Assessment of the Protective Role of Green Tea Extract in the Mandibular Condylar Cartilage Matrix Against Chondrotoxicity Induced by Ciprofloxacin
in Albino Rats
Ameera Kamal Khaleel1,Ramizu Bin Shaari2, MohamadArifAwang Nawi3, Ali Mihsen Hussein Al-yassiri4
1,2,3School of Dental Sciences, Health Campus, UniversitiSains Malaysia, KubangKerian, 16150 Kota Bharu,
Kelantan, Malaysia
4 Alhilla University College, Iraq
ABSTRACT
Ciprofloxacin is antibacterial drug with a broad-spectrum activity, it can induce chondrotoxicity which is of great clinical concern. Green tea (Camellia Sinensis) is one of the healthiest beverages which have many health benefits. The present study was aimed to investigate the preventive potential of green tea extract on mandibular matrix chondrotoxicity induced by ciprofloxacin in juvenile Wistar rats.
Twenty male rats were used and divided into four equal groups, the Saline/Water (S/W), Saline/Green tea (S/G), Ciprofloxacin/Water (C/W) and Ciprofloxacin / Green tea (C/G) groups. On day 32 of age, all the animals in C/W and C/G treated groups were subcutaneously injected by ciprofloxacin as two subcutaneous injections of 600 mg/kg of body weight, eight hours apart, while the S/W and S/G groups were subcutaneously injected by saline. The S/G and the C/G treated groups were intragasticallygavaged by green tea extract in an oral dose of 300mg/kg/day, eight days before the subcutaneously injection of saline or ciprofloxacin and continues for ten days. On day 34, all the animals were anaesthetized and mandibular condylar samples were taken for histochemical and immunohistochemical analysis.
In the S/W and the S/G groups, the rat’s mandibular condylar cartilages showed evenly distributed toluidine blue staining uptake indicating a high proteoglycans secretion. In the C/W group, the rat’s mandibular condylar cartilage showed a severe reduction of proteoglycans content, while in case of C/G group, a much more intense stain by toluidine blue was seen. Statistical analysis showed that green tea can cause a significant decrease in proteoglycan breakdown of the cartilage matrix in comparison with the C/W group (p<0.05).
In S/W and S/G groups, the positive immune reaction for collagen II were observed mainly in the extracellular matrix of intermediate zone. In the C/W group, the positive immune reaction for collagen II was mainly seen in the proliferative and upper mature zone, while in case of C/G group, it was seen in the proliferative and mature zone. Statistical analysis showed a non-significant increase in collagen II immune expression in C/G group in comparison with the C/W group (p>0.05). The beneficial effect of green tea may be by improving the body's anti-oxidation ability.
This study provides the first evidence that green tea can decrease the chondrotoxic effects of ciprofloxacin in a rat mandibular condyle matrix.
Keywords:
Green tea, Collagen II, Ciprofloxacin, Condylar cartilage, Histochemistry.
Introduction
Mandibular condylar cartilage has extraordinary mechanical properties and considered as the center of the growth in the craniofacial complex and is associated with the temporomandibular joint function and craniofacial skeletonmorphogenesis [1]. Histological structure is composed of the articular, proliferative, chondrocytic (mature), and hypertrophic cells layers. The extracellular matrix consists mainly from water, collagens (90–95% type II), and proteoglycans (10–15% of the wet weight of the articular cartilage) which is formed by chondrocytes and secreted extracellularly and representing the second-largest group of macromolecules in the extracellular matrix. It contains a protein core and covalently attached sulfated glycosaminoglycans. The core protein can have in excess of 100 glycosaminoglycan molecules laterally bound to it in a bottle
brush-like arrangement [2]. The extracellular matrix in mature cells layer appears hematoxylinphilic with the use of toluidine blue staining, and this represent the active formation of cartilage matrices [3]. The synthesis of collagen and proteoglycans is high in this zone [4].
Ciprofloxacin hydrochloride is consisting of 1-cyclopropyl-6-fluoro-1,4-dihydro-4-oxo-7-1- piperazinyl-3-quinolinecarboxylic acid with a molecular weight of 331.4 [5]. Greater bioavailability and the greater susceptibility of pathogens to ciprofloxacin was the causes for its use [6,7]. Ciprofloxacin is used for treatment of different types of infections [8,9,10,11,12], it can interrupt the DNA replication and prevent bacterial cell division [13]. Depending on the toxicological results in animals during postnatal growth, previous reported cases found that ciprofloxacin can induced chondrotoxicity and tendinopathy [14], and must not be use as first- line of treatment in children [15]. It can cause matrix degeneration and cleft formation in the center of articular cartilage with necrosis of the chondrocytes [16].
The polyphenolic flavonoids present in Camellia Sinensis plant are the (-)-epicatechin (EC), (-)- epigallocatechin (EGC), (-)-epicatechingallate (ECG), and (-)-epigallocatechingallate (EGCG) [17]. The other constituents present in green tea are: Vitamins like vitamin E, Minerals, Proteins, Amino acids, Carbohydrates, Lipids, Xanthine, and others [18]. Researchers found that green tea showed a multiple health benefits for a variety of disorders [19]. The reactive oxygen species (ROS) can be inhibited by the phenol rings in EGCG structure, which can also act as scavengers of the free radicals [20]. Aggrecan is a large aggregating proteoglycan, and its degradation has mainly due to aggrecanases. Consumption of green tea or EGCG inhibited the degradation of human cartilage proteoglycan and selectively inhibited the activities of some mammalian aggrecanases [21]. It was also found that the supplementation with vitamin E can diminished fluoroquinolone induced chondrotoxicity through excess formation of collagen, inhibition of free radicals’ formation or prevent the over expression of COX-2, which is considered as important enzyme in prostaglandin synthesis and chondrocyte apoptosis [14,22].
Previous researches uncover the importance of the dietary therapy for prevention of the chondrotoxic effect of ciprofloxacin. Since green tea contain different important constituents, the prevention of chondrotoxicity by green tea extract treatment can facilitate the use of ciprofloxacin during pregnancy and children. The objective of the present study was to assess the preventive potential of green tea extract in mandibular chondrotoxicity induced by ciprofloxacin in juvenile Wistar albino rats using histochemical analysis to detect proteoglycans, and immunohistochemical analysis to detect collagen II immune expression. They are the major constituents of the mandibular condylar cartilage matrix.
METHODS
A total number of the animals used in the study was 20 male Wister Albino rats. They were aged 24 days, weighing 35-45 g. The source of the animals was from the Laboratory Animal Research Center, College of Pharmacy, University of Karbala, Iraq. The cage size is (30) cm in width, (45) cm in length, and (15) cm in height. Animals were maintained in 25± 6°C, 12-hour light and dark cycle, and fed with a standard rat chow and allowed to drink water ad libitum. The research project was approved by the USM Institutional Animal Care and Use Committee (USM IACUC).
Experimental design: The animals were divided into four equal groups:
Saline/Water group (S/W): On day 32 of age, all the animals in this group were subcutaneously injected by physiological saline (0.9% NaCl) in the same manner like ciprofloxacin, and intragasticallygavaged by distilled water eight days before the subcutaneously injection of
Saline/Green tea group (S/G): On day 32 of age, all the animals in this group were subcutaneously injected by physiological saline (0.9% NaCl) in the same manner like ciprofloxacin, and intragasticallygavaged by green tea extract eight days before the subcutaneously injection of physiological saline and continues for ten days (day 24 to day 34).
Ciprofloxacin/Water group (C/W): On day 32 of age, all the animals in this group were subcutaneously injected by ciprofloxacin, and treated by intragastric gavage of distilled water eight days before the subcutaneously injection of ciprofloxacin and continues for ten days (day 24 to day 34).
Ciprofloxacin /Green tea group(C/G): On day 32 of age, all the animals in this group were subcutaneously injected by ciprofloxacin, and treated by intragastric gavage of green tea extract eight days before the subcutaneously injection of ciprofloxacin and continues for ten days (day 24 to day 34).
All rats of the C/W and C/G groups were received ciprofloxacin hydrochloride (Bactiflox, Switzerland, 750 mg) as two subcutaneous injections of 600 mg/kg of body weight, eight hours apart [22]. To prepare the injection solution, ciprofloxacin tablets were dissolved in 0.9% NaCl (7500 mg of ciprofloxacin in 100 ml of 0.9% NaCl yield a final ciprofloxacin strength of 600 mg/
8 ml, which is corresponding to an injection volume of 8 ml/kg).
Preparation of green tea extract: The extract was prepared by adding 2.5 g of green tea (Alwazah, Sri Lanka) to 50 ml boiling water in porcelain tea pot and steeped for 20 minutes until cooled to room temperature then filtered. The extract was daily prepared to prevent the degradation of important constituents [23] and used in oral dose of 300mg/kg/day [24,25]. The 2500 mg of green tea in 50 ml of boiling water yield a final green tea extract strength of 300 mg/6 ml which is corresponding to an intragastric volume of 6 ml/kg.
Rats anesthesia and dissection: Rats were first anaesthetized and dissection was performed by using surgical instruments. Previous study found that there were no differences in the histological or molecular properties of the mandibular condyle samples from the left and right sides [26]. So, after separation of the mandible, the right and left condylar heads were collected. The right condyles were used for immunohistochemistry and the left condyle were used for histochemistry.
Tissue processing, embedding, sectioning and staining: Condylar specimens were collected and fixed by 4% neutral buffered formalin for one day at 40C. Then decalcified with 10%
ethylene diamine tetra acetic acid containing dimethyl sulfoxide (pH 7.2) for two weeks, processed, sectioned and stained. Toluidine blue staining (Sigma) was used to detect proteoglycans in mandibular condylar cartilage. Sections were deparaffinized by xylene for 12 min, hydrated by serial alcohol to distilled water for four min, stained in 0.04% toluidine blue (prepared in 0.2 M acetate buffer and mixed on magnetic stirrer and filtered) for 10 min (The pH around 3.75 and less than 4.25), washed in tap water for one min, warm air dry for 9 min, cleared in xylene for 5 minutes, and cover slipped [27].
Two sagittal sections/each animal were stained by toluidine blue. The histomorphometrical measurements were made in a blinded and nonbiased manner using objective micrometer (EW10X/20/Japan). Three measurements were done / each section for the toluidine blue darkly stained area thickness, one in the middle of the thickest portion of the middle third, and one 200µm anterior and one 200µm posterior to this line. The resulting of three measurement per each section of condyle x100 magnification, resulted in a total of six measurement for each animal, and the mean value was then calculated. Figure-1 shows the toluidine blue darkly stained zone thickness measurement.
Immunohistochemistry: Sections was deparaffinized by xylene, hydrated in a series of alcohols and then distilled water. The endogenous peroxidase activity was quenched by 3% H2O2 for 15 min at room temperature, after that the sections were covered with trypsin as antigen retrieval solution and incubate for 10 minutes at 37 °C in humidified chamber.
Nonspecific protein staining was blocked by 1.5% goat serum for (30) min, and then incubated overnight with primary antibody at 4 °C (COL2A1 Monoclonal mouse antibody, Elabscience, USA, Clone No. 2F12D3) which was diluted in phosphate buffered saline in 1:300. Free antibodies were removed by washing the samples in phosphate-buffered saline, after that by the biotinylated secondary antibody followed by a horseradish peroxidase-conjugated streptavidin- biotin. The 3,3′ -diaminobenzidine (DAB) tetrahydrochloride (Sigma) was used to visualize the immunoreactivity. Mayer’s Hematoxylin was used as nuclear counter stain [28].
The negative control tissue specimens were used with each batch of stain. It indicates a tissue specimen, processed by using a non-immune serum and applying the antibody diluents only. Two immunohistochemical sections from each animal were blindly assessed by two workers, and the staining intensity was estimated using a modified semiquantitative method [29] as seen in Table- 1.
Figure-1: Photomicrographs of the rat’s mandibular condylar cartilage stained with toluidine blue to detect proteoglycans in the cartilage matrix. (A) represent the thickness of the deeply stained zone on a line drawn perpendicular to the cartilage surface in the middle of the thickest
portion of condylar middle third. 1, articular; 2, proliferative; 3, mature; and 4, hypertrophic cells layers (Toluidine blue x100).
Table -1: Assessment of the staining intensity after immunohistochemical staining for type II collagen.
Evaluation Criterion of evaluation
0 No staining.
0.5 Traces of staining mainly in pericellular sites.
1 Definitive staining restricted to the superficial layer and upper mid zone
2 Diffuse staining of the superficial and mid zones with pericellular staining of the upper deep zone.
3 Staining 2 that extended throughout the depth of deep zone.
Statistical analysis: The results were given as mean ± standard deviation. Using ANOVA test, the potential differences among groups were evaluated. SPSS computer programs (Statistical Package for the Social Science; SPSS Inc., version 24) was used for statistical calculations. The non-parametric statistical Kruskal-Wallis test was used to analyzed the significant differences on histochemical and immunohistochemical data. P - value less than or equal to 0.05 was considered statistically significant.
RESULTS
A. Histochemical results: Toluidine blue was used to detect proteoglycans in the condylar matrix. In the S/W and the S/G groups, the rat’s mandibular condylar cartilages showed evenly distributed toluidine blue staining uptake indicating a high proteoglycans secretion. In the C/W group the sagittal section of the rat’s mandibular condylar cartilage showed a severe reduction of proteoglycans of the intermediate zone. In the C/G group, the toluidine blue staining was seen much more intense than that of the C/W group (Figure-2). Statistical analysis showed that the proteoglycans content in the C/G group was significantly higher than the proteoglycans content of cartilage matrix in the C/W group (Table-2).
B. Immunohistochemical results: In S/W and S/G groups, the positive immune reaction for collagen II were observed mainly in the extracellular matrix of intermediate zone. The C/W group showed immune reactivity mainly in the proliferative and upper mature zone.
But in case of C/G group it was mainly seen in the proliferative and mature zone (Figure- 3; Figure -4). Statistical analysis showed a significant decrease in collagen II immune expression in the C/W and C/G groups in comparison with the S/W and S/G groups (p<9.05). Statistical analysis also showed a non-significant increase (p>0.05) in collagen II immune expression in the C/G in comparison with the C/W group (Table-2).
A B C D
Figure-2: Photomicrographs of the rat’s mandibular condylar cartilage: In the S/W (A) and S/G (B) groups, the toluidine blue staining is found in the middle and deep zones. (C) The C/W group
shows severe reduction in toluidine blue stain in the intermediate zone of the mandibular
condylar cartilage. (D) Toluidine blue staining appears diffuse in the middle zones of the articular cartilage of C/G group (Toluidine blue x 400).
A B C
Figure-3: Photomicrographs of the rat’s mandibular condylar cartilage: In (A) Lower magnification photomicrograph of the middle third of the mandibular condylar cartilage near the
thinnest portion of articular disc showing immune reactivity of collagen II in S/W group (Immunohistochemistryx40). Higher magnification photomicrograph of the middle third of the
mandibular condylar cartilage in S/W (B) and S/G (C) groups showing immune reactivity of collagen II (Immunohistochemistryx400).
.
A B C
Figure-4: Photomicrographs of the rat’s mandibular condylar cartilage: (A) Immune reactivity of collagen II in C/W group showing degradation of collagen II. (B) Collagen present in C/W group
in superficial and upper mid zone. (C) Immune expression of collagen II in C/G group (Immunohistochemistryx400).
Table-2: Histochemical analysis for proteoglycan content and immunohistochemical analysis for collagen II immune expression in rats’ mandibular condylar cartilage for the different groups of
the study.
*Significant p<0.05
Discussion
A. Proteoglycan content of the mandibular condylar cartilage: Present study showed that ciprofloxacin can causes a significant decrease in proteoglycan content of mandibular condylar cartilage. Fluoroquinoloneschondrotoxicity is a multifactorial event. Previous researches attributed the cartilage damage to the presence of oxidative stress and the DNA oxidative damage of chondrocytes, and then the proteoglycan synthesis, and this collectively result in a modification of the integrity of extracellular proteins [30,31]. Other study revealed that ciprofloxacin can decreased the content of the articular matrix with areas of decreased proteoglycan [14]. IL-6, IL-1Ƀ, and tumor necrosis factor – α can promote the imbalance between the cartilage destruction and repair processes by induction of ROS and different inflammatory mediators [32]. The IL-1β can also stimulates the condylar cartilage degradation, and then inhibits the proteoglycan and collagen synthesis. It can also increase the production of different proteolytic enzymes such as inducible nitric oxide synthase (iNOS), cyclooxygenase-2 (COX2), production of IL-6, IL-8, and prostaglandin E2 [33], and can significantly reduce aggrecans production by chondrocytes [34]
The present study showed a non-significant effects of green tea extract was seen in the S/G group, and the green tea extract can significantly decrease the ciprofloxacin toxicity. The EGCG in green tea can act as a good scavenger of the free radicals and reduce the toxicity caused by the oxidative stress [35,36], increase the levels of rat serum catalase (CAT) and superoxide dismutase (SOD), reduce the production of malondialdehyde (MDA) [37], and enhances aggrecan core
Groups
Mean± SD with level of significance
Toluidine blue P-value Collagen II P-value
S/W S/G
116.6±5.16 109.2±3.41
0.075 3±0
2.83±0.37
0.601 S/W
C/W
116.6±5.16 40.20±4.44
0.009* 3±0
1.1±0.49
0.009*
S/W C/G
116.6±5.16 64.37±1.32
0.009* 3±0
1.6±0.489
0.009*
S/G C/W
109.2±3.41 40.20±4.44
0.009* 2.83±0.372 1.1±0.49
0.008*
S/G C/G
109.2±3.41 64.37±1.32
0.009* 2.83±0.37 1.6±0.49
0.013*
C/W C/G
40.20±4.44 64.37±1.32
0.009* 1.1±0.489 1.6±0.489
0.210
protein synthesis in human articular chondrocytes [38] and thus it can decrease the damage to the matrix. Adcocks et al studied the effects of green tea on the extracellular matrix components of the cartilage and found that catechins in green tea were effective in inhibiting the proteoglycan breakdown. The ECG can also inhibit the IL-1 stimulated proteoglycan breakdown and no any toxic effects of catechins were found [39]. Other study found that the protein production of matrix metalloproteinase (MMP-1 and MMP-13) were seen suppressed in the human chondrocytes which were pre-treated with EGCG [40].
B. Collagen II content of the mandibular condylar cartilage:In S/W group, positive immune reaction for collagen II were observed in the extracellular matrix of intermediate zone.Others study found that the staining for type II collagen was mainly seen in the mature and hypertrophic cell layers [41,42]. Mizoguchi et al found also the same result [43]. Visnapuu et al study found that the proliferative cells can secrete type II collagen only during the first two weeks of the age of rats [44]. To provide space for the increasing cell population, Breckon et al study indicated that the enzymatic degradation of collagen is concentrated in the area undergoing active proliferation [45]. In the mandibular condylar cartilage of the growing rat, the regional differences in the expression of type II collagens is probably due to the local biomechanical environments and not due to the growth mode of condylar cartilage.
A significant decrease in immune reactivity of collagen II was seen in C/W group of the present study. The pathogenesis of fluoroquinolone induced chondrotoxicity is a multifactorial event. One of these factors is its inhibitory effects on collagen synthesis [31,46]. Lan et al study explored the changes of expression of collagen II on fetal articular cartilage of eight pregnant women treated with ciprofloxacin undergoing termination between 19 to 34 weeks, and compared with that of normal fetal cartilage. Results of the study showed that in the normal group, the collagen II was homogeneously distributed in the matrix of articular cartilage, while in the ciprofloxacin group, a markedly decreased staining of collagen II was found in the cartilage, and these changes were a results of cartilage lesions [47]. Williams et al study found that ciprofloxacin caused a statistically significant decrease in collagen synthesis compared with controls in all the studied fibroblast cultures [48].
A non-significant increase in immune reactivity of collagen II was seen in C/G group in comparison with the C/W group. Huang et al studied the EGCG effect on chondrocytes in vitro and found that it can significantly increase the collagen II expression [49]. Collagenase enzyme can hydrolyze the three-dimensional helix structure of collagen and affecting its biological activity. The collagen treated with EGCG can resist the action of collagenase by maintaining its three-helix structure and thus resist the hydrolysis action of collagenase [50]. Madhan et al study also found that 95% of collagen can resist the degradation of collagenase by EGCG treatment.
The hydrogen bonding between EGCG and collagenase can inhibit the activity of collagenase [51].
Adcocks et al study found that catechins were effective in inhibiting type II collagen breakdown in extracellular matrix components in vitro model [39]. Leong et al study found that EGCG administration was also shown to exert chondroprotective effects by reduction in the degradation of type II collagen in cartilage matrix after posttraumatic osteoarthritis in a mouse model [52].
Vankemmelbeke et al also found that EGCG can inhibit the degradation of cartilage type II collagen in human, and selectively cause inhibition of ADAMTS-1, -4 and -5[53]. Ahmed et al and Singh et al suggest that green tea polyphenols can causes a marked reduction of COX-2 and TNF-α as a collagen-induced inflammatory mediator in arthritic joints [54,55]. Green tea
beneficial effects were attributed to its antioxidant, anti-inflammatory and anti-proteinase properties [56].
Conclusions
Green tea extract administration can reduce the mandibular condylar cartilage matrix chondrotoxicity induced by ciprofloxacin by significant decrease in proteoglycan degradation and a non-significant increase in type II collagen immune expression in articular cartilage matrix.
CONFLICT OF INTEREST -Non.
SOURCE OF FUNDING -Self
ACKNOWLEDGMENTS
We would like to thank the dean of School of Dental Sciences, Health Campus, UniversitiSains Malaysia, and all the stuff in the college for their support.
REREFERENCES
[1] Nicke JC, Iwasaki LR, Gonzalez YM, Gallo LM,Yao H. Mechanobehavior and ontogenesis of the temporomandibular joint. J Dent Res,2018; 97(11): 1185–1192.
[2] Xia Y, Momot KI, Chen Z, Chen CT, Kahn D, Badar F. Biophysics and Biochemistry of Cartilage, Introduction to Cartilage, by NMR and MRI. 2016; pp 1-43.
[3] Hinton RJ, Jing J, Feng JQ. Genetic influences on temporomandibular joint development and growth. Curr Top Dev Biol,2015; 115:85–109.
[4] Bellinghen XV,Idoux-Gillet Y,Pugliano M,Strub M,Bornert F,Clauss, F et al. Temporomandibular joint regenerative medicine. Int J Mol Sci,2018; 19(2): 446-455.
[5] Tamma PD, Cosgrove SE, Maragakis LL. Combination therapy for treatment of infections with gram-negative bacteria. ClinMicrobiol Rev, 2012; 25(3): 450–470.
[6] Sharma RC, Jain A, Jain S,Pahwa R,ShaharYaM . Ciprofloxacin: review on developments in synthetic, analytical, and medicinal aspects. J Enzyme Inhib Med Chem, 2010; 25(4): 577- 589.
[7] Rodriguez CA, Agudelo M, Zuluaga AF, Vesga O. Impact on resistance of the use of therapeutically equivalent generics: the case of ciprofloxacin. Antimicrob Agents Chemother, 2015; 59(1): 53–58.
[8] Suh B, Lorber B. Quinolones. Med Clin North Am, 1995; 79(4), 869-894.
[9] Hamer DH, Gorbach SL. Use of the Quinolones for the Treatment and Prophylaxis of Bacterial Gastrointestinal Infections, in The Quinolones, (edit., Andriole, V.T.), Academic Press, New York NY, 1998; pp. 267-285.
[10] Oliphant CM, Green GM. Quinolones: a comprehensive review. Am Fam Physician, 2002;
65(3): 455–464.
[11] Yu VL, Vergis E. New macrolides or new quinolones as monotherapy for patients with community acquired pneumonia. Chest, 1998;113(5), 1158-1159.
[12] Prajapati BS, Prajapati RB, Patel BS. Advances in management of urinary tract infections.
Indian J Pediatr, 2008; 75(8), 809-812.
[13] Fief CA, Hoang KG, Phipps SD, Jessica L, Wallace JL, Deweese JE. Examining the impact of antimicrobial fluoroquinolones on human DNA topoisomerase IIα and Iiβ. ACS Omega, 2019;
4(2), 4049−4055.
[14] Halawa AM. Effects of ciprofloxacin on the articular cartilage and epiphyseal growth plate cartilage in the growing albino rats and the possible protective role of vitamin E (alpha- Tocopherole): A histological and morphometric study. Egypt J Histol, 2010;33(3):1-5.
[15] Sendzik J, Shakibaei M, Schafer-Korting M, Stahlmann R. Fluoroquinolones cause changes in extracellular matrix, signaling proteins, metalloproteinases and caspase-3 in cultured human tendon cells. Toxicology, 2005;212(1):24-36.
[16] Kato M. Chondrotoxicity of Quinolone Antimicrobial Agent. J Toxicol Pathol,2008; 21:123- 131.
[17] Reto M, Figueira ME,Mota-Filipe H, Almeida CMM. Chemical composition of green tea (Camellia sinensis), infusions commercialized in Portugal. Plant Foods for Human Nutrition, 2008; 62(4):139-144.
[18] Musial C, Kuban-Jankowska A, Gorska-Ponikowska M. Review beneficial properties of green tea catechins. Int J Mol Sci,2020; 21(1744):1-11.
[19] Khaleel AK, Bin Shaari R, AwangNawi MA, AlRefai AS. Health benefits of green tea and green tea catechins with an overview on their anti-cancer activity. Int J Pharm Res, 2020;
12(4): 1703-1711.
[20] Tipoe GL, Leung TM, Hung MW, Fung M. Green tea polyphenols as an anti-oxidant and anti- inflammatory agent for cardiovascular protection. CardiovascHematolDisordDrugTargets, 2007; 7(2): 135–144.
[21] Cheng X, Kuzuya M, Kanda S, Maeda K, Sasaki T, Wang QL et al. Epigallocatechin-3-gallate binding to MMP-2 inhibits gelatinolytic activity without influencing the attachment to extracellular matrix proteins but enhances MMP-2 binding to TIMP-2. Arch Biochem Biophys,2003; 415(1): 126–132.
[22] Pfister K, Mazur D, Vormann J, Stahlmann R. Diminished ciprofloxacin-induced chondrotoxicity by supplementation with magnesium and vitamin E in immature rats.
Antimicrob Agents Chemother, 2007; 51(3): 1022–1027.
[23] Rahman AF, Purwanto DA, Isnaeni A. The effect of vitamin C addition on epigallocatechingallate (EGCG) stability in green tea solution. Journal Farmasi Dan IlmuKefarmasian Indonesia, 2019; 6 (2): 62-68.
[24] Arteel GE, Uesugi T, Bevan LN, Gäbele E, Wheeler MD, McKim SE et al. Green tea extract protects against early alcohol induced liver injury in rats. Biol Chem,2002; 383 (3-4), 663–670.
[25] Abdel-Raheem IT, El-Sherbiny GA, Taye A. Green tea ameliorates renal oxidative damage induced by gentamicin in rats. Pak J Pharm Sci,2010; 23(1): 21–28.
[26] Wang YL, Zhang J, Zhang M, Lu L, Wang X, Guo M, et al. Cartilage degradation in temporomandibular joint induced by unilateral anterior crossbite prosthesis. Oral Dis, 2014;
20: 301–6.
[27] Schmitzy N, Lavertyz S, Krausx VB, Aigner. Basic methods in histopathology of joint tissues.
Osteoarthritis and Cartilage, 2010; 18: S113eS116.
[28] Zhou S, Xie Y, Li W, Huang J, Wang Z, Tang J et al. Conditional deletion of Fgfr3 in chondrocytes leads to osteoarthritis-like defects in temporomandibular joint of adult mice.
Scientific Reports, 2016; 6:1-13.
[29] Khazaeel K, Mazaheri Y, Tabar MH, Najafzadeh H, Morovvati H, hadrdan A. Effect of enrofloxacin on histochemistry, immunohistochemistry and molecular changes in lamb
[30] Mao X, Gu C, Chen D, Yu B, He J. Oxidative stress-induced diseases and tea polyphenols.
Oncotarget, 2017; 8(46):81649-81661.
[31] Maślanka T, Jaroszewski JJ, Chrostowska M. Pathogenesis of quinolone-induced arthropathy:
a review of hypotheses. Pol J Vet Sci,2004; 7(4): 323-331.
[32] Malemud CJ. Matrix metalloproteinases: role in skeletal development and growth plate disorders. Front Biosci, 2006;11(1):1702-1715.
[33] Kapoor M, Martel-Pelletier J, Lajeunesse D, Pelletier JP, Fahmi H. Role of proinflammatory cytokines in the pathophysiology of osteoarthritis. Nat Rev Rheumatol,2011; 7(1):33–42.
[34] Akhtar N, Haqqi TM. Epigallocatechin-3 gallate suppresses the global interleukin-1beta induced inflammatory response in human chondrocytes. Arthritis Res Ther,2011;13(3): R93.
[35] Kochman J, Jakubczyk K , Antoniewicz J, Mruk H, KatarzynaJanda K. Health benefits and chemical composition of matcha green tea: A review. Molecules, 2021; 26(1): 785-792.
[36] Luo Q, Zhang J, LiH,Wu D,Geng F, Corke H et al. Green extraction of antioxidant polyphenols from green tea (Camellia sinensis). Antioxidants 2020, 9(9):1 15.
[37] Ahmed NA, Radwan NM, AboulEzz HS, Salama NA. The antioxidant effect of green tea mega EGCG against electromagnetic radiation-induced oxidative stress in the hippocampus and striatum of rats. ElectromagnBiol Med,2017;36(1): 63-73.
[38] Andriamanalijaona R, Kypriotou M, Baugé C, Renard E, Legendre F, Raoudi M et al.
Comparative effects of 2 antioxidants, selenomethionine and epigallocatechin-gallate, on catabolic and anabolic gene expression of articular chondrocytes. J Rheumatol.
2005;32(10):1958–1967.
[39] Adcocks C, Collin P, Buttl DV. Catechins from green tea (Camellia sinensis) inhibit bovine and human cartilage proteoglycan and type II collagen degradation in vitro. J Nutr,2002; 132 (3): 341-346.
[40] Ahmed SA, Wang N, Lalonde M, Goldberg VM, Haqqi TM. Green tea polyphenol epigallocatechin-3gallate (EGCG)differentially inhibits interleukin-1beta induced expression of matrix metalloproteinase 1 and-13 in human chondrocytes. J PharmacolExpTher, 2004;308(2): 767–773.
[41] Mizoguchi I, Nakamura M, Takahashi I, Kagayama M, Mitani, H. An immunohistochemical study of localization of type I and type II collagens in mandibular condylar cartilage compared with tibial growth plate. Histochemistry, 1999; 93:593-599.
[42] Chen J, Utreja A, Kalajzic Z, Sobue T, Rowe D, Wadhwa S. Isolation and characterization of murine mandibular condylar cartilage cell populations. Cells Tissues Organs, 2012;195(3):232- 243.
[43] Mizoguchi I, Toriya N, Nakao Y. Growth of the mandible and biological characteristics of the mandibular condylar cartilage. Jpn Dent Sci Rev,2013; 49(4): 139—150.
[44] Visnapuu V, Peltomäki T, Isotupa K, Kantomaa T, Helenius H. Distribution and characterization of proliferative cells in the rat mandibular condyle during growth. Eur J Orthod,2000; 22(6): 631–638.
[45] Breckon JW, Hembry RM, Reynolds JJ, Meikle MC. Regional and temporal changes in the synthesis of matrix metalloproteinases and TIMP-1 during development of the rabbit mandibular condyle. J Anat, 1994;(1): 99-110.
[46] Li P, Cheng NN, Chen BV, Weng YM. In vivo and in vitro chondrotoxicity of ciprofloxacin in
juvenile rats. Arch Pharmacol Sin,2004; 25(10): 1262-1266.
[47] Lan H, Qu F, Jian SC, Wang SS. Immunohistochemistry study of ciprofloxacin on fetal cartilage. Chin J Antibiot,2001; 26(4): 298-303.
[48] Williams RJ, Attia E, Wickiewicz TL, Hannafin JA. The effect of ciprofloxacin on tendon, paratenon, and capsular fibroblast metabolism. Am J Sports Med,2001; 28(30): 364-369.
[49] Huang H, Liu Q, Liu L, Wu H, Zheng L. Effect of pigallocatechin‑3‑gallate on proliferation and phenotype maintenance in rabbit articular chondrocytes in vitro. ExperTher Med,2015;
9(1): 213-218.
[50] Jackson JK, Zhao J, Wong W, Burt HM. The inhibition of collagenase induced degradation of collagen by the galloyl containing polyphenols tannic acid, epigallocatechingallate and epicatechingallate. J Mater Sci Mater Med, 2010;21(5):1435–1443.
[51] Madhan B, Krishnamoorthy G, Rao JR, Nair BU. Role of green tea polyphenols in the inhibition of collagenolytic activity by collagenase. Int J Biol Macromol,2007; 41(1):16–22.
[52] Leong DJ, Choudhury M, Hanstein R, Hirsh DM, Kim SJ, Majeska RJ et al. Green tea polyphenol treatment is chondroprotective, anti-inflammatory and palliative in a mouse posttraumatic osteoarthritis model. Arthritis Res Ther, 2014;16(6):1-11.
[53] Vankemmelbeke MN, Jones GC, Fowles C, Ilic MZ, Handley CJ, Day AJ. Selective inhibition of ADAMTS-1, -4 and -5 by catechingallateesters.Eur J of Biochem, 2003; 270(11):2394–
2403.
[54] Ahmed S, Rahman A, Hasnain A, Lalonde M, Goldberg VM, Haqqi TM. Green tea polyphenol epigallocatechin-3-gallate inhibits the IL-1β-induced activity and expression of cyclooxygenase-2 and nitric oxide synthase-2 in human chondrocytes. Free Radic. Biol Med,2002; 33(8):1097-1105.
[55] Singh R, Ahmed S, Islam N, Victor M, Goldberg VM, Haqqi M. Epigallocatechin-3- gallate inhibits interleukin-1β-induced expression of nitric oxide synthase and production of nitric oxide in human chondrocytes: suppression of nuclear factor κB activation by degradation of the inhibitor of nuclear factor κB. Arthritis Rheum,2002; 46(8): 2079-2086.
[56] Frei B, Higdon V. Antioxidant activity of tea polyphenols in vivo: evidence from animal studies. J Nutr, 2003; 133(10): 3275S-84S.
