Senescence of skeletal stem cells and their contribution to age-related bone loss

Human aging is linked to bone loss, resulting in bone fragility and an increased risk of fractures. This is primarily due to an age-related decline in the function of bone-forming osteoblastic cells and accelerated cellular senescence within the bone microenvironment. Here, we provide a detailed discussion of the hypothesis that age-related defective bone formation is caused by senescence of skeletal stem cells, as they are the main source of bone forming osteoblastic cells and influence the composition of bone microenvironment. Furthermore, this review discusses potential strategies to target cellular senescence as an emerging approach to treat age-related bone loss.


Age-related bone loss is caused by impaired bone formation
The human skeleton is renewed and regenerated throughout life, by a cellular process known as bone remodeling that consists of cycles of resorption of old bone containing fatigue microfractures by osteoclastic cells, and its replacement with new bone with better biomechanical properties by osteoblastic cells.Under normal conditions, bone mass is maintained by a balanced activity of osteoclasts and osteoblasts.However, with aging bone formation is impaired as demonstrated by several histomorphometric studies performed on human bone biopsies (Brockstedt et al., 1993;Delaisse et al., 2020;Parfitt et al., 1995).Bone formation is the function of osteoblastic cells, which are differentiated from skeletal stem cells, also known as marrow stromal cells (MSCs) present in the bone marrow microenvironment.There is an increasing recognition that age-related impairment of bone formation is caused by age-related changes in biological characteristics and functions of MSC (collectively termed as MSC cellular senescence).This review focuses on the underlying mechanisms of cellular senescence of MSC, how MSC cellular senescence contributes to impairment of bone formation, and provides an update regarding the emerging therapeutic approach of targeting senescent MSC to enhance bone formation and reduce the rate of age-related bone loss.

What is cellular senescence?
Cellular senescence refers to a permanent state of cell cycle arrest that occurs when cells experience various types of stress e.g., irradiation, oxidative stress or oncogene activation, or reach the end of their replicative lifespan.Senescent cells exhibit characteristic alterations e.g., changes in gene expression, metabolism, chromatin organization, induction of anti-apoptotic pathways, and production of characteristic secretome known as "senescence-associated secretory phenotype" SASP (Campisi and d'Adda di Fagagna, 2007;Coppé et al., 2008).While it was initially descried as an in vitro phenomenon affecting diploid cells during long-term subculturing and hence named "replicative senescence" (Hayflick and Moorhead, 1961), cellular senescence is also observed during in vivo aging in many tissues including bone (Farr and Khosla, 2019).
Senescent cells are identified by several biomarkers that vary in terms of sensitivity and specificity e.g., positive staining for senescence-associated β-galactosidase (SA-β-gal) (Dimri et al., 1995), changes in p16 Ink4a , p21 Cip1 , p38 MAPK, and p53 gene expression (Davan-Wetton et al., 2021), loss of Lamin B1 immune reactivity due to impaired nuclear membrane integrity, formation of senescence-associated heterochromatin foci (SAHF) identified by immunostaining of MacroH2A, Heterochromatin protein 1 (HP1), Lysine 9 di-or tri-methylated histone H3 (H3K9Me2/3), and evidence of DNA damage visualized by γ-H2AX staining (McCauley and Dang, 2014).In addition, to the above-mentioned biomarkers that directly identify individual senescent cells, the total burden of senescent cells can be estimated indirectly by measuring the SASP factors in conditioned media of in vitro cultured cells or in serum.The SASP is composed of several factors that include interleukins, chemokines, and growth factors, proteases, including matrix metalloproteinases (MMPs) and serine proteases and their inhibitors, as well as insoluble proteins/extracellular matrix components, such as fibronectin or nonprotein molecules like reactive oxygen species (ROS) (Coppé et al., 2010).Recent studies have identified a common SASP signature.For example, a proteomic study carried out on senescent fibroblasts and epithelial cells has identified a core soluble SASP which encompasses 143 proteins that included growth/differentiation factor-15, MMP-1, stenniocalcin-1, and serine protease inhibitors (Basisty et al., 2020).Another transcriptomic study identified a senescence gene set of 125 genes encompassing predominantly SASP factors (n = 83) but also transmembrane (n = 20) and intracellular (n = 22) proteins, termed "SenMayo" (Saul et al., 2022).While the core senescence factors is thought to be universal, there exists differences in the composition of the SASP factors depending on the tissue and the context e.g., replicative senescence vs. senescence induced by irradiation or oncogenic activation (Coppé et al., 2008)

Cellular senescence of skeletal stem cells (MSC)
MSC are multipotent, self-renewing cells capable of differentiating into mesodermal cell types such as osteoblasts, chondrocytes, and adipocytes.Upon in vivo transplantation, MSC can form bone, cartilage, marrow adipocytes, and hematopoietic-supporting stroma (Sacchetti et al., 2007).MSCs exhibit a replicative senescence phenotype during long-term in vitro culture, which is influenced by the donor's age.Cultures established from elderly donors (65-80 years) display more accelerated cellular senescence compared to those from young donors (20-30 years), as evidenced by a decreased maximal in vitro lifespan and an increased rate of senescent cell accumulation (Stenderup et al., 2003).The accumulation of senescent cells has also been observed in vivo with aging, both in mice and human bones and within MSC, osteoprogenitor, osteoblast, and osteocyte populations (Farr et al., 2016).Therefore, skeletal aging is associated with the accumulation of senescent cells within the bone and this is the origin of the hypothesis that they contribute to age-related skeletal deterioration and bone loss (Kassem and Marie, 2011;Khosla et al., 2022).

Mechanisms underlying cellular senescence of MSC
In the following, we focus on the mechanisms that have been reported to induce cellular senescence (López-Otín et al., 2023) in MSC (Fig. 1) which include oxidative stress, mitochondrial dysfunction, defective extracellular vesicles involved in cell-cell communication, defective autophagy and mitophagy, telomere shortening, and epigenetic changes that induce loss of cellular integrity.

Oxidative stress
Reactive oxygen species (ROS) are molecules with oxidizing properties either with free radicals; such as superoxide anion (O 2 • − ), nitric oxide ( • NO), and hydroxy (HO • ), or without free radicals such as peroxynitrite (ONOO‾) and hypochlorous acid (HOCl) (Sies and Jones, 2020).ROS are needed for MSC self-renewal, proliferation, and differentiation under physiological conditions (Sart et al., 2015), however, their accumulation that arises when the release of ROS exceeds cellular  ability for its detoxification (Sies, 1997) can induce cellular senescence through four cellular mechanisms (Zhu et al., 2018): (1) DNA damage with subsequent activation of p53 and upregulation of p21 (Rai et al., 2009).
(2) Activation of the p38 MAPK pathway due to the upregulation of p19 protein expression and limitation of the cellular self-renewal (Ito et al., 2006;Tormos et al., 2013).
(3) Activation of nuclear factor κB (NF-κB) pathway that mediates SASP production (Lee et al., 2010;Lopes-Paciencia et al., 2019;Salminen et al., 2012).( 4) Accumulation of senescence-associated microRNAs (miRNAs) (Houri et al., 2020;Li et al., 2009;Xiao et al., 2022).In cultured mice and human MSC treatment with H 2 O 2 leads to cellular senescence and decreased osteogenic capacity that was reversed by treatment with an antioxidant melatonin (Fan et al., 2020;Lee et al., 2018).Similarly, treatment with antioxidant selenomethionine reduced ROS production and oxidative stress-related apoptosis and restored alkaline phosphatase (ALP) activity in senescent rat MSC cultures (Li et al., 2021).Also, the relationship between oxidative stress, miRNA, and bone cellular senescence is illustrated by the presence of low serum levels of senescence-associated miR-29a in patients with osteoporosis and reduced age-related bone loss due to accumulation of anti-oxidative proteins OXR1 and FOXO3 and reduced cellular senescence in miR-29a-transgenic mice (Lian et al., 2021).

Mitochondrial dysfunction
Age-related mitochondrial dysfunction is well documented and interventions aiming at improving mitochondrial function have been shown to promote healthspan (Amorim et al., 2022;Lima et al., 2022;López-Otín et al., 2023).Age-related mitochondrial dysfunction has also been reported in cultured MSC and osteoblastic cells as well as in bone.
In the following, we will describe changes in mitochondrial morphology and functions observed as a consequence of increased cellular senescence within the bone, and strategies that have been tested to improve mitochondrial functions and consequently bone health.
Changes in mitochondrial morphology with tissue aging have been reported.Senescent MSC exhibit large tubular mitochondria, due to defective mitochondrial fission (Li et al., 2019).These large abnormal mitochondria exhibit inappropriately high ROS production (Li et al., 2019).
Aging is also associated with impaired mitochondrial quality control (MQC), a mechanism needed to preserve mitochondrial homeostasis by removing damaged mitochondria through mitophagy, and generating new mitochondria termed mitochondrial biogenesis (Pickles et al., 2018;Yan et al., 2023).Senescent MSC exhibit impaired mitophagy and treating the cells with mitophagy activator CCCP (carbonyl cyanide m-chlorophenyl hydrazone) improved osteoblastic differentiation and functions (Guo et al., 2021).Similarly, activation of mitophagy with genisteinan isoflavonetreatment of cultured MSC established from ovariectomized rats was associated with reduced oxidative stress and cellular senescence (Li et al., 2023).
Age-related low levels of NAD + , a coenzyme playing a critical role in cellular energy metabolism, contribute to mitochondrial dysfunction, impaired cellular metabolism, and cellular senescence (Barilani et al., 2022;Wang et al., 2022;Yuan et al., 2020).Increasing NAD + levels through administration of NAD + precursors e.g., nicotinamide mononucleotide (NMN) or nicotinamide riboside (NR), decreased the burden of senescent MSC and bone loss in aged mice (Kim et al., 2021;Lu et al., 2023;Wang et al., 2022).Interestingly, NAD + levels also regulate the activity of NAD-dependent histone deacetylases called sirtuins, i.e., low levels of NAD + lead to low levels of, for example, the mitochondria-located sirtuin 3 (SIRT3) as shown in MSC cultured from aged rats (Denu, 2017;Ma et al., 2020).SIRT3 regulates cellular energy metabolism, ROS production, and MQC (Hu and Wang, 2022).It is reduced in aged (15-18 months old) rat MSC, in which SIRT3 replenishment restored cellular antioxidant capacity, and reduced the burden of senescent DNA damage and MSC senescence (Denu, 2017;Ma et al., 2020).Similarly, in MSC cultured from elderly individuals, SIRT3 overexpression enhanced mitochondrial antioxidant enzyme manganese superoxide dismutase activity (Wang et al., 2014).Pharmacologically enhancement of SIRT3 activity using hydrogen sulfide (NaHS) treatment of cultured murine MSC reduces burden of cellular senescence (Liu et al., 2023).
In addition to its role in energy metabolism, a recent area of investigation is the role of age-related mitochondria dysfunction in initiating SASP production which has been demonstrated to take place via mitochondria-to-nucleus signaling pathway including forming and shedding cytoplasmic chromatin fragments (Vizioli et al., 2020) or through the release of mitochondrial DNA into the cytosol (Victorelli et al., 2023).
Overall, these studies indicate that age-related mitochondrial dysfunction impairs MSC functions, accelerates MSC cellular senescence, and promotes SASP production.

Extracellular vesicles contribute to senescence in MSC
Extracellular vesicles (EVs) are nano-sized particles derived from the cell membrane and are released from different cell types including MSC (Cosenza et al., 2017;Lu et al., 2019;Phetfong et al., 2022;Wu et al., 2021).EVs contribute to cellular senescence in MSC through release of miRNA, lipids, and proteins.Similarly, in cultured MSC transfection with EVs isolated from bone marrow interstitial fluid of aged mice which are enriched in miR-183-5p induced cellular senescence (Davis et al., 2017).
Transfecting EVs isolated from H 2 O 2 oxidative-stress-induced-senescent C2C12 mouse myocytes and primary human myotubes that are enriched in miR-34a into cultured MSC induced cellular senescence phenotype (Fulzele et al., 2019).Also, the sphingolipid ceramide induces a senescence phenotype through inhibition of DNA synthesis and mitogenesis (Venable et al., 1995).Ceramide content is increased in serum EVs obtained from aged compared to young women, and long chain ceramide C24:1 loaded-EVs promotes cellular senescence in cultured MSC (Khayrullin et al., 2019).
In sera obtained from elderly osteoporotic patients (age 63.8 ± 4.0 years), EVs contain lower levels of proteins that are implicated in integrin-mediated adhesion and mechano-sensation, compared to healthy age-matched controls (Xie et al., 2018).Interestingly, the protein content of EVs isolated from older mice (20 months old) cortical bone showed higher expression of SASP proteins such as TGF-β2, OPG, MMP9, TIMP1, MIF1, PRDXs, and IGFBP3, compared to young mice (3 months old) (Zhang et al., 2019).MSC can also influence the senescence phenotype of other cells as demonstrated by the development of cellular senescence phenotype in mouse muscle satellite cells that have received EVs derived from senescent mouse MSC (Dai et al., 2022).
These studies demonstrate that EV content changes with age to a more senescence-prone constituents.To revert cellular senescence through targeting the EVs, one study has administered human EVs derived from embryonic stem cells to male senescence-prone SAMP8 mice in vivo and to cultured MSC in vitro.The authors report reversal of the cellular senescence, prevention of age-related bone loss as well as evidence for upregulation of "anti-aging" genes, genes associated with stem cell proliferation and osteoblastic differentiation (Gong et al., 2020).

Autophagy in senescent MSC
Several recent studies have demonstrated that changes in cellular autophagy are associated with increased burden of senescent MSC.In cultured human MSC, radiation-induced senescence was accompanied by reduced autophagic flux (Alessio et al., 2015).In aged male mice, cultured MSC expressed lower autophagy activity levels as estimated by the LC3-II/LC3-I ratio, which was associated with cellular senescence (Gonçalves et al., 2023).Rejuvenation of MSC from aged mice through exposure to paracrine factors released by MSC from young mice, was accompanied by increased autophagy activity (Hung et al., 2022), while MSC from aged male L2G85 reporter transgenic mice showed higher autophagosome formation, indicating less autophagy due to accumulation of autophagosomes, which was associated with increased burden of cellular senescence (Yang et al., 2018).Insulin-like growth factor 1 knockdown enhanced autophagy in aged MSC and thus protected the cells from undergoing apoptosis (Yang et al., 2018).The relationship between cellular senescence and autophagy is an emerging area of investigation in the MSC biology field and the preliminary data demonstrate that lower autophagy is observed in aged MSC and associated with increased cellular senescence burden.

Epigenetic changes in senescent MSC and osteoprogenitor cells
Age-related epigenetic changes involve DNA methylation, histone modifications, chromatin remodeling, and post translational changes.In human MSC isolated from aged donors, invariant and differentially methylated CpGs compared to young donors are described, specifically, hypermethylation of transcription factor binding sites such as SMARCC1, STAT5, ZNF274, NFE2, JUNB, and hypomethylation of REST and ZBTB33 have been reported (Pasumarthy et al., 2017).Also, additional epigenetic changes such as differentially expressed genes and miRNAs as well as dysregulated alternative splicing events were reported in cultured human MSC derived from elderly donors (Peffers et al., 2016).
Epigenetic changes may cause cellular senescence as shown by knock-down experiments of long noncoding RNA LINCO1638 that led to DNA damage, genomic instability, and accelerated cellular senescence (Gordon et al., 2023).Furthermore, age-related changes in MSC miRNA expression have been described.For example, in adult MSC miR-196a was up-regulated, and inhibited gene expression of HOXB7 gene known to have positive effects on proliferation and for its "anti-aging" effects (Candini et al., 2015).Interestingly, treatment with a noncovalent DNA methyltransferase inhibitor RG108 up-regulated telomerase reverse transcriptase (TERT) mRNA expression and counteracted the cellular senescence phenotype in senescent MSC (Oh et al., 2015).

Telomere shortening
Telomere shortening is a principle mechanism inducing cellular senescence and results from incomplete replication of linear chromosomes by DNA polymerase (López-Otín et al., 2023).Short telomere length has been associated with age-related organ dysfunction in several studies including bone changes of age-related low bone mass and increased fracture risk (Valdes et al., 2007).This was observed in a mouse model of shortened telomeres, Terc deficient mice, which had decreased bone mass and impaired bone formation and was suggested to be mediated by accelerated senescence of bone marrow MSC and their progeny (Saeed et al., 2011).Also, telomere shortening was observed in human cultured MSC from aged donors, which presented with lower proliferation capacity and higher burden of cellular senescence per population doubling compared to cells obtained from young donors (Stenderup et al., 2003).In contrast, when the human telomerase reverse transcriptase gene (hTERT) was overexpressed in cultured human MSC, telomeres were elongated, which was associated with elimination of cellular senescence phenotype and enhancement of in vivo bone formation capacity (Abdallah et al., 2005;Simonsen et al., 2002) as well as a rejuvenated molecular signature (Saeed et al., 2015;Twine et al., 2018)

Cellular senescence and bone fragility in metabolic bone diseases
Metabolic diseases, such as obesity and type 2 diabetes (T2D) lead to the accumulation of senescent cells in metabolically active tissues, i.e., adipose tissue, liver, skeletal muscle, pancreas, and cardiovascular tissue (Schafer et al., 2017).Young and healthy mice injected with a low number of senescent cells exhibited accelerated aging and tissue dysfunction (Xu et al., 2018).A number of murine experimental studies from our group, have demonstrated that obesity and T2D lead to decreased bone mass and impaired bone formation (Ali et al., 2022;Figeac et al., 2022;Tencerova et al., 2019) as well as impaired fracture healing (Figeac et al., 2022), an observation caused by increased senescent cell burden of MSC and its bone microenvironment.This was confirmed by an independent study which reported similar results (Alessio et al., 2020) that corroborated that metabolic dysfunction leads to accelerated MSC senescence and impairs bone formation.
The contribution of accelerated skeletal cellular senescence in obesity and T2D induced bone fragility was also evident in humans, where MSC isolated from bone marrow aspirates of young obese men exhibited an increased mRNA expression of senescence associated markers (TP53, CDKN2A, CDKN1A) and oxidative stress markers (HMOX1, SOD2, ALDH1A1), increased β-gal staining and activity, and increased intracellular ROS production as compared to those isolated from lean donors (Tencerova et al., 2019).

Targeting MSC and osteoprogenitor cellular senescence to improve bone health
Several anti-osteoporosis treatments are currently available to target age-related bone loss and either suppress bone resorption (anti-catabolic or anti-resorptive) or stimulate bone formation (anabolic).However, rebound bone mass loss after halting treatment, and concerns for side effects led to a substantial number of patients being untreated and stimulated the interest in alternative treatment options (Doolittle et al., 2021).Additionally, osteoporosis is associated with multiple age-related co-morbidities, such as cardiovascular diseases, diabetes, neurodegeneration, and osteoarthritis which exacerbate bone fragility independent of bone mass.These co-morbidities are mostly treated individually regardless of bone health.Targeting senescent cells has been proposed as a general "anti-aging" approach for treating not only osteoporosis but other age-related diseases.

Senotherapeutics
Senotherapeutics are pharmacological agents that aim at either eliminating senescent cells (senolytics) or inhibiting their biological products, i.e., SASP components, without inducing cell death (senomorphics) (Niedernhofer and Robbins, 2018;Zhang et al., 2023).Numerous preclinical studies confirmed the potency of those treatments to decrease the rate of skeletal aging and are now being tested in clinical trials (https://clinicaltrials.gov/).

Senolytics
As the first group of senolytics dasatinib (D), a protein tyrosine kinase inhibitor, and quercetin (Q), a plant flavonoid (Zhu et al., 2015), were identified and used to reinduce apoptosis in senescent cells by targeting their antiapoptotic mechanisms.
In vitro and in vivo studies have demonstrated the ability of senolytics to exert direct effects on MSC in mice and humans.Quercetin treatment for 48 hours decreased senescent cell burden in MSC population isolated from aged mice (Xing et al., 2023) and D+Q treatment cleared senescent cells within the osteoblastic cell populations defined as cells expressing high p16 levels within the CD24 high osteolineage, CD24+/RUNX2+, and late osteoblast/osteocyte clusters (Doolittle et al., 2023).
In a mouse model, combining D+Q improved the osteogenic differentiation capacity of aged MSC in vitro and in vivo (Zhou et al., 2021).Zhang et al., have also demonstrated that Q improved proliferation and osteoblast differentiation of rat MSC while inhibiting adipogenesis (Zhang et al., 2020).In an aged murine model, D+Q treatment decreased the burden of senescent osteocytes and osteoclasts, and led to increased cortical and trabecular bone mass (Chandra et al., 2020;Farr et al., 2017;Zhu et al., 2015).Other senolytics like fisetin, a polyphenol, has been reported to protect against OVX-and inflammation (LPS-)-induced bone loss in mice (Léotoing et al., 2013).A panel of 10 flavonoid polyphenols were tested for senolytic activity, using cultured senescent murine and human fibroblasts in vitro.Fisetin was identified as the most potent senolytic (Yousefzadeh et al., 2018).Giving a diet containing fisetin resulted in reduced senescence burden in old mice as evaluated by expression of senescence and SASP markers in multiple tissues (fat, spleen, liver, and kidney) (Yousefzadeh et al., 2018).
It is also plausible that part of the positive effect on bone mass following systemic senolytic treatment is caused by reducing the burden of senescent cells in non-skeletal tissues as indicated by a more effective reduction in RankL through systemic clearance of senescent cells (Farr et al., 2023).
Eighteen clinical trials testing the effects of senolytic treatment on several age-related diseases, are currently being conducted.Five clinical trials are employing fisetin or D+Q combination and focus on agerelated bone loss, including several bone-related outcomes such as changes in bone turnover markers and bone mass (NCT05371340, NCT04313634, NCT06018467), as well as outcomes such as inflammation markers and gait speed (NCT03675724, NCT03430037).The results of one of the clinical trials has just been reported and showed higher bone formation rate as estimated by changes in bone turnover markers at 2 and 4 weeks following intermittent D+Q treatment in aged women with high-burden of senescent cells in peripheral blood as measured by T-cell p16 mRNA levels.Interestingly, higher radius BMD at week 20 was observed in treated women with high cellular senescence burden (Farr et al., 2024).The results of additional clinical trials are needed to determine the magnitude of the effects as well as the feasibility of this approach in treating patients with osteoporosis.

Senomorphics
Senomorphics are pharmacological agents that counteract the negative effects of senescent cells by inhibiting factors included in the SASP, by either action on specific intracellular pathways needed for SASP production or by monoclonal antibodies that block the SASP factors (Fadini et al., 2010).Among the first senomorphics employed are corticosteroids, metformin, resveratrol, rapamycin, and aspirin.Newer and more specific senomorphics include ruxotinib and JQ1, which are re-purposed from cancer therapy.More recently, monoclonal antibodies like siltuximab and tocilizumab have been investigated for their capacity to target proinflammatory cytokines or their receptors.
The efficacy of senomorphics have been demonstrated in several studies.In an aged mouse model (20-to 22-month-old mice), 2-4 months treatment with JAKi, ruxolitinib, can improve bone mass, bone strength, and bone microarchitecture as compared to vehicle-treated mice (Farr et al., 2017).
Rapamycin, clinically approved as immunosuppressant for preventing transplant rejections and as adjuvant for treating certain types of cancer (Romashkan et al., 2021), is known as an inhibitor of the mTOR signaling pathway, regulating cell growth, survival, and metabolism (Zhang et al., 2023).In multiple species, from nematodes to mice, rapamycin treatment led to reduced burden of cellular senescence and suppressed SASP factor production and extended healthspan as well as lifespan (Selvarani et al., 2021).Interestingly, Gu et al., demonstrated that in a mice model of lupus nephritis (chronic autoimmune disease involving several organs) rapamycin can alleviate the impact of the disease, reversing cellular senescence, and immune dysregulation in both human and murine MSC cells (Gu et al., 2016).In several clinical trials analogs of rapamycin (rapalogs) have demonstrated their ability to boost influenza vaccine response in the elderly population, by targeting immunosenescence (Mannick et al., 2014).The effects of rapamycin on age-related bone loss in humans have not been studied.

Combination therapies
Senescent cells upregulate multiple anti-apoptotic pathways requiring combined therapy targeting more than one genetic pathway to improve senescent cell clearance as senolytics target only single senescent cell anti-apoptotic pathways (Chaib et al., 2022).Similarly, the diverse SASP factors and their multiple down-stream effector pathways limits the efficiency of senomorphics targeting specific SASP factor (Zhang et al., 2023).
Thus, future studies should consider examining the effects of combining multiple senoltyics or senomorphic agents on the effects on clearing senescent cells and abolishing age-related tissue dysfunction as well as delineating the potential side effects of this approach.

Telomerase activators
Another strategy to enhance tissue functions during aging is to activate telomerase gene to elongate the telomeres or prevent their shortening during age.One approach used is treatment with a plant extract of the green pericarp of the pistachio (pistachio vera) identified as telomerase gene activator which was found to enhance cell proliferation and reduce senescent cell burden in cultured aged rat MSC (Askai et al., 2023) .
Another promising molecular treatment option is the miRNA-based therapy.MiR-195 has been shown to bind the 3'-UTR region of the mouse Tert gene and thus knock-down of miR-195 production increased telomere length, reduced cellular senescence, and increased expression of "anti-aging" genes such as Sirt-1 and pro-survival markers p-Akt and Bcl-2 (Okada et al., 2016).Direct effects of telomerase activation on age-related bone loss were observed in 2-year-old mice treated with adeno-associated virus carrying murine Tert in telomerase gene therapy protocol.Tert mRNA expression and telomerase activity were increased in the treated mice accompanied by improved bone mass compared to age-matched control mice (Bernardes de Jesus et al., 2012).In a health maintenance program, the telomerase-activating small molecules TA-65, cycloastragenol, which is present in the Astragalus species, was administered to 59 human subjects for 12 months.In the treatment group, fewer cells with short telomeres were observed in the peripheral blood lymphocytes, and granulocytes (Harley et al., 2011), but this resulted in limited clinical bone, metabolic, and immunological effects (Harley et al., 2013).Thus, further studies are needed to determine the feasibility of this approach.

Final remarks
In this review, we have explored the mechanisms contributing to cellular senescence within the bone microenvironment, with a particular focus on skeletal stem cells (MSC) senescence and its role in skeletal deterioration and age-related bone loss.We have also provided an update regarding recent interest in testing therapeutics targeting cellular senescence in humans, motivated by the significant positive effects observed in preclinical animal models.These therapies hold potential not only to enhance bone health but also to mitigate the burden of agerelated degenerative diseases.