Update on the correlation between mitochondrial function and osteonecrosis of the femoral head osteocytes

ABSTRACT

Mitochondrial health is maintained in a steady state through mitochondrial dynamics and autophagy processes. Recent studies have identified healthy mitochondria as crucial regulators of cellular function and survival. This process involves adenosine triphosphate (ATP) synthesis by mitochondrial oxidative phosphorylation (OXPHOS), regulation of calcium metabolism and inflammatory responses, and intracellular oxidative stress management. In the skeletal system, they participate in the regulation of cellular behaviors and the responses of osteoblasts, osteoclasts, chondrocytes, and osteocytes to external stimuli. Indeed, mitochondrial damage or dysfunction occurs in the development of a few bone diseases. For example, mitochondrial damage may lead to an imbalance in osteoblasts and osteoclasts, resulting in osteoporosis, osteomalacia, or poor bone production, and chondrocyte death and inflammatory infiltration in osteoarthritis are the main causes of cartilage degeneration due to mitochondrial damage. However, the opposite exists for osteosarcoma, where overactive mitochondrial metabolism is able to accelerate the proliferation and migration of osteosarcoma cells, which is a major disease feature. Bone is a dynamic organ and osteocytes play a fundamental role in all regions of bone tissue and are involved in regulating bone integrity. This review examines the impact of mitochondrial physiological function on osteocyte health and summarizes the microscopic molecular mechanisms underlying its effects. It highlights that targeted therapies focusing on osteocyte mitochondria may be beneficial for osteocyte survival, providing a new insight for the diagnosis, prevention, and treatment of diseases associated with osteocyte death.

1. Introduction

Osteonecrosis of the femoral head (ONFH) remains a formidable challenge in orthopedic medicine, often referred to as the ‘undead cancer.’ This complex multifactorial disease, with a pathogenesis not yet fully elucidated, is characterized by microenvironmental abnormalities in the femoral head, leading to osteocyte death and subsequent bone tissue collapse [1–3]. Literature suggests various contributing factors, including excessive alcohol consumption, steroid hormone use and certain diseases, such as systemic lupus erythematosus (SLE) and sicklemia [4–8]. ONFH predominantly affects middle-aged individuals, and without effective intervention, approximately 80% of patients require total hip arthroplasty [9]. The femoral head's structural and functional integrity is crucial for maintaining normal locomotion and daily activities, directly impacting quality of life and physical health. Bone tissue comprises stem cells (bone progenitor cells), osteoblasts, osteoclasts, and osteocytes. Among these, only osteocytes reside within the bone tissue, while the other three cell types are located at the bone tissue margins. Mature osteoblasts can transform into osteocytes, contributing to a healthy bone structure [10,11]. Consequently, ONFH is characterized by alterations in osteocyte population.

Mitochondria are enclosed organelles found in cells and can be divided from outside to inside into four regions: outer membrane (OMM), intermembrane space, inner membrane (IMM) and mitochondrial matrix. As individuals age, alterations in mitochondrial dynamics, autophagy, reactive oxygen species (ROS) content, and metabolites lead to a natural decline in mitochondrial function, facilitating the development of age-associated diseases [12]. Consequently, mitochondrial dysfunction is recognized as a hallmark of cellular senescence, death, and degenerative diseases [13]. In osteocyte-related diseases, mitochondria in pathological states often play a critical role in osteocyte death. Recent studies indicate that mitochondrial damage may be involved in ONFH, highlighting it as a potential treatment target [9]. Healthy mitochondria serve as the primary source of ATP, directly providing the energy required for osteocyte metabolism [14]. Additionally, mitochondria are involved in regulating bone tissue-related metabolic processes and maintaining bone microenvironmental homeostasis, providing a stable microenvironment for osteocyte growth and development and ensuring a healthy bone structure. Given the critical physiological roles of mitochondria, research should focus on maintaining mitochondrial OXPHOS function, calcium metabolism, inflammatory responses, and reducing ROS levels to preserve mitochondrial homeostasis [15]. Indeed, maintenance of mitochondrial homeostasis and function can influence the fate of osteocytes [10]. Kitase et al. demonstrated that reducing ROS production and mitochondrial disruption in osteocytes enhanced osteocyte viability [16].

The significance of mitochondria in cellular biology is well-established [17]. As primary energy producers in cells, mitochondria are integral to nearly all aspects of cellular activity and function. Mounting evidence underscores the potential role of mitochondrial dysfunction in the development of various conditions, including degenerative and metabolic diseases, type 2 diabetes, Parkinson's disease, metastatic tumors, and neuromuscular disorders [18–24]. Furthermore, research indicates that mitochondrial damage is closely associated with osteocyte senescence and apoptosis [25]. In vivo experiments have demonstrated that inhibiting mitochondrial damage prevents osteocyte death, potentially impeding the progression of osteonecrosis [26]. Current investigations into the relationship between mitochondria and osteocytes have opened new avenues in the study of ONFH pathogenesis. This review highlights the crucial potential of mitochondrial physiological function in promoting osteocyte survival, offering valuable insights and directions for future research and the development of novel therapies for ONFH.

2. Mitochondrial physiological functions and potential factors for impairment

2.1. Mitochondria synthesize ATP: to support osteocyte survival

Mitochondria are essential organelles in eukaryotes responsible for oxidative metabolism and the final oxidation of major nutrients, including carbohydrates, lipids, and proteins. This process involves glycolysis, the tricarboxylic acid cycle (TCA cycle), and OXPHOS. ATP synthesis in mitochondria is a sophisticated system comprising three phases, with OXPHOS being the primary mode of ATP production in vivo (Figure 1), primarily occurring on the IMM. The mitochondrial respiratory chain (MRC) generates significantly more ATP than alternative methods. For instance, certain single-celled organisms that have lost OXPHOS capacity or mitochondria produce ATP through fermentation, a complex and inefficient process yielding [27–31]. In OXPHOS, five enzyme complexes on the MRC collaborate with two mobile electron carriers to facilitate electron transfer. Complexes I, II, III, and IV sequentially transfer electrons, ultimately to oxygen, while simultaneously pumping protons (H⁺) from the mitochondrial matrix into the intermembrane space, creating an H⁺ concentration gradient. Subsequently, protons diffuse back into the matrix via ATP synthase (complex V), following the concentration gradient, to convert ADP to ATP, sustaining normal cellular functions and metabolism [13,32–34]. Morphologically, the IMM forms ‘cristae’ through inward folding [17]. Well-developed mitochondria possess more complex cristae, increasing the IMM surface area and accommodating more membrane proteins for OXPHOS, thus maximizing ATP synthesis [34,35,36]. Additionally, the cristae structure guides the proper assembly of the MRC protein complex, determining respiratory efficiency and further optimizing ATP production [37]. Consequently, well-developed mitochondria are generally considered more efficient in ATP production compared to damaged or immature mitochondria. The viability of osteocytes is crucial for maintaining bone tissue integrity and function. Any factor interfering with osteocytes may negatively impact bone tissue development, potentially leading to bone-related disorders [13]. Our discussion of osteocytes encompasses osteoblasts and bone mesenchymal stem cells (BMSCs) osteogenesis, processes requiring substantial ATP [38]. Numerous studies have demonstrated ATP's importance in cell growth, differentiation, information transfer, and metabolic senescence [39,40], underscoring aerobic respiration as a necessary process for complex cellular evolution. Osteocyte mitochondria provide a respiratory membrane that scales either linearly or super-linearly with osteocyte volume or surface area [41], ensuring adequate energy supply to large-volume osteocytes. This characteristic exemplifies the advantage of mitochondria in supplying energy to oxygen-demanding organisms. Although osteocytes can utilize glycolysis to satisfy a small portion of their energy needs in early stages, they rely on an intact OXPHOS system in later stages [42]. Studies have shown that approximately 80% of ATP for osteocyte physiological activity is produced through aerobic respiration [43,44], emphasizing the critical importance of maintaining mitochondrial integrity and homeostasis. Damage or reduction in osteocyte mitochondria, or absence of enzyme complexes on the MRC, impairs OXPHOS completion, resulting in decreased ATP levels and metabolic rates in osteocytes. This accelerates osteocyte aging, ultimately leading to reduced osteocyte function or death. Furthermore, in osteonecrosis ONFH, osteocyte mitochondria exist in a hypoxic microenvironment. The accumulation of lactic acid from persistent anaerobic respiration can directly stimulate rapid osteocyte death [45]. Simultaneously, osteocyte mitochondria have a significantly shorter lifespan when exposed to this microenvironment, exacerbating the condition.

Figure 1.

OXPHOS electron transport processes and ATP synthesis. Complexes I, II, III, IV and two electron carriers on the MRC transfer electrons sequentially to oxygen, and protons are synthesized into ATP via complex V. Abbreviations: OXPHOX: oxidative phosphorylation; ATP: adenosine triphosphate; Complex I: NADH dehydrogenase; Complex II: succinate dehydrogenase; Complex III: Cytochrome c oxidoreductase; Complex IV: Cytochrome c oxidase; Complex V: ATP synthase; MRC: mitochondrial respiratory chain; H⁺: protons; Cyt c: Cytochrome c; ROS: reactive oxygen species.

2.2. Mitochondrial regulation of calcium metabolism and osteocyte apoptosis

Ca²⁺ is a crucial signaling molecule that functions as an essential second messenger capable of regulating cellular physiological processes. Intracellular calcium fluctuations significantly impact cell signaling, metabolism, and cell fate. Historically, mitochondria were considered the primary organelle for regulating calcium homeostasis, with large amounts of calcium sequestered in the mitochondrial matrix as cytoplasmic calcium concentration increased [46]. Research has also demonstrated that mitochondria maintain the oxidative metabolism of organisms by providing a continuous flow of calcium into the mitochondrial matrix through mitochondrial calcium metabolism. This process regulates Ca²⁺-dependent enzymes in the TCA cycle, enhances aerobic respiration to increase ATP production [47,48], and more efficiently provides energy to the cell. However, this does not imply that higher concentrations of Ca²⁺ in organelles or cells are always beneficial. Conversely, if the Ca²⁺ threshold is exceeded, this overloaded calcium microenvironment can negatively affect growth, differentiation, and division, necessitating tight control to prevent potentially toxic responses to high calcium levels. Fleckenstein et al. discovered that the series of physiological changes occurring after the entry of overloaded Ca²⁺ into cardiomyocytes is a potential mechanism leading to myocardial fiber necrosis, which has sparked widespread interest in Ca²⁺ as a death-triggering factor [49]. Existing studies have also demonstrated that intracellular Ca²⁺ overload or perturbation of intracellular Ca²⁺ compartmentalization can lead to cytotoxic effects and thus trigger apoptosis or cell death [50–52]. Indeed, mitochondrial calcium overload is one of the pro-apoptotic mechanisms inducing mitochondrial swelling, OMM perturbation or rupture [53], and is a major factor causing premature apoptosis or cell death [50]. Generally, in a healthy state, osteocyte mitochondria can accomplish calcium transport via the mitochondrial calcium uniporter (MCU), or can be achieved via Na⁺/Ca²⁺ exchangers (NCX) [46,54]. Under physiological conditions, MCU has a low affinity for Ca²⁺, but when osteocytes are stimulated, the Ca²⁺ concentration in the cytoplasm increases, and the mitochondria can accumulate a large amount of Ca²⁺. This ensures that there is no overloading of calcium in the microenvironment for osteocyte survival (Figure 2), which prevents apoptosis or osteocyte death due to Ca²⁺ overloading. Consequently, the uptake of Ca²⁺ by mitochondria has long been considered a safety mechanism in cases of intracellular Ca²⁺ overload [50]. When mitochondria are damaged or dysfunctional, leading to a loss of calcium ion storage and uptake capacity, calcium ions can efflux from the mitochondria into the cytoplasm or peripheral environment, activating Caspase activity [55]. Studies have shown that Caspases are highly active in ONFH femoral head tissues, with a significantly reduced number of osteocytes compared to healthy femoral head tissues [56]. This suggests that a caspase-induced osteocyte apoptosis underlies this phenomenon. Conversely, when the mitochondrial volume of osteocytes is exceeded, the locally overloaded Ca²⁺ interacts with cardiolipin in the IMM, leading to decreased lipid fluidity of the membrane and the formation of cardiolipin-enriched structural domains and protein aggregates [57]. This enriched structure leads to increased ROS production by MRC, promoting the oxidation of membrane phospholipids and proteins, thus increasing membrane permeability [50], and inducing the opening of a high conductance channel in the OMM of osteocyte mitochondria called the mitochondrial permeability transition pore (mPTP). The formation of mPTP disrupts the integrity of the mitochondrial membrane and exacerbates damage to osteocyte mitochondria [46,58], leading to the collapse of the proton gradient and ATP production on the MRC [46]. Due to mPTP formation, which enlarges the OMM gap, cytochrome c and other pro-apoptotic proteins are more readily released from the mitochondria into the cytoplasm, further activating downstream Caspases and initiating apoptotic signaling [53,59]. In fact, the release of proteins from mitochondria can lead to the activation of different modes of cell death [60]. Additionally, the rapid uptake of Ca²⁺ by mitochondria stimulates Ca²⁺ sensitive matrix dehydrogenase, a key site for NADH production by MRC, thus stimulating mitochondrial energy metabolism [46]. Therefore, mitochondria, in addition to their established function of producing most of the cellular ATP, play a crucial role in apoptosis [53].

Figure 2.

Mitochondrial regulation of calcium metabolism in osteocytes. The MCU and NCX positioned on the IMM, facilitate the transport of calcium. When mitochondria are damaged or dysfunctional, they lose their capacity to transport calcium effectively. Consequently, upon stimulation of osteocytes, the mitochondria situated within these cells are unable to regulate calcium transport adequately. This results in the establishment of a high-calcium microenvironment, which triggers the opening of the mPTP on the mitochondrial membrane. This, in turn, disrupts the integrity of the mitochondria. Abbreviations: MCU: mitochondrial calcium uniporter; NCX: Na⁺/Ca²⁺ exchangers; mPTP: mitochondrial permeability transition pore.

2.3. Mitochondria and inflammatory response

Inflammation has been proposed as one of the potential mechanisms in the etiology of ONFH [61,62], which also involves neurological disorders, cardiovascular disease, renal disease, liver disease, tumors and fractures, osteonecrosis and arthritis [63–68], with a significant potential for exacerbation by uncontrolled inflammation. Interestingly, inflammation may affect specific disease processes in contrasting ways, such as NLRP3-induced intestinal inflammation modulating the integrity of intestinal homeostasis and shaping the innate immune response [69]. Recent studies have demonstrated that mitochondria play a crucial role in regulating the inflammatory response [70–72]. Inflammation is often initiated by the activation of pattern recognition receptors (PRR) expressed by immune and non-immune cells [68,70], including Toll-like receptors (TLR) and NOD-like receptors (NLR). Notably, mitochondrial metabolites as endogenous damage-associated molecular patterns (DAMPs) and certain viral and bacterial molecules (pathogen-associated molecular patterns, PAMPs) can activate PRR and subsequently trigger inflammation. The occurrence of DAMPs signals tissue damage [73], and high mobility group protein 1 (HMGB 1) is a classical DAMP and an important late inflammatory factor compared to early inflammatory factors such as TNF-α and IL-6. Necrotic cells can passively release HMGB 1 from the nucleus into the extracellular space to induce an inflammatory response. In contrast, HMGB 1 in apoptotic cells binds tightly to nucleosomes, preventing its release and thus avoiding inflammation. Some mitochondrial components bear a strong resemblance to bacterial molecules [74], which can be regarded as PAMPs and function as PRR ligands, such as cyclic mtDNA [17,68]. Additionally, mtDNA, N-formyl peptide, cardiolipin, mtRNA and fumarate can all act as mitochondrial metabolites (DAMPs) to activate PRR and elicit an inflammatory response [75,76]. Indeed, mtDNA can cause an inflammatory response in bone tissue through multiple pathways. When mitochondria are damaged, this can lead to mtDNA leakage into the cytoplasm and activation of the cGAS-STING pathway [75,77]. The cGAS-STING pathway, as a driver of acute or chronic inflammation, can activate the NF-κB signaling pathway, and activated NF-κB induces the production of pro-inflammatory stimulants, including TNF-α, IL-1β and IL-6 (Figure 3) [78]. Secondly, mtDNA is rich in CpG islands, a structure that can be recognized by TLR9 and can directly activate the NF-κB signaling pathway without passing through the cGAS-STING pathway, thus triggering inflammation. Interestingly, activated NF-κB is also able to induce elevated expression of NLRP3 inflammasomes, and NLRP3 recruitment is able to convert the inactive form of pro-Caspase-1 into active Caspase-1, which promotes the release of pro-inflammatory cytokines such as IL-Iβ and IL-6 [79–81]. Furthermore, mtDNA lacks histone protection and has no damage repair system compared to nuclear DNA, resulting in mtDNA being more susceptible to mutation or oxidation. Relevant studies have found that the incidence of mtDNA mutation is 10–100 times higher than that of nuclear DNA [82]. Importantly, mutated mtDNA accumulates in human tissues, ultimately disrupting mitochondrial homeostasis, exacerbating mitochondrial damage [83,84], and leading to the inability of mitochondria in bone tissues to accurately control the course of the inflammatory response. The presence of persistent inflammation in bone tissue can impede the activation of the immune system through various mechanisms, potentially leading to the accumulation of inflammatory metabolites [85], which behave in a biologically disruptive manner, thereby deteriorating the bone tissue. In the case of bone tissue, mutated mtDNA not only deprives bone tissue mitochondria of control over inflammation, but it also has the ability to essentially and directly reduce bone content in bone tissue [86]. Importantly, the mitochondrion is a stable organelle with a particularly strong outer wall, and such a structure results in its contents not easily leaking into the surrounding environment [77]. This discussion of mitochondria as a major regulator of the inflammatory response is not solely due to mitochondria containing several DAMPs/PAMPs or PRRs ligands [68], but also because the outer wall of mitochondria provides a microscopic framework that directly influences whether the inflammatory process ‘persists’ or ‘resolves’ in bone tissue.

Figure 3.

Mitochondria-mediated inflammation regulation in osteocytes. In the absence of DNA, cGAS remains in a perpetual state of autoinhibition. Upon binding to DNA, cGAS becomes activated, catalyzing the synthesis of cGAMP from GTP. cGAMP acts as a second messenger to bind the ER membrane protein interferon STING and induces conformational changes that activate STING. Activated STING activates IKK, which phosphorylates the IκB, and the subunit on IκB is specifically recognized and degraded by E3 ubiquitin ligase, releasing NF-κB. Free NF-κB enters the nucleus and acts in conjunction with interferon regulatory factors such as IRF3 to induce the expression of inflammatory factors such as TNF-α, IL-1β, and IL-6, ultimately resulting in inflammation. Abbreviations: cGAS: cyclic GMP-AMP synthase; cGAMP: cyclic GMP-AMP; GTP: guanosine triphosphate; ER: endoplasmic reticulum; STING: stimulator of interferon genes; NF-κB: nuclear factor-kappa B; IKK: inhibitor of kappa B kinase; IκB: inhibitor of NF-κB; IRF3: interferon regulatory factor 3.

2.4. ROS disrupt the bone microenvironment: disrupt bone metabolic processes and cause ischemia

The femur is the primary weight-bearing bone in humans, and the acetabulum encases the femoral head to facilitate the range of motion of the lower extremities, including abduction, external rotation, adduction, internal rotation, flexion and posterior extension. The femoral head is crucial for maintaining overall health, and its proper function relies on its structural integrity and a healthy bone microenvironment [13]. ROS are inevitable metabolites of mitochondrial aerobic respiration. Specifically, the enzyme complex on the MRC undergoes electron transfer and ultimately transfers electrons to oxygen, including the TCA cycle and the OXPHOS process, which synthesizes substantial amounts of ATP and releases minimal quantities of ROS. Additionally, under physiological conditions, the complex may experience electron leakage during electron transfer, and small amounts of oxygen are directly reduced to oxygen radicals [13,87], such as superoxide anion (O₂^-), hydrogen peroxide (H₂O₂), hydroxyl radical (OH^-), ozone (O₃), and singlet oxygen (¹O₂), which are ROS [88]. The former is the predominant process involving oxygen, accounting for approximately 95% or more of the total oxygen consumption. Generally, physiological levels of ROS produced by osteocyte mitochondria do not cause adverse effects on the organism, and in some instances can even stimulate osteocyte generation. For example, low levels of ROS increase Nrf2 activity during osteogenesis, which promotes the expression of primary osteocytes and osteoblasts specifically, and enhances the role of osteoblasts in differentiation and proliferation [89]. However, when cellular mitochondria are damaged, the incomplete MRC results in incomplete electron transfer to oxygen, causing most of the oxygen to be directly reduced to large amounts of toxic ROS [87], leading to oxidative stress and disrupting the intracellular oxidation–reduction balance. Furthermore, ROS levels in bone tissue can be increased by certain drugs, such as glucocorticoids, though the exact mechanism remains unclear [90]. Notably, toxic ROS also attack the mitochondria themselves, affecting the activity of the enzyme complexes on the MRC and reducing its efficiency, which in turn impacts mitochondrial OXPHOS and leads to a reduction in ATP synthesis [13].

Bone metabolism imbalance arises from the disruption of the physiological equilibrium between osteoblasts and osteoclasts, leading to discrepancies in the coupling of bone formation and resorption [91]. This imbalance alters the microstructure of bone tissues, resulting in bone-related disorders. Bone metabolism is a lifelong dynamic process, with a healthy skeletal structure relying on close coordination between bone formation and resorption [92]. In advanced stages of ONFH, the femoral head's tissue structure undergoes significant alterations. Extensive osteocyte death in the femoral head tissue impairs its ability to provide adequate support, causing the femoral head surface to collapse. This event largely depends on osteocyte numbers, including osteoblast maturation and osteogenic differentiation of BMSCs. Specifically, excessive ROS production can increase p66(shc) phosphorylation levels, identified as a crucial mediator in osteocyte apoptosis [93,94]. P66(shc) phosphorylation at Ser-36 triggers its mitochondrial translocation, initiating an oxidative stress response and cytochrome c release [95–98]. Cytoplasmic cytochrome c then activates the Caspase cascade, leading to osteocyte apoptosis [60]. In bone tissue, the number of surviving osteocytes significantly decreases in the presence of excess ROS, failing to maintain a healthy and robust bone structure. Notably, p66(shc) also functions as an oxidoreductase, further amplifying mitochondrial ROS production [95,99]. It is important to note that osteoblast numbers in bone tissue depend not only on their maturation but also on the osteogenic differentiation of BMSCs. The Wnt/β-catenin pathway has been found to directly promote BMSCs’ directional differentiation into osteoblasts and accelerate osteoblast maturation [100]. Stably expressed β-catenin enters the nucleus, binds to intranuclear transcription factors TCF/LEF, and initiates the expression of early osteoblast differentiation marker genes Runx2 and Osterix, followed by osteoblast generation and maturation. However, ROS overproduction can cause another transcription factor, FoxO, to be retained in the nucleus. FoxO can bind with β-catenin to prevent its binding with TCF/LEF [101], thus impeding osteoblastogenesis (Figure 4). Conversely, this type of ROS stimulates osteoclast activity and promotes proliferation [102], and studies have shown that scavenging ROS content inhibits receptor activator of NF-κB ligand (RANKL)-induced osteoclastogenesis [103]. It is now evident that the reduction in osteoblast numbers disrupts bone metabolism, resulting in a bone resorption rate that exceeds the bone formation rate [92]. This clearly does not provide an optimal bone tissue microenvironment for achieving satisfactory bone mass. Therefore, enhancing cellular mitochondrial function to effectively prevent ROS overproduction is crucial for maintaining bone tissue health and treating bone metabolism-related diseases.

Figure 4.

Regulation of the bone microenvironment by osteocyte mitochondria: implication for bone metabolism and blood supply conditions. In the nucleus, excessive ROS production prompts FoxO to competitively bind β-catenin with TCF/LEF, inhibiting osteoblastogenesis. Furthermore, this ROS directly stimulates RANKL to induce osteoclastogenesis, disrupting bone metabolism equilibrium. Concurrently, ROS directly damages the vascular wall and reduces blood supply. The emergence of antioxidant stress factor (Nrf2) mitigates the direct interaction of ROS with the vascular wall. Instead, by interfering with Nrf2 ubiquitination, followed by Nrf2 translocation to the nucleus, the formation of Nrf2-Maf through dimerization with sMAF, and binding to AREs of target genes, a series of cytoprotective genes are expressed, protecting endothelial cells. Additionally, ROS directly disrupt lipid metabolism, stimulating PPAR and RXR binding to form PPAR-RXR dimers, followed by PPRE binding. At this stage, the co-activator participates synergistically, ultimately regulating the transcription of target genes and adipocytes. The resulting adipocyte mass production eventually obstructs blood vessels and impairs blood supply. Abbreviations: RANKL: receptor activator of nuclear factor-κB ligand; sMAF: small Maf; AREs: antioxidant response elements; RXR: retinoid X receptor; PPRE: PPAR response element; co-activator: nuclear receptor coactivation cofactor.

The medial femoral circumflex artery is a crucial trophic vessel for the femoral head, branching off from the circulatory system and entering the medullary cavity of the bone tissue [104]. While osteocytes require substantial ATP for survival [38], a robust blood supply is equally vital for their physiological activity, with healthy blood vessels ensuring a constant supply of oxygen and nutrients to the bone tissue [4]. Research indicates that various risk factors, including smoking, hypercholesterolemia, diabetes, and hypertension, contribute to ROS production within the vascular wall, increasing oxidative stress [105,106]. This oxidative stress impairs endothelial cell regulation of vascular function, tone, and smooth muscle proliferation, leading to endothelial dysfunction that promotes vasospasm, atherosclerotic thrombosis, and vascular inflammation [107]. Studies have demonstrated that Nrf2 signaling acts as a key transcription factor in regulating oxidative stress resistance and is a major inducer of cellular defenses. Activated during oxidative stress states, it increases the expression of antioxidant genes such as glutathione (GSH), NAD(P)H: quinone oxidoreductase 1 (NQO1), glutathione-S-transferses (GSTs), glutamate cysteine ligase catalytic (GCLC), and heme oxygenase-1 (HMOX1), effectively protecting endothelial cells from ROS-induced damage (Figure 4) [108–111]. Endothelial cells are crucial in maintaining normal vascular endothelium structure and function [112,113]. Endothelial dysfunction directly disrupts blood flow regulation and vasoactive molecule release [114], disturbing the delicate balance between vasoconstriction and dilation. This disruption results in an inability to meet the bone tissue's urgent blood requirements, causing ischemia. Modulation of the vascular system is critical for initiating the angiogenesis-osteogenesis coupling necessary for full regeneration and repair of osteocytes in necrotic regions [4]. Notably, disturbed blood flow in damaged vessels transitions from advection to turbulence, promoting thrombus formation while delaying fibrin dissolution in clots, blocking vascular patency and exacerbating ischemia [115]. Additionally, ROS can stimulate PPAR transcriptional machinery, inducing adipocyte formation [116,117]. This adipocyte formation signifies the onset of lipid metabolism disorder. The small arteries in the medullary cavity are highly susceptible to blockage by adipocytes, leading to localized ischemia and increased medullary cavity pressure, disrupting bone microenvironment homeostasis. Under physiological conditions, blood and oxygen are inextricably linked. Consequently, ischemic areas due to ROS are inevitably oxygen-deficient. Hypoxic stress in bone tissue induces mitochondrial calcium overload, mitochondrial fragmentation, and osteocyte apoptosis [59], triggering a cascade of events. In response, a series of processes must be initiated to restore impaired blood flow and provide a healthy, stable internal microenvironment for osteocyte growth.

2.5. Potential causes of mitochondrial damage

The role of mitochondrial physiological functions in health and disease is gaining increasing attention [118]. In fact, the physiological functions of mitochondria can be summarized into four aspects: synthesizing ATP from nutrients through the OXPHOS system to provide energy, regulating Ca²⁺ concentration to control the process of apoptosis, modulating inflammatory responses, and controlling the synthesis and clearance of ROS through the electron transport chain system in mitochondria to maintain the redox balance within cells. The bone microenvironment discussed here encompasses not only bone metabolism and blood supply but also energy levels, inflammation, and Ca²⁺ concentration. Indeed, disruption of mitochondrial physiological functions can lead to disturbances in the bone microenvironment. On one hand, due to natural aging, mitochondria gradually degenerate as the human body ages, leading to mitochondrial dysfunction, which is inevitable [119]. During the aging process, mtDNA mutations become more frequent and may disrupt mitochondrial dynamics and bioenergetics over time, ultimately resulting in metabolic changes and increased glycolysis [120,121]. Consequently, mitochondrial damage in brain tissue caused by aging accumulates in the brain, disrupting cellular bioenergetics, reducing ATP production, and lowering metabolic efficiency. Simultaneously, the increase in glycolysis can also elevate ROS production, which is a primary cause of neurodegenerative diseases [122]. On the other hand, research has identified various factors that can trigger mitochondrial dysfunction, including viral or bacterial infections and physical inactivity [118]. Sepsis and septicemia can directly damage mitochondrial structures, leading to a decline in mitochondrial function. Mitochondrial transcription factor A (TFAM) plays a crucial role in maintaining mitochondrial function, energy metabolism, and cell survival. Studies have shown that TFAM levels in mitochondria are significantly reduced in sepsis and septicemia compared to control groups [118,123]. Currently, low cardiopulmonary fitness is considered one of the leading causes of mortality [124]. A study demonstrated that after four weeks of exercise, the activity of mitochondrial respiratory chain complexes I-IV in the trained legs of 10 healthy men increased by 46-61% compared to the untrained legs [125]. In fact, exercise can improve mitochondrial health by increasing mitochondrial number, enhancing the transcriptional activity of mitochondrial proteins, and reducing ROS production [118]. Some unhealthy lifestyle habits can also impair mitochondrial health, such as high-sugar and high-fat diets, smoking, and alcohol consumption. Each damaged mitochondrion exhibits reduced respiratory capacity and decreased mitochondrial membrane potential, often accompanied by abnormal ROS formation. Moreover, the accumulation of such defective mitochondria in the body can accelerate inflammation and amplify oxidative stress [126]. As mentioned earlier, excessive ROS can attack mitochondria themselves, leading to dysfunction.

3. Mitochondrial homeostasis achieved through mitochondrial fusion, fission and autophagy provides a stable bone microenvironment

Mitochondria are highly dynamic organelles that execute a variety of fundamental biological processes. They can modify their morphology, size, quantity, and quality by regulating mitochondrial fusion, fission, and autophagy. This process achieves functional complementarity of mitochondria and constructs a stable internal environment, known as mitochondrial homeostasis. This homeostasis is essential for maintaining mitochondrial health and physiology, ultimately supporting cell survival and physiological activities [13]. Recent studies have revealed that mitochondrial homeostasis is involved in the onset and development of osteocyte differentiation. Disruption of osteocyte mitochondrial homeostasis may lead to a series of morphological and functional abnormalities in various bone-associated cells [127–129]. Mitochondria possess a double-membrane structure, and mitochondrial fusion involves the combination of outer membrane fusion and inner membrane fusion. This process facilitates the exchange of contents, including proteins, lipids, and mtDNA [32,130]. Research has shown that mtDNA exchange during mitochondrial fusion is tightly regulated rather than occurring freely, which aids in eliminating mutated mtDNA and protecting mitochondrial health [131,132]. Mitochondrial fusion optimizes its physiological role by better coordinating mitochondrial function and promoting the repair of damaged mitochondria [133]. Key proteins involved in mitochondrial fusion include mitofusins 1 and 2 (Mfn 1 and Mfn 2) and optic atrophy 1 (Opa 1) [132,134]. The inverse action of mitofusins on the OMM is an early step in fusion, facilitating OMM fusion [135]. Opa 1, a dynamin-associated GTPase present in the mitochondrial intermembrane space, plays a crucial role in IMM fusion, thereby facilitating mitochondrial fusion [136]. Studies have found that inactivation of Opa 1 can cause mitochondrial morphological changes leading to incapacitation, such as mitochondrial swelling and contraction and cristae swelling [137]. Mitochondrial fission, the opposite process of fusion, cleaves tubular mitochondria into smaller units, facilitating the removal of damaged or dysfunctional mitochondria through autophagy and maintaining a healthy mitochondrial population [130]. Dynamics related protein 1 (Drp 1) is a key regulator of mitochondrial fission, and dysfunctional fission can disrupt mitochondrial quality control, leading to the accumulation of damaged mitochondria and the release of pro-apoptotic factors [130]. Generally, Drp 1 is predominantly present in the cytoplasm, translocating to the OMM during fission. It interacts with Drp 1 receptors, including mitochondrial fission protein 1 (Fis 1), mitochondrial fission factor (Mff), and mitochondrial dynamics proteins of 49 kDa (MiD 49) and 51 kDa (MiD 51), forming helical oligomers that encircle and compress mitochondria to complete fission. Subsequently, Drp 1 returns to the cytoplasm for reuse. Under normal physiological conditions, precise coordination between mitochondrial fission and fusion maintains mitochondrial number and function, ensuring mitochondrial homeostasis [138]. As mitochondria are present in various cell types and influence cell growth and differentiation, during the differentiation of mouse MSCs into osteocytes, an increase in the expression of Mfn 1 and Mfn 2 was observed, with MSCs mitochondria gradually fusing into tubular shapes. Conversely, mitochondrial fragmentation and increased expression of Drp 1 and Fis 1 were observed during differentiation into chondrocytes [139]. Notably, the bioenergetics of the cell differentiation process remains dependent on the OXPHOS process. Mitochondrial homeostasis is maintained by regulating mitochondrial dynamics, ensuring that the cell is filled with a sufficient number of healthy mitochondria. This regulation ensures that the OXPHOS system functions properly and provides sufficient ATP. Additionally, mitochondrial dynamics can alter the migration and proliferation behavior of endothelial cells in bone tissue [140]. Drp 1-mediated mitochondrial fission enhances the differentiation of EPCs into endothelial cells, induces the formation of plate pseudopods, accelerates the migration of endothelial cells, and induces neovascularization [141].

Mitochondrial autophagy, another form of mitochondrial homeostasis, is a crucial process controlling mitochondrial quality. It mitigates cellular stress caused by noxious stimuli by eliminating damaged or dysfunctional mitochondria and excess proteins, promoting continuous mitochondrial renewal, and maintaining healthy and functionally active mitochondria [25]. Under pathological conditions, osteocyte mitochondria experience MRC electron leakage, which releases ROS, increasing intracellular ROS levels in osteocytes and leading to osteocyte apoptosis [13]. To counteract the excessive accumulation of oxygen free radicals, mitochondrial autophagy is initiated. This process maintains osteocyte mitochondrial homeostasis by degrading damaged osteocyte mitochondria, thereby reducing ROS levels and protecting osteocytes from apoptosis [13]. Essentially, maintaining mitochondrial homeostasis in osteocytes ensures the stability of the internal environment within bone tissue. In a study investigating potential mechanisms of osteonecrosis ONFH, mouse osteocytes exposed to glucocorticoids in a hypoxic environment triggered osteocyte apoptosis. The study found that overexpression of hypoxia inducible factor 1α (HIF-1α) in the hypoxic environment triggered the expression of its downstream marker BNIP3, which reduced the inhibitory effect of glucocorticoid-induced mitophagy in osteocytes, thus protecting osteocytes from apoptosis [142]. Emerging research demonstrates that the use of autophagy regulators can modulate mitophagy in osteocytes, favoring osteocyte survival [143], including MTOR and AMPK regulators. Chen et al. found that Apelin-13, an endogenous adipokine involved in bone homeostasis, ameliorates oxidative stress by activating the mitophagy program in BMSCs, which in turn leads to the restoration of osteogenic function through AMPK-α phosphorylation [144]. In another experiment, MTOR was observed to activate PINK1/Parkin-mediated mitophagy, and this activation reduced osteocyte apoptosis by eliminating ROS and damaged mitochondria [145]. PINK1 and Parkin interact with each other, with PINK1 acting as an autophagy signal for mitochondria and also as a detector to measure mitochondrial damage [146]. Parkin acts to amplify this signal, and together they regulate the mitophagy process to maintain mitochondrial quality and ensure mitochondrial homeostasis.

4. Targeting osteocyte mitochondria within the bone microenvironment

In fact, osteocyte mitochondrial damage or altered function, including regulation of ATP synthesis, calcium metabolism, and inflammatory responses, as well as oxidative stress states released by the mitochondria, may explain some mechanisms inducing osteocyte death during ONFH. To comprehend the pathogenesis of ONFH, the microenvironment of osteocyte survival in the femoral head tissue warrants consideration. The bone microenvironment is a complex biological system where abnormal alterations often occur interconnectedly and synergistically. Current research recognizes that strategies targeting the bone microenvironment are crucial for osteocyte survival. Mitochondria, organelles present in cells, directly influence the tissue microenvironment. Targeting osteocyte mitochondria aims to restore a healthy bone microenvironment by eliminating damaged mitochondria and repairing mitochondrial function. Notably, different cell types in bone tissue (including bone-associated cells, immune cells, endothelial cells, etc.) share the same microenvironment and play a vital role in bone formation [147]. The bone microenvironment can be categorized into three parts based on functions and components: physiological, chemical, and physical [147]. Physiologically, osteocytes undergo optimal growth and development supported by mitochondrial functions, such as ATP synthesis, calcium metabolism, and regulation of inflammatory responses. Recent studies have shown that M2-type macrophages in bone tissue can convert the inflammatory environment into an anti-inflammatory microenvironment, improving the physiological microenvironment at the injury site and promoting osteocyte survival [148]. Chemically, excess ROS produced by damaged or dysfunctional mitochondria stimulate vascular endothelial cells and various signaling molecules, increasing oxidative stress and contributing to an ischemic hypoxic microenvironment. Some physical factors, such as electrical stimulation, have been found to induce mitochondrial autophagy and maintain mitochondrial health [149]. Due to the dynamic interaction of the abnormal bone microenvironment [147], addressing a single factor is insufficient to maintain a healthy bone microenvironment, necessitating the identification of the origin of abnormal microenvironmental changes. Osteocyte mitochondria can achieve homeostasis through dynamics and autophagic processes, thereby ensuring a healthy bone microenvironment.

5. Conclusions and perspective

To date, limited research has been conducted on mitochondrial pathology in ONFH and other orthopedic diseases, with even fewer studies exploring their molecular mechanisms. Currently, ONFH diagnosis relies on patient-reported hip pain symptoms and MRI; however, most patients with ONFH are asymptomatic in early stages, resulting in missed opportunities for optimal intervention. Existing therapies primarily address ONFH clinical symptoms and alleviate pain, but they are ineffective in slowing or reversing ONFH progression. Therefore, a more comprehensive understanding of ONFH pathogenesis and the development and evaluation of ameliorative therapies are essential. The pathogenesis of ONFH is primarily attributed to various factors leading to an abnormal microenvironment of the femoral head, ultimately resulting in premature osteocyte senescence, apoptosis, and death, which compromises bone microstructure and leads to manifestations such as femoral head collapse. Mitochondria have become a significant focus in biomedical research due to their crucial role in various human diseases. Initially perceived as fluid cytoplasmic particles or symbiotic bacteria, mitochondria were later recognized as ‘bean-like’ organelles responsible for aerobic respiration, with enzyme complexes on the MRC synthesizing most of the cell's ATP via OXPHOS. Mitochondria were once considered the ‘energy factories of the cell’. Subsequently, they were identified as matrilineally inherited organelles with their own separate genome, expanding the field of mitochondrial medicine due to the potential for mtDNA mutations or deletions to cause disease. Recent studies have demonstrated that targeting mitochondria can indeed affect conditions in other orthopedic diseases, including intervertebral disc degeneration, osteoarthritis, rheumatoid arthritis, and osteoporosis [143,150–152], providing new perspectives for the prevention and treatment of bone diseases. Despite limited research on mitochondrial regulation of osteocytes, it is evident that physiological regulation of osteocyte mitochondria significantly impacts overall bone tissue physiology. Damaged mitochondria influence disease progression through various modes of injury, notably oxidative stress, calcium homeostasis imbalance, impaired ATP synthesis, and uncontrolled inflammatory responses [153]. These modes of injury are often interrelated, with one mode positively affecting others, leading to acute or chronic irreversible apoptosis or death. Thus, targeting mitochondria can be effective in improving patient prognosis, even if mitochondrial dysfunction is not the primary driver of disease development but rather an aggravating factor. As modern medicine advances, disease studies have shifted from cells to various subcellular organelles, with mitochondria gaining attention due to their role as signaling centers coordinating overall organism function. In the field of disease diagnosis, the detection of indicators such as mtDNA mutations and changes in membrane potential has provided new methods for the early diagnosis of diseases. Research has found that in the pathogenesis of major depressive disorder (MDD), mtDNA acts as an inflammatory trigger, released from dysfunctional mitochondria in the central nervous system into the peripheral circulation, thereby contributing to the neuroinflammatory disease pathway. By reviewing the neuroinflammation theory in MDD, researchers have demonstrated that mtDNA can serve as an innovative clinical biomarker for MDD and holds potential for treating the disorder [154]. In fact, mitochondrial proteomics studies have identified 23 proteins with significantly altered expression [17]. Unsurprisingly, changes in the expression of mitochondrial proteins hint at the onset of diseases. Hopefully, with continuous advancements in medicine, powerful and high-throughput technologies will enable the measurement of a larger number of mitochondrial proteins and metabolites, thereby unlocking the clinical application value of mitochondria. Moreover, clinical data on biomarkers can provide medical professionals with early and accurate insights into the progression of diseases. In the future, more detailed research should be conducted on the specific mechanisms of mitochondrial proteins and metabolites in diseases. In the field of treatment, several studies have shown that targeting mitochondria has potential in treating osteonecrosis-associated diseases. Electromagnetic field therapy stimulates the mitochondrial OXPHOS system of osteocytes, promoting osteosynthesis [155]. Artificial mitochondrial transplantation, which upregulates OXPHOS activity and increases ATP production, has shown potential in enhancing BMSC function and promoting bone defect repair [156]. In recent years, the emergence of nanotechnology has significantly enhanced the effectiveness of treatments. Precision-designed nanoparticles can capture overloaded Ca²⁺ around MSC mitochondria, modulating mitochondrial calcium flux, restoring mitochondrial and MSC function, and treating inflammation-associated bone diseases [157]. Deng et al. reported that porous Se@SiO2 nanocomposites effectively reduce ROS damage and protect osteocytes by mitigating oxidative stress [158]. The TBP@CeO₂ nanozyme, which possesses mitochondrial targeting capability, exhibits polyene enzyme activity and demonstrates robust ROS scavenging ability. It can effectively eliminate ROS and ensure sufficient ATP production, thereby restoring mitochondrial function [159]. In fact, nanozymes are nanomaterials with intrinsic enzyme-like properties and serve as important alternatives to natural enzymes. As emerging biocatalysts, nanozymes hold great potential in mimicking the activities of natural enzymes, enhancing mitochondrial function, and providing novel therapeutic strategies for aging-related diseases [160]. These experimental results demonstrate that enhancing mitochondrial function is a powerful approach to maintaining bone health. Ongoing research explores specific methods to enhance mitochondrial function, including traditional chinese medicine intervention and gene therapy [161]. Zuogui Pill (ZGP) exhibits significant antioxidant and anti-apoptotic effects in iron overload-induced osteoporosis by scavenging ROS, thereby mitigating mitochondrial damage [162]. Wen-Shen-Tong-Luo-Zhi-Tong Decoction (WSTLZTD) can enhance mitochondrial energy metabolism and osteogenic differentiation in aged BMSCs, subsequently treating bone diseases [163]. Notably, appropriate exercise and a healthy dietary lifestyle represent the least costly and minimally side-effect strategies for humans. These approaches not only strengthen mitochondria themselves but also facilitate self-improvement. Enhancing mitochondrial function is a key strategy to prevent the accumulation of dysfunctional mitochondria, which is crucial for cellular homeostasis and longevity [164]. It is important to note that these mitochondrial interventions are intended to ensure normal mitochondrial function, and focus should be placed on targeting bone microenvironmental therapies to reduce osteocyte death for preventive treatment.

Mitochondrial dysfunction in ONFH osteocytes potentially compromises MRC integrity and ATP synthesis, while also leading to disturbances in calcium metabolism, production of mutated mtDNA, ROS, and pro-inflammatory factors. These alterations disrupt the bone microenvironment and may induce detrimental effects on osteocytes, both directly and indirectly. Targeted mitochondrial interventions show promise in preserving mitochondrial function, potentially stabilizing the bone microenvironment and addressing osteocyte death-related diseases. Such strategies may pave the way for advancements in precision and personalized medicine.

Funding Statement

This work was supported by grants from the National Natural Science Foundation of China [grant number 82202768]; the Fundamental Research Funds for the Central Universities [grant number 2242020K40156]; Open Project Programme of the Key Base for Standardized Training for General Physicans, Zhongda Hospital, Southeast University [grant number ZDZYJD-QK-2022-16]; Jiangsu Entrepreneurship and Innovation Doctors [grant number 202030338]; Zhongda Hospital Affiliated to Southeast University, Jiangsu Province High-Level Hospital Construction Funds [grant number GSP-LCYJFH20].

Disclosure statement

No potential conflict of interest was reported by the author(s).

Availability of data and materials

All data and resources used in the paper have been cited and indicated.