Targeting cellular senescence in progenitor cells as a strategy to enhance bone regeneration by cell therapies: a systematic review of pre-clinical investigations

Bone regeneration therapy is becoming increasingly important as the world's population ages with increasing life expectancies. According to United Nations' "World Population Prospects 2022", by 2050, the global population of 65 years old or older will more than double, reaching over 1.5 billion globally [1]. Maintaining bone health in an aging society is directly linked to improving quality of life. In elderly individuals, the decrease in bone density and the increased risk of fractures compromise their independence, making bone regeneration therapy a critical, yet unmet clinical need [2].

In orthopaedics and dentistry, progress in bone regeneration therapy enables safer and more effective treatments for elderly patients and those with impaired healing potential, leading to improvements in functional outcomes such as ambulation, autonomy, and masticatory efficiency, ultimately enhancing patients' overall quality of life [3 –5].

Recently, significant attention has been given to the clinical application of mesenchymal stromal cells (MSCs) in the field of bone regeneration [6, 7]. These self-renewing multipotent cells can differentiate into bone [8], cartilage [9], and adipose tissue [10], as well as other cell types under in vitro conditions. Interestingly, recent findings have highlighted that MSCs transplanted in vivo promoted bone regeneration primarily through paracrine effects by secreting immunomodulatory factors contributing to the polarization of M0 macrophages to the M2 phenotype [11, 12]. The advantages of using MSCs include self-renewal, multipotency, and immunomodulatory capabilities, thus reducing the risk of immune rejection with enhanced safety compared to other treatments [13]. However, age-related decline in MSC function and differentiation capacity remain major challenges.

Cellular senescence (or aging) presents a significant obstacle in bone regeneration therapy. Senescent cells secrete substances broadly defined as senescence-associated secretory phenotype (SASP). It may contain soluble signaling factors such as interleukins, chemokines, growth factors, proteases, insoluble proteins or ECM components. These substances negatively affect surrounding cells, causing inflammation and tissue dysfunction, thereby impacting the bone regeneration process. Senescence markers such as p16Ink4a, p21 and p53 that are cell cycle inhibitors, are also known to play crucial roles in cellular senescence [14 –16]. Expression of these factors are generally induced in response to DNA damage and stress, thus leading to cell cycle arrest and senescence [17].

Moreover, the accumulation of reactive oxygen species (ROS) and mitochondrial dysfunction are involved in the mechanisms of cellular senescence. ROS are naturally generated within cells. However, excessive accumulation of ROS causes damage to DNA, proteins, and lipids [18]. When mitochondrial function declines, energy production becomes inefficient, and ROS production increases, creating a vicious dysregulated cycle. These factors all contribute to the progression of cellular senescence that eventually impairs tissue regenerative capacity [19].

The aim of this systematic review is to survey existing literature to address the question whether cellular senescence signified by associated senescence markers such as p16, p21, p53, ROS, and SASP, play a role in the efficacy of progenitor cell therapies specifically on bone regeneration; and if targeting cellular senescence would improve treatment efficacies.

This systematic review highlights the effects of cellular senescence in impairing the regenerative processes of bone. It is important to recognize the considerable variability in MSCs based on their anatomical source, culture conditions, donor characteristics, and methods of cell isolation. This heterogeneity can significantly impact functional outcomes, such as differentiation capacity, immunomodulatory properties, and anti-inflammatory effects, which may lead to inconsistencies in therapeutic efficacy [45]. Moreover, the variability in physiological environments across different age groups requires further investigation to ensure that treatment strategies are effective across a broad population.

Cellular senescence in MSCs significantly reduces their ability for osteogenesis. Senescent MSCs lose their typical fibroblast-like shape and adopt a flatter, elongated morphology, with a notable increase in size and granularity [46 –48]. Using human cells, four methods of inducing senescence (replicative senescence, H₂O₂ treatment, etoposide treatment, and irradiation) were compared, with irradiation proving to be the most effective [24]. The study showed that senescent MSCs exhibit increased inflammation and altered differentiation, being more prone to adipogenesis than osteogenesis. Senescent MSCs exhibit increased expression of senescence markers such as p16Ink4a, p21, and p53, which contribute to the arrest of cell growth and diminished osteogenic capacity [25, 27, 28, 30, 32, 36, 37, 49]. Additionally, SASP creates a local inflammatory microenvironment that hinders bone regeneration. The accumulation of senescent cells leads to the development of SASP. This promotes inflammation (including increased levels of IL-1β, IL-6 and TNFα), protein degradation, and tissue dysfunction, further inducing senescence in nearby cells and tissues [50]. Additionally, SASP impairs the maintenance, regeneration, and repair of musculoskeletal tissues [51 –53]. In summary, it is generally reported that cellular senescence characterized by the increase in senescence markers expression is associated with decreased osteogenic capacity of MSCs.

Out of 13 studies, two examined SASP factors. Gresham et al. observed that senescent MSCs exhibit elevated levels of IL-1β, IL-6, and IFN-γ, particularly in stress-induced senescence models like etoposide and irradiation [24]. Zheng et al. demonstrated that TNFα, MMP-3, and MMP-13 were significantly increased in the diabetic osteoporosis (DOP) group, where the osteogenic ability of hJBMMSCs was notably reduced in the DOP environment [30]. These SASP factors were shown to promote chronic inflammation, enhance osteoclast activity, and disrupt the bone matrix, ultimately impairing bone healing and regeneration [54, 55].

By understanding the biology of senescence markers in MSCs, potential strategies directly targeting these markers to modulate cellular senescence may be developed to favorably impact clinical outcomes. Out of the 13 papers, 5 focused on gene modification as a therapeutic approach. When p53, p21, and p16 expression levels are elevated, cells are prone to cell cycle arrest, senescence, or programmed cell apoptosis. The accumulation of senescent cells expressing high levels of p21 or p16 contributes to a pro-inflammatory environment, disrupting tissue homeostasis and impairing functions essential for processes like osteogenesis and other reparative mechanisms. This chronic inflammatory environment exacerbates tissue dysfunction, hindering effective regeneration and repair. Therefore, current literature generally supports the theory that cellular senescence characterized by elevated expression of the above senescence markers is associated with a chronic low level inflammatory microenvironment.

Among these markers, p53 plays a pivotal role in the regulation of cellular senescence, apoptosis, and the broader senescence process. However, the cellular context such as the tissue or cell type, the cell's proliferative capacity, and the nature of the damage, determine whether p53 induces senescence or apoptosis. In cases of severe damage or stress, p53 triggers apoptosis via factors such as Bax and PUMA [56, 57] However, in response to mild DNA damage or stress, p53 induces cell cycle arrest, which is associated with the increased expression of other senescence markers, such as p16Ink4a and p21. This ultimately leads to the cessation of cell growth and the onset of senescence. As a senescence marker, p53 activation triggers the production of reactive oxygen species (ROS), contributing to cellular stress and the induction of the SASP, as well as matrix metalloproteinases such as MMP3. These factors contribute to the arrest of cell growth and the overall decline in regenerative potential, including diminished osteogenesis. In senescent cells, the role of p53 is crucial in regulating the cellular response to stress and damage, and it plays a significant part in the overall senescence process. This includes influencing the decline in osteogenesis capacity and limiting the regenerative potential of tissues. Thus, targeting p53 is a potential therapeutic strategy to prevent cellular senescence and promote bone regeneration. The studies referenced in this paper suggest that while p53 knockdown and inhibition of p53 via regulation of the p53-p21 pathway can enhance osteogenic differentiation and reduce cellular senescence, potential drawbacks may arise due to the broader regulatory roles of p53 in cellular homeostasis. As a critical tumor suppressor, downregulation or mutation of p53 may impair the cell's ability to arrest the cell cycle in response to DNA damage, thereby potentially increasing the risk of uncontrolled cell proliferation or malignancy. However, it has been reported that loss or inactivation of p53 alone is insufficient to initiate tumorigenesis; while it facilitates the development of a pro-tumorigenic environment, additional genetic or environmental insults are typically required to drive malignant transformation [58, 59]. Furthermore, p16 and p21, which are downstream effectors of p53 and traditionally recognized as tumor suppressors, have also been implicated in tumor-promoting activities under specific conditions, particularly through enhancing the chemotactic activity of monocytic myeloid-derived suppressor cells (Mo-MDSCs). These CDK inhibitors upregulate CX3CR1 expression via SMAD3 signaling, thereby promoting Mo-MDSC infiltration into tumors and facilitating immune evasion and tumor progression [60]. This paradoxical role highlights the complexity of their functions in cancer biology and underscores the need for further investigation to elucidate their context-dependent effects on tumorigenesis. Zheng et al. emphasize the need for careful management of p53's effects on cell differentiation and proliferation. Similarly, in Choi et al.'s study, while inhibition of the p53-p21 pathway via superoxide dismutase (SOD1) conjugates showed efficacy against oxidative stress-induced senescence, the effects of p53 inhibition on cellular stress responses and long-term stability have not been fully explored. Thus, there is an indication that complete p53 suppression may not be optimal, warranting caution in clinical applications. The expression of p16Ink4a is another hallmark of senescent cells with ongoing research into targeting these cells for selective removal aiming to rejuvenate tissues [61, 62]. This approach holds the potential to prevent tissue dysfunction and slow the progression of age-related diseases caused by the accumulation of senescent cells. In summary, our review has identified that these most common senescence markers being proposed as direct interventional targets to manipulate the progression of cellular senescence. However, many of the primary studies have missed to incorporate sampling from different age groups thus dampening the power of the studies.

In our review, we have also identified other senescence-related biological pathways that are commonly targeted to achieve the goal of bone regeneration.

It is well reported that MSCs undergo senescence with aging, leading to diminished regenerative capacity. In the context of bone regeneration, several cell source-related factors are associated with increased MSC senescence and impaired function. Advanced age is a primary factor contributing to cellular aging, which results in reduced proliferation, migration, and osteogenic differentiation potential of MSCs [63]. Additionally, disease states such as diabetes mellitus have been shown to exacerbate MSC dysfunction through mechanisms involving chronic inflammation, impaired angiogenesis, and metabolic dysregulation. Oxidative stress is another critical contributor, as elevated reactive oxygen species (ROS) levels promote DNA damage and senescence, further impairing the regenerative potential of MSCs. Thus, many of the anti-senescence interventions tend to target oxidative stress or ROS accumulation. Of the 13 studies included in this review, 7 reported that reducing ROS levels effectively attenuated cellular senescence. These findings underscore the pivotal role of oxidative stress in senescence and highlight the potential therapeutic benefit of targeting ROS reduction to mitigate age-related cellular dysfunction and enhance bone regeneration. ROS produced during mitochondrial energy metabolism contribute directly to oxidative stress, accelerating cellular dysfunction and the onset of senescence. Therefore, maintaining mitochondrial integrity is essential for sustaining bone regeneration. In this context, therapeutic strategies aimed at reducing oxidative stress and improving mitochondrial function offer a promising approach to counteracting the negative effects of cellular senescence and promoting bone regeneration.

Additionally, therapeutic strategies targeting cellular senescence known as senotherapeutics represent a highly active area of research in bone regeneration and tissue repair. Among the agents covered in this review, senolytic agents like fisetin have demonstrated efficacy in selectively eliminating senescent cells and enhancing cellular function, while other methods and agents are also being explored as promising candidates for applications in regenerative medicine. Preclinical studies have demonstrated that fisetin effectively reduces cellular senescence in aged C57BL/6 mice, extending the lifespan of 85-week-old mice by approximately 3 months and leading to improved health outcomes [64]. Based on these findings, clinical trials have been initiated to assess the efficacy of fisetin in elderly populations, with a focus on markers of frailty, inflammation, insulin resistance, and bone metabolism. In addition to fisetin, other senotherapeutic such as ABT-263, dasatinib, and quercetin as senolytics, and metformin, rapamycin, resveratrol, and NAC as senomorphics are expected to become key focuses of future research aimed at evaluating their potential clinical applications. In addition, the therapeutic effects of other senolytics on cells particularly the cocktail therapy of dasatinib and quercetin (D་Q) have recently attracted attention, with expectations for further therapeutic benefits [65]. D་Q has been reported to improve the osteogenic capacity of aged BMSCs in vitro and in vivo in mouse models [66], but studies on human BMSCs remain limited, and further investigation in this field is anticipated for bone regeneration.

Although the primary focus of this review was senescence markers, and therefore studies using human iPSCs were not directly included in our analysis, it is noteworthy that within the 101 studies categorized under the proportion of cell sources used as intervention strategies in bone regeneration, some reports investigated iPSC-based approaches. Among them, Spitzhorn et al. demonstrated that iPSCs reprogrammed from aged donors and differentiated into iPSC-derived MSCs (iMSCs) acquire a rejuvenation-associated gene signature, including INHBE, DNMT3B, POU5F1P1, CDKN1C, and GCNT2, and regain a fetal-like secretome. These changes enable iMSCs to circumvent age-related declines in osteogenic potential [67]. This study emphasized that the regenerative effects of MSCs are thought to rely largely on paracrine mechanisms mediated by secreted factors rather than directly by the cells themselves. In a complementary approach, Hanetseder et al. showed that extracellular matrices generated from iPSC-derived mesenchymal progenitors (iECM) significantly enhanced osteogenesis of bone marrow stromal cells from both young and aged donors. Strikingly, aged MSCs cultured on iECM exhibited restored mineralization capacity absent on standard culture substrates, highlighting the promise of iPSC-derived matrices to provide a rejuvenated microenvironment [68].

Reprogramming into iPSCs induces an epigenetic reset leading to cellular rejuvenation. However, only a limited number of studies have systematically investigated how senescence markers are altered during this process. Demonstrating changes in the expression of such markers would provide critical evidence for the reliability and safety of iPSC-based approaches in regenerative medicine. In the context of bone regeneration, verifying whether senescence markers are indeed reduced in iMSC- or iECM-based therapies is indispensable for assessing the durability of therapeutic effects, the risk of persistent SASP-driven inflammation, and the potential for tumorigenesis or abnormal differentiation. Moreover, as a bridge to anti-aging research, understanding the behavior of senescence markers is particularly important. While iPSC-based therapies are often challenged by concerns regarding tumorigenic risk and difficulties in controlling lineage-specific differentiation, incorporating the dynamics of senescence markers would help determine whether these therapies truly represent "anti-aging" interventions or merely reset the cellular phenotype of aging.

The primary aim of this systematic review was to identify senescence markers associated with bone regeneration. To minimize the risk of overlooking potentially relevant but less frequently investigated markers, we did not restrict our search to a predefined set. Although markers such as p19^ARF and H3K9me3 have been reported in the broader literature, no studies to date have examined their roles in bone regeneration [69].

Notably, the majority of included studies induced cellular senescence in vitro using experimental manipulations, rather than assessing cells derived from physiologically aged donors. While a limited number of studies utilized cells from elderly individuals, only one of the 13 studies directly compared aged and young cells, and this investigation was constrained by a small sample size. Salamanna et al. reported that the expression levels of SASP factors (IL1α, IL1β, IL-6, IL-8, TNFα, MCP-1, CCL4, and CXCL2) in MSCs derived from clotted vertebral bone marrow aspirate from younger and older donors showed no significant differences. They concluded that age-related exacerbation of SASP is unlikely to impair bone regenerative capacity, and that these cells retain sufficient potential for bone regeneration therapy [70]. These findings underscore the need for future large-scale studies employing physiologically aged human cells to more accurately elucidate the role of cellular senescence in bone regeneration.

With the progression of an aging society, elderly individuals represent the primary target population for regenerative therapies. However, age-related declines in the regenerative capacity of MSCs limit their effectiveness in bone regeneration. Thus, novel therapeutic strategies that counteract MSC senescence, such as senolytics or iMSC/iECM-based approaches, hold considerable clinical significance. In this context, elucidating senescence markers is particularly important, as they provide critical insights into treatment efficacy and safety. This review has highlighted that current studies on senescence markers in bone regeneration remain largely confined to the in vitro stage, with relatively few in vivo evaluations. To advance toward clinical application, future research should include validation studies in animal models, particularly aged models that incorporate senescence-associated factors, and explore combinatorial strategies with existing biomaterials.

The data for this review were exclusively sourced from PubMed, Web of Science, Embase and Scopus, potentially omitting relevant studies from other databases. Despite this potential shortcoming, a comprehensive understanding of the fundamental mechanisms of cell senescence in bone regeneration could provide new insights into improving regenerative therapies in our aging population. This systematic review presents several novel contributions to the field of stem cell research and therapy by targeting cellular senescence in progenitor cells. Unlike previous narrative reviews, it systematically and quantitatively evaluates the relationship between senescence markers and bone regeneration outcomes, with a unique focus on human-derived cell sources. The review is the first to map the distribution of these cell sources in senescence research, providing foundational data for future clinical translation. It identifies specific senescence markers (such as p16, p21, p53, SA-β-Gal, and ROS) as proposed therapeutic targets and critically appraises common study design flaws, particularly the lack of age- and disease-diverse samples. The paper clarifies the mechanisms of cell therapy, distinguishing paracrine effects from direct engraftment, and expands the discussion on the potential and challenges of iPSC-based therapies, including safety and standardization issues. We attempted to provide comprehensive tabulation of findings and a focused discussion on clinical translation and future research directions to offer a new perspective and roadmap for advancing the field.