Green tea extract rich in epigallocatechin gallate impairs alveolar bone loss in ovariectomized rats with experimental periodontal disease

Abstract

Periodontal disease and osteoporosis are characterized by bone resorption, and researchers have shown an association between these two diseases through increasing loss of systemic bone mass and triggering alveolar bone loss. Green tea is a common and easily accessible beverage, and evidences show that flavonoid epigallocatechin gallate (EGCG) could decrease bone loss in pathologies such as osteoporosis and periodontal disease. In order to verify its possible effects and apply them in the treatment and prevention of these diseases, this investigation aimed to evaluate the influence of green tea extract (GTE) on bone metabolism of ovariectomized rats after experimental periodontal disease (EPD) by histological, morphological and microtomographic parameters. Wistar female rats were divided into Sham, Sham + EPD, Sham + EPD + GTE, OVX, OVX + EPD and OVX + EPD + GTE groups. Immediately after surgery, gavage administration of 50 mg/kg of green tea extract (GTE) was performed for 60 days, with subsequent induction of periodontal disease by ligature 15 days before euthanasia. Mandible and femur samples were collected for histological, morphometric and microtomographic analysis. The results were analysed by means of statistical software with significance set at 5%. Histological and morphometric analysis showed a significant decrease in alveolar and femoral trabecular bone loss in groups that received GTE. Microtomographic results showed that trabecular thickness and bone surface density values in alveolar bone interradicular septum of the OVX + EPD + GTE groups were similar to the Sham group. The results obtained suggest that green tea extract may improve bone metabolism in osteoporotic rats with periodontal disease.

1. INTRODUCTION

Bone turnover consists of two opposite activities which are resorption of old bone by osteoclasts and formation of new bone by osteoblasts. ¹ Disturbances in bone remodelling may occur through both systemic and local factors such as hormonal and inflammatory changes, inducing osteoporosis and periodontal disease occurrence. Both are characterized by bone resorption, being silent and asymptomatic. ² , ³ , ⁴ , ⁵ The pathogenesis of periodontal disease is a complex process involving individual immune response to subgingival biofilm, associated with increased kappa nuclear factor receptor ligand B (RANKL) and decreased levels of osteoprotegerin in gingival tissue and biological fluids, including saliva and crevicular gingival fluid, resulting in an increased RANKL/OPG ratio. ⁶ The involvement of the RANKL and OPG system is also well established in postmenopausal osteoporosis, and studies showed an increase in serum levels of RANKL in postmenopausal women with periodontal disease. ⁷ The deficiency of oestrogenic hormones that occurs in the menopause promotes a reduction in bone mineral density, and may also contribute to the imbalance of the RANK‐RANKL‐OPG system in periodontal structures, stimulating the increase in serum inflammatory mediators (IL‐1, IL‐6 and TNF), thus promoting reduction or even loss of alveolar bone insertion. ⁸ , ⁹

The importance of treatment and prevention of osteoporosis and periodontal disease has been reported in the literature, with the use of natural compounds with promising results. ¹⁰ Green tea is one of the most popular beverages in the world and its principal compound is the flavonoid epigallocatechin‐3‐gallate (EGCG), which is considered to be responsible for protective effects against diabetes, hypertension and cardiovascular diseases, as well as for possessing antibacterial properties. ¹¹ , ¹² , ¹³

In recent years, a positive relationship between green tea consumption and bone mineral density in postmenopausal women has been established, mainly attributed to EGCG. ¹⁴ Some articles suggest that EGCG may protect against bone loss and thus prevent the deterioration of its microarchitecture in ovariectomized rats, ¹⁵ while microbiological evaluations of green tea and periodontal disease association observed by means of radiographic and histological parameters resulted in a decrease in periodontal lesions in the absence of osteoporosis. ¹² , ¹⁶ , ¹⁷ , ¹⁸ , ¹⁹ , ²⁰ , ²¹ , ²² , ²³ Thus, because of the lack in the literature of studies involving the association among osteoporosis, periodontal disease and GTE intake, the purpose of this investigation was focused on the hypothesis that green tea extract might influence positively bone metabolism of ovariectomized rats with experimental induction of periodontal disease, impairing bone loss.

2. MATERIALS AND METHODS

2.1. Experimental Protocol

Female Wistar rats were used for this study. The research protocol was approved by the Animal Research Committee of FORP‐USP (permit number 2016.1.594.58.7). The animals were randomly assigned to the following groups: Sham, Sham with experimental periodontal disease (Sham + EPD), Sham with experimental periodontal disease and green tea administration (Sham + EPD + GTE), ovariectomized (OVX), ovariectomized with experimental periodontal disease (OVX + EPD) and ovariectomized with experimental periodontal disease and green tea administration (OVX + EPD + GTE).

2.2. Ovariectomy procedure

The animals were weighed and anaesthetized with xylazine 10 mg/kg (Dopaser, Hertape Calier SA) and ketamine 75 mg/kg (Ketamine Agener; Agener União). After anaesthesia, the animals were submitted to trichotomy, and after asepsy with iodinated alcohol (PVPI), bilateral cutaneous incisions were performed to expose and/or excise the ovaries followed by suture of the tissues with silk thread 4.0 (Ethicon; Johnson & Johnson). ²⁴ Each animal received a single dose of penicillin 24 000 IU/kg (Pentabiotico Veterinário Porte—Fort Dodge^®) and flunixin meglumine anti‐inflammatory and analgesic 1.1 mg/kg (Banamine Pet Injection; Schering‐Plow Coopers Brazil). The success of the ovariectomy procedure was confirmed by oestrous cycle and the macroscopic analysis of the uterine horns. ²⁵

2.3. In vivo administration of green tea extract (GTE)

Green tea extract (GTE) used in this investigation was Sunphenon EGCG, produced by Taiyo International, certified by FDA (www.sunphenon.com/formulator-resources) and kindly provided by R & S Blumos do Brasil. This extract is characterized by containing not <94% of EGCG and <0.1% caffeine. After being dissolved in distilled water, the extract was administered daily with a gastric probe at a concentration of 50 mg/kg ²⁶ , ²⁷ for a period of 60 days immediately after ovariectomy procedure. Sham animals received water instead of the extract.

2.4. Induction of periodontal disease

Forty‐five days after osteoporosis induction and 15 days prior to euthanasia, the animals were weighed and anaesthetized as described previously. The surgical procedure of ligature was carried out with the insertion of a cotton thread in the cervical region of the lower first molars to induce periodontal disease. ²⁸

2.5. Euthanasia

Euthanasia was performed by means of conventional doses of anaesthesia followed by decapitation. The femurs and hemimandibles were removed and stored in formaldehyde for all further experiments.

2.6. Morphometric analysis of alveolar bone loss

The hemimandibles were immersed for 4 hours in hydrogen peroxide, followed by staining with 1% methylene blue for demarcation of the cemento‐enamel junction (CEJ). Each hemimandible was photographed with the use of a stereoscopic magnifying glass. Measurements were made by the lingual face in ImageJ software and the linear distance (mm) between the CEJ and the alveolar bone collar was measured, with reference to the long axes of the roots of the first lower molar, with a total of three measurements corresponding to the number of roots of each tooth.

2.7. Histological analysis

Femur and hemimandible samples were immersed in 10% buffered formaldehyde for 24 hours. Then, samples were decalcified in 0.5 mol/L EDTA + TRIS, and solutions were renewed every 2 days. After 30 days of decalcification, it was performed a neutralization with 5% sodium sulphate solution for 24 hours. After this period, the samples were dehydrated in increasing series of alcohols, diaphanized in xylol and included in paraffin. Longitudinal cuts of 5 µm thickness were performed, followed by staining with haematoxylin and eosin, which allowed qualitative and quantitative morphological evaluation. A Leica DM 4000B light microscope (Leica) coupled to a Leica digital camera DFC310FX (Leica) was used to capture the images that were analysed in 20× magnifications. For the hemimandibles, five images were collected for each animal and quantitative analysis of bone loss in the furcation region of the first molar was performed using ImageJ software. ²⁹ For quantitative analysis of femoral proximal epiphysis, a grid of 266 points using ImageJ software (version 1.46 plugin/grid) was applied in 40 images per experimental group in a 20× magnification. The points located in trabecular bone and adipocytes were counted. The number of points counted was multiplied by the total points of the test system and represented in percentage (%).

2.8. Acquisition of images by computerized microtomography

Microtomography (micro‐CT) of the mandibles and femurs was performed using a SkyScan Model 1174 Microtome (Bruker), set at 50 Kv and 800 mA, image resolution of 1024 × 1024 × 1024 pixels (10.2 µm pixel size for the mandible and 20.4 µm for the femur). The pixel dimensions were used to scan the alveolar bone support region (interradicular septum) of the lower molars in the mandible and the proximal femoral epiphysis. To perform the morphometric analysis of structures of interest, the scanned images were imported into NRecon software (Bruker) for image reconstruction greyscale pattern. To obtain morphometric parameters, the software CT Analyzer (Bruker, Belgium) was used to perform the morphometric analysis. The parameters evaluated were bone surface (BS‐mm²), bone volume (BV‐mm³), trabecular thickness (Tb.Th‐mm), trabecular number (Tb.N‐1/mm), trabecular separation (mm), connectivity density (Conn.Dn‐1/mm³), bone surface density (BS/TV‐1/mm) and total porosity (Po(tot) ‐%).

2.9. Statistical analysis

Data obtained were submitted to normality tests so that parametric data were analysed with analysis of variance (ANOVA), followed by the post‐test for multiple comparisons between the groups. Non‐parametric data were analysed with the Kruskal‐Wallis test followed by adequate post‐test. The level of significance was 5% using GraphPad Prism 5.0e software.

3. RESULTS

3.1. Ovariectomy

Macroscopic evaluation of the uterine horns showed that in the Sham group, the uterine horns present a normal diameter surrounded by adequate amount of adipose tissue. Nevertheless, in the OVX group it was observed the atrophy of uterine horns with excessive deposition of fatty tissue along their walls.

3.2. Macroscopic morphometric analysis of alveolar bone loss

Linear morphometric analysis was performed by measuring the distance from the CEJ to the alveolar border. While no significant difference was observed between the Sham and OVX groups (P = .2051), the Sham + EPD and OVX + EPD groups showed a significant increase in alveolar bone loss when compared to the other groups (P < .0001), thus showing the success of the periodontal disease induction. The OVX + EPD group had the highest bone loss of all experimental groups (P < .0001). The groups with green tea administration, Sham + EPD + GTE and OVX + EPD + GTE, showed promising results with significatively less alveolar bone loss when compared to the Sham + EPD and OVX + EPD groups (P < .0001). There was no statistically significant difference between the groups that received GTE when compared to the Sham and OVX groups (P = .6382) (Figure 1).

FIGURE 1.

Qualitative and quantitative morphometric analysis of the mandibles of the groups: (A) Sham, (B) ovariectomized (OVX), (C) Sham with experimental periodontal disease (Sham + EPD), (D) ovariectomized with experimental periodontal disease (OVX + EPD), (E) Sham with experimental periodontal disease with green tea extract administration (Sham + EPD + GTE) and (F) ovariectomized with experimental periodontal disease with administration of green tea extract (OVX + EPD + GTE), stained with methylene blue to reveal the cementoenamel junction (ACJ). Different letters indicate significant statistical difference between groups

3.3. Mandibular histological analysis

Qualitative and quantitative analysis of histological images showed a higher bone loss in the furcation region of the mandibular first molar in the OVX + EPD group when compared to the other groups. The Sham + EPD and OVX + EPD groups had a higher bone loss compared to the other groups (P < .0001), which was significantly higher for the OVX + EPD group (P < .0001). However, there was no statistically significant difference between the Sham + EPD + GTE and Sham and OVX groups (P = .4466) (Figure 2).

FIGURE 2.

Qualitative and quantitative histological analysis of alveolar bone in first molar furcation. (A) Sham, (B) ovariectomized (OVX), (C) Sham with experimental periodontal disease (Sham + EPD), (D) ovariectomized with experimental periodontal disease (OVX + EPD), (E) Sham with experimental periodontal disease with green tea extract administration (Sham + EPD + GTE) and (F) ovariectomized with experimental periodontal disease with administration of green tea extract (OVX + EPD + GTE). d, dentin, lp, periodontal ligament, a, alveolar bone, p, pulp. Haematoxylin and eosin staining. Increase 20×. Different letters indicate significant statistical difference between groups

3.4. Femoral histological analysis

Histological images showed a higher presence of adipocytes in the osteoporotic groups as well as less trabecular bone, when compared to the Sham group and the groups that received GTE. Quantitative analysis of proximal epiphysis allowed was performed to obtain the percentages of trabecular bone and adipocytes for each experimental group. The percentage of trabecular bone was significantly higher for Sham, Sham + EPD, Sham + EPD + GTE and OVX + EPD + GTE groups. The latter group, which received GTE, had the highest percentage (P < .0001). The percentage of adipocytes was higher for the OVX and OVX + EPD groups (P < .0001), and among the other groups, there was no statistical difference (Figure 3).

FIGURE 3.

Qualitative and quantitative histological analysis of the femoral epiphysis. Groups: (A) Sham, (B) ovariectomized (OVX), (C) Sham with experimental periodontal disease (Sham + EPD), (D) ovariectomized with experimental periodontal disease (OVX + EPD), (E) Sham with experimental periodontal disease with green tea administration (Sham + EPD + GTE) and (F) ovariectomized with experimental periodontal disease with administration of green tea (OVX + EPD + GTE). to, trabecular bone, mo, bone marrow, ad, adipocytes. Haematoxylin and eosin staining. Increase 20×. Different letters show significant statistical difference

3.5. Microtomographic analysis of alveolar bone

The images of the alveolar bone at the mandibular molars interradicular septum show a rarefaction in the bone microarchitecture mainly in the Sham + EPD and OVX + EPD groups (Figure 4) compared with the other experimental groups. From all the morphometric parameters analysed, bone surface (BS) and trabecular number (Tb.N) did not show significant differences (P = .6895 and P = .0987 respectively). The connectivity density (Conn.Dn) was statistically higher in the Sham group when compared to all other experimental groups (P < .0001), which showed similarity to each other. Bone volume (BV) was significantly higher in the Sham group when compared to the Sham + EPD, Sham + EPD + GTE, OVX and OVX + EPD + GTE groups (P = .0005). The trabecular thickness (Tb.Th) and bone surface density (BS/TV) were higher in the Sham group when compared to the Sham + EPD, Sham + EPD + GTE and OVX + EPD groups (P = .0002 and P < .0001 respectively), but similar to the OVX + EPD + GTE group (P = .1024 and P = .1543 respectively). The total porosity (Po(tot)) was significantly lower in the Sham group when compared to the Sham + EPD, Sham + EPD + GTE, OVX + EPD and OVX + EPD + GTE groups (P = .0007). Lower trabecular thickness (P = .0238), lower bone surface density (P = .0006) and greater trabecular separation (Tb.Sp) were observed in the OVX + EPD group (P = .0010) when compared to the OVX group. There was similarity in the parameters of trabecular thickness and bone surface density (P = .1024 and P = .1543 respectively) between the Sham and OVX + EPD + GTE groups (Table 1).

FIGURE 4.

3D femoral neck reconstruction and 3D first molar interradicular septum reconstruction of the experimental groups: (A) Sham, (B) ovariectomized (OVX), (C) Sham with experimental periodontal disease (Sham + EPD), (D) ovariectomized with experimental periodontal disease (OVX + EPD), (E) Sham with experimental periodontal disease with green tea administration (Sham + EPD + GTE) and (F) ovariectomized with experimental periodontal disease with administration of green tea (OVX + EPD + GTE)

Table 1.

Morphometric parameters obtained by means of microtomographic analysis of the first molar interradicular septum: bone surface (BS‐mm²), bone volume (BV‐mm³), trabecular thickness (Tb.Th‐mm), trabecular number (Tb. (Tb.Sp‐mm), connectivity density (Conn.Dn‐1/ mm³), bone surface density (BS/ TV‐1/ mm) and total porosity ((Po) ‐%)

Morphometric parameters	Sham	Sham + EPD	Sham + EPD + GTE	OVX	OVX + EPD	OVX + EPD + GTE
BS	11.29 ± 3.496	9.05 ± 2.63	8.78 ± 1.97	8.95 ± 1.33	9.15 ± 1.87	9.91 ± 1.27
BV	0.92 ± 0.153	0.40 ± 0.14 a	0.49 ± 0.10 a	0.64 ± 0.10	0.43 ± 0.14 a	0.54 ± 0.15 a
Tb.Th	0.30 ± 0.012	0.18 ± 0.03 a	0.20 ± 0.02 a	0.26 ± 0.05 b	0.17 ± 0.01 a	0.23 ± 0.03
Tb.N	2.59 ± 0.065	1.95 ± 0.33	2.27 ± 0.64	2.68 ± 0.12	1.89 ± 0.43	2.30 ± 0.47
Tb.Sp	0.11 ± 0.011	0.29 ± 0.03 a	0.24 ± 0.06 a	0.10 ± 0.04 b	0.28 ± 0.02 a	0.25 ± 0.07 a , c
Conn.Dn	236.41 ± 45.01	105.65 ± 9.51 a	147.85 ± 12.29 a	140.17 ± 17.84 a , b	104.69 ± 11.81 a	129.81 ± 15.12 a
BS/TV	11.70 ± 0.42	8.13 ± 0.86 a	8.92 ± 1.93 a	11.91 ± 0.48	7.84 ± 1.34 a	9.68 ± 0.56
Po(tot)	20.54 ± 2.65	63.74 ± 7.07 a	59.02 ± 16.88 a	23.21 ± 7.97	54.53 ± 17.32 a	55.44 ± 24.06 a

^aStatistical difference in relation to the Sham group.

^bDifference between OVX and OVX + EPD.

^cDifference between OVX + EPD and OVX + EPD + GTE.

3.6. Femoral microtomographic analysis

The femurs obtained from experimental groups were submitted to microtomographic analysis, and three‐dimensional reconstruction of femoral neck showed a rarefaction in bone microarchitecture mainly in the OVX + EPD group (Figure 4). The morphometric parameters analysed did not show statistically significant differences between the experimental groups, except for total porosity (P = .0024). In this parameter, it was observed that in the OVX + EPD and OVX + EPD + GTE groups the total porosity was statistically higher when compared to the Sham group (indicated by * in Table 2). The total porosity in the OVX + EPD + GTE group was also statistically higher than in the Sham + EPD and OVX groups (indicated by the symbols + and # in Table 2 respectively).

Table 2.

Morphometric parameters obtained by means of microtomographic analysis of the femoral epiphysis: bone surface (BS‐mm²), bone volume (BV‐mm³), trabecular thickness (Tb.Th‐mm), trabecular number (Tb. (Tb.Sp‐mm), connectivity density (Conn.Dn‐1/ mm³), bone surface density (BS/ TV‐1/ mm) and total porosity ((Po) ‐%)

Morphometric parameters	Sham	Sham + EPD	Sham + EPD + GTE	OVX	OVX + EPD	OVX + EPD + GTE
BS	96.78 ± 11.02	106.57 ± 14.89	92.41 ± 4.36	86.07 ± 18.32	74.69 ± 11.09	104.99 ± 3.94
BV	7.32 ± 0.43	7.07 ± 1.55	5.62 ± 0.52	6.21 ± 1.72	6.07 ± 1.78	5.47 ± 0.73
Tb.Th	0.32 ± 0.03	0.27 ± 0.01	0.27 ± 0.02	0.25 ± 0.02	0.25 ± 0.02	0.24 ± 0.009
Tb.N	2.31 ± 0.25	2.12 ± 0.23	2.54 ± 0.15	2.66 ± 0.21	2.47 ± 0.08	2.49 ± 0.24
Tb.Sp	0.17 ± 0.001	0.16 ± 0.01	0.16 ± 0.009	0.17 ± 0.009	0.19 ± 0.02	0.19 ± 0.01
Conn.Dn	39.23 ± 3.69	48.51 ± 1.11	64.72 ± 5.63	44.99 ± 4.04	45.98 ± 0.14	81.21 ± 16.75
BS/TV	9.25 ± 0.80	10.24 ± 0.35	10.87 ± 0.67	10.23 ± 0.60	10.04 ± 0.19	10.77 ± 0.82
Po(tot)	29.90 ± 2.19	32.99 ± 4.48	33.89 ± 5.36	32.36 ± 1.87	39.66 ± 6.09 a	43.26 ± 0.82 a , b , c

^aStatistical difference in relation to the Sham group.

^bDifference between Sham + EPD and OVX + EPD + GTE.

^cDifference between OVX and OVX + EPD + GTE.

4. DISCUSSION

Considering the importance of maintaining bone health and thus contributing to increased life expectancy, there is a substantial need for the discovery of new therapies for the treatment of skeletal diseases. The investigations carried out focusing on the association between green tea extract (GTE) and bone metabolism are increasing, thus emphasising the importance of this proposed theme for study. ³⁰ , ³¹

The association of green tea extract with periodontal disease also brings results that suggest that catechins present in GTE offer a promising perspective for the development of a new treatment for periodontitis, due to the ability of the extract to decrease the secretion of metalloproteinases, enzymes responsible for tissue destruction. ¹² We have not yet found in the current literature the simultaneous association of GTE with periodontal disease and osteoporosis, a situation proposed in this study.

In the present investigation, osteoporosis was induced by the removal of the ovaries, a technique widely described in the literature. ³² , ³³ , ³⁴ , ³⁵ This surgical procedure aims to decrease the levels of oestrogenic hormones of animals and thus simulate the postmenopausal osteoporosis of adult women.

After the establishment of osteoporosis, periodontal disease was induced by a ligature method, inserting a cotton thread around the mandibular first molar, a technique widely described and used in the literature since 1960. ³⁶ In order to cause significant alveolar bone loss, the cotton thread was kept in position for 15 days, according to the protocol already described. ²⁸ The establishment of two experimental models, one local and the other systemic, permitted the isolated and associated evaluation of bone loss from alveolar and femoral sites.

In the results that assessed alveolar bone loss by morphological and morphometric parameters it was observed that the OVX + EPD group had a higher bone loss when compared to the OVX group. However, in the analysis of femoral epiphyses, no significant difference was observed between the OVX and OVX + EPD groups, indicating that isolated periodontitis does not induce bone loss in femur, but osteoporosis may exacerbate alveolar bone loss. These data corroborate the observations of Anbinder et al, who after using an experimental model of rats with osteoporosis and periodontal disease found that animals with periodontal disease had an increase in the local levels of IL‐6, IL‐17 and TNF‐α, but no alteration of the systemic parameters such as IL‐4 and osteocalcin. ⁹ Studies in humans found no association between periodontitis and development of osteoporosis in men, whereas in women over 50 years old, a positive association was observed depending on the severity of periodontitis. ³⁷

Thus, the aim of this investigation was to confirm whether the green tea extract rich in EGCG would be an alternative to prevent and/or decrease the progression of periodontal disease in the presence of osteoporosis. The literature reports that the polyphenols present in green tea have the capacity to improve bone density, thus reducing its loss in situations of local or systemic diseases. ³⁸ , ³⁹ It is believed that the protective effect of EGCG is linked to its antioxidant characteristic, preventing and/or reducing the inflammatory response and bone loss by inhibiting osteocyte apoptosis, decreasing the activity of osteoclasts and increasing the activity of osteoblasts and generating the balance of the RANKL/OPG relationship. ⁴⁰

The association between GTE and periodontal disease has been evaluated both with in vivo animal models and in humans. It has already been observed that the local irrigation of GTE at sites of experimental induction of periodontal disease with the technique of ligature in rats was an adjuvant in the reduction in inflammation, osteoclast activity and alveolar bone loss by histological analysis. ⁴¹ A randomized clinical study by Taleghani et al evaluated the effect of green tea consumption after brushing in patients with depth of probing ≥5 mm, showing that the consumption of green tea accompanied by basic periodontal treatment decreased the depth of probing without decreasing the biofilm index. ²³ Systematic reviews, such as that of Gartenman et al, analysed the articles that used the topical application of GTE as an adjunct to conventional mechanical therapy, concluding that local application of GTE along with conventional therapy significantly reduced the progression of alveolar bone loss. ⁴² In addition, a meta‐analysis performed with the inclusion of 14 articles concluded that green tea consumption increased bone mineral density in patients with osteoporosis. ⁴³

In vitro studies have shown the positive effect of EGCG on osteogenic differentiation of bone marrow mesenchymal stem cells from mice and humans, in addition to promoting the reduction in osteoclastogenesis and RankL/OPG ratio at concentrations of 1‐10 µmol/L. ³⁸ Lin et al observed that EGCG increased the expression of Runx2, Bmp2, Alpl, Bglap and osteonectin genes in addition to observing greater ALP activity and mineralization in human bone marrow stromal osteoprogenitor cells at doses of 1‐10 µmol/L, with the highest dose showing better results. These data point to effects on osteoblastic functional activity that reflect a better quality of bone matrix in the presence of osteoporosis. ¹³

Daily administration of 50 mg/kg GTE protocol is in agreement to concentrations already established in the literature. ²⁶ Other authors, such as Wu et al, administered higher oral doses of GTE (60, 120 and 370 mg/kg) for shorter periods (13 days) to ovariectomized rats, also observing an increase in bone mass and an inhibition trabecular bone loss, decreasing some signs of osteoporosis. Nevertheless, positive effects achieved at lower doses may prevent signs of systemic toxicity that may occur. ³¹

The linear morphometric analysis of the distance between the CEJ and the gingival region of the first molar on the histological slides showed a higher bone loss in the OVX + EPD group, and similar values for the OVX + EPD + GTE Sham group, confirming the protective effect of GTE. A quantitative analysis of the trabecular bone at the femoral epiphyses trabecular bone was also performed. There was a similarity between trabecular loss on the OVX and OVX + EPD groups when compared to the Sham group. In this case, it is also evident in the lower bone loss in the ovariectomized groups that received the GTE. These results suggest the differentiation of mesenchymal cells from bone marrow into osteoblasts, considering that the percentage of adipocytes in the GTE groups was similar to the Sham group and significantly lower than in the OVX and OVX + EPD groups.

As a last parameter, we performed the quantitative analysis of the bone architecture by calculating morphometric indices with the help of microtomography. ⁴⁴ The obtained data showed that this microarchitecture was not modified in the neck of the femur in all the experimental groups, except for total porosity parameter. According to Bouxsein et al, the total porosity Po (tot) corresponds to the percentage of porosity found in the region of interest. ⁴⁴ The highest values of Po (tot) were found in the groups OVX + EPD and OVX + EPD + GTE, suggesting that the extract does not prevent the increasing porosity in the bone tissue with the association between osteoporosis and periodontal disease.

In the interradicular septum, the differences were relevant, with a significant loss of microarchitecture in the Sham + EPD and OVX + EPD groups. It should be noted that GTE administration was more efficient for the ovariectomized groups when compared to the Sham groups, especially for trabecular thickness and bone surface density, where the values were similar to the Sham group, suggesting a protective effect of GTE. These two parameters were significantly decreased with the presence of periodontal disease in both the Sham group and the OVX group, confirming the presence of thinner trabeculae and less bone surface regarded to the tissue volume, promoted by the experimentally induced inflammatory lesion, with even greater damage altering the microarchitecture of the alveolar bone with the association of osteoporosis/periodontal disease. GTE administration in ovariectomized rats with periodontal disease brought trabecular thickness and bone surface density to values similar to the Sham group, suggesting that the extract prevented at least in part a deterioration of bone microarchitecture. Anwar et al observed in ovariectomized monkeys by means of microtomographic analysis that oestrogen deficiency led to the fragility of the alveolar bone, inversely proportional to the lumbar bone mineral density. ⁴⁵ Studies such as this confirm the effect of osteoporosis on the trabecular area of the alveolar bone. Nevertheless, there are no data in the literature that can be used to correlate with our microtomographic findings in the region of interest assessed, that is the interradicular septum after GTE administration.

The experimental models used in this study were satisfactory to confirm the damage of osteoporosis and periodontal disease in the bone metabolism of mandibular and long bones, verified by several study parameters. GTE has been shown to have positive and protective effects on bone loss in a situation of osteoporosis associated with periodontal disease, confirming the initial hypothesis of this investigation. These are promising results that should lead to further research to confirm our findings.

ETHICAL APPROVAL

The research protocol was approved by the Animal Research Committee of School of Dentistry of Ribeirão Preto, University of São Paulo/FORP‐USP (Permit Number 2016.1.594.58.7).

CONFLICT OF INTEREST

The authors declare that there is no conflict of interest.

Vargas‐Sanchez PK, Pitol DL, de Sousa LG, et al. Green tea extract rich in epigallocatechin gallate impairs alveolar bone loss in ovariectomized rats with experimental periodontal disease. Int J Exp Path 2020;101:277–288. 10.1111/iep.12379