Vitamin C (Vit C), a water-soluble vitamin, has a six-carbon lactone in structure. Some mammals synthesize Vit C from glucose in their bodies. As there is no L-gulonolactone oxidase enzyme in humans, ascorbic acid synthesis does not occur. Therefore, the necessary amount of Vit C for the human body must be met fully through daily intake⁷. The daily recommended amount varies between men and women, but is approximately 70-90 mg/day and it increases in pregnancy and lactation. Although Vit C is generally known as an antioxidant and oxidative stress scavenger due to its protective effects in cardiovascular diseases, obesity and neurodegenerative diseases, cancers and inflammation processes. It was discovered that the first positive effect of Vit C on the cardiovascular system was on endothelial dysfunction. The results obtained from this discovery have provided other positive effects on the cardiovascular system in association to coronary artery disease and related deaths in patients with low Vit C levels⁸. On the other hand it has been observed that patients with cancers such as gastric cancer generally have lower levels of Vit C than healthy adults. Vit C also decreases oxidative stress on the living tissue⁹. The role of Vit C in inflammation can be summarized as the differentiation and function of immune cells and epithelial barrier cells. Low levels of Vit C have been noted in inflammation, just as in patients with cancer or cardiovascular diseases¹⁰. Moreover Vit C stimulates the synthesis of collagen, which is the most important component of the extracellular bone matrix, and the formation of osteoblasts, which are bone-forming cells. It also scavenges free radicals that are harmful to bone health and reduces oxidative stress. Thus, it is thought to protect bones against osteoporosis by preventing bone resorption¹¹. Vit C, whose positive effects on bone tissue have been determined, has also taken its place in dentistry and dental implant studies, which have become very popular recently. Platelet-rich fibrin with ascorbic acid has been reported to enhance gingival tissue gain and fill radiographic defect, in periodontitis patients¹². Cho et al.¹³ reported that guided bone regeneration (GBR) was used to stimulate bone growth in tissue defects adjacent to dental implants. The GBR adjacent to the implant triggers active bone modeling for the rapid filling of new bone tissue at the defect area in the early post-implantation period. Another effect of Vit C that will increase implant bone connection, is its positive effect on wound healing. Ascorbic acid stimulates wound closure by collagen turnover and by manipulating the state of the inflammation. In a study comparing ascorbic acid and zinc, it was found that Vit C was 4 times stronger than zinc oxide in wound closure¹⁴. Li et al. reported that Vit C supplementation improved postoperative recovery following dental implant surgery in patients with chronic periodontitis and patients treated with GBR¹⁵. This study aimed to evaluate the effect of dietary supplementation of Vit C on the osseointegration after implant insertion surgery with GBR.

Animals and Study Design: The experiments have been carried out in accordance with the Declaration of Helsinki. 30 female Sprague Dawley rats which were aged 3 to 5 months were obtained from the Fırat University Experimental Research Center (Elazig, Turkiye). The mean bodyweight of the rats was measured approximately 280 to 300 gr. The rats were held in cages with 12-hour light and 12-hour dark cycle and a standard temperature 24±2 C. They were fed standard chow and water ad libitum. Vaginal swabs were taken for standardization and all rats were in the same oestrus period.

The number of rats required for the experimental setup was determined by the power analysis; It was calculated that there should be at least 8 rats in each group when the deviation was 8%, type 1 error (α) was 0.05 and type 2 error (β) (Power=0.80). The study was started with 10 rats in each group in case the rats died during the surgical procedures and during the experimental setup.

Control graft group (n=10): As in all groups, the right tibial metaphysis corticocancellous part was reached surgically, in the control group. Bone cavities were opened with proper surgical drills under steril serum perfusion. And titanium implants with a diameter of 2.5 mm diameter and the length of 4 mm were loaded in cavities. The bone graft (Cerabone, Botiss Biomaterials, Zossen Germany) was placed in the bone defect that can be defined as the area correspponding to 2 mm of the implant length in the neck region. No other surgical intervention or drug administration were applied to the control group rats until the end of 8 weeks when the procedure was completed.

Vit C dose 1 graft group (n=10): The same surgical procedure was performed as applied to the control graft group. However, 5 mg/kg of Vit C was administered during the 8 weeks period (three days a week) by oral gavage¹⁵.

Vit C dose 2 graft group (n=10): The same surgical procedure was performed as applied to the control graft group. This time, unlike dose 1 group, 10 mg/kg Vit C was administered. At the end of 8 weeks, all rats were sacrificed and the experiment was terminated. Then the implants and surrounding bone tissue were removed gently and subjected to biomechanical analyse tests that evaluate bone implant connection and bone regeneration¹⁵.

Surgical Procedures: All surgical procedures were performed in environments complying with sterility conditions and under general anesthesia. The rats have fasted for 8 hours before anesthesia. General anesthesia was achieved with intramuscular injections of 40 mg/kg ketamine hydrochloride (Ketasol®, Richter Pharma, Austria) and 5 mg/kg xylazine hydrochloride (Rompun®, Bayer, Germany). Mepivacaine hydrochloride (0.3 mL/kg, scandicaine epinephrine 1:100,000 to 2%; Septodont, France) infiltration was used to reduce hemostasis at the wound site. The surgical area to be operated was shaved. then it was stained with povidone iodide for sterilization. After making an incision of approximately 1.5 cm over the tibial crest with a No. 15 scalpel, a periosteal elevator was used to reach the proximal tibia. Cavities were opened in the right tibial metaphyseal corticocancellous bones of the rats (Figure 1). Half of the length of the cavity was opened with a diameter of 2.5 mm, while the upper half was opened with a diameter of 4.5 mm. Titanium implants with a diameter of 2.5 mm and a length of 4 mm (Implance Dental Implant System, AGS Medical Corporation, Istanbul, Turkiye) were placed in these cavities. And a bone graft was placed into the bone defect corresponding to 2 mm of the implant length in the neck region. The surgical procedure was completed by closing the flaps using absorbable sutures (4/0 vicryl; Ethicon, Inc., Somerville, NJ, USA) for the soft tissues and monofilament sutures for the skin (nylon 4.0; Ethicon, Inc.) ^2,3. In postoperative chemotherapy, antibiotics (50 mg/kg penicillin) and analgesics (0.1 mg/kg tramadol hydrochloride) were administered intramuscularly every 24 hours for three days. No additional application was made during the eight-week experiment period. During the postoperative period, the rats were followed daily for possible complications such as pain, separation, infection, restricted movement and weight loss. All rats were decapitated after an eight-week recovery period and the experiment was terminated. Implants were taken for biomechanical analysis together with the surrounding bone tissues.

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Figure 1: Surgical integration of the 2.5 mm diameter and 4 mm length titanium implants.

Biomechanical Analysis: The experimental groups were started with ten rats to be prepared for the possibility of rat loss during the surgical applications and experimental period. Two rats from the control group were excluded from the study because their implants did not fit properly. The implant parts taken together with the surrounding bone tissues were prepared to be tested as blocks and they were kept in 10% buffered formalin solution. All implants were placed in polymethylmethacrylate blocks immediately to prevent dehydration. A rotating apparatus was placed to measure the torque of the implants. An extraction force was applied manually, slowly and incrementally counterclockwise using a digital torque instrument (Mark 10, NY, USA) (Figure 2)³. As soon as the dental implant returned to the bone socket, the procedure was terminated immediately. The highest torque force (Ncm) measured during the first rotation of the implant in the socket was automatically recorded.

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Figure 2: Biomechanical (Mark 10, NY, USA); reverse torque analysis, of the implants.

Statistical Analysis: Statistical analysis was performed using IBM SPSS Statistics (Version 22.0). The appropriateness of the normal distribution of the parameters was assessed by the Kolmogorov–Smirnov test. A one-way analysis of variance (one-way ANOVA) was performed for the parameters, showing a normal distribution. Tukey’s Honestly Significant Difference test was used to detect the group causing the difference. Continuous data were presented as mean ± standard deviation values, and numerical measurements were presented as median, minimum, and maximum values. A statistical significance level of 95% (P<0.05) was adopted.

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Table 1: Biomechanical reverse torque analysis in of the groups after guided bone regeneration

The experience of the physician and the quality of the implant are often not sufficient for perfect osseointegration. Nastri et al.¹⁹ stated that for perfect osseointegration, the immunological status of the host and its nutritional content and balance should be taken into account. It is noteworthy that the effects of daily nutrients, including vitamins and minerals, may be more than expected, and more studies are being conducted in this area. Because of its direct effects on bone metabolism, vitamin D has been the most researched vitamin in studies. It is known that vitamin D deficiency slows down implant osseointegration and increases the risk of graft infection²⁰. Liu et al. reported that vitamin D supplementation has increased the fixation of titanium implants in the case of chronic renal failure, which has negative effects on bone metabolism ²¹. In another experimental study, the effect of vitamin supplementation was measured by using immediate implants instead of extracted premolar teeth of dogs. It was remarkable that vitamin D supplementation in these dogs without vitamin D deficiencies reduced crestal bone loss and increased osseointegration²². In one of the above-mentioned studies, Vit C supplementation in addition to vitamin D, increased the success of dental implant surgery in patients with vitamin D deficiency¹⁹. However, although the literature evaluating the effects of Vit C on bone tissue has been reached, there are very few studies that have observed its effects on dental implants and osseointegration. A study of Vit C on the gene level showed that Vit C improves trabecular bone formation by influencing the bone matrix gene expression of osteoblasts ²³. In a study comparing the data of the fifth lumbar vertebra and femur in ovarectomized rats, it was found that Vit C increased bone density ²⁴. Malmir et al. conducted a meta-analysis on clinical researches ¹¹. They stated that higher dietary Vit C intake resulted in a lower risk of hip fracture and osteoporosis, as well as a higher bone mineral density in the femoral neck and lumbar spine. Vit C contributes to the normal function of bones and teeth by enhancing osteoblastogenesis and inhibiting osteoclastogenesis through Wnt/β-catenin signaling. In this study, it has been shown for the first time that Vit C improves bone regeneration and teeth health²⁵. Thereafter, the European Food Safety Authority's opinion of Vit C was shaped by its contribution to the normal function of bones and teeth. All these findings and opinions of the authorities support our results that a positive effect was observed at the 5 mg/kg dose of Vit C. There is no definite indication for the use of Vit C in wound healing. However, supplementation with Vit C for 10 days after extraction of premolar teeth has been shown to improve wound healing²⁶. In a cell culture study by Li et al., it was stated that gelatins doubly modified with Vit C and β-glycerophosphate salt can be scaffolds for versatile bone tissue engineering²⁷. With its effectiveness in both bone tissue and non-bone tissue, Vit C seems to be essential for osseointegration following dental implant surgery to be at the desired level. These effects were confirmed by a randomized controlled trial, in which 128 patients with chronic periodontitis were treated with Vit C after dental implant surgery with bone grafts or GBR like in our study. It was observed that the wound healing of the patients who took vitamin C supplements for 14 days after the surgery were better than the patients who did not. Another conclusion of the same study is that vitamin C supplementation provides a more significant improvement especially in patients with chronic periodontitis who underwent implant surgery¹⁵. The most recent systematic review that was published in 2022 stated that Vit C accelerated bone healing without causing any side effects²⁸. This review also supports the more remarkable result of our study, that with an application dose above the plateau (10 mg), the osseointegration values remained almost the same as those of the control group without causing any advers effects in the reverse torque.

Reverse torque analysis is used in in vivo and in vitro research to assess osseointegration. The reverse torque analysis method cannot be used clinically. Reverse torqueing of the implant in the bone tissue into which it is placed provides a biomechanical measurement of the force required to break the bone-implant connection. It is an objective method for evaluating bone-implant attachment. Reverse torque analysis is a method that allows the evaluation of the interaction of all bone tissue around the titanium implant with the implant surface. A very thin section is required for histological analysis. In our study, reverse torque test was used to evaluate the effect of different postoperative vitamin C doses on osseointegration according to the literature. Although numerical differences were detected between the groups, no statistical difference was detected²⁹.

Research on the role of Vit C supplementation in patients with dental implants continues with interest. It is not difficult to predict that Vit C, which is essential for the maturation of collagen and the normal function of bone tissue, is a virgin area in dentistry studies and will be examined as much as vitamin D in the future. Based on the limited results of this study, the difference in ossointegration levels in the experimental groups at the numerical level, although not statistically, is promising in terms of the Vit C and bone tissue regeneration relationship. Further studies are needed to examine the relationship between osseointegration and Vit C.

Acknowledgement
The authors wish to thanks Implance Dental Implant System, AGS Medical Corporation, Istanbul, Turkiye for providing the titanium implants.

Competing interests: The authors declerate there is no competing interests. The authors do not have any financial interests, either directly or indirectly, in the products or information listed in the paper.

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