The morphological changes in aged bone and osteocytes
Based on the morphological evaluation and observation of young and aged long bones (Figs. 1 and 2), the main phenotypes of aged cortical bone can be generally summarized as (1) loss of bone mass, (2) changes in bone matrix (structure and composition), and (3) poor osteocyte status. We assume that poor osteocyte status may be the root cause of bone ageing phenotypes, and molecular evidence that can link the senescence of osteocyte network to bone ageing urgently needs clarification.
Acquisition of biological information from bone cells and its limitation
In this study, the biological information was obtained directly from the cortical bone. So, the key issue is ensuring that osteocytes are the most abundant cells in the sampling site. In this context, we took a small segment of cortical bone where the osteocytes showed highly differentiated morphology with abundant dendrite connectivity. Heart perfusion can effectively remove the blood cells from bone tissue. The pretreatment of the cortical bone surface by a quick flush with TRIzol reagent before RNA extraction and by a short digestion with trypsin and collagenase before exosome extraction can reduce the influence of adherent cells, including osteoblasts (OBs), lining cells, and osteoclasts (OCs). Nevertheless, it is difficult to remove the vascular endothelial cells (VECs) in the bone matrix. Research on the quantitative morphometry of the bone vasculature has shown that the vascular volume density accounts for 00.13% of the cortical total volume in the region of the murine femoral mid-diaphysis [33]. Additionally, histological observations of serial sections in our preliminary experiment suggested that the proportion of osteocytes was more than 90% (90.49%) of the total number of cells in the middle 3-mm segments of the cortical bone (Additional file 1: Figure S1 and S2). Although the osteocytes are the majority of cells in our sampling site, the information we obtained was not from pure osteocytes but a mixture, which we consider as the limitation of this study. More accurate sampling methods and cutting-edge sequencing methods, such as laser microdissection based on hard tissue and single-cell sequencing, may be helpful to optimize the methods of this study.
DEGs involved in bone mechanosensation
The expression of many key factors involved in the bone mechanosensation is downregulated in the aged cortical bone. First, the critical structural molecules that allow osteocytes to bear mechanical force are downregulated (Fig. 4): integrins and related factors (e.g., Itga10, Itgb4, Chad, Fap), components of focal adhesions (e.g., Lamb2, Thbs2/4, Tnc, Tnn, Pdgfra), cytoskeleton proteins (e.g., Actn2, Cav1, Flnc, Myl2, Mylk4), and the main calnexin (Cx43/Gja1) that composes the gap junction between osteocytes. Second, key genes whose products involve in mechanical stimulation induced second messenger production: key enzymes involved in NO, PGE2, and ATP synthesis (e.g., NOS1, COX1-3, ND1-5), factors involved in the regulation of homeostasis and the intracellular flow of calcium ions (Casq1, Jph2, Trdn, Jsrp1, Hrc, Capn3, Ryr1), and Sost, an osteocyte-specific marker that integrates osteocyte mechanotransduction and bone mass by antagonizing Wnt/beta-catenin signalling [34, 35]. Additionally, downstream signalling elicited by mechanical force-induced messengers, such as cGMP-PKG, PI3K-AKT, and WNT signalling, is downregulated (Fig. 4a). Taken together, these lines of evidence tend to show the attenuation of the mechanical sensing of aged bone cells.
Hormone receptors
Here, the gene expression of some hormone receptors, including Pth1r, VDR, Fgf1r, and Fgf2r, was downregulated in aged cortical bone. Parathyroid hormone (PTH), secreted by the parathyroid glands, plays a central role in maintaining bone metabolic homeostasis by binding to the PTHR, a G protein-coupled membrane-spanning receptor. Activation of PTH1R increases both bone formation and resorption by inhibiting Sost expression and elevating the RANKL/OPG ratio, respectively [13]. Vitamin D binds to vitamin D receptor (VDR), as is the case with nuclear receptors. Its main function is to mediate calcium and phosphorus metabolism by regulating the expression of the molecules (e.g., DMP1, MEPE, PHEX) involved in the synthesis and secretion of inorganic pyrophosphate and the regulation of bone matrix mineralization [15]. The expression of FGF23, the core regulator of body calcium and phosphorus [14], has been demonstrated to be influenced by both PTH/PTHR and vitamin D/VDR signalling through the fibroblast growth factor receptor (FGFR) [13, 36]. Given their important role in mediating the calcium phosphate homeostasis between the bone and the circulatory system, downregulation of these receptors may alter how cells perceive and respond to hormones during ageing.
DEGs involved in bone formation and resorption
Many key osteogenic molecules and pathways are downregulated in aged group, including members of the BMP and WNT signalling pathways (Tgfb2, Bmp2, Wnt5b, Wnt16, Lrp4, Lrp5) and their target genes (Gremlin, Twist, Ocn, Postn, Gja1, Opg, Cdh2/11/15, and Alp1) (Fig. 4b). Moreover, many regulators of these two pathways are also downregulated; some are positive regulators, e.g., Col1a1, Bambi, Cav1, Fgfr2, and Kank1, while some are inhibitors, including Twist, Dkk1, Nbl1, and Cdh2. Downregulation of these regulatory factors indicates that the dominant role of bone cells in regulating bone formation weakens during ageing.
Regarding the formation and maintenance of bone matrix, gene expression levels are downregulated for a variety of collagens (e.g., COL1, COL2, COL3, COL5, COL8, COL11), molecules involved in collagen fibril organization (e.g., LUM, P3H1, GREM1, LOX), and many non-collagenous bone matrix proteins (OCN, ON, POSTN, DMP1, MEPE, PHEX) (Fig. 4). The lack of these molecules could affect the mineralization and mechanical properties of the extracellular matrix.
It has been demonstrated that the mediation of osteoclastic bone resorption depends mainly on the receptor activator of nuclear factor kappa-Β ligand (RANKL)/osteoprotegerin (OPG) ratio. RANKL, produced by osteocytes, is the major osteoclast differentiation factor [37, 38]. Although our data showed no significant difference in RANKL expression, downregulation of Opg/Tnfrsf11b may increase the RANKL/OPG ratio, which could be the reason for the excessive bone resorption in the aged bone. In addition, the upregulation of several key genes of osteoclast stimulating factors, such as Adam8, Tyrobp, Sbno2, and Ccr1, could promote osteoclast formation. Furthermore, higher expression levels of genes involved in regulating proinflammatory cytokine responses are positively associated with osteoclast activation in the aged bone.
DEGs related to immunity
From our data, it is obvious that the proinflammatory state is the most significant feature of bone aging, as the upregulated genes were enriched in many inflammatory GO terms involved in both innate and adaptive immunity. In innate immunity, pattern recognition receptors (PRRs), a group of receptors that activate the formation of inflammasomes by identifying pathogen-associated molecular patterns (PAMPs) and damage-associated molecular patterns (DAMPs) in the environment, play important roles in regulating pathogen recognition and activation of the intracellular inflammatory response [39]. In our data, pattern recognition-related inflammatory molecules and pathways were upregulated in the aged group (Fig. 5), including (1) two important pattern recognition receptor (PRR) pathways—the Toll-like receptor and NOD-like receptor signalling pathways (Fig. 5a); (2) molecules and signalling pathways downstream of PRRs that trigger innate immunity, including NF-κB signalling (Fig. 5a) and genes related to interferon production (Irf1, Ptpn22, Myd88, Cd14, Pycard); (3) NLRP inflammasome components (Nlrp3, Pycard, Card11); and (4) molecules that are regulated in response to viruses, fungi, and lipopolysaccharide (Fig. 5a). These lines of evidence suggest that pathogen infections may be the cause of the enhanced proinflammatory state in aged cortical bone. In addition, a large number of upregulated DEGs were enriched in GO terms involved in adaptive immunity, such as regulation of cytokine production and secretion, tumour necrosis factor (TNF) production, lymphocyte proliferation, differentiation, and migration (Fig. 5b). It is worth mentioning that some known regulators of adaptive immunity were found in OLCS exosomes (see below in the “Immune-associated proteins” section), and most of them were upregulated in the aged group, which might be further evidence for the involvement of aged bone cells in the regulation of adaptive immunity.
On the other hand, there are many receptors of proinflammatory cytokines whose gene expression levels were increased in the aged group: some of them are involved in the mediation of immunosuppressive signals, such as Lilrb4, Pilra, Il10ra, and Il1r2; some play pathogenic roles in inflammatory and autoimmune diseases such as rheumatoid arthritis, for example, Il17ra; and Il18rap and Il21r, whose products are involved in the activation of signalling pathways associated with inflammation, including NF-κB, MAPK8, and JAK-STAT. Changes in the expression levels of these molecules could affect the responses of aged bone cells to inflammatory factors. Notably, molecules negatively regulating JAK-STAT signalling were downregulated (Fig. 4b), while molecules positively regulating the JAK-STAT cascade were upregulated in the aged group (Fig. 5b). Given that JAK-STAT signalling is considered to have the capacity to mediate the responses of target cells to inflammatory cytokines and plays an important role in regulating cell apoptosis [40], the active regulation of JAK-STAT signalling might be the link between cell responses to inflammatory cytokines and cell death.
DEGs involved in cellular energy metabolism
Mitochondrial and endoplasmic reticulum (ER) insufficiencies are widely considered to be putative hallmarks of cell senescence [41]. Here, signs of decline in energy metabolism in the aged bone cells were evident: first, in the aged group, of the 69 downregulated transcripts whose products serve mitochondrial functions, many are involved in ATP production (tricarboxylic acid cycle, respiratory chain, and oxidative phosphorylation). Downregulation of these key genes may directly reduce the cellular energy supply. In addition, several downregulated molecules are key components of complex I (ND1-4) and complex IV (COX1-3) in the respiratory chain, whose dysfunction could cause electron leakage and increase the production of reactive oxygen species (ROS), one of the most important factors for cell senescence. Finally, the gene expression of several key enzymes that respond to cellular stress was downregulated: for example, LONP, which can protect cells from various stress conditions by regulating mitochondrial metabolism and repairing mitochondrial DNA [42], and PINK, which plays an irreplaceable role in mediating mitochondrial autophagy [43]. In addition, the products of some DEGs located in the ER were downregulated. FKBP9, CRTAP, and FKBP10 function as protein-folding factors mediated by chaperones [44]. MNF2 and VDAC are typical key factors on the ER–mitochondria interface [45, 46]. These changes in energy metabolism could be the root causes of bone ageing.
Changes in some cellular metabolism pathways are also important clues for determining the underlying mechanisms of bone ageing. The FoxO signalling pathway is regulated by and counteracts external changes that disturb homeostasis, including metabolic stress, oxidative stress, and growth factor deprivation [47]. According to the KEGG analysis, the FoxO signalling pathway was upregulated in the aged group, which may be further corroboration of the severe cellular stress in aged bone cells. Furthermore, FoxO signalling may have special cellular functions in the bone: it has been demonstrated that activation of FoxO in osteoblasts can hinder bone formation, as it can competitively inhibit the WNT pathway by diverting β-catenin from TCF- to FoxO-mediated transcription [48]. Moreover, the FoxO pathway is normally negatively regulated through phosphorylation mediated by the PI3K-AKT pathway in diverse organisms [49]. Meanwhile, PI3K-AKT is responsible for mechanical force-induced osteogenesis in the bone [8]. Consistently, in our data, FoxO signalling was upregulated (Fig. 5a) and accompanied by the downregulation of PI3K-AKT and WNT signalling (Fig. 4a, b). Thus, the crosstalk among these three pathways could be the molecular explanation and underlying link between the bone loss and decline in cell function that occur during ageing.
The loss- and gain-of-function of OLCS exosomes with age indicated by GO analysis among exosomal proteins
The results revealed that the skeleton could secrete large amounts of regulatory proteins involved in regulating a wide range of cellular activities, from regulating energy metabolism and protein transportation to cell differentiation, morphogenesis, and movement (Fig. 6d). Additionally, biological processes involving the maintenance of cell homeostasis and anti-senescence were included, such as molecules involved in the regulation of telomere maintenance, protein folding and stabilization, tissue regeneration, lysosome organization, and proteins in response to ROS and hormones (Fig. 6d, e).
In clear contrast to young controls, the aged OLCS exosomes lacked functional proteins for cellular activities, including maintenance of the morphological properties of cells, maintenance of cell homeostasis, and regulation of cellular metabolism and transportation (Table 1). Moreover, the results revealed a functional decline in the regulation of DNA conformation, telomere maintenance, protein complex assembly, and localization (Table 1). In addition, proteins involved in lysosome organization and cellular responses to hormones (parathyroid, angiotensin, and mineralocorticoid) were absent in the aged group (Table 1). On the other hand, the enriched proteins in the aged group were mainly associated with activation of immune responses, cell damage, and cell death (Table 1). Most of the features mentioned above mirrored the findings at the RNA level; some of them have been demonstrated or categorized in existing studies linked to cell senescence [41] and could elucidate both the causes and consequences of bone ageing.
Proteins identified in OLCS exosomes
Senescence-associated secretory phenotype
Studies have demonstrated that aged cells can secrete proinflammatory factors, growth factors, chemokines and proteases to mediate age-related inflammatory responses, wound healing, and even tumour progression; this condition is now referred to as the senescence-associated secretory phenotype (SASP) [50, 51]. Here, several classical SASP proteins, including TGF-β2, OPG, MMP9, TIMP1, MIF1, PRDXs, and IGFBP3, were found to be upregulated in aged OLCS exosomes. Among them, TGF-β2 and OPG are important mediators of bone metabolism; serum levels of TGF-β2 and OPG have shown significant positive correlations with bone turnover markers in Chinese women [52]. TIMP1, a tissue inhibitor of metalloproteinases (MMPs), is expressed in both osteocytes and osteoblasts and maintains the balance of bone matrix degradation by regulating MMP levels [53]. Moreover, TIMP/MMPs can also accommodate the immune influx in the extracellular matrix [54]. MIF1 is known to activate several transcription factors and stress kinases that can mediate inflammatory and tumourigenic signalling, e.g., NF-κB and AMPK [55]. Thus, the increased expression of these SASP proteins in senescent OLCS exosomes may explain how aged bone cells contribute to the environmental effects of adjacent and distal cells and systems.
Immune-associated proteins
Our analyses of the immune-associated proteins in OLCS exosomes have shown interesting findings regarding the cross-regulation between the skeleton and the immune system: First, approximately 10% (85 out of 1019 proteins identified in the young group, and 76 out of 700 proteins in the aged group) of the identified proteins found in OLCS exosomes were enriched in immune-related GO terms, and approximately 80% (67 out of 85/76 in young/aged group) of them overlapped between the young and aged groups. Although most of them were found to have a higher LFQ intensity in aged OLCS exosomes than in young ones, their overall composition in exosomes was relatively constant across age. To some extent, these results indicate that there is a certain group of molecules in OLCS exosomes, both aged and young, that may serve an immune regulatory function. Second, among these overlapping immune-associated proteins were some key factors that have been shown in existing studies to regulate the activities of both immune cells and bone cells, including CD44, CD47, CD59, TGF-β2, and GSN. Among these, CD44 is a novel marker of osteocytic differentiation in bone [56]; meanwhile, it is also widely expressed in many kinds of immune cells (T cells, granulocytes, monocytes) and is critical for their maturation [57, 58]. CD47, known as a “don’t eat me” signal to macrophages, plays a critical role in immune cell activation [59, 60], and it also has a profound effect on skeletal remodelling and bone maintenance through its actions on both osteoblasts and osteoclasts [61]. For CD59, this molecule was recently demonstrated to be a regulator of bone growth and homeostasis by interfering with the complement system in innate immunity [62]. TGF-β, a multifunctional cytokine, plays integral roles in the regulation of adaptive immunity, mainly through activating T cells and effector and regulatory T cells [63]; in the bone, it is a critical regulator coupling of bone formation and resorption [64]. All these proteins, except CD44, showed increased expression in aged OLCS exosomes. Similarly, many other immunoregulatory molecules were upregulated in aged OLCS exosomes, including several complement molecules and regulators: C3, C1QA, C1QB, C1QC, C4A, C8B, MBL1, MASP2, FCNB; positive regulators of T cell/B cell activation: SLC4A1, SPN, TFRC, ANXA1, CORO1A, FLOT, PNP, PTPRC; and regulators of leukocyte proliferation, migration, and aggregation: S100A9, S100A8, PNP, PLG, MIF, ECM1, SERPINE1, SLC4A1, GSTP1, ELANE.
On the other hand, only a small number of immune-associated proteins were found to have higher intensities in the young group relative to the aged group, including M-CSF (CSF-1), CD1d, Galectin1 (LGALS1), CD90 (THY1), and RHOA. Among them, M-CSF (macrophage colony-stimulating factor 1) is a haematopoietic growth factor involved in the proliferation, differentiation, and survival of monocytes and macrophages; in the bone, it is one the most important cytokines for the maturation and function of osteoclasts. CD1d is a key molecule necessary to activate CD1d-dependent natural killer T cells that regulate certain tissue-specific immune activities [65]. Galectin1, a homodimeric galactose-binding lectin, is able to selectively bind with a T cell surface receptor (NP-1) and inhibit T cell proliferation [66, 67]. CD90, also known as the T lymphocyte differentiation antigen, is a conserved cell surface protein with a single V-like immunoglobulin domain; CD90 can activate the T cell receptor [68] and is expressed as a surface marker for bone marrow stem cells [69, 70] and osteoblasts [71].
Collectively, a large number of immune regulatory factors for both innate and adaptive immunity were found in OLCS exosomes. These findings may provide clues for exploring the mutual regulation between the skeleton and immune system and the mechanism of bone ageing.
Bone remodelling-associated proteins in OLCS exosomes
Proteins that are involved in osteoblast differentiation (e.g., COL-1/5, LRP, MMP-2, DCN, CX43, β-CATENIN, TGF-β, ALP) and osteoclast differentiation (e.g., M-CSF, ANXA, FBN1, SLC9, SRC, ATP6AP) were found to be upregulated in the young group relative to the aged group (Fig. 7a, c), indicating that exosomes may be one of the possible mechanisms of bone secretion in the regulation of bone remodelling and decrease with age.
Antioxidative proteins in OLCS exosomes
Since cell ageing is identified as a decline in various functions of the cell, almost all evidence that supports an increase in harmful effects or a loss of beneficial effects in the aged cell is reasonable. However, there is an exception: some exosomal proteins involved in maintaining cell redox homeostasis and removal of ROS are upregulated in aged OLCS exosomes relative to young ones (Fig. 7b, d), including several key molecules, such as peroxiredoxins (PRDXs), superoxide dismutase 2 (SOD2), thioredoxin 1 (TRX1), glutathione S-transferase P1 (GSTP1), and haptoglobin, which manifests in aged OLCS may exert a positive effect on surrounding or even distant cells to resist oxidative damage and ageing.