Effects of Epigallocatechin-3-O-Gallate on Bone Health

Abstract

Tea is one of the most consumed beverages in the world, belonging to the category of compounds known as tannins and flavonoids. One of the polyphenols found in large amounts in green tea leaves (Camellia sinensis) is epigallocatechin-3-O-gallate (EGCG). Though EGCG has shown some pharmacological effects, to date, it has not been utilised as a therapeutic agent. This is attributed to the fact that EGCG lacks adequate stability, and it is known to degrade through epimerization or auto-oxidation processes, especially when it is exposed to light, temperature fluctuations, some pH values, or the presence of oxygen. Consuming green tea with EGCG can alleviate the effects of bone diseases, such as osteoporosis, and support faster bone regeneration in the case of fractures. Therefore, this review focuses on the current state of research, highlighting the effects of EGCG on bone biology, such as enhancing osteoblast differentiation, promoting bone mineralisation, improving bone microarchitecture, and inhibiting osteoclastogenesis through the modulation of the RANK/RANKL/OPG pathway. Additionally, EGCG exerts antioxidant, anti-inflammatory, and dose-dependent effects on bone cells. It also downregulates inflammatory markers (TNF-α, IL-1β, and COX-2) and reduces oxidative stress via the inhibition of reactive oxygen species generation and the activation of protective signalling pathways (e.g., MAPK and NF-κB). Studies in animal models confirm that EGCG supplementation leads to increased bone mass and strength. These findings collectively support the further exploration of EGCG as an adjunct in the treatment and prevention of metabolic bone diseases. The authors aim to present the relationship between EGCG and bone health, highlighting issues for future research and clinical applications.

1. Introduction

Whilst green tea (Camellia sinensis) contains various classes of chemical compounds, such as catechins, phenolic acids, polyphenolic acids, amino acids, proteins, and lipids, in this paper, we will focus on catechins as represented by flavonoids. This is significant because the following catechins are all found in green tea: epigallocatechin gallate (EGCG), epicatechin gallate (ECG), epigallocatechin (EGC), and epicatechin (EC). This fact makes green tea much richer in catechins than black tea (one cup of brewed green tea contains about 200–300 mg of EGCG, whereas black tea contains only about 18 mg per cup), and subsequently, its antioxidant capacity is directly proportional to the catechin content [1,2,3,4,5]. The number and proportion of active compounds depend on various factors related to the conditions of cultivation, including soil, climate, light, geography, microorganisms, and temperature. During black tea processing, fermentation takes place, and most catechins present in fresh green tea leaves undergo oxidation to theaflavins. These substances are the origin of the characteristic features of black tea—its dark colour and astringent taste [6].

EGCG, the major catechin with a polyphenolic structure, which was first isolated from green tea, has become a popular subject of research due to its intriguing properties [7]. The number of studies on this compound has proportionally begun to increase since the early 2000s. EGCG can be found in other plants such as Camellia gymnogyna (Baiyacha), a wild tea plant species closely related to C. sinensis. Research has shown that Baiyacha contains high levels of methylated EGCG derivatives, such as 3″-methyl-epigallocatechin gallate (EGCG3″Me), which are rarely observed in regular tea plants [8]. While Camellia sinensis is by far the richest and most studied source, trace amounts of EGCG or structurally related galloylated catechins have been detected in the leaves, skins, or seeds of some fruits (e.g., apples, plums, blackberries, cranberries), nuts (hazelnuts and pecans), and other plants (onions), but these levels are generally much lower [9]. EGCG has the ability to quench reactive radicals and chelate transition metal ions, avoiding the formation of reactive oxygen species (ROS) [7]. Its varied therapeutic properties have made it a popular bioactive ingredient and drug carrier in novel formulations. Due to its antioxidant, antivirus, and anti-inflammatory effects, it seems to be a promising compound for potential use in the treatment of many diseases [2,3,8,9]. Interestingly, epigallocatechin-3-gallate has been found to influence neuroprotection, including relapsing–remitting multiple sclerosis [10,11,12,13,14]. However, EGCG exhibits several limitations, such as poor solubility in lipids, low bioavailability, rapid decomposition and a short half-life. In order to overcome these limitations, it would be necessary to either use its highly effective dose in vivo to get the expected pharmacological effect and/or modify the EGCG structure. As far as structural modification is concerned, Liu et al., in their study, presented its impact on the properties of EGCG derivatives. Changes in the chemical structure contributed to improved stability, uptake by tumour cells, solubility in lipids, and the ability to penetrate cells [14].

EGCG can effectively neutralise ROS and, therefore, prevent oxidative damage in healthy cells. It reveals chemopreventive properties as it contributes to apoptosis and stops cancer cells from growing. These effects are mediated by the modulation of cell cycle regulatory proteins, the activation of killer caspases, and the suppression of oncogenic transcription and pluripotency maintenance factors. EGCG has demonstrated its ability to affect many signal transduction pathways and block telomerase activity, causing the stimulation of telomere fragmentation. Clinical studies indicate that EGCG consumption reduces tumour incidence and multiplicity in the liver, stomach, skin, lung, mammary gland, and colon [2,3]. EGCG is also considered a potential therapeutic agent able to prevent the carcinogenesis of oral potentially malignant disorders (OPMDs) that most often precede the diagnosis of oral squamous cell carcinoma. EGCG may block the progression of cancer by disrupting the cell cycle and precancerous lesions of the oral cavity [13].

EGCG also exhibits neuroprotective abilities, as it is assumed to diffuse and localise in the brain. Additionally, EGCG shows potential for the prevention of Alzheimer′s disease to reduce the amino acid form of β-amyloid Aβ and Τ (tau) protein toxicity and inhibit apoptosis. Moreover, ECGG may be a promising natural neuroprotective agent for the treatment of Parkinson’s disease by reducing and preventing dopamine loss as well as inhibiting oxidative stress [15]. In addition, EGCG seems to be a promising therapeutic for reducing neuroinflammation and preventing nerve damage after intracerebral haemorrhage (ICH). Bao et al. 2023 [16] investigated the effects of EGCG pretreatment on neuroinflammation-mediated neuronal pyroptosis in experimental ICH in mice. They observed the alleviation of microglial pyroptosis and neuroinflammation and an improvement in neurological function [16]. EGCG has also been reported to inhibit high glucose-induced senescence in H9C2 cardiomyocytes, as well as inflammation, and oxidative stress response and to trigger peroxisome proliferator-activated receptor gamma-dependent signalling pathways, thereby preventing diabetic cardiomyopathy [17].

The advantageous effects of EGCG on bone condition have been widely researched [1,11]. Based on the results of many studies, EGCG has been shown to play a significant regulatory role in the immune response in osteoporosis, fracture healing, and periodontitis [18,19,20]. Moreover, green tea phytochemicals were found to improve various bone loss models associated with ageing, estrogen deficiency, and chronic inflammation [21,22]. The regular consumption of green tea is associated with improved bone health. Although a higher bone mineral density (BMD) and a lower hip fracture rate are associated with a higher consumption of green tea, the relationship remains unclear [18,23]. EGCG can suppress receptors for advanced glycation end product (RAGE) expression by epigenetic regulation, which leads to chondrocyte protection against osteoarthritis [24]. Interestingly, EGCG, as a substance capable of regulating bone metabolism, also affects periodontal remodelling during orthodontics, which was studied by Zou et al. [25]. In a study on rats, it reduced orthodontic tooth movement and orthodontic-induced root resorption. Furthermore, EGCG impaired osteoclastogenesis on the pressure side and promoted osteogenesis on the tension side. Summarising, EGCG is a non-toxic, naturally occurring compound with diverse and promising biological properties and a diversity of beneficial health effects, which is worth discussing in more detail.

Considering the aforementioned properties of EGCG, this review explores the current state of research, emphasising its effects on bone density, mineralisation, and the regulation of bone metabolism, from two perspectives: functional and biochemical effects. It aims to clarify the connection between EGCG and bone health, while highlighting key areas for future research and potential clinical applications.

The literature search was performed using the following databases: PubMed and Google Scholar. Searches were carried out using combinations of the following keywords: “epigallocatechin-3-gallate”, “EGCG”, “green tea polyphenols”, “bone health”, “bone metabolism”, “osteoblast”, “osteoclast”, “osteoporosis”, “bone regeneration”, and “bone density”. The authors have included scientific publications from 1 January 1985 to 30 March 2025. In the first stage of the review, the titles of the articles were carefully checked. If the title clearly indicated that the article was outside the scope of our review, the reference was rejected. If the title suggested the presence of data consistent with our interests, the publication was subject to further analysis. In the next step, the abstracts were assessed and, if still possible, the full texts were analysed. Following an initial screening of titles and abstracts, the full texts of potentially relevant studies were reviewed for eligibility. A total of 162 articles met all inclusion criteria and were included in the final review.

2. Functional Effects of EGCG

EGCG can exert various functional effects as illustrated in Figure 1. Most of these are positive, although a few adverse ones have also been reported. EGCG supplementation has been demonstrated to influence various aspects of bone formation and maintenance in both in vitro and animal trials. One of its positives is the upregulation of alkaline phosphatase (ALP), which stimulates bone mineralisation [26]. Additionally, EGCG has been shown to modulate osteoblast function by selectively inhibiting the extracellular signal-regulated kinase (ERK; p42/p44) branch of the mitogen-activated protein kinase (MAPK) pathway, thereby reducing PGD2-induced HSP27 expression in osteoblasts. This process may influence cellular stress responses rather than directly promote osteoblast proliferation or differentiation [27]. EGCG has also shown the potential to induce bone regeneration through mechanisms such as enhancing bone callus formation and by modulating the collagen membrane structure and function [21]. However, in certain cases, EGCG supplementation may require co-administration with α-calcium triphosphate to effectively promote the regeneration [28]. Furthermore, the concentration levels of EGCG play a crucial role in how effective it is. High dosages of EGCG can inhibit the proliferation of mesenchymal cells involved in osteogenesis in periodontitis, while intermediate concentrations can promote it. It has been shown that an intermediate dose of 4 and 6 µM promotes human periodontal ligament cell (hPDLC) osteogenesis by increasing ALP activity and the mRNA and protein expression levels of the osteogenic markers type I collagen (COL1), runt-related transcription factor 2 (RUNX2), osteopontin (OPN), and Osterix (OSX). Specifically, quantitative real-time PCR analysis demonstrated that EGCG treatment significantly upregulated the mRNA levels of COL1, RUNX2, OPN, and OSX, indicating enhanced osteogenic differentiation at these concentrations [29]. Yet another study proved that the therapeutic effects of EGCG are dose-dependent. Mah et al. conducted research in which immunodeficient mice were transplanted in vivo with human alveolar bone-derived cells (hABCs) that had been pretreated with 10 µM of EGCG and mixed with calcium phosphate carrier combined with EGCG (0.1, 0.5, or 1.5 mg) [30]. The results indicated that EGCG up to 10 µM slightly increased osteogenic differentiation, while at higher concentrations above 25 µM, the proliferation and migration of hABCs were reduced. Hard tissue formation efficiency was enhanced when the low concentration in the 0.1 mg EGCG group was used, whereas EGCG levels exceeding 0.5 mg significantly reduced hard tissue formation.

In some instances, such as human bone marrow mesenchymal stem cells (hBM-MSCs) and human mesenchymal stem cells (hMSCs), additional osteoinductive agents or mechanical stretching were found to induce this effect by increasing the osteogenic differentiation of MSCs and accelerating osteogenic differentiation, respectively [31,32]. Notably, EGCG prevents the TNF-α-mediated inhibition of osteogenic differentiation in hBM-MSCs while concurrently modulating osteoimmunological pathways. It suppresses TNF-α-induced IL-6 synthesis in osteoblasts via the attenuation of p44/p42 MAP kinase and stress-activated protein kinases/Jun amino-terminal kinases (SAPK/JNK) pathways, thereby reducing RANKL expression and shifting the RANKL/OPG balance towards bone formation [33]. EGCG does not solely impact tissue formation but contributes to improving bone microarchitecture and preventing bone loss [34,35]. Nakagawa et al. proved that this molecule hampers bone resorption by inducing apoptosis in osteoclast cells [36]. Interestingly, one study suggested that bone resorption might be stopped by the combined actions of osteoblasts and osteoclasts, without involving apoptosis [37]. Additionally, EGCG inhibits osteoclastogenesis through multiple pathways, notably by downregulating key osteoclastogenic transcription factors, such as c-Fos and the nuclear factor of activated T cells 1 (NFATc1), and by suppressing RANKL-induced JNK and NF-κB signalling, thereby mitigating bone resorption. Moreover, EGCG treatment decreases osteoclast formation by suppressing key osteoclastogenic factors, downregulating c-Fos and NFATc1 expression, and inhibiting JNK and NF-κB signalling in preosteoclasts, thus directly reducing osteoclastogenesis [32,33,34,38]. These processes that impact bone homeostasis are also dependent on dosage, particularly during the early stages of osteoclastogenesis [35,38]. Osteoblast migration plays a key role both in the physiological bone metabolism and in pathological bone processes. Research performed on cloned osteoblast-like MC3T3-E1 cells derived from a newborn mouse by Kawabata et al. [39] showed that the compound inhibited insulin-like growth factor 1 (IGF-I)-induced osteoblast migration by attenuating IGF-I-induced p44/p42 MAP kinase phosphorylation.

The effects described above were researched using various cell lines, whose characteristics and relevance to physiological processes are outlined below. Human mesenchymal stem cells (hBM-MSCs) are the most relevant model in the context of human physiology, as they are directly derived from humans and reflect the properties of human stem cells [26,31,32,33,39,40,41]. The MC3T3-E1 line, although widely used in the in vitro studies of osteoblasts, originates from mice and does not fully replicate the processes occurring in human bone tissue [27,32,34,42,43]. Similarly, the RAW264.7 line, a murine model of macrophages and osteoclasts, is valuable for studying bone remodelling; however, interspecies differences limit the direct translation of findings to human physiology [23,43,44,45,46,47]. In summary, hBM-MSCs appear to provide the most physiologically relevant model for human studies. The MC3T3-E1 and RAW264.7 cell lines remain useful for fundamental and mechanistic research, but their applicability to directly model human physiological processes is limited.

2.1. Stimulating Mineralisation and Enhancement of Bone Formation

One of the main effects influencing bone structure is the stimulation of bone metabolism, leading to the enhancement of mineralisation. Chen et al. [26] demonstrated in their study on the D1 cell line (a murine bone marrow mesenchymal stem cell line) that the administration of EGCG at a dose of 1 or 10 µmol/L promotes mineralisation by increasing the osteogenic gene expressions and upregulation of ALP activity. In another study, Vali et al. observed that treatment with 1–5 µM of EGCG in SaOS-2 cells (human osteoblast (HOB)-like cells) led to an increased formation of mineralised bone nodules by enhancing osteoblast differentiation, along with a decrease in RUNX2 protein levels [48].

The improvement in bone structure and the enhancement of bone formation following EGCG supplementation have also been observed in various studies. Yamauchi et al. demonstrated that it can modulate osteoblast cell function towards bone formation by inhibiting p44/p42 MAP kinase in cloned osteoblast-like MC3T3-E1 cells derived from newborn mouse calvaria [27]. Various bone defects and fractures can be alleviated by this compound. Rodriguez et al. observed in parietal bone rats that the combination of EGCG and α-calcium triphosphate (whose derivative is hydroxyapatite, responsible for mechanical bone strength) reduced bone defects by inducing maximum bone regeneration [28]. EGCG itself enhanced the healing of bone defects by de novo bone formation, as noted by Lin et al. [44]. One of the possibilities of facilitating the healing of bone fractures by EGCG may be its influence on enhancing bone callus formation. The study performed by Lin et al. on the right tibial bones of rats indicated that it acts by increasing the expression of bone morphogenetic protein 2 (BMP-2), which subsequently enhances the bone mechanical properties [21]. Chu et al. demonstrated in the in vivo study on the dorsal part of the rat cranium that EGCG can also be used to modify collagen membranes, which in turn induces bone formation [45]. They indicated that an increase in the number of macrophages (M2) promoted the secretion of the vascular endothelial growth factor (VEGF) and BMP-2 and upregulated the expression of RUNX-2 and osteopontin (OPN) after implantation of the EGCG-modified collagen membranes. EGCG was also found to be an anti-angiogenic agent. The study performed by Takita et al. on rats indicated the reduction in the activity of ALP and calcium content, accompanied by an increase in the content of type II collagen, which enables the simultaneous inhibition of ectopic osteogenesis and the enhancement of chondrogenesis [49].

2.2. Prevention/Alleviation of Bone Loss and Inhibition of Bone Resorption

The preservation of proper bone structure can occur through several mechanisms beyond the promotion of osteogenic differentiation or mineralisation processes. Hayashi et al. [34] showed that EGCG can prevent bone loss due to the suppression of the SAPK/JNK pathway in cloned osteoblast-like MC3T3-E1 cells derived from newborn mouse calvaria. In contrast, Chen et al. [35] proved that EGCG treatment ameliorates bone loss and improves bone microarchitecture by enhancing osteogenesis after BMP-2 expression in the in vivo study conducted on ovariectomy-induced osteopenic rats (their proximal tibia and third lumbar spine were compared).

The main cell population responsible for bone resorption is the osteoclast. Under physiological conditions, they maintain the proper mechanical strength of bones. Sometimes, under the influence of various factors, a pathological increase in bone resorption may occur. This can be prevented, for example, by inducing osteoclast death, thereby preventing/reducing bone resorption. Nakagawa et al. [36] found that EGCG treatment leads to the inhibition of bone resorption by inducing the apoptotic death of crude murine osteoclast-like multinucleated cells (OCLs), which involves the Fenton reaction affecting caspase-3 activity. Also, Yun et al. [46] noted an apoptotic osteoclast cell death in the murine monocyte/macrophage cell line, RAW 264.7-cell-derived osteoclasts treated with EGCG. This process was caused by DNA fragmentation and caspase-3 activation. On the other hand, Yun et al. [37] showed that the expression of matrix metalloproteinases-9 (Mmp-9) genes derived from primary osteoblastic mouse cells stimulated by Porphyromonas gingivalis could be inhibited by EGCG. This study suggested that bone resorption may be inhibited not only through the induction of osteoclast apoptosis but also by modulating osteoblast–osteoclast signalling pathways.

2.3. Inhibition of Osteoclastogenesis and Osteoclast Differentiation

According to conclusions drawn from studies by Lee et al. [50] on mouse bone marrow cells, EGCG inhibited osteoclast differentiation from bone marrow cells and primary osteoblast co-cultures induced by IL-1, TNF-α, and vitamin D₃ plus PGE₂ by reducing c-Fos and the nuclear factor of activated T cells 1 NFATc1 expression, thereby inhibiting the RANKL-induced activation of the NF-κB and JNK/c-Jun signalling pathways. Several years later, Xu et al. [47] came to a similar conclusion that RANKL-induced osteoclastogenesis and F-actin ring formation were inhibited by EGCG through the downregulation of NFATc1 and c-Fos in mouse macrophage RAW 264.7 cells. They also found that the derivative of EGCG exerted a stronger effect than the parent molecule. The effects of RANKL are reversed by osteoprotegerin (OPG). Chen et al. [19] showed that the co-culture of RAW 264.7 cells and the feeder cells ST2 treatment with EGCG resulted in an increase in OPG mRNA expression and a decrease in RANKL mRNA expression, and therefore, the RANKL/OPG ratio decreased. Moreover, a decrease in osteoclastogenesis by TRAP-positive (tartrate-resistant acid phosphatase) osteoclasts and TRAP activity, was noted.

Osteoclast differentiation is physiologically stimulated by, among others, osteoblasts. The inhibition of this process may cause a reduction in the number of osteoclasts. Kamon et al. [38] reached this conclusion after observing that EGCG decreased ALP activity and suppressed the mRNA expression of ALP and osteocalcin (OCN) in MC3T3-E1 cells, as well as in the co-cultures of osteoblasts from neonatal mouse calvaria and bone marrow cells. The treatment of pluripotent mesenchymal cells D1 with EGCG increased OPG expression and thus inhibited osteoclast differentiation and activity. Such conclusions were made by Chen et al. [26,42]. Several years later, Fujita et al. [43], using cloned osteoblast-like MC3T3-E1 cells derived from newborn mouse calvaria, showed an inhibition of osteoclast differentiation and activation by increasing OPG release and the levels of OPG mRNA expression. Many factors can influence the conversion of monocytes to osteoclasts. One of them is NFATc1. Morinobu et al. [51] showed in the antibody-induced arthritis model of male mice that NFATc1 expression was limited by EGCG, which prevented the CD14+ monocytes from converting to osteoclasts. Simultaneously, catechin did not affect the expression of NF-κB, c-Fos, and c-Jun. In turn, Lin et al. observed on RAW 264.7 (a murine preosteoclast cell line) that osteoclast differentiation and bone resorption activity at an early stage of osteoclastogenesis were inhibited by EGCG in a dose-dependent manner through its effects on NF-κB-mediated transactivation and RANKL signalling via intranuclear NF-κB translocation [52]. In contrast, Oka et al. found in rat osteoclast precursor cells and mature osteoclasts that EGCG had an inhibitory effect on osteoclast formation and differentiation by inhibiting the enzymatic activity of MMP-2 and MMP-9 and the levels of MMP-9 mRNA in osteoclast precursor cells [53].

Major conclusions are as follows:

• EGCG influences bone health by upregulating alkaline phosphatase to stimulate bone mineralisation [26].
• It modulates osteoblast function through MAP kinase inhibition, contributing to bone tissue formation [27].
• It can potentially induce bone regeneration by enhancing bone callus formation and modifying collagen membranes [21,45].
• Concentration matters: high EGCG dosages inhibit mesenchymal cell proliferation in osteogenesis, while intermediate concentrations promote it [29].
• It also prevents the inhibition of osteogenic differentiation by affecting TNF-α, and it has various effects on bone microarchitecture and osteoclasts [33,34,35,36,37].

3. Biochemical Effects of EGCG

The effects of EGCG on various biochemical pathways and potential pharmacological effects are summarised in Figure 2.

EGCG administration led to a reduction in osteoclast differentiation and activation, achieved through an increased expression of OPG mRNA [19] and the inhibition of RANKL production [54]. Additionally, it showed a greater ability to enhance OPG mRNA expression in pluripotent stem cells compared to other green tea catechins [42,43]. Numerous in vitro studies demonstrated that its supplementation was capable of suppressing RANKL-induced osteoclastogenesis [44,47,50]. As mentioned previously, another crucial factor in physiological bone growth is ALP, which could be significantly activated by it. This effect was attributed to the upregulation of Runx2 gene expression and the inhibition of acid phosphatase mRNA expression [26,29,33,40,41,55]. Runx2, a key gene involved in osteoblast differentiation, was efficiently activated by it at low concentrations (typically up to 5 µM) [29,33,40,41]. Furthermore, in vitro studies showed that EGCG could overexpress other genes related to bone development, such as Osx and Bmp-2, at concentrations of up to 10 µM [26,29,31,32,33,35,40,41].

The compound also promoted the synthesis of VEGF [34,56], stimulating bone growth by influencing the SAPK/JNK pathway and reducing interleukin 6 (IL-6) synthesis, which is responsible for bone resorption [54,57,58,59]. EGCG was not only implicated in osteogenesis processes but also in the treatment of bone-related neoplasms, such as chondrosarcoma, through the activation of caspase-3 [60]. Finally, it affected MAP kinases involved in the regulation of osteoblast proliferation and differentiation, either attenuating or inhibiting their activity [27,39,57,58]. It was also demonstrated that it could impede bone resorption by inducing a Fenton-type reaction, leading to the production of radicals that trigger osteoclast apoptosis [36]. Conversely, EGCG protected the osteogenic differentiation of human bone marrow-derived mesenchymal stem cells by mitigating hydrogen peroxide-related adverse effects [40].

3.1. RANK/RANKL/OPG Path Regulation

RANKL, the receptor activator of nuclear factor-κB (RANK), and OPG belong to the TNFs and their receptor superfamilies. As a TNF superfamily molecule, RANKL forms a homotrimer and binds to its receptors. RANK and OPG act as monomers and homodimers, respectively. The RANKL/RANK/OPG is a crucial system for bone resorption [61]. RANK/RANKL/OPG form the essential cytokine system that is capable of regulating all ranges of osteoclast functions (proliferation, differentiation, fusion, activation, and apoptosis). The balance in bone resorption depends on the local ratio of RANKL to OPG [62]. The central regulation of osteoclast proliferation and differentiation is controlled by OPG, RANKL, and the macrophage colony-stimulating factor (M-CSF) [63]. RANK is found in osteoclasts and their precursors. RANKL is produced in both soluble and membrane-bound forms by osteoblasts and stromal cells in the bone marrow and has a pivotal role in osteoclastogenesis, differentiation, activation, and the apoptosis of osteoclasts. OPG is a soluble product of stromal cells and osteoblasts, and it plays the role of a decoy receptor of the RANK ligand. The function of OPG is to block the RANK-RANKL interactions, which are required for the differentiation and activation of osteoclasts [64,65].

The inhibition of the RANKL–RANK signalling pathway in bone can increase bone mass by preventing osteoclastic bone resorption [66]. The activities of RANKL are mediated by its binding to RANK. The interaction between RANKL and RANK is the preliminary key step in the osteoclastogenesis pathway. The inhibition of RANKL binding with RANK by OPG can therefore suppress bone resorption and favour bone formation [65,67]. The connection of RANKL to RANK is followed by the recruitment of the TNF receptor-associated factor (TRAF). Although several of these proteins (TRAFs 1, 2, 3, 5, and 6) are involved in the RANK signalling pathway, TRAF-6 seems to be the crucial protein for RANKL signalling in osteoclasts [61,65,68]. TRAF proteins are believed to serve as second messengers that activate multiple downstream signalling pathways, including those involving transcription factors like NF-κB and c-Jun. When activated, NF-κB increases the expression of c-Fos [69,70], which subsequently combines with the NFAT-c1 to stimulate the transcription of genes involved in osteoclast activity [65]. The RANKL/RANK/OPG system, therefore, acts as a common final pathway for several cytokines and hormonal factors involved in bone resorption [61,65].

The inhibition of RANKL synthesis caused by EGCG treatment was also noted by Ishida et al. [54] on NRG cells (a mouse marrow-derived fibroblast-like cell line with osteoblast-like characteristics) and was accompanied by a reduction in the production of IL-6, which resulted in the inhibition of inflammation in the treatment of osteomyelitis. Green tea catechins (EC, EGC, ECG, EGCG) can enhance the expression of OPG mRNA in pluripotent mesenchymal cells, but of these, EGCG has the strongest effect, which leads to the inhibition of osteoclast differentiation and activity [42]. OPG production is stimulated by bone morphogenetic protein 4 (BMP-4) [71]. By controlling this protein expression in osteoblasts, Fujita et al. [43] examined the effects of EGCG and coffee polyphenol chlorogenic acid (CGA) on OPG synthesis in cloned osteoblast-like MC3T3-E1 cells derived from newborn mouse calvaria. The findings demonstrated that EGCG considerably inhibits osteoclast differentiation and activation through increasing the release of OPG triggered by BMP-4, whereas CGA has no discernible impact. Lin et al. in their study performed on RAW 264.7 indicated that by preventing RANKL-mediated NF-κB transcriptional activity and the nuclear transport of NF-κB, the inhibition of RANKL-induced differentiation of osteoclasts and the formation of resorption pits in RAW 264.7 cells and primary bone marrow macrophages (BMMs) were observed [52]. In another study, the inhibition of the JNK/c-Jun and NF-κB pathways by EGCG in mouse BMMs obtained from the femurs and tibias of 5-week-old ICR mice led to the inhibition of RANKL-induced osteoclast differentiation and an induction of c-Fos and NFATc1 [50]. Another mechanism of the inhibition of RANKL-induced osteoclastogenesis and the formation of F-actin rings was related to downregulating NFATc1 and c-Fos by EGCG in mouse macrophage RAW 264.7 cells [47].

3.2. Increasing Alkaline Phosphatase Activity

ALP is a metalloenzyme that plays a crucial role in the formation of hard tissue and serves as one of the bone maturation markers [72,73]. The form of phosphatase that acts within bone tissue is the liver/bone/kidney isoenzyme, also known as tissue-nonspecific alkaline phosphatase (TNSALP) [72,74,75]. ALP increases the local concentrations of inorganic phosphate, promoting mineralisation while simultaneously reducing the extracellular levels of pyrophosphate, a known inhibitor of mineral formation [72]. Signalling pathways such as the wingless-related integration site (Wnt), BMP-2, fibroblast growth factor (FGF), and IGFBP/IGF have been reported to control ALP expression. In particular, the Wnt-dependent activation of RUNX2 and LEF1/TCF transcription factors enhances ALP expression. The elevated serum ALP activity occurs during physiological bone growth. The activation of BMP-BMPR-Smads and/or FGF-FGFR-MAPK signalling increases ALP expression and osteoblast differentiation. IGFs are powerful ligands that activate the PI3K/Akt pathway to promote ALP expression and osteoblast differentiation [76]. The additional control of ALP expression is exerted through the actions of 1,25-(OH)₂-vitamin D, retinoic acid, and parathormone [73]. ALP activity is detectable in the osteoid areas of new bone formation but not in the calcified bone matrix [77]. This is because ALP acts in the initial phase of cartilage calcification [73]. ALP plays a role in the hydrolysis of inorganic pyrophosphate (PPi) to produce inorganic phosphate (Pi). The ratio between the levels of PPi and Pi is important for bone mineralisation [72,73,75].

In many in vitro studies, the influence of EGCG on ALP activity has been discussed (

Table 1. EGCG influence on alkaline phosphatase (ALP) activity.

Ref.	Effects in Cytobiochemistry	Cell Line	Functional Changes and Methodology	Cell Culture Data
	Influence	EGCG Conc.
[26]	Increasing the expression of osteogenic genes and elevating ALP activity	D1	ALP activity assayed by chemiluminescence ALP activity (4 days): increased	Solvent: DMSO, diluted in culture medium Osteogenic substrate/status: Cells cultured in DMEM, FBS, β-glycerophosphate Transcriptional changes: mRNA expressions (Cbfa1/Runx2, Osx, Alp, osteocalcin) measured by RT-PCR
	Stimulating mineralization	1–10 µM
[29]	Increasing ALP activity, mRNA, and protein expression of COL1, RUNX2, OPN, and OSX	hPDLCs	ALP activity assayed quantitatively and qualitatively ALP activity (7 days): Significantly increased at 2 and 4 µM of EGCG compared to control; 10 µM showed decreased ALP	Solvent: distilled water, diluted in culture medium Osteogenic substrate/status: Cells cultured in osteogenic differentiation medium containing α-MEM, FBS, L-ascorbic acid, dexamethasone, β-glycerophosphate
	Promoting hPDLC osteogenesis	4, 6 µM
[33]	Increasing ALP activity/mineralization and the expression of the RUNX2 and OSX	hBM-MSCs	ALP activity assessed by staining and colorimetric assay Functional osteogenesis: EGCG at 5 and 10 μM promoted ALP activity and mineralization; 20 and 40 μM had no effect	Solvent: EGCG added directly to culture media at specified concentrations Osteogenic substrate/status: Cells cultured in osteogenic differentiation medium containing β-glycerol phosphate, dexamethasone, ascorbic acid 2-phosphate
	Preventing TNFα inhibition of survival, osteogenic differentiation of hBM-MSCs (EGCG low conc.)	5 µM
[41]	Increasing the expression of BMP-2, RUNX2, ALP, ONC, and OCN mRNA, ALP activity, and mineralization	hBM-MSCs	Transcriptional changes: mRNA levels of RUNX2, BMP-2, ALP, osteonectin, and osteocalcin measured by RT PCR Protein/functional changes: ALP activity measured by chemiluminescent assay mRNA expression (qPCR): ALP increased ALP activity: Increased	Solvent: EGCG in DMSO, diluted in culture medium Osteogenic substrate/status: Cells cultured in DMEM, β-glycerophosphate
	Enhancement of osteogenic differentiation of hBM-MSCs	1, 10 µM
[55]	Increasing ALP activity and inhibition of mRNA expression of acid phosphatase	UMR-106 RAW 264.7	Protein/functional changes: ALP activity in UMR-106 cells assessed by colorimetric assay at day 4 EGC increased ALP activity by approximately 39.3% at 10 μM and 78.7% at 20 μM relative to control GC and GCG showed slight, insignificant decreases in ALP activity	Solvent: Catechins (EGC, GC, GCG) dissolved in DMSO, diluted in culture medium Osteogenic substrate/status: UMR-106 cells cultured in DMEM with FBS
	Promoting osteoblastic activity and inhibiting osteoclast differentiation	5–20 µM
[38]	Suppressing the expression of mRNA of ALP and OCN	MC3T3-E1	ALP activity and gene expression (Alp, osteocalcin) measured at 4, 7, and 10 days of continuous EGCG exposure Protein/functional changes: ALP activity in MC3T3-E1 cells decreased dose-dependently with EGCG (1–10 μM) over time	Solvent: EGCG sourced from Mitsui Nourin and added directly to culture medium (α-MEM, FBS) Osteogenic substrate/status: MC3T3-E1 cells cultured in α-MEM with FBS, ascorbic acid; for mineralisation assay, cells treated with β-glycerophosphate after initial differentiation
	Decreasing the formation of osteoclasts	10 µM

Abbreviations: ALP—alkaline phosphatase; BMP-2—bone morphogenetic protein; COL1—collagen type 1; D1—murine bone marrow mesenchymal stem cell line; DMSO—dimethyl sulfoxide; FBS—fetal bovine serum; hBM-MSCs—human bone marrow–mesenchymal stem cells; hPDLCs—human periodontal ligament cells; α-MEM—minimum essential medium, alpha modification; OCN—osteocalcin; ONC—osteonectin; OSX—Osterix; RT-PCR—real-time PCR; RUNX2—runt-related transcription factor 2; TNFα—tumour necrosis factor-α.

). In the majority of them, the increased expression of osteogenic proteins was observed and was accompanied by stimulating mineralisation, osteogenesis promotion, and the enhancement of osteogenic differentiation.

3.3. Effects on RUNX2 Protein Level

Runx2, a member of the runt-domain gene family, regulates the differentiation of osteoblasts from multipotent mesenchymal stem cells [48] and plays a fundamental role in osteoblast maturation and homeostasis [78,79,80,81]. RUNX2 enhances osteoblast differentiation at an early stage but suppresses osteoblast maturation at late stages [82]. The RUNX2 transcription factor operates by binding to the osteoblast-specific cis-acting element 2 (Ose2), located in the promoter region of all major osteoblast-related genes, where it regulates their expression. Its level and/or activity are dictated by a number of different external cues, while multiple signalling pathways that affect osteoblast function merge to and are integrated by RUNX2 [78]. The ectopic expression of RUNX2 in mesenchymal cell lines leads to the upregulation of osteoblast-specific genes like Ocn, Alp, collagenase-3 (Col3), matrix metalloproteinase-13 (Mmp-13), bone sialoprotein, and collagen type Iα1 [78,83]. The Type I isoform of RUNX2 seems to be mainly involved in the intramembranous bone formation, while the Type II isoform has an exclusive role in the endochondral bone formation [84]. During the intramembranous bone formation, the Type I isoform is widely expressed in osteoprogenitor cells and in active osteoblasts, whereas the Type II isoform expression is stringently restricted to cells lining mineralised bones [84]. RUNX2 also plays a crucial role in chondrocyte maturation by the direct regulation of Indian Hedgehog (Ihh) expression. Ihh is a signalling molecule that regulates cartilage development and bone growth. RUNX2 induces the expression of Sp7 (specificity protein 7). Sp7 (also known as Osterix) is a transcription factor essential for osteoblast differentiation and bone formation. Additionally, the cooperation among RUNX2, Sp7, and canonical Wnt signalling, a pathway important for cell proliferation and differentiation, is essential for the differentiation of preosteoblasts into immature osteoblasts [80,81]. It also induces the proliferation of osteoprogenitors by the direct regulation of fibroblast growth factor receptors like FGFR-2 and FGFR-3 expression. These receptors are involved in signalling pathways that influence the proliferation and differentiation of osteoprogenitor cells [80].

In many in vitro studies, the influence of EGCG on RUNX2 protein expression was extensively examined, revealing variable outcomes depending on the cell type and experimental conditions (

Table 2. EGCG influence on the RUNX2 protein level.

Ref.	Effects in Cytobiochemistry	Cell Line	Functional Changes and Methodology	Cell Culture Data
	Influence	EGCG Conc.
[48]	Increase in osteoblast differentiation and decrease in RUNX2 protein levels	SaOS-2	Protein changes: RUNX2 protein level analysed by Western blot. No change at 6 or 24 h; significant decrease (~65% reduction) at 48 h with 5 µM of EGCG relative to control	Solvent: PBS; EGCG directly dissolved in culture medium Osteogenic substrate/status: Cells cultured in Ham’s F-12 medium with FBS, dexamethasone, ascorbic acid, β-glycerophosphate (added to induce mineralisation)
	Increasing the formation of mineralised bone nodules from HOB-like cells by enhancing osteoblast differentiation	5 µM
[33]	Increasing ALP activity/mineralisation and increasing the expression of the RUNX2 and OSX	hBM-MSCs	Transcriptional changes (qPCR): Runx2 and Osx mRNA measured at day 16 of osteogenic differentiation. TNFα (5–20 ng/mL) decreased Runx2 and Osx expression by up to ~20% at low doses and more at higher doses; EGCG at low doses (5 and 10 µM) reversed TNFα-induced suppression, restoring mRNA levels close to control	Solvent: EGCG was added directly to the culture medium Osteogenic substrate/status: Cells induced to differentiate with osteogenic medium containing β-glycerophosphate, dexamethasone, ascorbic acid 2-phosphate
	Preventing TNFα inhibition of survival and osteogenic differentiation of human hBM-MSCs (at low conc. of EGCG)	5 µM
[32]	Increasing the expression of RUNX2, BMP-2, and VEGF	hMSCs	RUNX2 (osteogenic marker): EGCG alone significantly increased expression over control; mechanical stretch alone increased both RUNX2 and myocardin; EGCG with mechanical stretching induced the highest RUNX2 expression	Solvent: EGCG added to basal culture media Osteogenic substrate/status: Cells cultured on fibronectin-coated elastomeric PDMS membranes; osteogenic differentiation induced by mechanical stretching and/or EGCG treatment in basal media without other osteogenic supplements EGCG added 24 h after seeding; mechanical stretch applied 24 h after seeding for 4 h per day, repeated for 4 consecutive days; cells harvested immediately after last stimulation (total ~5 days culture)
	Acceleration of osteogenic differentiation	25 µM
[41]	Increasing the expression of BMP-2, RUNX2, ALP, ONC, and OCN mRNA as well as ALP activity and mineralisation	hBM-MSCs	Runx2 mRNA increased by 57% and 85% at 24 h; 169% and 203% at 48 h for 1 and 10 µM, respectively	Solvent: EGCG powder dissolved in DMSO stock, diluted in culture medium before use Osteogenic substrate/status: Cells cultured in standard medium supplemented with β-glycerophosphate
	Enhancement of osteogenic differentiation of hBM-MSCs	1 and 10 µM
[29]	Increasing ALP activity, mRNA, and protein expression levels of COL1, RUNX2, OPN, and OSX	hPDLCs	Transcriptional changes (qRT-PCR, after 7-day treatment): RUNX2, COL1, OSX, and OPN mRNA levels increased significantly at 4 to 8 µM of EGCG RUNX2, COL1, and OSX mRNA peaked at 6 µM; OPN peaked at 8 µM Described as a remarkable increase compared to control Protein changes (Western blot, after 14 days of treatment): RUNX2 protein significantly increased only at 4 µM	Solvent: EGCG dissolved in distilled water to prepare a stock solution, diluted in prewarmed growth or osteogenic differentiation medium before use Osteogenic substrate/status: Cells cultured in osteogenic differentiation medium (α-MEM, FBS, antibiotics) supplemented with L-ascorbic acid, dexamethasone, β-glycerophosphate; medium renewed every 3 days
	Promoting hPDLC osteogenesis	4 and 6 µM

Abbreviations: ALP—alkaline phosphatase; BMP-2—bone morphogenetic protein; COL1—collagen type 1; D1—murine bone marrow mesenchymal stem cell line; DMSO—dimethyl sulfoxide; FBS—fetal bovine serum; hBM-MSCs—human bone marrow–mesenchymal stem cells; hMSC—human mesenchymal stem cells; hPDLCs—human periodontal ligament cells; α-MEM—minimum essential medium, alpha modification; OCN—osteocalcin; ONC—osteonectin; OPN—osteopontin; OSX–Osterix; RUNX2—runt-related transcription factor 2; SaOS-2—human osteoblast (HOB)-like cells; TNFα—tumor necrosis factor-α; VEGF—vascular endothelial growth factor.

). In SaOS-2 cells, EGCG enhanced osteoblast differentiation and mineralised nodule formation, despite a reduction in RUNX2 protein levels [48], suggesting a RUNX2-independent mechanism of osteogenesis in this context. In contrast, most studies reported an upregulation of RUNX2 expression following EGCG treatment. For example, in hBM-MSCs, EGCG increased RUNX2 and OSX expression while preventing the TNFα-induced suppression of osteogenic differentiation [33]. In hBM-MSCs, EGCG reversed H₂O₂-induced damage and promoted Runx2 gene expression along with other osteogenic markers such as ALP, OSX, β-catenin, and cyclin D1 [40]. Similarly, in hMSCs, RUNX2 expression was elevated alongside BMP-2 and VEGF, contributing to enhanced osteogenic differentiation [32]. EGCG also upregulated Runx2 and other osteogenic genes in hBM-MSCs [41] and hPDLCs [29], further supporting its role in activating RUNX2-driven pathways.

3.4. Increasing Expression of the Osterix

OSX is an osteoblast-specific transcription factor required for bone formation [85,86,87,88]. It activates a repertoire of genes during the differentiation of preosteoblasts into mature osteoblasts and osteocytes and decreases osteoblast proliferation [85]. There are two main pathways through which Osx gene expression is induced: directly or indirectly [89]. RUNX2 plays a key role during bone development by directly initiating the expression of other osteogenic genes, such as Alp and Col1 in the early stage and Opn and Osx in the later stage [90]. For the RUNX2-dependent pathway, OSX inhibits chondrocyte differentiation in RUNX2-expressing precursor osteoblasts. RUNX2 expression is initially detected in prechondrogenic mesenchymal cells. As development progresses, OSX expression is induced, leading to the differentiation of RUNX2-positive cells into osteoblast precursors [86,87]. In both membranous and endochondral skeletons, OSX-null preosteoblasts are unable to differentiate into osteoblasts, which means that without OSX, mature osteoblasts are not formed [85]. Moreover, OSX inhibits the Wnt pathway [85,87]—a RUNX2-independent pathway. The Wnt pathway modulates bone formation through the control of progenitor cell proliferation and differentiation [87].

The influence of EGCG on the expression of OSX was examined in many in vitro studies, with consistently positive effects observed across various cell types (

Table 3. EGCG influence on Osterix expression.

Ref.	Effects in Cytobiochemistry	Cell Line	Functional Changes and Methodology	Cell Culture Data
	Influence	EGCG Conc.
[26]	Increasing the expression of osteogenic genes and elevating ALP activity	D1	Transcriptional changes (semi-quantitative RT-PCR): Osx mRNA increased by 66% (1 µM) and 137% (10 µM)	Solvent: EGCG dissolved in DMSO stock, diluted in culture medium before use Osteogenic substrate/status: Cells cultured in DMEM, FBS, sodium ascorbate, and antibiotics; β-glycerophosphate for ALP and mineralisation assays
	Stimulating mineralization	1–10 µM
[29]	Increasing ALP activity and mRNA and protein expression levels of COL1, RUNX2, OPN, and OSX	hPDLCs	Transcriptional changes: RUNX2, COL1, and OSX mRNA expression increased significantly with 4–8 μM of EGCG, peaking at 6 μM for RUNX2, COL1, and OSX Protein expression changes (Western blot): OPN and OSX proteins upregulated at 6 μM of EGCG; 10 μM of EGCG decreased the OSX protein compared to control	Solvent: Dissolved in distilled water as stock, diluted in culture medium before use Osteogenic substrate/status: Cells cultured in osteogenic differentiation medium containing α-MEM, FBS, antibiotics, L-ascorbic acid, dexamethasone, β-glycerophosphate
	Promoting hPDLC osteogenesis	4 and 6 µM
[33]	Increasing ALP activity/mineralisation and increasing the expression of the RUNX2 and OSX	hBM-MSCs	Transcriptional changes: TNFα (1–20 ng/mL) dose-dependently decreased Runx2 and Osx expression up to ~20% at 5 ng/mL more at higher doses; EGCG (5 and 10 μM) reversed TNFα-induced suppression of Runx2 and Osx mRNA expression at day 16	Solvent: EGCG used at specified micromolar concentrations diluted in culture/differentiation media Osteogenic substrate/status: Cells induced to differentiate using osteogenic medium containing β-glycerol phosphate, dexamethasone, L-ascorbic acid 2-phosphate
	Preventing TNFα inhibition of survival and osteogenic differentiation of human hBM-MSCs (at low conc. of EGCG)	5 µM

Abbreviations: ALP—alkaline phosphatase; COL1—collagen type 1; D1—murine bone marrow mesenchymal stem cell line; DMSO—dimethyl sulfoxide; FBS—fetal bovine serum; hBM-MSCs—human bone marrow–mesenchymal stem cells; hPDLCs—human periodontal ligament cells; α-MEM—minimum essential medium, alpha modification; OPN—osteoponin; OSX—Osterix; RT-PCR—real-time PCR; RUNX2—runt-related transcription factor 2; TNFα—tumour necrosis factor α.

). In D1 murine mesenchymal stem cells, EGCG enhanced osteogenic differentiation by upregulating osteogenic gene expression, which included increased OSX levels [26]. In hPDLCs, EGCG significantly elevated both the mRNA and protein expression of OSX, indicating its direct role in promoting osteogenesis [29]. Similarly, in hBM-MSCs, low concentrations of EGCG stimulated OSX expression while protecting against the TNFα-induced inhibition of osteogenic differentiation [33]. In hBM-MSCs, EGCG reversed oxidative damage induced by H₂O₂, leading to a marked increase in OSX expression and enhanced mineralization capacity [40].

The most important class of biomolecules in bone regeneration is the BMP [91]. These proteins can regulate the function and differentiation of cells involved in bone formation and healing [92,93,94]. BMP-2 is an exceptionally potent growth factor that acts as a chemoattractant for mesenchymal stromal cells, rapidly inducing their differentiation into osteoblasts while also supporting their viability. BMP-2 is the most potent molecule in osteoinduction, essential for new bone formation [91]. The differentiation of mesenchymal cells into osteoblast precursors is induced by BMP-2 and has a beneficial effect on osteoblast maturation by increasing the expression of RUNX2 and osteoblast marker genes [95]. Xiong et al. [92] reported that besides inducing bone growth, BMP-2 and other BMPs stimulated the increase of VEGF in the bone formation process. During skeletal development, BMPs and Wnts have been implicated in the specification of osteo/chondro progenitors and remain involved in each of the subsequent stages of endochondral ossification through the regulation of RUNX2 and OSX expression [93]. BMPs can activate RUNX2, and BMP-2 has been shown to activate OSX in RUNX2 mutant cells, but whether this is direct or mediated through β-catenin is not certain [93,96]. Another possible mechanism by which BMP-2 may act is through the levels of Smad1, a BMP-specific transcription factor. In Xenopus embryos, the binding of BMP to the BMP receptor complex triggers three sequential phosphorylations in Smad1 that control the timing of the BMP signal [97]. BMP activity has also been shown to initiate transcription factor Sox9 expression as cell condensation takes place during embryonic skeletogenesis [93,94,98]. What is more, BMP-2 induces the synthesis of OPG in human osteoblasts [99].

Takita et al. studied whether the inhibition of angiogenesis enhances chondrogenesis and inhibits osteogenesis using EGCG [49]. Bone morphogenetic protein-induced osteo- and chondrogenesis was chosen as the experimental system to elucidate the biochemical mechanism of bone formation. In the study, a fibrous glass membrane (FGM) was used as a BMP carrier, mixed with 1.2 µg of rhBMP-2 and 1 or 10 µg of EGCG, and subcutaneously implanted in rats. The results showed that its supplementation increases cartilage formation and reduces bone formation. The FGM, coupled with BMP and implanted subcutaneously in rats, produced only cartilage in the membrane after 1 and 2 weeks. Increasing the dose of EGCG resulted in a decrease in the activity of alkaline phosphatase and calcium content, while it increased the content of type II collagen [49].

In numerous studies, the effect of EGCG on the expression of bone morphogenetic protein 2 has been investigated, consistently demonstrating its role in promoting osteogenesis (

Table 4. EGCG influence on the expression of BMP-2.

Ref.	Effects in Cytobiochemistry	Cell Line/Animal	Functional Changes and Methodology	Cell Culture Data
	Influence	EGCG Conc.
[35]	Attenuation of oestrogen deficiency induced decreases in BMD, BV/TV, TbTh, and TbN in the proximal tibia, an increase in TbSp in the proximal tibia, and an increase in BV/TV and TbTh in L3, increasing the synthesis of BMP-2	12-week-old female Sprague–Dawley ovariectomy-induced osteopenic rats	Protein changes: Immunohistochemistry for BMP-2 expression in proximal tibial trabecular bone showed An increase from 31% positive area in the OVX group to 53% in OVX with the 10 mg/kg/day of EGCG group (approximately 70% relative increase)	Solvent: EGCG administered intraperitoneally, dissolved appropriately for injection Osteogenic substrate/status: N/A (in vivo bone tissue analysis post-treatment)
	Mitigation of bone loss and improvement in bone microarchitecture	10 µM, 3.4 mg/kg/day
[31]	Increasing the expression of BMP-2 (EGCG with osteoinductive agents)	hBM-MSCs	Protein changes (immunohistochemistry): BMP-2 protein expression increased dose-dependently with EGCG treatment, visible at days 7, 14, and 21, but less than positive control	Solvent: EGCG dissolved in DMSO stock; later culture medium Osteogenic substrate/status: Cells cultured either in pure culture medium (no osteogenic supplements) or osteogenesis-induced medium (positive control: α-MEM, FBS, ascorbic acid, β-glycerophosphate, dexamethasone)
	Increasing the osteogenic differentiation of hMSCs	5 µM
[32]	Increasing the expression of RUNX2, BMP-2, and VEGF	hMSCs	Intracellular signalling genes Bmp-2 and Vegf were significantly upregulated by stretch, further enhanced synergistically by stretch with EGCG; TGF-β1 also increased with stretch and stretch with EGCG	Solvent: EGCG added directly to culture medium (DMEM-LG, FBS) Osteogenic substrate/status: Basal culture medium only; no osteogenic supplements used
	Acceleration of osteogenic differentiation	25 µM
[41]	Increasing the expression of BMP-2, RUNX2, ALP, ONC, and OCN mRNA as well as ALP activity and mineralization	hBM-MSCs	Transcriptional changes (qPCR): Bmp-2 mRNA increased ~459% (1 µM) and 502% (10 µM) at 48 h	Solvent: EGCG stock dissolved in DMSO, diluted in culture medium before use Osteogenic substrate/status: Cells cultured in DMEM, β-glycerophosphate
	Enhancement of osteogenic differentiation of hBM-MSCs	1 and 10 µM
[21]	Increasing the expression of BMP-2 and enhancing bone callus formation	Right tibial bones of rats	Key molecular/protein changes: BMP-2 protein expression (IHC) in callus tissue increased with EGCG treatment	Solvent: DMSO Osteogenic substrate/status: Local percutaneous injection at fracture site
	Facilitating the healing of fractures of the tibia	10 µM, 0.52 µg/kg in total

Abbreviations: ALP—alkaline phosphatase; BMP-2—bone morphogenetic protein 2; COL1—collagen type 1; D1—murine bone marrow mesenchymal stem cell line; DMEM—dulbecco’s modified eagle medium, low glucose; DMSO—dimethyl sulfoxide; FBS—fetal bovine serum; hBM-MSCs—human bone marrow–mesenchymal stem cells; hMSCs—human mesenchymal stem cells; α-MEM—minimum essential medium, alpha modification; OCN—osteocalcin; ONC—osteonectin; RUNX2—runt-related transcription factor 2; TNFα—tumour necrosis factor- α; VEGF—vascular endothelial growth factor.

). In an in vivo model using ovariectomy-induced osteopenic rats, EGCG mitigated bone loss by improving bone microarchitecture parameters, including BMD and trabecular bone structure, while also upregulating BMP-2 synthesis in both the proximal tibia and lumbar spine [35]. In vitro, EGCG was shown to enhance BMP-2 expression in hBM-MSCs, particularly when combined with osteoinductive agents, thereby promoting osteogenic differentiation [31]. Similarly, in hMSCs, EGCG increased BMP-2 expression alongside RUNX2 and VEGF, contributing to accelerated osteogenic commitment [32]. Additional studies confirmed that EGCG upregulated BMP-2 mRNA levels in hBM-MSCs, along with markers such as RUNX2 and ALP, leading to enhanced mineralisation and osteogenic differentiation [41]. Furthermore, in a rat tibial fracture model, EGCG treatment increased BMP-2 expression and promoted callus formation, supporting its role in fracture healing [21].

3.5. Increasing the Level of Vascular Endothelial Growth Factor

The inactivation of VEGF completely inhibits the invasion of blood vessels with the simultaneous impairment of trabecular bone formation and the expansion of the hypertrophic chondrocyte zone in the mouse tibial epiphyseal growth plate [100]. Within the VEGF family of proteins, the following can be found: VEGF-A, VEGF-B, VEGF-C, VEGF-D, and PIGF. These proteins are bound by the receptors VEGFR1 and VEGFR2. VEGFR1 is activated by VEGF-A, -B, and PIGF, while VEGFR2 is activated by VEGF-A, -C, and -D [101]. VEGF-A is one of the critical mediators of blood vessel invasion into the cartilaginous mould. During typical distraction osteogenesis, the expression of both VEGFR1 and VEGFR2 is induced [102,103]. Furthermore, blocking VEGF-A activity in the distraction gap through the antibody-mediated inhibition of VEGFR1 and VEGFR2 significantly reduces bone formation and decreases the number of blood vessels [102,104]. Another hypothesis, termed the “spontaneous rupture theory”, was proposed by Pufe et al. [105]. According to them, VEGF increases MMP expression in endothelial cells and in tendon fibroblasts themselves. The invasion of vessels and the MMP expression lead to a weakening of the normal tendon structure, which further leads to a decrease in the mechanical strain and subsequently to the spontaneous rupture. However, Hu and Olsen [106] in their study came to a different conclusion. They found that the effects of extracellular VEGF are dose-dependent: high levels of VEGF inhibit regenerative cell infiltration and the osteoinduction of mesenchymal progenitor cells, resulting in reduced bone formation. The proper levels of VEGF produced by osteoblastic cells are crucial for the coupling of angiogenesis and osteogenesis during the healing of small bone defects. VEGF produced by osteoblasts is important for the early angiogenic response and the infiltration of macrophages during the initial phase of inflammation. In wound healing and tumour progression, macrophage infiltration occurs before angiogenesis, and VEGF acts as a chemotactic signal for macrophages and monocytes. This is why the proangiogenic effects of osteoblast-derived VEGF involve macrophages, in addition to directly targeting endothelial cells. Optimal amounts of VEGF are crucial for therapeutic outcomes. Not only a dose dependency but also a mechanism of delivery were noted by Rumney et al. [107].

In an in vitro study performed on cloned osteoblast-like MC3T3-E1 cells derived from newborn mouse calvaria, it was shown that the enhancement of SAPK/JNK activation in osteoblast-like MC3T3-E1 cells could lead to an increase in PGF_2α-stimulated VEGF synthesis in osteoblast-like MC3T3-E1 cells and thus regulate capillary endothelial cell proliferation [56]. In another study performed on hMSCs with EGCG combined with mechanical stretching, it was shown that increasing the expression of RUNX2, BMP-2, and VEGF leads to an acceleration of osteogenic differentiation [32]. In another in vivo study, it was presented that the implantation of EGCG-modified collagen membrane results in an increase in the number of macrophages (M2), promotes the secretion of VEGF and BMP-2, and increases the expression of RUNX-2 and OPN. It is interesting because the collagen membrane could be used in guided bone regeneration (GBR), acting as a barrier to prevent the migration of epithelial cells and connective tissue, as well as it could maintain space for bone regeneration, and promote important biological activities including cell adhesion, proliferation, migration, and differentiation [45].

3.6. Inhibition of Interleukin-6 Synthesis

Cytokines are short-lived proteins with a low molecular mass of approximately 15–20 kDa and play crucial roles in autocrine, paracrine, and endocrine signalling. These molecules play a key role in the development and activity of the immune system. Interleukin-6 (IL-6) family cytokines are classified based on the common signalling receptor subunit glycoprotein 130 kDa (gp130). Currently, this class includes eight cytokines: IL-6, IL-11, ciliary neurotrophic factor (CNTF), leukaemia inhibitory factor (LIF), oncostatin M (OSM), cardiotrophin-like cytokine (CLC), cardiotrophin 1 (CT-1), and IL-27 [108]. These cytokines demonstrate unique cell-surface expression by interacting with the specific transmembrane IL-6 receptor (mIL-6R) or its soluble forms (sIL-6R) and the signal-transducing subunit molecule gp130 [109]. In the case of the treatment of different autoimmune diseases, suppressing the IL-6 family of cytokines becomes advantageous. Furthermore, recent advances in cytokine blocking have helped reduce adverse effects related to metabolic dysfunction and bacterial infections [108].

Regarding interleukin-6, the presence of both anti-inflammatory and pro-inflammatory activity is induced by different biological mechanisms and sources [110]. It is well known that interferon-β2 (IFN-β2), hepatocyte-stimulating factor, and hybridoma/plasmacytoma growth factor (also known as IL-6) are mainly produced by monocytes and macrophage cells [111]. Moreover, B and T cells, hepatocytes, endothelial cells, fibroblasts, keratinocytes, mesangial cells, adipocytes, and several tumour cells can produce IL-6 constitutively or after stimulation [109,110,112]. IL-6 plays a significant role in the proliferation and differentiation of cells in humans. Additionally, the immunopathogenic role of IL-6 in tumour development, metastasis, angiogenesis, apoptosis, and therapeutic resistance has been proven in recent publications [109,113,114,115]. It also exerts a significant physiological effect on a wide range of functions, such as promoting B cell differentiation [58]. When osteoblasts are affected by IL-1 or IL-6, the expression of RANKL, an osteoclast differentiation factor, is induced, resulting in bone resorption [116,117,118].

According to Kaur et al. [111], triggering the trans-signalling mechanism with IL-6 promotes various pathological pathways, including those that could contribute to multiple sclerosis, rheumatoid arthritis, anaemia, inflammatory bowel disease, Crohn’s disease, Alzheimer’s disease, and cancer. Among these routes are JAK/STAT3, Ras/MAPK, and PI3K–PKB/Akt, and the regulation of CD4+ T cells and VEGF levels. The research of Tanaka et al. [117] provided an example of tocilizumab, an IL-6 receptor (IL-6R)-neutralising monoclonal antibody. It was approved in more than 100 countries for the treatment of autoimmune diseases induced by IL-6. Additionally, patients with rheumatoid arthritis demonstrated that a blockade of IL-6 activity was observed to be at least as efficient as the blockade of the tumour necrosis factor [117]. Normally, an average person′s buffer of IL-6 in the blood is in the range of 1–5 pg/mL. The concentration of cytokines rapidly increases several thousand times during inflammatory states, and under lethal septic stages, it can even reach levels of several mg/mL. Soluble sIL-6R concentration usually stays within 40–75 ng/mL, and the levels of sgp130 reach approximately 250–400 ng/mL [108]. In NRG cells, suppressing the production of interleukin 6 and RANKL by EGCG led to the inhibition of inflammation in osteomyelitis treatment [54]. The reduction of IL-6 synthesis by EGCG on osteoblast-like MC3T3-E1 cells is caused by different mechanisms, such as the attenuation of the p44/p42 MAP kinase pathway and/or p38 MAP kinase [58,59], as well as the inhibition of the PDGF-BB-induced phosphorylation of SAPK/JNK [57].

3.7. Induction of Caspase-3

Both programmed cell death mechanisms in the human body—apoptosis and pyroptosis (an inflammatory form of cell death)—are caspase-dependent pathways. Caspases are a family of cysteine-dependent proteases that are synthesised as inactive zymogens. They are activated by dimer formation (e.g., apoptotic initiator caspases-8, -9) or by cleavage (e.g., apoptotic effector caspase-3) [119]. Caspase-3 cleaves a series of substrates and activates an endonuclease, leading to DNA fragmentation that is the hallmark of apoptosis [120]. In their overview of caspases, Svandova et al. [121] state that these proteases play not only a key role in apoptosis and inflammation, but also are multifunctional enzymes with lethal and non-lethal functions. According to recent findings, caspases are localised subcellularly. These enzymes have to be transported into the nucleus for central caspase-3 to regulate the apoptotic disintegration of the nucleus. In fact, it was discovered that activated caspase-3 is localised in the nucleus of cells during apoptosis [122]. In the case of non-apoptotic processes, it is proposed that caspase activation is locally held in subcellular compartments, leading to the availability of specific substrates. Caspase-3 is typically localised in the cytoplasm of intact cells. Another method of non-apoptotic engagement could involve sub-lethal caspase activation. This may be related to a phenomenon known as mitochondrial outer membrane permeabilization (MOMP); specifically, a subtype referred to as “minority MOMP”. This effect supposes that a part of the cell mitochondria is permeable. Initially, the phenomenon was discovered to trigger apoptosis via rapid caspase activation. Minority MOMP reduced caspase activation, which is insufficient to trigger cell death [123]. Instead, this caspase activity damages DNA, resulting in increased genomic instability [121,124].

The therapeutic use of caspase-3 in the treatment of several illnesses, including cancer, heart failure, and neurological disorders, has significantly increased to date [125]. Zhang et al. [126], in the review article, provided follow-up data about the remarkable impact of caspase-3 ferment inhibition by EGCG due to the reduction of cell apoptosis and restoration of mitochondrial membrane potential. Therefore, EGCG could be useful for preventing cardiovascular diseases characterised by endothelial dysfunction [127], as well as neurological diseases and models with injuries in primary retinal pigment epithelial cells [128,129].

Chondrosarcoma is a malignant tumour of cartilage tissue. Islam et al. showed in an in vitro study that EGCG significantly reduced the viability of HTB-94 human chondrosarcoma cells by activating caspase-3/CPP32-like proteases and promoting apoptosis [60]. In another study performed on the murine monocyte/macrophage cell line, Raw 264.7, it was noted that DNA fragmentation and the induction of the activation of caspase-3 in RAW 264.7 cell-derived osteoclasts could lead to osteoclast apoptosis caused in part by caspase-3 and thus the prevention of alveolar bone resorption [46].

3.8. Influence on SAPK/JNK Pathway

The family of SAPKs plays a crucial role as intracellular signalling components to transduce the various signals of agonists [56]. SAPKs are induced by protein kinase cascades that have an association with sensors or receptors on the cell surface. This leads to the dual phosphorylation of SAPKs on adjacent tyrosine and threonine residues [130]. Meanwhile, c-Jun N-terminal kinases (JNKs) can be induced mainly by pro-inflammatory cytokines or exposure to environmental stress. The JNK subfamily consists of three kinase types: JNK1, JNK2, and JNK3. These three isoforms are encoded by the Jnk1, J2, and Jnk3 genes, respectively. The first two kinases are expressed in various tissues, whereas the third one is mainly found in the brain, heart, and testes [131].

In the human body, JNK regulates various cell processes such as cell differentiation, proliferation, survival, and metabolic reprogramming, best known for its role in programmed cell death [131,132,133]. According to Yung and Giacca, these kinases respond to various cellular stress signals triggered by cytokines, free fatty acids, as well as hyperglycaemia [134]. It also stands as a key mediator in the shift from obesity to type 2 diabetes. Various disorders in humans, such as neurological conditions like neurodegenerative disease, chronic inflammatory conditions (rheumatoid arthritis and inflammatory bowel disease), infectious disorders, and cancer, have connections with dysregulated JNK signalling [131].

Several studies on the influence of EGCG on the SAPK/JNK pathway were performed on cloned osteoblast-like MC3T3-E1 cells derived from newborn mouse calvaria. Tokuda et al. noted that enhanced SAPK/JNK activation in osteoblast-like MC3T3-E1 cells promoted PGF2α-stimulated VEGF synthesis by enhancing SAPK/JNK activation in osteoblast-like MC3T3-E1 cells and thus regulating capillary endothelial cell proliferation [56]. Takai et al. studied the inhibition of the PDGF-BB-induced phosphorylation of SAPK/JNK and found that the inhibition of PDGF-BB stimulated IL-6 synthesis via the suppression of the SAPK/JNK pathway in osteoblasts [57]. Hayashi et al. noted that the reduction in the TGF-β-stimulated induction of HSP27 in osteoblasts prevents the suppression of bone loss [34].

3.9. Attenuation of p44/p42 MAP Kinase or p44/p42 MAP Kinase and p38 MAP Kinase

The main characteristic of enzymes named kinases involves the transference of the γ-phosphate from ATP to serine, threonine, or tyrosine amino acid residues of a downstream protein substrate, therefore ensuring a communication cascade that is fundamental to eukaryotic cells [135]. Generally, kinases are dually phosphorylated at serine and threonine residues and, consequently, named Ser/Thr kinases [133,136]. MAPKs constitute a group of serine/threonine protein kinases consisting of three major subfamilies: (i) ERK p42/p44 ERK, also known as p42/p44 MAPK; (ii) c-Jun N-terminal kinases; and (iii) p38 MAPKs. The p42/p44 MAPKs are mainly involved in the regulation of cell proliferation and differentiation, while the JNKs and p38 MAPKs are involved in apoptosis, inflammation, and responses to environmental stress [137]. Rehfeldt et al. [133] described numerous interconnections between the MAPK pathways, which can occasionally make it difficult to differentiate between them. For example, the JNK and p38 pathways are commonly linked to inflammation and cellular death and are both initiated by cytokines as well as additional cellular stressors [138,139,140]. At the same time, both enzymes may not always work the same way; they can sometimes induce anti-apoptotic processes. A recent study indicates that p38 has a detrimental effect on JNK in some cellular situations [141].

The EGCG influence was studied on the cloned osteoblast-like MC3T3-E1 cells, derived from newborn mouse calvaria. It was found that the attenuation of the p44/p42 MAP kinase pathway led to a reduction in the ET-1-stimulated IL-6 synthesis [58]. Studies performed on the same cell line also showed that the inhibition of p44/p42 MAP kinase led to the suppression of PGD2-stimulated HSP27 induction through the inhibition of p44/p42 MAP kinase and thus the modulation of osteoblast cell function towards bone formation [27]. In a similar study, the attenuation of p44/p42 MAP kinase and p38 MAP kinase led to a reduction in FGF-2-stimulated IL-6 synthesis at least partly via the attenuation of p44/p42 MAP kinase and p38 MAP kinase [59]. The suppression of p44/p42 MAP kinase can also lead to the suppression of IGF-I-induced osteoblast migration [39].

3.10. Inhibitory Effect on Matrix Metalloproteinases

Matrix metalloproteinases (MMPs) include the family of zinc-dependent endopeptidases commonly classified on the basis of their substrates and the organization of structural domains, including collagenases (MMP-1, -8, -13, and -18), gelatinases (MMP-2 and -9), stromelysins (MMP-3 and -10), matrilysins, membrane-type (MT) MMPs, and other metalloproteinases. This enzyme family has the combined ability to degrade the organic components of the connective tissue matrix [53]. Generally, the structure of these enzymes includes a propeptide sequence, a hinge region or linker peptide, a hemopexin domain, and a catalytic metalloproteinase domain containing a catalytic zinc ion [142].

Shifts in MMP expression and activity are observed during common biological processes like pregnancy and wound healing. Moreover, such changes have also been implicated in cardiovascular diseases such as atherosclerosis, aneurysms, and varicose veins, musculoskeletal disorders such as osteoarthritis and bone resorption, and various cancers [142]. Therefore, changes in the profiles of MMPs can be used as an important biomarker to track cardiovascular disease, musculoskeletal disorders, and cancer development in the human body.

The regeneration of bones and their repair depend on many regulated mechanisms. MMP regulation plays a key role in this process. MMPs, produced by bone cells in an active state, are essential for chondrocyte formation and development processes and the viability and functionality of osteoclasts, osteoblasts, and osteocytes [143]. Particularly, MMP-9 or gelatinase-B is also a type IV collagenase. It is primarily produced by cell types such as osteoblasts, dendritic cells, fibroblasts, keratinocytes, T-cells, macrophages, and granulocytes [142,144]. The expression of MMP-9 in osteoclasts is significantly higher than in other cell types [53]. According to Khoswanto, MMP-2, MMP-9, and MMP-13 play especially significant roles in bone tissue [143]. MMP-2 appears to alter bone growth by inducing osteoclast and osteoblast activity and differentiation. Animals deficient in MMP-9 exhibited abnormal bone formation mainly during endochondral ossification.

In an in vitro study, EGCG was shown to inhibit osteoclast formation and reduce MMP-9 expression in osteoblasts, which contributed to the suppression of bone resorption [37]. Additionally, in cultures of rat osteoclast precursor cells and mature osteoclasts, the inhibition of MMP-2 and MMP-9 enzymatic activity was noted, and along with decreased MMP-9 mRNA expression in precursor cells, it significantly impaired both the formation and differentiation of osteoclasts [53].

3.11. Antioxidant Properties of EGCG

EGCG reveals antioxidant activity [145,146]. However, it has also been reported that the production of hydroxyl radicals is increased by flavonoids [147]. In the Fenton reaction (Figure 3), hydroxide ions (OH⁻) and hydroxyl radicals (OH^•) are formed in the reaction of iron(II) (Fe²⁺) with hydrogen peroxide (H₂O₂) [148]. A study by Nakagawa et al. performed on crude murine osteoclast-like multinucleated cells (OCLs) showed that the reduction of Fe(III) to Fe(II) was promoted by EGCG. EGCG promoted the reduction process, and single-strand DNA breakage in a cell-free system was induced by the combination of EGCG/Fe(III)/H₂O₂. Single-strand DNA damage was induced by EGCG in a dose-dependent manner in the presence of both Fe(III) and H₂O₂, which indirectly indicates the presence of hydroxyl radicals, which are difficult to detect directly. It led to the inhibition of bone resorption by inducing the apoptotic death of murine osteoclast-like cells [36]. In contrast, Wang et al. [40] proved that the viability of hBM-MSCs improved after treatment with EGCG. The H₂O₂-induced adverse effect was nullified by treatment with EGCG, as evidenced by ALP activity, calcium deposition, and the gene expressions of Runx2 and Osx. EGCG also enhanced the osteogenic differentiation of hBM-MSCs.

3.12. Hormetic Properties and Toxicity of EGCG

EGCG exhibits a biphasic dose–response relationship in cellular systems: at low concentrations, it demonstrates antioxidant, anti-inflammatory, and osteoprotective properties, whereas at higher concentrations, it can exert cytotoxic effects. This dual activity is attributed to the pro-oxidative potential of EGCG under specific cellular and environmental conditions [149].

In a study conducted on astrocytes, it was shown that EGCG at a high concentration (50 μM) induced mitochondrial membrane depolarisation and activated the opening of the mitochondrial permeability transition pore (mPTP). This led to a sharp decline in the inner mitochondrial membrane potential, increased permeability to ions and small molecules, and mitochondrial swelling, thereby increasing the risk of membrane rupture. Concurrently, pro-apoptotic proteins, such as cytochrome c, were released into the cytoplasm. In comparison, the effect at a concentration of 1 μM was significantly weaker [150]. Similar phenomena were observed in studies on human cancer cell lines and rat hepatocytes [151].

EGCG itself can generate reactive oxygen species (ROS) via auto-oxidation at high concentrations, particularly through Fenton-like reactions in the presence of elevated levels of iron ions (Fe²⁺), such as in the liver or tumour tissues, and copper ions (Cu⁺), which may accumulate in certain inflammatory conditions [148,152,153,154]. Additionally, EGCG can enhance oxidative stress when combined with cytotoxic agents (doxorubicin and bortezomib), heavy metals (e.g., cadmium), ultraviolet radiation, or H₂O₂ [155,156,157,158,159].

Furthermore, excess EGCG has been shown to induce endoplasmic reticulum (ER) stress and disrupt proper protein folding, leading to the activation of the unfolded protein response (UPR). In mouse-derived MRPE cells, EGCG reduced the expression of chaperones and UPR markers such as GRP78, CHOP, PERK, IRE1α, and caspase-12 [128]. Similarly, in studies on the model organism Caenorhabditis elegans, EGCG modulated cadmium-induced ER stress via the PERK–eIF2α–ATF4 signalling pathway [160].

However, at higher concentrations (≥50 μM), the increased expression of UPR markers was observed, indicating an activation of the stress response and the potential induction of cell death [161]. A review by the European Food Safety Authority (EFSA) indicates that a daily oral dose of EGCG ≥800 mg may lead to hepatotoxicity, highlighting the importance of defining safe dose ranges and confirming the hormetic properties of this compound. In a mouse model, EGCG at a dose of 1500 mg/kg induced acute liver injury, while doses in the range of 400–800 mg/kg were toxic under conditions of dietary restriction (Figure 4) [162].

Major conclusions are as follows:

• EGCG reduces osteoclast differentiation and activation: EGCG administration leads to a reduction in osteoclast differentiation and activation by increasing OPG mRNA expression and inhibiting RANK ligand production [19,36,42].
• It enhances OPG mRNA expression: EGCG exhibits a stronger capacity to enhance OPG mRNA expression in pluripotent stem cells compared to other green tea catechins, contributing to its role in bone health [42].
• It influences bone growth and osteogenesis: EGCG promotes bone growth by stimulating VEGF synthesis, affecting the SAPK/JNK pathway, and reducing IL-6 synthesis [34,56,57,58,59].

4. Conclusions and Future Research Directions

Research on the effects of EGCG, the primary polyphenol found in green tea, on bone growth and development provides substantial evidence of its potential application in the treatment of skeletal system disorders. The findings summarised in this study suggest that it plays an important role in regulating bone metabolism, offering new perspectives for the treatment of bone diseases such as osteoporosis, and significantly contributing to bone tissue regeneration. The effects on bone health are summarised in Figure 5.

EGCG demonstrates the ability to stimulate bone mineralisation by enhancing ALP activity and regulating the expression of osteogenic genes such as Runx2, Osx, and Bmp-2. In vitro studies on cell lines have shown that this compound promotes osteoblast differentiation and the formation of mineralised bone nodules. Furthermore, it supports bone regeneration by increasing bone callus formation and modifying collagen membranes. However, its effects are dose-dependent, and high concentrations (above 25 μM) may inhibit the proliferation of mesenchymal cells involved in osteogenesis, whereas intermediate concentrations (1–10 μM) promote these processes [26,28,30,48]. EGCG also exerts a protective effect on bone tissue by inhibiting bone resorption. This mechanism involves the induction of osteoclast apoptosis through its influence on the Fenton reaction and caspase-3 activation. Additionally, by regulating the RANK/RANKL/OPG pathway, it reduces osteoclastogenesis. EGCG increases OPG expression while lowering the RANKL/OPG ratio, resulting in decreased osteoclast differentiation and activity, thus helping to prevent the loss of bone mass. Moreover, EGCG improves bone microarchitecture, enhancing parameters such as BMD and trabecular thickness [19,44].

It is interesting that EGCG inhibits the production of IL-6 and the activation of MAPK pathways (e.g., p44/p42 and JNK), thereby reducing inflammation and bone resorption [54]. At the same time, it stimulates the synthesis of VEGF, supporting angiogenesis and osteogenesis. Similar to most flavonoids, it exhibits antioxidant properties, neutralising reactive oxygen species. As a result, it protects bone cells from oxidative stress and chronic inflammation, key risk factors for osteoporosis and other skeletal disorders, helping to maintain healthy bone structure and enhance regenerative processes [108,109,110,112]. Additionally, it may influence calcium metabolism by modulating the expression of specific calcium transporters and receptors responsible for maintaining calcium homeostasis. Optimising calcium transport and storage in bone cells promotes more effective mineralisation and improves bone tissue quality (Figure 6) [30,40,49].

However, it is worth noting that the stability of this compound is limited because it undergoes rapid degradation on the way to epimerization and auto-oxidation, influenced by factors such as pH, temperature, and oxygen exposure [154]. This poses a challenge to its therapeutic application. Despite the fact that we already know a lot about this topic, there is still a need for more well-designed and controlled clinical trials to validate the results from preclinical studies. It is especially important for individuals at risk of osteoporosis to determine the effectiveness and safety of EGCG supplementation. What is more, research about the molecular mechanisms underlying its effects on bone cells is necessary. This is very important to provide insights into its potential therapeutic applications through the comprehension of specific pathways. Establishing a proper dose to determine the optimal influence and adverse effects for bone health is also a future research direction. Last but not least, research for the long-term effects of EGCG supplementation is necessary to assess its impact on bone mineral density, fracture risk, and overall bone quality.