosis and increase the anoxidant capacity, which has an important place in the osseointegraon mechanism. The possive effect differences than our study can be explained by the supplementaon amount. In another study that we can evaluate in terms of osseointegraon and bone metabolism Zhu et al. [19], reported that vitamin C intake following ovariectomy in rats prevented low- cycle bone loss. The researchers stated that the posive effects on bone mineral density and micro-computed tomography parameters obtained from the subjects were due to the smulaon of bone formaon with vitamin C supplementaon. They claimed that bone formaon parameters were obtained 8 weeks aſter ovariectomy, similar to controls, and that vitamin C could be a skeletal anabolic agent. The different results from our study may be explained by the rats used in this study were ovariectomised and their possible metabolic alternaons. Sarısözen et al. [20], determined stascaly significant increase (P<0,05) in fracture healing process of rats. The difference from this study may be due to the their high supplementaon of vitamin C (200 mg/kg) than ours (5 and 10mg/kg) and analysis method. In addion, Dethlefs-Canto et al. [21], reported in their review that vitamin C applicaon aſter tooth extracon reduces postoperave pain, inflammaon, probing pocket depth at the extracon site, and mesiodistal length of the socket, and has the potenal to accelerate alveolar bone healing aſter tooth extracon. Abdulhameed and his colleagues reported in an in vitro study that vitamin C may have a posive effect on ossificaon by increasing mineralizaon in cells [22]. CONCLUSION In the light of all these data, our study results indicate that; 5 and 10mg/kg vitamin C supplementaon has no posive or negave effects on osseointegraon. We can explain this in two ways; the first, the dosages of vitamin C used in this study is not sufficient to create an effect. Second, the endogenous synthesis of this vitamin is enough even though under these stress condions. Further studies need to be performed to determine these two aspects. Conflicts of Interest The authors declare that there are no known conflicts of interest. Acknowledgement This study was supported by Firat University Scienfic Project Foundaon Department with the project number DHF.21.02. The authors wish to thanks Implance Dental Implant System, AGS Medical Corporaon, Istanbul, Turkiye for providing the tanium implants. BIBLIOGRAPHIC REFERENCES [1] Barrios-Garay K, Toledano-Serrabona J, Gay-Escoda C, Sánchez-Garcés MÁ. Clinical effect of vitamin C supplementaon on bone healing: A systemac review. Med. Oral Patol. Oral Cir. Bucal. [Internet]. 2022; 27(3):e205-e215. doi:hps://doi.org/pxjz [2] Olsen E, Wagenhoffer Z, Hetényi N. Nutrion, health and welfare of Norwegian pet guinea pigs. Acta Vet. Hung. [Internet]. 2025; 73(2):73-80. doi:hps://doi.org/pxj2 [3] Akinmoladun OF. Stress amelioraon potenal of vitamin C in ruminants: a review. Trop. Anim. Health Prod. 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