Effect of lycopene on titanium implant osseointegration in ovariectomized rats

Implant-based treatment has been widely used in the fields of orthopaedics and dentistry. Regarding patient characteristics, aside from smoking and periodontitis as negative risk factors [1], the successful fixation and stability of dental implants depend on the implant site (mandible vs. maxillary) [2, 3] and bone quality and quantity around the implant [4]. Osteoporosis is a systemic skeletal metabolic disease characterized by low bone mass and micro-architectural bone deterioration, with a high risk of fragility fracture [5]. Although osteoporosis is not considered as a risk factor for dental implant failure [6, 7], initial implant stability can be influenced by both local and skeletal bone densities, and osteoporotic patients need more healing time [8, 9]. Proper implant osseointegration, i.e. direct contact between the implant and surrounding bone, is desired for mechanical fixation of the implant [10]. In a rat model, the quantity and quality of osseointegration remained significantly impaired under osteopenic conditions12 weeks after implantation, leading to decreased implant fixation [11]. Various therapeutic agents have been investigated and verified to be effective in promoting implant osseointegration in osteopenic bone [12]. Although adverse events are rare among approved osteoporosis treatment agents, in some cases, bisphosphonates and denosumab could cause osteonecrosis of the jaw, raloxifene may increase the risks of thrombus and stroke and teriparatide may cause nausea and other side effects [13]. Thus, new safe, cost-effective drugs that can accelerate implant osseointegration under osteoporotic conditions are still in demand.

The bone remodelling process is similar between osseointegration and fracture healing [10]. Some researchers have proposed that oxidative stress and antioxidants might play a role in fracture healing [14, 15]. Immediately after fracture, oxidative stress significantly increases due to severe bone loss and ischaemia [14]. Reactive oxygen species (ROS) can cause cell DNA damage, accumulate in the injury site and have detrimental effects on bone healing [15]. During normal healing, the antioxidants in the body increase correspondingly to scavenge excessive ROS [14]. However, in cases when the antioxidant system is compromised, elevated ROS activity leads to oxidative stress, which inhibits osteogenesis and can reduce bone regenerative capacity [15]. Thus, an imbalance between excessive ROS generation and an insufficient antioxidant defence mechanism makes these fractures difficult to heal. Bednarek-Tupikowksa et al. demonstrated higher oxidative biomarkers and reduce d antioxidative potency in postmenopausal women than in premenopausal women [16]; these findings are associated with compromised bone healing capacity due to osteoporosis. However, antioxidant treatment promoted bone healing in rats with severe bone loss [17]. Furthermore, an antioxidant-based diet prevented bone loss in menopausal women [18], and a case-control study showed that the antioxidant intake score was inversely associated with hip fracture risk in ever-smokers but not in never-smokers [19]. Therefore, antioxidant therapy might be useful in promoting osseointegration under osteoporotic conditions.

Lycopene, a carotenoid found in red fruits and vegetables, is one of the most potent natural antioxidants and has the highest scavenging capacity for singlet oxygen molecules among all carotenoids [20]. Lycopene has been used for decades to prevent chronic diseases [21]. Lycopene exerts its antioxidant effect mainly through activating the antioxidant response element/electrophile response element (ARE) transcription system by disrupting the cytosolic interactions between the major ARE-activating transcription factor, nuclear factor erythroid 2-related factor 2 (Nrf2) and its inhibitor, Kelch-like ECH-associated protein 1 (Keap1) [22]. Recently, Mackinnon et al. showed that a lycopene-restricted diet significantly decreased circulating lycopene and decreased biomarkers of oxidative stress and bone resorption in healthy postmenopausal women [23]. In addition, in a pilot study using a small group of healthy postmenopausal women, the same group showed that lycopene supplementation significantly decreased the detection of oxidative stress markers and a bone resorption marker [24]. These findings suggest that lycopene has a positive effect on bone metabolism by reducing the rate of bone resorption. Interestingly, a prospective cohort study of 946 men and women reported that lycopene intake is inversely associated with the risk of osteoporotic hip fracture [25]. Moreover, lycopene supplementation prevents bone loss and maintains bone strength in ovariectomized (OVX) rats [26]. However, the role of lycopene in implant osseointegration under osteopenic conditions remains unknown.

OVX rats have been widely used as a model of postmenopausal osteoporosis in humans [27]. OVX rats have also been used in many studies on implant osseointegration under osteopenic conditions [12]. Implant osseointegration could be achieved in OVX rats, but according to histomorphometric and functional analysis, less osseointegration occurred in OVX rats than in sham rats within 2–12 weeks [11, 28, 29]. These results indicated that a longer healing time was needed due to compromised healing capacity under osteopenic conditions, which is similar to the implant healing process of humans.

We hypothesized that lycopene prevents delayed implant osseointegration under osteopenic conditions. In our study, we used OVX rats as an osteopenic model and analysed the effect of lycopene on titanium-based implant osseointegration.

Osteoporosis, a common disease in elderly people, is characterized by low BMD and loss of structural and biomechanical properties [33]. The compromised bone healing capacity under osteopenic conditions shows adverse effects on implant fixation and osseointegration 2–12 weeks after implantation [11, 28, 29] and affects the initial stability and healing time of the implant in osteoporotic patients [8, 9]. Implant fixation can be mostly explained by the degree of osseointegration [34]. In this study, we histomorphometrically and functionally investigated the effects of lycopene on implant osseointegration in osteopenic rats. As expected, the sham group had more mechanical implant fixation, BC and bone mass than did the OVX group, indicating the destructive effect of osteopenia on implant osseointegration. Furthermore, as expected, lycopene maintained mechanical implant fixation, BC and bone mass around the implant in the OVX + lycopene group to almost the same level as that in the sham group. These results indicate that lycopene could prevent bone loss and maintain osseointegration in OVX rats 12 weeks after implantation. These findings suggest that lycopene might be a new therapeutic agent for preventing delayed osseointegration under osteopenic conditions.

The OVX rat model is most commonly used to simulate postmenopausal osteoporosis [35]. In our study, compared with sham rats, OVX rats showed significantly decreased BMD and compromised bone tissue micro-structure surrounding the implant. These results indicate that OVX can induce osteopenia in rats, in accordance with published data on the effects of OVX on bone mass [11]. In addition, osseointegration, implant fixation and micro-structure of the bone surrounding the implant were significantly lower in OVX rats than in sham rats. These results were in accordance with those of other studies [11, 32, 36, 37], indicating the adverse impact of OVX on osseointegration in rats.

Lycopene is a natural potent antioxidant that is considered a human nutritional supplement by the United States Food and Drug Administration (US FDA) [38]. The recommended dosages for humans are 5–7 mg/day to prevent chronic diseases and 35–75 mg/day to treat cancer and cardiovascular diseases [21]. The largest dosage reported for rats was 3000 mg/kg/day for 13 weeks without any adverse effects observed [39]. The only side effects in cases of excessive intake of lycopene were alterations in the skin and liver colouration, which were non-toxic and reversible [40]. Ardawi et al. [26] demonstrated the dose-dependent effect of lycopene on bone loss prevention in OVX rats. In their study, the highest dosage of the OVX + lycopene group, which was 45 mg/kg/day, showed the best effect on bone mass in OVX rats. In our study, 50 mg/kg/day lycopene was given to OVX rats by gavage for 12 weeks, and no adverse effects were observed. Therefore, lycopene is a relatively safe medication.

A number of studies have demonstrated the beneficial effects of lycopene on bone metabolism [23, 24, 26, 41‐43]. In vitro studies have shown that lycopene promotes proliferation and differentiation of osteoblasts [26, 44] and suppresses osteoclastogenesis and differentiation of osteoclasts in normal and OVX rats [26, 45]. Lycopene treatment can increase the expression of bone formation biomarkers in rats [42] and can decrease the expression of bone resorption biomarkers in osteoporotic patients [24, 41]. Taken together, these findings indicate that lycopene has both anabolic and anti-catabolic effects on bone metabolism. In our study, histomorphometric and micro-CT analyses showed significantly increased BA, BMD and the micro-structure of the bone surrounding the implants in OVX rats, demonstrating the treatment effects of lycopene on local osteopenic bone. Our findings were consistent with the results of Ardawi et al. [26] and Iimura et al. [43], which both reported that lycopene prevented bone loss in OVX rats. Therefore, lycopene could be an effective therapeutic agent for osteoporosis.

Several drugs reportedly promote osseointegration and implant fixation by improving the micro-structure of the bone surrounding the implants in rats [36, 37, 46]. At the tissue level, osseointegration could be affected by the quantity and quality of the bone around the implant. Li et al. [11] reported impaired implant osseointegration and fixation in OVX rats with compromised peri-implant bone micro-structure. In our study, lycopene increased BA, BV/TV, BMD and Tb.Th and decreased Tb.Sp of peri-implant bone in OVX rats. Consistent with the results of the aforementioned studies, these results revealed that the role of lycopene in osseointegration might be mediated by altering the peri-implant bone mass and trabecular micro-architecture.

Our study has several limitations. First, the experimental situation is not representative of clinical situations. Second, measuring the decalcification of bone tissue might not be the best method for performing a histological analysis of the osseointegration rate. The interfaces of the bone remained intact by visual inspection after the implants were pushed out from the femur, as they did after embedding and sectioning. One possible reason why the interface between the implant and adjacent tissues was not apparently damaged is that the surface of the implant had no threads and was basically smooth. Another reason is that damage might occur only at small interfacial points and did not affect the calculation of BA and BC. However, the bone tissue on the slides was deformed to different degrees during the process of staining, possibly because the decalcified bone tissue was prone to exfoliation from the slides, making the inner boundary of the bone irregular and complicating the calculation of BA and BC. Based on this study, applying non-decalcified bone to the slide to maintain the original shape of the bone for better analysis of the osseointegration rate might be more appropriate. Third, we did not examine the condition of the bone at the time of implant placement. Lycopene-treated rats had better osseointegration than did OVX + vehicle rats at 12 weeks post-implant placement. This difference may have occurred due to the prevention of further bone loss between 12 and 24 weeks post-OVX or through a positive effect of lycopene on the healing process. Because we did not examine the condition of the bone at the time of implant placement, we cannot know whether lycopene improved or maintained osseointegration in the lycopene-treated group. Fourth, we did not record the uterine weights or examine oestrogen production levels before and after lycopene treatment (lower body weight was observed in lycopene-treated rats than in OVX + vehicle rats after 12 weeks of gavage, but the difference was non-significant; data not shown). Several authors have found that oestrogen production is stimulated by treatment with lycopene and other carotenoid derivatives [42, 47]. This stimulation is reflected in uterine hypertrophy [26] and could be responsible for the positive bone effects of lycopene treatment, making the effects of lycopene indirect. Due to a lack of recording of the variation in uterine weights and oestrogen production levels before and after the treatment, we cannot determine whether lycopene performed its role through oestrogen production. If lycopene’s mode of action is through oestrogen, then it will not have similar effects on male rats or non-OVX-induced osteopenic rats. Therefore, the specific mechanism of how lycopene promotes osseointegration under osteopenic conditions needs further study.

We evaluated the influence of systematically administered lycopene on implant osseointegration in an OVX rat model. The results indicated that lycopene significantly increased implant osseointegration, fixation and bone mass in OVX rats to the level of those in sham rats. Within the limitations of the study, lycopene may be an effective medication for preventing delayed osseointegration under osteopenic conditions. Further research is warranted to determine the specific mechanism of how lycopene prevents delayed osseointegration under osteopenic conditions.