The Cytokine and Bone Protein Expression by Ellagic Acid-Hydroxyapatite in Bone Remodelling Model

Objective Ellagic acid, a phenolic compound with anti-inflammatory potential, can be used to accelerate the bone healing process and affect human health, while hydroxyapatite is the most commonly used bone graft material. Using a combination of the two materials results in reduced inflammation and increased osteogenesis. This study aimed to determine the effects of combining ellagic acid and hydroxyapatite in bone marker remodelling by analysing the expression of tumour necrosis factor-α (TNF-α), interleukin 10 (IL-10), bone morphogenetic 4 protein (BMP-4), and osteopontin (OPN). Methods Thirty Wistar rats were used in the study. A defect was created in each animal's femur using a low-speed diamond bur. In the control group, the bone was then treated with polyethylene glycol (PEG). In one of the other groups, the bone was treated with hydroxyapatite, and in the other, with ellagic acid-hydroxyapatite. The femur was biopsied 7 days after the procedure and again 14 days after the procedure, and an indirect immunohistochemical (IHC) examination was performed for TNF-α, IL-10, BMP-4, and OPN expression. Results The ellagic acid-hydroxyapatite decreased TNF-α expression in the bone tissue after 7 days and again after 14 days (p < 0.05). On the other hand, it increased IL-10, BMP-4, and OPN expression (p < 0.05) during the same time periods. Conclusion Ellagic acid-hydroxyapatite plays a role in bone marker remodelling by decreasing the expression of TNF-α and increasing the expression of IL-10, BMP-4, and OPN. This hydroxyapatite combination can therefore be recommended for use as bone graft material.


Introduction
Te alveolar bone is morphologically and functionally different from other bones. It is sensitive to mechanical stress and bone loss stimuli because it contains signifcantly different mineralisation rates than other bones in the body [1]. Alveolar bone defects occur easily and can necessitate several dental procedures, including tooth extractions. Tooth extractions commonly lead to further bone defects, especially in the anterior region of the maxilla where the cortical bone is thinner [2]. Alveolar bone retrieval procedures, such as odontectomies, also carry a risk of alveolar bone defects [3]. Excessive orthodontic pressure due to orthodontic treatment is another risk factor [4] as are pathological processes, such as periodontitis [5] and cleft lip and palate [6]. Te aforementioned factors can result in bone defects that not only impede prosthetic reconstruction but that also cause aesthetic and masticatory problems [7]. As with other bone defects, alveolar defects can be repaired using a bone graft material.
Hydroxyapatite is commonly used as a bone graft material [8][9][10] because its chemical composition and crystal structure are similar to that of bone. In addition, hydroxyapatite has osteoconductive properties-it increases osteoblast activity while inhibiting excessive osteoclast activity [11]. Te physical, chemical, and mechanical properties of hydroxyapatite, as well as the biocompatibility and bioactivity of the material, make it the most promising bone graft material in the feld of modern dentistry [12,13].
When prolonged infammation occurs, the properties and efects of hydroxyapatite are no longer maximal. Prolonged infammation leads to bone resorption activity due to osteoclast activity modulated by proinfammatory cytokines, such as TNF-α [14]. Terefore, adding an anti-infammatory to the graft is necessary to suppress the infammatory process. Ellagic acid may be used for this purpose. Te dominant properties of ellagic acid are that it is an antioxidant and an anti-infammatory [15][16][17][18]. For this reason, it is expected to reduce infammation and stimulate bone remodelling [14,19]. Te study showed that ellagic acidhydroxyapatite increases osteoblast activity, decreases osteoclast activity, and stimulates bone remodelling [20]. Te decrease in osteoclast activity refects the end stage of infammation and the initial stage of bone deposition. During bone remodelling and deposition, anti-infammatory cytokines and growth factors, such as interleukin 10 (IL-10), bone morphogenetic 4 protein (BMP-4), and osteopontin (OPN), are required to accelerate osteoblast maturation [21].
Due to the anti-infammation properties of ellagic acid and the bone stimulation properties of hydroxyapatite, the aim of this research is to prove the efcacy of ellagic acidhydroxyapatite in assisting and increasing bone marker remodelling. Tis will be achieved by analysing the expression of TNF-α, IL-10, BMP-4, and OPN. Te results of the study will contribute to the research in the feld of biomaterials and tissue regeneration, particularly as it relates to the orofacial region of the body.

Animals.
Tirty healthy male Wistar rats (Rattus norvegicus), each weighing 200-250 grams, were divided into three groups of fve rats each.
A 10 mm incision was made in each animal's lateral femur. Te distance from the joint was 50 mm between the tibia and femur. Te defect was created using a 0.84 mm round bur (801G/018, Meisinger, Germany), which rotated with a low-speed engine. Te defect was 2 mm in diameter and depth. A saline solution was used for irrigation during the procedure.
After the bone defect was created, one of three materials was applied to the bone, depending on which group the animal was in. In one of the control groups, PEG was used; in another, hydroxyapatite was used; and in the experimental group, ellagic acid-hydroxyapatite was used.
After application, the tissue over the bone defect was sutured using nylon (Nylus nylon, nonabsorbable sutures, Lotus Surgicals, India), and the animals were given oral gentamicin (2-4 mg/kg body weight) every 24 hours. Te animals received standard postsurgical care.
Te femur of each animal was biopsied after 7 days and again after 14 days. An immunohistochemical (IHC) examination was also performed to analyse the defective bone tissue.

Data Analysis.
Te data were analysed using the Shapiro-Wilk test for data distribution and Levene's test for data homogeneity. Te diferences in expression between the control, hydroxyapatite, and ellagic acid-hydroxyapatite groups were analysed using the one-way analysis of variance (ANOVA) and Tukey's honest signifcant diference (HSD) tests. For the latter test, a test result of p < 0.05 was considered a signifcant diference.

TNF-α Expression.
TNF-α expression was present in the bone tissue, as per Figure 1. Te ellagic acid-hydroxyapatite group had lower TNF-α expression than the control group after seven days (p � 0.001). After 14 days, the ellagic acidhydroxyapatite group had lower TNF-α expression than the hydroxyapatite and control groups (p � 0.001; p � 0.012) (See Figure 2(a)).

IL-10
Expression. IL-10 expression was present in the bone tissue, as per Figure 3. Te ellagic acidhydroxyapatite group had a higher expression than the control group after 7 days and again after 14 days (p � 0.001) (See Figure 2(b)).

BMP-4 Expression.
BMP-4 expression was present in the bone tissue, as per Figure 4. Te ellagic acidhydroxyapatite group had a higher expression than the control group after 7 days and again after 14 days (p � 0.001) (See Figure 2(d)).

OPN Expression.
OPN expression was present in the bone tissue, as per Figure 5. Te ellagic acid-hydroxyapatite group had the highest expression of all the groups after 7 days and again after 14 days (p < 0.05) (See Figure 2(c)).

Discussion
Infammation is a normal part of the bone healing process. However, prolonged infammation has numerous negative efects on the body, one being a prolonged healing process. Bone remodelling using only hydroxyapatite causes more intense infammation and prolongs the healing process [22,23] by producing TNF-α [24]. To decrease infammation, hydroxyapatite should be combined with ellagic acid. Tis serves to control and suppress infammation and predict the bone remodelling occurrence [25]. Tis condition proves that for the purposes of this research, ellagic acid-hydroxyapatite decreases TNF-α expression more efectively than hydroxyapatite. Uncontrolled TNF-α expression, due to prolonged infammation, stimulates osteoclast diferentiation, destroys the extracellular matrix, and results in bone resorption [26]. Tis indicates that ellagic acid reduces TNF-α expression by suppressing the activation of the nuclear factor kappa B (NF-κB) pathway [27][28][29][30].
Ellagic acid in the NF-κB pathway inhibits IKK activation to prevent degradation and reduce translocation to the nucleus. Tis process results in reduced proinfammatory cytokines, particularly TNF-α [31]. Inhibiting the production of proinfammatory cytokines increases anti-infammatory cytokines and growth factors such as IL-10 and BMP-2 [31,32].
IL-10 is a potent anti-infammatory cytokine, which actively reduces and regulates other proinfammatory cytokines, such as TNF-α and interleukin 1β (IL-1β) [33,34]. Tis study showed that IL-10 expression was higher in the bone tissue after 7 days and 14 days. Te function of IL-10 is to regulate infammation, which reduces proinfammatory cytokines, namely TNF-α. Te higher proinfammatory cytokines (TNF-α) trigger osteoclast genesis and cause increased osteoclast diferentiation, resulting in bone resorption. Increased IL-10 expression triggers osteoblast genesis by increasing osteoclast diferentiation. Tis supports faster bone remodelling and accelerates all phases of bone healing. IL-10 appears to be an important regulator of bone homeostasis and infammatory conditions [35,36].
Other growth factors, such as the transforming growth factor, support bone remodelling, called BMP-4. BMP-4 regulates the migration and diferentiation of mesenchymal stem cells during bone remodelling, induces osteogenesis, and rolls the remodelling of the bone matrix into mature bone [37]. In this study, BMP-4 expression increased as IL-10 expression increased. BMP-4 and IL-10 increased osteoblast activity's diferentiation due to ellagic acid's antiinfammatory properties [33]. Te hydroxyapatite in the ellagic acid-hydroxyapatite combination also helps to increase bone remodelling. Te hydroxyapatite releases calcium ions to support bone remineralisation [32].
Te current research shows that the combination of ellagic acid-hydroxyapatite can reduce the main   Te Scientifc World Journal proinfammatory cytokine of bone infammation, TNF-α, and increase bone growth factors, BMP-4 and OPN, through anti-infammatory cytokine and IL-10. Tis mechanism is fundamental to initiate bone regeneration, to constitute the proportional ratio between osteoblast and osteoclast, as the primary cell in bone formation before cellular processes occur. Te decreased TNF-α, in this research, also provides the fnding that this cytokine may play a role in the decrease of osteoclast activity, as stated in the previous study [20]. Consequently, the decreased osteoclast activity will increase osteoblast activity, fostered by the anti-infammatory properties of the ellagic acid and also lead to an increase in the bone protein proliferation factor, OPN. It is believed that OPN can induce mesenchymal stem cell (MSC) migration to defect sites and initiate diferentiation into chondrocytes and osteoblasts [38]. Tis study also proved that ellagic acid-hydroxyapatite could increase OPN expression. Te hydroxyapatite acts as a matrix to provide cell adhesion during remodelling [39]. Ellagic acid is several plants' polyphenolic compound, a secondary metabolite that is easy to obtain and commercially available as naturaceutical [40]. Te source of ellagic acid is pomegranate (Punica granatum L.) and in the wood and bark of some tree species [41]. Of this availability, it will be easy to use and obtain and promising to use as a bone substitute with hydroxyapatite. Te limitation of the study, it only analyzes one proinfammatory cytokine, namely TNF-α. Although other cytokines, such as IL-6 and IL-1β, also play a role. Te TNF-α itself is representative to answer the purpose of this study because it is a marker of infammation in general.

Conclusion
Current research shows that the combination of ellagic acidhydroxyapatite combination is able to reduce the main proinfammatory cytokine of bone infammation, TNF-α so that it can increase bone growth factors, BMP-4 and OPN, through IL-10. Tis mechanism is fundamental to initiate bone regeneration, before the constitution of osteoblast and osteoclast. Tis combination of materials can be safely recommended for bone grafting material to promote bone regeneration. Further study is needed, to implement this combination in humans, especially in bone fracture and   orofacial bone defects, including alveolar bone defects, cleft lip, and palate. Regarding safety, some studies have shown such low toxicity that the chances of use are even greater.

Data Availability
Te data will be available upon personal request to address to the corresponding author Intan Nirwana (intan-n@ fkg.unair.ac.id).

Additional Points
Ellagic acid-hydroxyapatite plays a role in bone marker remodelling by decreasing the expression of TNF-α. Ellagic acid-hydroxyapatite also plays a role in bone marker remodelling by increasing the expression of IL-10, BMP-4, and OPN.

Ethical Approval
Te Ethics Committee for the Faculty of Dental Medicine at Airlangga University (495/HRECC.FODM/XI/2020) registered and approved the research protocol.

Conflicts of Interest
Te authors declare that they have no conficts of interest.