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Ageing characteristics of bone indicated by transcriptomic and exosomal proteomic analysis of cortical bone cells

Ageing characteristics of bone indicated by transcriptomic and exosomal proteomic analysis of... Background: Degenerative changes in the skeleton play an important role in ageing. As the foremost sensors and orchestrators of bone remodelling, osteocytes contribute significantly to the health of the skeleton. Embedded in a mineralized bone matrix, the osteocyte network and the surrounding lacunar canaliculae work together as a functional syncytium—the osteocytic lacunar-canalicular system (OLCS). However, changes in the OLCS during ageing and related mechanisms cannot be fully understood by using traditional histological analysis. Methods: To link the phenotypes of aged osteocytes and their functional changes during ageing, we analysed the changes in the gene expression profiles of bone cells and the proteomic profiles of OLCS exosomes derived from aged and young cortical bone. Results: Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed genes (DEGs) suggested that a decline in cell energy metabolism and an increased level of the proinflammatory state are major characteristics of bone ageing. Moreover, some DEGs were key regulators of bone mechanical sensation and bone remodelling, which are indicative of reduced bone-specific function with age. Further, the identified proteins in OLCS exosomes showed potential changes in the secretory function bone. Compared with young controls, the decreased functional proteins in aged OLCS exosomes were enriched mainly in GO terms that included regulating bone development and remodelling, cell-matrix adhesion, and cell clearance and homeostasis. Notably, several functions of exosomal proteins of the aged group revealed potential new roles, such as regulating innate and adaptive immunity, wound healing, and angiogenesis and eliminating oxidative stress. Conclusion: The information obtained from bone cells and OLCS exosomes will help us discover new features of bone ageing. Keywords: Bone ageing, Gene expression profiling, Exosome, OLCS Background lacunar-canalicular system (OLCS) [1–3], underscoring Osteocytes, the most numerous cell populations in bone, that the function of osteocytes cannot be implemented are responsible for sensing mechanical force and acting without cell-to-cell connections and their 3D physio- as the “hub” of the regulatory network for bone remod- logical environment. elling. The osteocyte network and the lacunar canalicu- Mechanical stress is an effective stimulus for osteo- lae constitute a functional syncytium—the osteocytic cytes to release messengers such as ATP, nitric oxide (NO), and prostaglandin E2 (PGE2) [4–6], which then * Correspondence: yaosun@tongji.edu.cn transport through the lacunar-canalicular system (LCS) Department of Oral Implantology, School and Hospital of Stomatology, and gap junctions between adjacent cells to the osteo- Tongji University, Shanghai Engineering Research Center of Tooth genic surface to prompt osteoblast-mediated bone for- Restoration and Regeneration, 399 Middle Yanchang Road, Shanghai 200072, China mation and directly inhibit osteoclast activation [7–9]. Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhang et al. Journal of Orthopaedic Surgery and Research (2019) 14:129 Page 2 of 17 Additionally, since osteocytes account for more than group) were scanned by μCT50 (Scanco, Switzerland), 90% of bone cells [10], the large number of processes in 10 μm per slice. The parameters included the bone vol- the osteocyte network possesses an immense interface ume to total volume ratio (BV/TV), trabecular number for the exchange of information and substances between (Tb.N) and cortical bone thickness (CBT). For histo- cells and the extracellular environment [11, 12]. Thus, logical analysis, 4-μm-thick paraffin sections of speci- steroids, hormones, and cytokines in the environment mens were prepared. First, the sections were stained can affect the regulatory function of osteocytes by bind- with hematoxylin and eosin. For observation of bone ing to their specific receptors, for example, the parathy- matrix, the sections were stained with Toluidine Blue, roid hormone (PTH) [13], fibroblast growth factors Sirius Red (Sigma, USA), and Masson Trichrome (IHC (FGFs) [14], and vitamin D3 [15]. World, USA) as per the manufacturer’s instructions. For Osteocytes are also responsible for the secretion of the transmission electron microscopy (TEM), observations bone. Recently, the endocrine function of the bone has were conducted using H-7650 transmission electron gradually gained more attention, as bone-derived hor- microscope (Hitachi, Japan). mones and substances (OCN, FGF23, and LCN2) have been identified as regulating the homeostasis of metabol- Sample preparation ism, calcium phosphorus, and even innate immune re- For RNA sample preparation, three 3-month-old (sam- sponses and cognition [16–20]. Abnormal serum levels ples Y1, Y2, and Y3, weighing from 25 to 27 g) and three of these known hormones and substances are considered 20-month-old (samples O1, O2, and O3, weighing from risk factors for several degenerative diseases, including 32 to 37 g) female C57BL/6 mice were anaesthetized by cardiovascular disease (CVD) [21, 22], chronic kidney intraperitoneal infusion with 5% sodium pentobarbital. disease (CKD) [23–25], type 2 diabetes [26], and obesity Then, the animals were perfused through the left ven- [27–29]. Furthermore, osteoimmunology studies have tricle at a constant flow of 20 ml/min with ice-cold shown cross-regulatory mechanisms between bone and physiological saline (PBS) for 60 s. Additionally, 3-mm the immune system, especially in skeleton degenerative segments of the cortical bone in the mid-shaft of the diseases such as osteoporosis and osteoarthritis. The femur and tibia diaphysis were isolated and sampled. above evidence prompts us to probe the correlation be- The bone marrow was first washed away with ice-cold tween bone secretion and ageing. PBS. To remove the cell components on the surface, the Since ageing is a time-dependent process, and osteo- cortical bone was cut into halves lengthwise, the inside cytes are the longest-lived cells in bone, we hypothesized and outside surfaces were vigorously flushed with PBS, that ageing should have great influence on gene tran- and then with ice-cold TRIzol® reagent (Ambion, USA) scription and cell secretion of osteocytes. Herein, by twice. The cortical bone segments derived from a single combining transcriptome-wide and proteome-wide de- mouse were then smashed in TRIzol reagent, and the scriptions of functional changes, the importance of total RNA was extracted per the manufacturer's instruc- osteocyte/bone cell senescence during bone ageing is tions. For exosome sample preparation, two emphasized. 3-month-old (samples young1 and young2, weighing 305 and 312 g) and two 20-month-old (samples old1 and Methods old2, weighing 399 and 376 g) female SD rats were Animals anaesthetized by intraperitoneal infusion with 5% so- SPF C57BL/6 mice and SD rats were obtained from the dium pentobarbital. The heart was perfused with 200 ml medical laboratory animal center of Tongji Medical Uni- of PBS through the left ventricle before sacrifice. The versity, Shanghai, China. Before experiments, the ro- long bones were isolated under sterile conditions. The dents were acclimatized to laboratory conditions for a soft tissue was carefully removed, and the mid-shaft cor- week, with a commercial standard cube diet (Xietong tical bone derived from the femur and tibia was sampled. Medical and Biological Engineering Co. Ltd., Jiangsu) Bone marrow was removed by flushing. Then, the cor- and water ad libitum. The animal experiment ethics tical bone was incubated in 0.25% trypsin at 37 °C for 10 committee of Tongji University approved the animal ex- min to remove the cellular components on the bone sur- periments (number: TJLAC-017-015). All methods were face. The bone segments were cut into 2 × 2-mm pieces carried out in accordance with the approved guidelines and incubated in 0.1% type I collagenase at 37 °C for 20 of Tongji University for Molecular Science. min. After digestion, bone segments were washed with PBS five times and centrifuged at 1000 rpm for 10 min. Morphological analysis and observations The precipitates were incubated in serum-free MEM at Three-month-old and 20-month-old C57BL/6 female 37 °C and 5% CO . After incubation for 48 h, the mice were sacrificed by cervical dislocation. For medium was collected and centrifuged at low speed micro-CT analysis, the femurs (six samples in each (300 g, 10 min; 2000 g, 30 min) and ultra-high speed Zhang et al. Journal of Orthopaedic Surgery and Research (2019) 14:129 Page 3 of 17 (10,000 g, 30 min; 150,000 g, 2 h at 4 °C) using a SW28 and that overlapped between two runs were selected and rotor (Beckman Coulter, USA) for exosome isolation. processed for statistical analysis using Perseus software Two replicates were performed for each group. (1.3.0.4). RNA sequencing Bioinformatic analysis Sequencing libraries were generated using the NEBNext® Differential expression analysis was performed using the UltraTM RNA Library Prep Kit for Illumina® (NEB, DESeq R package (1.10.1). The P values were adjusted USA) following the manufacturer’s recommendations, using Benjamini and Hochberg’s approach. Genes with an and library quality was assessed on the Agilent Bioanaly- adjusted P value less than 0.05 found by DESeq were con- zer 2100 system. Clustering of the index-coded samples sidered to be differentially expressed. For the LC-MS/MS was performed on a cBot Cluster Generation System analysis, proteins with an LFQ intensity ratio between the using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina) two groups (old and young) larger than 2 and overlapped according to the manufacturer’s instructions. The library between two replicants were selected for Gene Ontology preparations were sequenced on an Illumina HiSeq (GO) and Kyoto Encyclopedia of Genes and Genomes 2000/2500 platform, and 100-bp/50-bp single-end reads (KEGG) enrichment analysis performed by the clusterPro- were generated. Raw data in fastq format were first proc- filer R package in Bioconductor [32]. GO terms and essed through in-house Perl scripts. Clean data were ob- KEGG pathways with corrected P values less than 0.05 tained by removing reads containing adapters, reads were considered significantly enriched. containing poly-N, and low-quality reads from the raw data. At the same time, the Q20, Q30, and GC content Statistical analyses of the clean data were calculated. All downstream ana- All statistical analyses were performed with SPSS soft- lyses were based on high-quality clean data. Bowtie ware, version 20.0. When data sets adhered to a normal v0.12.9 was used to align the single-end clean reads to distribution, Student’s t test was used to evaluate the UniGene sequences. HTSeq v0.6.1 was used to count statistical differences between the two groups. The the read numbers mapped to each gene. The reads per Mann-Whitney U test was used for data that were not kilobase transcriptome per million reads (RPKM) of each normally distributed. P < 0.05 was considered significant. gene was calculated based on the length of the gene and All numerical data are expressed as the mean ± s.d. the read count mapped to the gene. Results Exosome identification The morphological changes in aged bone The morphology of OLCS exosomes was observed by Compared with young bones, aged bones displayed sig- transmission electron microscopy (Hitachi, Japan). Laser nificant decreases in bone mass indicated by the reduced scattering microscopy (ZetaView®, Germany) was used to micro-CT parameters of BV/TV, Tb.N, and CBT measure the particle size of the exosomes (Fig. 6b). The (Fig. 1a–g). Histological analysis showed a decreased expression of exosome markers (CD9 and CD81) was con- number of osteocytes and an increased number of empty firmed by Western blotting (Fig. 6c). Anti-CD9 and CD81 lacunae in aged relative to young cortical bone (Fig. 1l). antibodies were purchased from Abcam (Cambridge, UK). Compared with young bone, the inner surface of aged cortical bone was less smooth with more visible resorp- Liquid chromatography-mass spectrometry analysis tion pits, below which empty lacunae were easily ob- Exosomes were lysed in SDT buffer and ultrasonicated, served (Fig. 1h–k). To further investigate the condition and protein concentrations were subjected to SDS-PAGE. of the extracellular matrix, Toluidine Blue, Masson, and Proteins were then digested using the FASP protocol [30]. Sirius Red staining were performed. The results showed Two micrograms of enzymatic hydrolysis lysate (obtained abundant dendrites connecting into a network around by filter-aided sample preparation) were analysed using young osteocytes in young bone (Fig. 1m), while con- 150 μm × 20 mm Thermo EASY column SC001 traps tinuous dendrites were rare in aged cortical bone (Thermo, USA). Liquid chromatography-mass spectrom- (Fig. 1n). Masson staining revealed predominantly blue etry (LC-MS/MS) was performed using the QExactive staining in young cortical bone (Fig. 1o); in aged bone, platform in positive ion mode with a scanning range of however, bone matrix was mainly stained in red (Fig. 1p), 300-1800 m/z. Twenty fragment spectra (MS2 scan) were indicating a change in collagen composition in bone collected after each full scan. The resolution for MS1 and matrix during ageing. Sirius Red staining demonstrated MS2 at m/z 200 was set to 70000 and 17500, respectively. poor densification and loose tissue structure of collagen The raw data were analysed using MaxQuant software fibres in aged bone matrix (Fig. 1q, r). Further, morpho- (1.3.0.5). Label-free quantitation (LFQ) analysis [31] was logical abnormalities in the shapes of cell bodies, den- performed, and the proteins with a certain LFQ intensity drites, and nuclei were shown by TEM (Fig. 2). The Zhang et al. Journal of Orthopaedic Surgery and Research (2019) 14:129 Page 4 of 17 Fig. 1 Ageing-related changes of bone and bone matrix. a–f Representative micro CT images: overall views (a, b), trabecular views (c, d), and cross section views (e, f) of young and aged mouse femurs. g Micro CT measurements for bone volume to total volume ratio (BV/TV), trabecular number (Tb.N), and cortical bone thickness (CBT) in mouse femurs. h–k Representative images of H&E staining of mouse femurs: low magnification (h, i) and high magnification of cortical bone area (j, k), young and aged. White arrows (k) indicate the empty lacunae, and black arrows indicate the resorption pits (i, k). l The osteocyte and empty lacuna counts per unit area in the young and aged cortical bone. m, n Representative images of Toluidine Blue staining of cortical bone, young and aged. o–r Distribution of collagen in the bone indicated by Masson’s trichrome (o, p) and Sirius Red staining (q, r), young and aged. Data in all bar plots are shown as means ± SD, *P < 0.05 osteocytes of aged mice showed many morphological ab- Exosome identification and exosomal proteins identified normalities, such as cytoplasmic lysis, empty cytoplasm, by LC-MS/MS and abnormal nuclei (Fig 2d–l). Moreover, tethering ele- Given that secretory substances of the bone can regulate ments between osteocyte dendrites and the canalicular the functions of adjacent bone cells (OCs, OBs, MSCs) or wall were visible in young bone (Fig. 2c) but were absent even distant organs, exosomes, small membrane-bound in aged bone (Fig. 2i). vesicles that carry biological macromolecules to target sites, might be an effective mediator of bone secretion and GO and KEGG pathway enrichment analysis of the DEGs could show functional alterations from the perspective of between aged and young cortical bone cells bone secretion. In this context, OLCS exosomal proteins Differential expression analysis of the RNA-seq results of both groups were isolated for LC-MS/MS analysis. identified 271 upregulated and 477 downregulated differen- Three conventional experiments were performed to con- tially expressed genes (DEGs) between aged and young cor- firm the exosomal nature of isolated vesicles: visualization of tical bone cells (Fig. 3). The most enriched GO terms and the isolated vesicles by electron microscopy revealed the pres- KEGG pathways are listed in Figs. 4 and 5. With reference ence of spherical structures approximately 100 nm in size to the given functions of osteocytes, the key molecules in (Fig. 6a); particle size measurement indicated a size range be- our DEGs that can perform and represent these functions tween80and 200nm,withapeak size of 120.9nmin diam- are highlighted, as changes in the expression levels of these eter (Fig. 6b), and the expression of the general exosome functional molecules can partially reflect the characteristics markers CD9 and CD81 was validated by Western blotting of bone cell senescence. showing MWs of 25 and 20 kDa, respectively (Fig. 6c). Zhang et al. Journal of Orthopaedic Surgery and Research (2019) 14:129 Page 5 of 17 Fig. 2 Ageing-related morphological changes in osteocytes. a–c Micromorphology of osteocytes in young mouse cortical bone, shown by TEM. Osteocyte shape (a) and the ultrastructure of osteocyte processes (b, c). The connecting fibers between cell processes and pericanalicular matrix, indicated by a black arrow (c). In the aged bone, osteocyte dendrites lack fibers anchoring to the canalicular wall (i). d–l TEM images of osteocytes in aged mouse cortical bone. Aged osteocytes show a variety of abnormal cytomorphological features: cytoplasmic lysis (d, e), indicated by a white star; empty cytoplasm (f–h), indicated by a black star; and (j–l) abnormal forms of the nucleus, shrinkage (j), and swelling (k, l) Further, in subsequent LC-MS/MS analysis, a total of were enriched in the GO cellular component term “extra- 1019 proteins in young and 700 proteins in aged OLCS cellular exosomes” (data not shown), which was at the top exosomes were identified. A large number of of the list sorted by P value, and the following GO terms: exosome-associated proteins were found as evidence that focal adhesion, membrane, vesicles, extracellular matrix, authentic exosomes were obtained: generally, more than and membrane raft (data not shown), which are all 58% (592 out of 1019 in the young group) and 63% (444 exosome-related cellular components. Furthermore, many out of 700 in the aged group) of the identified proteins exosome markers were found both in young and aged Zhang et al. Journal of Orthopaedic Surgery and Research (2019) 14:129 Page 6 of 17 Fig. 3 Transcriptome analysis of aged and young mouse cortical bone cells by RNA-seq. a Heat map of differential gene expression between the young (Y) and aged (O) groups. b Volcano plots for the differentially expressed genes (DEGs), aged vs young. Numbers are given for the upregulated (red plots) and downregulated (green plots) DEGs OLCS exosomes, including the commonly studied exosome and young groups (the same GO terms between two markers tetraspanins (CD9, CD63, CD81), flotillin and cav- groups were eliminated) were preserved and are listed in eolin (FLOT1, FLOT2, CAV1), major histocompatibility Table 1. LFQ analysis identified 236 downregulated and complex protein (RT1.Alu, RT1-CE14, RT1-Bu alpha, 177 upregulated differentially expressed proteins (DEPs) RT1-Bu beta, RT1.Alu, RT1.Ab), and integrins (ITGA1, 2, 5 in OLCS exosomes of the aged group relative to that of and ITGB1,2,3,6). Moreover,manyother potential the young control. The top 50 DEPs (sorted by log2 fold markers were also included: annexins (ANXA1, 2, 3, 4, 5, 6, change in LFQ intensity) are listed by heatmap depiction 7, and 11), transcription factors (EF1A and EF2), heat shock in Figs. 7 a and b. The enriched GO terms among down- proteins (HSPA8, HSP90AA1, HSP90AB1, and HSPD1), regulated and upregulated DEPs are listed in Figs. 7c phosphatidylserine-binding protein (MFGE8/lactadherin), and d. Ageing characteristics of bone indicated by the and growth factor receptor (EGFR [only in the young results of this study were summarized in Fig. 8. group]). Last but not least, several proteins that are consid- ered to be absent or underrepresented in exosomes have Discussion not been identified, for example, Argonaute/RISC complex The morphological changes in aged bone and osteocytes (AGO) and golgin (GM130). Based on the morphological evaluation and observation of young and aged long bones (Figs. 1 and 2), the main GO analysis of DEPs in exosomes derived from young and phenotypes of aged cortical bone can be generally sum- aged cortical bone marized as (1) loss of bone mass, (2) changes in bone The top GO terms (BP) for the young group (with an in- matrix (structure and composition), and (3) poor osteo- put of 1019 proteins) are listed in Fig. 6d. Among them, cyte status. We assume that poor osteocyte status may the ageing- and bone remodelling-related GO terms and be the root cause of bone ageing phenotypes, and mo- associated proteins are displayed in Figs. 6 e and f, re- lecular evidence that can link the senescence of osteo- spectively. The differing GO terms between the aged cyte network to bone ageing urgently needs clarification. Zhang et al. Journal of Orthopaedic Surgery and Research (2019) 14:129 Page 7 of 17 Fig. 4 Top GO terms and KEGG pathways among downregulated DEGs. Enriched KEGG pathways (a) and GO terms (b) for downregulated DEGs, aged vs young. The abscissa represents DEG numbers. c Network of the most enriched GO terms (BP) and associated DEGs, downregulated, aged vs young. The sizes of the yellow nodes are proportional to the numbers of DEGs related to a given GO term Acquisition of biological information from bone cells and differentiated morphology with abundant dendrite con- its limitation nectivity. Heart perfusion can effectively remove the In this study, the biological information was obtained blood cells from bone tissue. The pretreatment of the directly from the cortical bone. So, the key issue is en- cortical bone surface by a quick flush with TRIzol re- suring that osteocytes are the most abundant cells in the agent before RNA extraction and by a short digestion sampling site. In this context, we took a small segment with trypsin and collagenase before exosome extraction of cortical bone where the osteocytes showed highly can reduce the influence of adherent cells, including Zhang et al. Journal of Orthopaedic Surgery and Research (2019) 14:129 Page 8 of 17 Fig. 5 Top GO terms and KEGG pathways among upregulated DEGs. Enriched KEGG pathways (a) and GO terms (b) for upregulated DEGs, aged vs young. The abscissa represents DEG numbers. c Network of the most enriched GO terms (BP) and associated DEGs, upregulated, aged vs young. The sizes of the yellow nodes are proportional to the numbers of DEGs related to a given GO term osteoblasts (OBs), lining cells, and osteoclasts (OCs). murine femoral mid-diaphysis [33]. Additionally, histo- Nevertheless, it is difficult to remove the vascular endo- logical observations of serial sections in our preliminary thelial cells (VECs) in the bone matrix. Research on the experiment suggested that the proportion of osteocytes quantitative morphometry of the bone vasculature has was more than 90% (90.49%) of the total number of cells shown that the vascular volume density accounts for in the middle 3-mm segments of the cortical bone 00.13% of the cortical total volume in the region of the (Additional file 1: Figure S1 and S2). Although the Zhang et al. Journal of Orthopaedic Surgery and Research (2019) 14:129 Page 9 of 17 Fig. 6 Exosome characterization and proteomics analysis of exosomal proteins. (a) Representative TEM image of exosomes isolated from young rat cortical bone (scale bar = 100 nm). b Particle size measurement of exosomes by dynamic light scattering. c Western blot analysis of CD9 and CD81 in exosomes. d The most enriched GO terms among the proteins identified in young rat OLCS exosomes. The abscissa represents protein numbers. e Network of the ageing-related GO terms and associated proteins identified in young rat OLCS exosomes. f Network of the bone remodelling-related GO terms and associated proteins identified in young rat OLCS exosomes. The sizes of the yellow nodes are proportional to the numbers of DEGs related to a given GO term osteocytes are the majority of cells in our sampling site, of this study. More accurate sampling methods and the information we obtained was not from pure osteo- cutting-edge sequencing methods, such as laser micro- cytes but a mixture, which we consider as the limitation dissection based on hard tissue and single-cell Zhang et al. Journal of Orthopaedic Surgery and Research (2019) 14:129 Page 10 of 17 Table 1 The most enriched GO terms (BP) among proteins identified in exosomes of both groups GO terms among proteins identified in GO terms among proteins identified in exosomes (aged) exosomes (young) Protein Establishment of protein localization to Protein depolymerization (11) membrane (35) Cellular protein complex disassembly (13) Protein peptidyl-prolyl isomerization (10) Positive regulation of protein localization to nucleus (14) Chaperone-mediated protein complex assembly Protein nitrosylation (4) (4) Positive regulation of protein secretion (24) DNA DNA conformation change (23/217) None Telomere maintenance (15/113) Cell appendage Lamellipodium organization (16) None morphogenesis Ruffle organization (10) Membrane raft assembly (4) Podosome assembly (6) Regulation of cell junction assembly (12) Metabolism Nitric oxide metabolic process (12) None Regulation of nitric oxide biosynthetic process (12) Tricarboxylic acid metabolic process (8) Cellular transportation Golgi vesicle transport (25) None Energy coupled proton transmembrane transport (12) Positive regulation of protein transport(45) Cell clearance Lysosome organization (5) None Protein targeting to lysosome (5) Cell homeostasis Regulation of pH (13) Negative regulation of homeostatic process (18) Cellular response Cellular response to peptide hormone stimulus Acute-phase response (10) (34) Response to fungus (7) Response to parathyroid hormone (6) Response to immobilization stress (7) Response to angiotensin (7) Response to lipopolysaccharide (34) Response to mineralocorticoid (10) Killing by host of symbiont cells (4) Cell stress Response to endoplasmic reticulum stress (23) Respiratory burst (5) Cellular response to oxidative stress (23) Cell death Negative regulation of apoptotic signaling Regulation of intrinsic apoptotic signaling pathway in response to pathway (15) DNA damage (6) Cytolysis (6) Membrane invagination (9) Immunologic process I-kappaB kinase/NF-kappaB signaling (24) Activation of immune response (27) Positive regulation of leukocyte-mediated immunity (13) B cell-mediated immunity (16) Positive regulation of inflammatory response (13) Neutrophil migration (11) Granulocyte chemotaxis (12) Positive regulation of T cell activation (16) Leukocyte migration (29) Cell-cell recognition (9) Complement activation, lectin pathway (4) Cell-matrix adhesion Regulation of cell-matrix adhesion (16) None Skeletal system Positive regulation of ossification (14) None Osteoclast differentiation (5) Nervous system Axon ensheathment (17) Negative regulation of nervous system development (23) Peripheral nervous system axon regeneration (5) Glial cell differentiation (25) Other systems/tissue Myeloid cell development (12) Fibroblast proliferation (12) Regulation of muscle contraction (18) Renal system development (25) Regulation of coagulation (16) Smooth muscle cell proliferation (14) The numbers in parentheses are the counts of proteins annotated for each GO term Zhang et al. Journal of Orthopaedic Surgery and Research (2019) 14:129 Page 11 of 17 Fig. 7 GO analysis of the differentially expressed exosomal proteins. a, b Heatmap depiction of the top 50 overlapping downregulated (a, green) and upregulated (B, red) proteins in OLCS exosomes, aged vs young. c, d The most enriched GO terms among downregulated (c) and upregulated (d) exosomal proteins, aged vs young. The numbers are DEP counts for each GO term sequencing, may be helpful to optimize the methods of downregulated (Fig. 4a). Taken together, these lines of this study. evidence tend to show the attenuation of the mechanical sensing of aged bone cells. DEGs involved in bone mechanosensation The expression of many key factors involved in the bone Hormone receptors mechanosensation is downregulated in the aged cortical Here, the gene expression of some hormone receptors, bone. First, the critical structural molecules that allow including Pth1r, VDR, Fgf1r, and Fgf2r, was downregu- osteocytes to bear mechanical force are downregulated lated in aged cortical bone. Parathyroid hormone (PTH), (Fig. 4): integrins and related factors (e.g., Itga10, Itgb4, secreted by the parathyroid glands, plays a central role Chad, Fap), components of focal adhesions (e.g., Lamb2, in maintaining bone metabolic homeostasis by binding Thbs2/4, Tnc, Tnn, Pdgfra), cytoskeleton proteins (e.g., to the PTHR, a G protein-coupled membrane-spanning Actn2, Cav1, Flnc, Myl2, Mylk4), and the main calnexin receptor. Activation of PTH1R increases both bone for- (Cx43/Gja1) that composes the gap junction between os- mation and resorption by inhibiting Sost expression and teocytes. Second, key genes whose products involve in elevating the RANKL/OPG ratio, respectively [13]. Vita- mechanical stimulation induced second messenger pro- min D binds to vitamin D receptor (VDR), as is the case duction: key enzymes involved in NO, PGE2, and ATP with nuclear receptors. Its main function is to mediate synthesis (e.g., NOS1, COX1-3, ND1-5), factors involved calcium and phosphorus metabolism by regulating the in the regulation of homeostasis and the intracellular expression of the molecules (e.g., DMP1, MEPE, PHEX) flow of calcium ions (Casq1, Jph2, Trdn, Jsrp1, Hrc, involved in the synthesis and secretion of inorganic Capn3, Ryr1), and Sost, an osteocyte-specific marker pyrophosphate and the regulation of bone matrix that integrates osteocyte mechanotransduction and bone mineralization [15]. The expression of FGF23, the core mass by antagonizing Wnt/beta-catenin signalling [34, regulator of body calcium and phosphorus [14], has been 35]. Additionally, downstream signalling elicited by demonstrated to be influenced by both PTH/PTHR and mechanical force-induced messengers, such as vitamin D/VDR signalling through the fibroblast growth cGMP-PKG, PI3K-AKT, and WNT signalling, is factor receptor (FGFR) [13, 36]. Given their important Zhang et al. Journal of Orthopaedic Surgery and Research (2019) 14:129 Page 12 of 17 Fig. 8 Ageing characteristics of bone indicated by transcriptomic and exosomal proteomic analysis of cortical bone cells. The scheme enumerates the main findings of this study. Up-and down-regulations of bone cell functions and signaling pathways are shown in pink and light green columns, respectively role in mediating the calcium phosphate homeostasis be- these molecules could affect the mineralization and tween the bone and the circulatory system, downregula- mechanical properties of the extracellular matrix. tion of these receptors may alter how cells perceive and It has been demonstrated that the mediation of osteo- respond to hormones during ageing. clastic bone resorption depends mainly on the receptor activator of nuclear factor kappa-Β ligand (RANKL)/oste- DEGs involved in bone formation and resorption oprotegerin (OPG) ratio. RANKL, produced by osteocytes, Many key osteogenic molecules and pathways are down- is the major osteoclast differentiation factor [37, 38]. Al- regulated in aged group, including members of the BMP though our data showed no significant difference in and WNT signalling pathways (Tgfb2, Bmp2, Wnt5b, RANKL expression, downregulation of Opg/Tnfrsf11b Wnt16, Lrp4, Lrp5) and their target genes (Gremlin, may increase the RANKL/OPG ratio, which could be the Twist, Ocn, Postn, Gja1, Opg, Cdh2/11/15, and Alp1) reason for the excessive bone resorption in the aged bone. (Fig. 4b). Moreover, many regulators of these two path- In addition, the upregulation of several key genes of osteo- ways are also downregulated; some are positive regula- clast stimulating factors, such as Adam8, Tyrobp, Sbno2, tors, e.g., Col1a1, Bambi, Cav1, Fgfr2, and Kank1, while and Ccr1, could promote osteoclast formation. Further- some are inhibitors, including Twist, Dkk1, Nbl1, and more, higher expression levels of genes involved in regu- Cdh2. Downregulation of these regulatory factors indi- lating proinflammatory cytokine responses are positively cates that the dominant role of bone cells in regulating associated with osteoclast activation in the aged bone. bone formation weakens during ageing. Regarding the formation and maintenance of bone DEGs related to immunity matrix, gene expression levels are downregulated for a From our data, it is obvious that the proinflammatory variety of collagens (e.g., COL1, COL2, COL3, COL5, state is the most significant feature of bone aging, as the COL8, COL11), molecules involved in collagen fibril upregulated genes were enriched in many inflammatory organization (e.g., LUM, P3H1, GREM1, LOX), and GO terms involved in both innate and adaptive immun- many non-collagenous bone matrix proteins (OCN, ON, ity. In innate immunity, pattern recognition receptors POSTN, DMP1, MEPE, PHEX) (Fig. 4). The lack of (PRRs), a group of receptors that activate the formation Zhang et al. Journal of Orthopaedic Surgery and Research (2019) 14:129 Page 13 of 17 of inflammasomes by identifying pathogen-associated DEGs involved in cellular energy metabolism molecular patterns (PAMPs) and damage-associated mo- Mitochondrial and endoplasmic reticulum (ER) insuffi- lecular patterns (DAMPs) in the environment, play im- ciencies are widely considered to be putative hallmarks portant roles in regulating pathogen recognition and of cell senescence [41]. Here, signs of decline in energy activation of the intracellular inflammatory response metabolism in the aged bone cells were evident: first, in [39]. In our data, pattern recognition-related inflamma- the aged group, of the 69 downregulated transcripts tory molecules and pathways were upregulated in the whose products serve mitochondrial functions, many are aged group (Fig. 5), including (1) two important pattern involved in ATP production (tricarboxylic acid cycle, re- recognition receptor (PRR) pathways—the Toll-like re- spiratory chain, and oxidative phosphorylation). Down- ceptor and NOD-like receptor signalling pathways regulation of these key genes may directly reduce the (Fig. 5a); (2) molecules and signalling pathways down- cellular energy supply. In addition, several downregu- stream of PRRs that trigger innate immunity, including lated molecules are key components of complex I NF-κB signalling (Fig. 5a) and genes related to interferon (ND1-4) and complex IV (COX1-3) in the respiratory production (Irf1, Ptpn22, Myd88, Cd14, Pycard); (3) chain, whose dysfunction could cause electron leakage NLRP inflammasome components (Nlrp3, Pycard, and increase the production of reactive oxygen species Card11); and (4) molecules that are regulated in re- (ROS), one of the most important factors for cell senes- sponse to viruses, fungi, and lipopolysaccharide (Fig. 5a). cence. Finally, the gene expression of several key en- These lines of evidence suggest that pathogen infections zymes that respond to cellular stress was downregulated: may be the cause of the enhanced proinflammatory state for example, LONP, which can protect cells from various in aged cortical bone. In addition, a large number of up- stress conditions by regulating mitochondrial metabol- regulated DEGs were enriched in GO terms involved in ism and repairing mitochondrial DNA [42], and PINK, adaptive immunity, such as regulation of cytokine pro- which plays an irreplaceable role in mediating mitochon- duction and secretion, tumour necrosis factor (TNF) drial autophagy [43]. In addition, the products of some production, lymphocyte proliferation, differentiation, DEGs located in the ER were downregulated. FKBP9, and migration (Fig. 5b). It is worth mentioning that CRTAP, and FKBP10 function as protein-folding factors some known regulators of adaptive immunity were mediated by chaperones [44]. MNF2 and VDAC are typ- found in OLCS exosomes (see below in the “Immune-as- ical key factors on the ER–mitochondria interface [45, sociated proteins” section), and most of them were up- 46]. These changes in energy metabolism could be the regulated in the aged group, which might be further root causes of bone ageing. evidence for the involvement of aged bone cells in the Changes in some cellular metabolism pathways are regulation of adaptive immunity. also important clues for determining the underlying On the other hand, there are many receptors of mechanisms of bone ageing. The FoxO signalling path- proinflammatory cytokines whose gene expression way is regulated by and counteracts external changes levels were increased in the aged group: some of that disturb homeostasis, including metabolic stress, oxi- them are involved in the mediation of immunosup- dative stress, and growth factor deprivation [47]. Ac- pressive signals, such as Lilrb4, Pilra, Il10ra, and cording to the KEGG analysis, the FoxO signalling Il1r2; some play pathogenic roles in inflammatory and pathway was upregulated in the aged group, which may autoimmune diseases such as rheumatoid arthritis, for be further corroboration of the severe cellular stress in example, Il17ra; and Il18rap and Il21r, whose prod- aged bone cells. Furthermore, FoxO signalling may have ucts are involved in the activation of signalling path- special cellular functions in the bone: it has been dem- ways associated with inflammation, including NF-κB, onstrated that activation of FoxO in osteoblasts can hin- MAPK8, and JAK-STAT. Changes in the expression der bone formation, as it can competitively inhibit the levels of these molecules could affect the responses of WNT pathway by diverting β-catenin from TCF- to aged bone cells to inflammatory factors. Notably, FoxO-mediated transcription [48]. Moreover, the FoxO molecules negatively regulating JAK-STAT signalling pathway is normally negatively regulated through phos- were downregulated (Fig. 4b), while molecules posi- phorylation mediated by the PI3K-AKT pathway in di- tively regulating the JAK-STAT cascade were upregu- verse organisms [49]. Meanwhile, PI3K-AKT is lated in the aged group (Fig. 5b). Given that responsible for mechanical force-induced osteogenesis in JAK-STAT signalling is considered to have the cap- the bone [8]. Consistently, in our data, FoxO signalling was acity to mediate the responses of target cells to in- upregulated (Fig. 5a) and accompanied by the downregula- flammatory cytokines and plays an important role in tion of PI3K-AKT and WNT signalling (Fig. 4a, b). Thus, regulating cell apoptosis [40], the active regulation of the crosstalk among these three pathways could be the mo- JAK-STAT signalling might be the link between cell lecular explanation and underlying link between the bone responses to inflammatory cytokines and cell death. loss and decline in cell function that occur during ageing. Zhang et al. Journal of Orthopaedic Surgery and Research (2019) 14:129 Page 14 of 17 The loss- and gain-of-function of OLCS exosomes with transcription factors and stress kinases that can mediate age indicated by GO analysis among exosomal proteins inflammatory and tumourigenic signalling, e.g., NF-κB The results revealed that the skeleton could secrete large and AMPK [55]. Thus, the increased expression of these amounts of regulatory proteins involved in regulating a SASP proteins in senescent OLCS exosomes may explain wide range of cellular activities, from regulating energy how aged bone cells contribute to the environmental ef- metabolism and protein transportation to cell differenti- fects of adjacent and distal cells and systems. ation, morphogenesis, and movement (Fig. 6d). Add- itionally, biological processes involving the maintenance Immune-associated proteins of cell homeostasis and anti-senescence were included, Our analyses of the immune-associated proteins in such as molecules involved in the regulation of telomere OLCS exosomes have shown interesting findings regard- maintenance, protein folding and stabilization, tissue re- ing the cross-regulation between the skeleton and the generation, lysosome organization, and proteins in re- immune system: First, approximately 10% (85 out of sponse to ROS and hormones (Fig. 6d, e). 1019 proteins identified in the young group, and 76 out In clear contrast to young controls, the aged OLCS of 700 proteins in the aged group) of the identified pro- exosomes lacked functional proteins for cellular activ- teins found in OLCS exosomes were enriched in ities, including maintenance of the morphological prop- immune-related GO terms, and approximately 80% (67 erties of cells, maintenance of cell homeostasis, and out of 85/76 in young/aged group) of them overlapped regulation of cellular metabolism and transportation between the young and aged groups. Although most of (Table 1). Moreover, the results revealed a functional de- them were foundtohaveahigherLFQ intensityinaged cline in the regulation of DNA conformation, telomere OLCS exosomes than in young ones, their overall compos- maintenance, protein complex assembly, and localization ition in exosomes was relatively constant across age. To (Table 1). In addition, proteins involved in lysosome some extent, these results indicate that there is a certain organization and cellular responses to hormones (para- group of molecules in OLCS exosomes, both aged and thyroid, angiotensin, and mineralocorticoid) were absent young, that may serve an immune regulatory function. Sec- in the aged group (Table 1). On the other hand, the ond, among these overlapping immune-associated proteins enriched proteins in the aged group were mainly associ- were some key factors that have been shown in existing ated with activation of immune responses, cell damage, studies to regulate the activities of both immune cells and and cell death (Table 1). Most of the features mentioned bone cells, including CD44, CD47, CD59, TGF-β2, and above mirrored the findings at the RNA level; some of GSN. Among these, CD44 is a novel marker of osteocytic them have been demonstrated or categorized in existing differentiation in bone [56]; meanwhile, it is also widely studies linked to cell senescence [41] and could elucidate expressed in many kinds of immune cells (T cells, granulo- both the causes and consequences of bone ageing. cytes, monocytes) and is critical for their maturation [57, 58]. CD47, known as a “don’t eat me” signal to macro- Proteins identified in OLCS exosomes phages, plays a critical role in immune cell activation [59, Senescence-associated secretory phenotype 60], and it also has a profound effect on skeletal remodel- Studies have demonstrated that aged cells can secrete ling and bone maintenance through its actions on both os- proinflammatory factors, growth factors, chemokines teoblasts and osteoclasts [61]. For CD59, this molecule was and proteases to mediate age-related inflammatory re- recently demonstrated to be a regulator of bone growth sponses, wound healing, and even tumour progression; and homeostasis by interfering with the complement sys- this condition is now referred to as the tem in innate immunity [62]. TGF-β, a multifunctional senescence-associated secretory phenotype (SASP) [50, cytokine, plays integral roles in the regulation of adaptive 51]. Here, several classical SASP proteins, including immunity, mainly through activating T cells and effector TGF-β2, OPG, MMP9, TIMP1, MIF1, PRDXs, and and regulatory T cells [63]; in the bone, it is a critical regu- IGFBP3, were found to be upregulated in aged OLCS lator coupling of bone formation and resorption [64]. All exosomes. Among them, TGF-β2 and OPG are import- these proteins, except CD44, showed increased expression ant mediators of bone metabolism; serum levels of in aged OLCS exosomes. Similarly, many other immuno- TGF-β2 and OPG have shown significant positive corre- regulatory molecules were upregulated in aged OLCS exo- lations with bone turnover markers in Chinese women somes, including several complement molecules and [52]. TIMP1, a tissue inhibitor of metalloproteinases regulators: C3, C1QA, C1QB, C1QC, C4A, C8B, MBL1, (MMPs), is expressed in both osteocytes and osteoblasts MASP2, FCNB; positive regulators of T cell/B cell activa- and maintains the balance of bone matrix degradation tion: SLC4A1, SPN, TFRC, ANXA1, CORO1A, FLOT, by regulating MMP levels [53]. Moreover, TIMP/MMPs PNP, PTPRC; and regulators of leukocyte proliferation, mi- can also accommodate the immune influx in the extra- gration, and aggregation: S100A9, S100A8, PNP, PLG, MIF, cellular matrix [54]. MIF1 is known to activate several ECM1, SERPINE1, SLC4A1, GSTP1, ELANE. Zhang et al. Journal of Orthopaedic Surgery and Research (2019) 14:129 Page 15 of 17 On the other hand, only a small number of Conclusion immune-associated proteins were found to have higher A global view of changes in the gene expression profiles of intensities in the young group relative to the aged group, bone cells and secretory proteins in OLCS exosomes sug- including M-CSF (CSF-1), CD1d, Galectin1 (LGALS1), gests key characteristics of bone ageing. Some changes con- CD90 (THY1), and RHOA. Among them, M-CSF sidered hallmarks of cell ageing have been reviewed (macrophage colony-stimulating factor 1) is a haemato- previously, e.g., reductions in energy metabolism, cellular poietic growth factor involved in the proliferation, differ- responses to hormones, DNA conformation, telomere entiation, and survival of monocytes and macrophages; maintenance, and an increased level of the proinflamma- in the bone, it is one the most important cytokines for tory state. Moreover, newly identified changes are indicative the maturation and function of osteoclasts. CD1d is a of reductions in bone-specific functions, e.g., mechanical key molecule necessary to activate CD1d-dependent sensation and bone remodelling regulation. Further, our natural killer T cells that regulate certain tissue-specific data suggest that exosomes of young OLCS have multiple immune activities [65]. Galectin1, a homodimeric molecules that could play roles in maintaining cellular galactose-binding lectin, is able to selectively bind with a homeostasis; however, these functions are weakened during T cell surface receptor (NP-1) and inhibit T cell prolifer- ageing. Nevertheless, the upregulated proteins in aged ation [66, 67]. CD90, also known as the T lymphocyte OLCS exosomes provide clues that these aged OLCS exo- differentiation antigen, is a conserved cell surface pro- somes could have additional functions, such as promoting tein with a single V-like immunoglobulin domain; CD90 wound healing and scavenging free radicals, that help the can activate the T cell receptor [68] and is expressed as a surrounding cells resist senescence. On the other hand, surface marker for bone marrow stem cells [69, 70] and aged cells are considered the principal promoters of sys- osteoblasts [71]. temic and local inflammation that characterize ageing and Collectively, a large number of immune regulatory promote age-related diseases. Herein, we first found that a factors for both innate and adaptive immunity were large number of immune-related DEGs were upregulated found in OLCS exosomes. These findings may provide in aged bone cells, and then, we determined that many clues for exploring the mutual regulation between the regulatory factors both for innate and adaptive immunity skeleton and immune system and the mechanism of are found in OLCS exosomes, indicating that exosomes bone ageing. may act as shuttles propagating inflammatory states from the aged cells to the immune cells. Taken together, this study provides new insights into a further understanding of Bone remodelling-associated proteins in OLCS exosomes bone ageing. Proteins that are involved in osteoblast differentiation (e.g., COL-1/5, LRP, MMP-2, DCN, CX43, β-CATENIN, Additional files TGF-β, ALP) and osteoclast differentiation (e.g., M-CSF, ANXA, FBN1, SLC9, SRC, ATP6AP) were found to be Additional file 1: Figure S1 and S2. Description of the upregulated in the young group relative to the aged sampling method of cortical bone cell in this study. (Fig. S1. Cell group (Fig. 7a, c), indicating that exosomes may be one composition of mouse cortical bone shown by H&E staining. Fig. S2. The sampling method of this study). (DOCX 21222 kb) of the possible mechanisms of bone secretion in the regulation of bone remodelling and decrease with age. Abbreviations BV/TV: Bone volume to total volume ratio; CBT: Cortical bone thickness; Antioxidative proteins in OLCS exosomes CKD: Chronic kidney disease; CVD: Cardiovascular disease; DAMPs: Damage- associated molecular patterns; DEGs: Differentially expressed genes; Since cell ageing is identified as a decline in various DEPs: Differentially expressed proteins; ER: Endoplasmic reticulum; GO: Gene functions of the cell, almost all evidence that supports Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; LC-MS/ an increase in harmful effects or a loss of beneficial MS: Liquid chromatography-mass spectrometry; LCS: Lacunar-canalicular system; LFQ: Label-free quantitation; MSCs: Mesenchymal stem cells; effects in the aged cell is reasonable. However, there OLCS: Osteocytic lacunar-canalicular system; PAMPs: Pathogen-associated is an exception: some exosomal proteins involved in molecular patterns; ROS: Reactive oxygen species; RPKM: Reads per kilobase maintaining cell redox homeostasis and removal of transcriptome per million reads; SASP: Senescence-associated secretory phenotype; Tb.N: Trabecular number; VECs: Vascular endothelial cells ROS are upregulated in aged OLCS exosomes relative to young ones (Fig. 7b, d), including several key mol- Acknowledgements ecules, such as peroxiredoxins (PRDXs), superoxide Not applicable. dismutase 2 (SOD2), thioredoxin 1 (TRX1), glutathi- one S-transferase P1 (GSTP1), and haptoglobin, which Funding This study was funded by the National Science Foundation of China (81470715 manifests in aged OLCS may exert a positive effect and 81771043), Y. Sun Key Project of Chinese National Programs for Research on surrounding or even distant cells to resist oxida- and Development (2016YFC1102705, Y. 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Ageing characteristics of bone indicated by transcriptomic and exosomal proteomic analysis of cortical bone cells

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Copyright © 2019 by The Author(s).
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Medicine & Public Health; Orthopedics; Surgical Orthopedics
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DOI
10.1186/s13018-019-1163-4
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Abstract

Background: Degenerative changes in the skeleton play an important role in ageing. As the foremost sensors and orchestrators of bone remodelling, osteocytes contribute significantly to the health of the skeleton. Embedded in a mineralized bone matrix, the osteocyte network and the surrounding lacunar canaliculae work together as a functional syncytium—the osteocytic lacunar-canalicular system (OLCS). However, changes in the OLCS during ageing and related mechanisms cannot be fully understood by using traditional histological analysis. Methods: To link the phenotypes of aged osteocytes and their functional changes during ageing, we analysed the changes in the gene expression profiles of bone cells and the proteomic profiles of OLCS exosomes derived from aged and young cortical bone. Results: Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis of differentially expressed genes (DEGs) suggested that a decline in cell energy metabolism and an increased level of the proinflammatory state are major characteristics of bone ageing. Moreover, some DEGs were key regulators of bone mechanical sensation and bone remodelling, which are indicative of reduced bone-specific function with age. Further, the identified proteins in OLCS exosomes showed potential changes in the secretory function bone. Compared with young controls, the decreased functional proteins in aged OLCS exosomes were enriched mainly in GO terms that included regulating bone development and remodelling, cell-matrix adhesion, and cell clearance and homeostasis. Notably, several functions of exosomal proteins of the aged group revealed potential new roles, such as regulating innate and adaptive immunity, wound healing, and angiogenesis and eliminating oxidative stress. Conclusion: The information obtained from bone cells and OLCS exosomes will help us discover new features of bone ageing. Keywords: Bone ageing, Gene expression profiling, Exosome, OLCS Background lacunar-canalicular system (OLCS) [1–3], underscoring Osteocytes, the most numerous cell populations in bone, that the function of osteocytes cannot be implemented are responsible for sensing mechanical force and acting without cell-to-cell connections and their 3D physio- as the “hub” of the regulatory network for bone remod- logical environment. elling. The osteocyte network and the lacunar canalicu- Mechanical stress is an effective stimulus for osteo- lae constitute a functional syncytium—the osteocytic cytes to release messengers such as ATP, nitric oxide (NO), and prostaglandin E2 (PGE2) [4–6], which then * Correspondence: yaosun@tongji.edu.cn transport through the lacunar-canalicular system (LCS) Department of Oral Implantology, School and Hospital of Stomatology, and gap junctions between adjacent cells to the osteo- Tongji University, Shanghai Engineering Research Center of Tooth genic surface to prompt osteoblast-mediated bone for- Restoration and Regeneration, 399 Middle Yanchang Road, Shanghai 200072, China mation and directly inhibit osteoclast activation [7–9]. Full list of author information is available at the end of the article © The Author(s). 2019 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated. Zhang et al. Journal of Orthopaedic Surgery and Research (2019) 14:129 Page 2 of 17 Additionally, since osteocytes account for more than group) were scanned by μCT50 (Scanco, Switzerland), 90% of bone cells [10], the large number of processes in 10 μm per slice. The parameters included the bone vol- the osteocyte network possesses an immense interface ume to total volume ratio (BV/TV), trabecular number for the exchange of information and substances between (Tb.N) and cortical bone thickness (CBT). For histo- cells and the extracellular environment [11, 12]. Thus, logical analysis, 4-μm-thick paraffin sections of speci- steroids, hormones, and cytokines in the environment mens were prepared. First, the sections were stained can affect the regulatory function of osteocytes by bind- with hematoxylin and eosin. For observation of bone ing to their specific receptors, for example, the parathy- matrix, the sections were stained with Toluidine Blue, roid hormone (PTH) [13], fibroblast growth factors Sirius Red (Sigma, USA), and Masson Trichrome (IHC (FGFs) [14], and vitamin D3 [15]. World, USA) as per the manufacturer’s instructions. For Osteocytes are also responsible for the secretion of the transmission electron microscopy (TEM), observations bone. Recently, the endocrine function of the bone has were conducted using H-7650 transmission electron gradually gained more attention, as bone-derived hor- microscope (Hitachi, Japan). mones and substances (OCN, FGF23, and LCN2) have been identified as regulating the homeostasis of metabol- Sample preparation ism, calcium phosphorus, and even innate immune re- For RNA sample preparation, three 3-month-old (sam- sponses and cognition [16–20]. Abnormal serum levels ples Y1, Y2, and Y3, weighing from 25 to 27 g) and three of these known hormones and substances are considered 20-month-old (samples O1, O2, and O3, weighing from risk factors for several degenerative diseases, including 32 to 37 g) female C57BL/6 mice were anaesthetized by cardiovascular disease (CVD) [21, 22], chronic kidney intraperitoneal infusion with 5% sodium pentobarbital. disease (CKD) [23–25], type 2 diabetes [26], and obesity Then, the animals were perfused through the left ven- [27–29]. Furthermore, osteoimmunology studies have tricle at a constant flow of 20 ml/min with ice-cold shown cross-regulatory mechanisms between bone and physiological saline (PBS) for 60 s. Additionally, 3-mm the immune system, especially in skeleton degenerative segments of the cortical bone in the mid-shaft of the diseases such as osteoporosis and osteoarthritis. The femur and tibia diaphysis were isolated and sampled. above evidence prompts us to probe the correlation be- The bone marrow was first washed away with ice-cold tween bone secretion and ageing. PBS. To remove the cell components on the surface, the Since ageing is a time-dependent process, and osteo- cortical bone was cut into halves lengthwise, the inside cytes are the longest-lived cells in bone, we hypothesized and outside surfaces were vigorously flushed with PBS, that ageing should have great influence on gene tran- and then with ice-cold TRIzol® reagent (Ambion, USA) scription and cell secretion of osteocytes. Herein, by twice. The cortical bone segments derived from a single combining transcriptome-wide and proteome-wide de- mouse were then smashed in TRIzol reagent, and the scriptions of functional changes, the importance of total RNA was extracted per the manufacturer's instruc- osteocyte/bone cell senescence during bone ageing is tions. For exosome sample preparation, two emphasized. 3-month-old (samples young1 and young2, weighing 305 and 312 g) and two 20-month-old (samples old1 and Methods old2, weighing 399 and 376 g) female SD rats were Animals anaesthetized by intraperitoneal infusion with 5% so- SPF C57BL/6 mice and SD rats were obtained from the dium pentobarbital. The heart was perfused with 200 ml medical laboratory animal center of Tongji Medical Uni- of PBS through the left ventricle before sacrifice. The versity, Shanghai, China. Before experiments, the ro- long bones were isolated under sterile conditions. The dents were acclimatized to laboratory conditions for a soft tissue was carefully removed, and the mid-shaft cor- week, with a commercial standard cube diet (Xietong tical bone derived from the femur and tibia was sampled. Medical and Biological Engineering Co. Ltd., Jiangsu) Bone marrow was removed by flushing. Then, the cor- and water ad libitum. The animal experiment ethics tical bone was incubated in 0.25% trypsin at 37 °C for 10 committee of Tongji University approved the animal ex- min to remove the cellular components on the bone sur- periments (number: TJLAC-017-015). All methods were face. The bone segments were cut into 2 × 2-mm pieces carried out in accordance with the approved guidelines and incubated in 0.1% type I collagenase at 37 °C for 20 of Tongji University for Molecular Science. min. After digestion, bone segments were washed with PBS five times and centrifuged at 1000 rpm for 10 min. Morphological analysis and observations The precipitates were incubated in serum-free MEM at Three-month-old and 20-month-old C57BL/6 female 37 °C and 5% CO . After incubation for 48 h, the mice were sacrificed by cervical dislocation. For medium was collected and centrifuged at low speed micro-CT analysis, the femurs (six samples in each (300 g, 10 min; 2000 g, 30 min) and ultra-high speed Zhang et al. Journal of Orthopaedic Surgery and Research (2019) 14:129 Page 3 of 17 (10,000 g, 30 min; 150,000 g, 2 h at 4 °C) using a SW28 and that overlapped between two runs were selected and rotor (Beckman Coulter, USA) for exosome isolation. processed for statistical analysis using Perseus software Two replicates were performed for each group. (1.3.0.4). RNA sequencing Bioinformatic analysis Sequencing libraries were generated using the NEBNext® Differential expression analysis was performed using the UltraTM RNA Library Prep Kit for Illumina® (NEB, DESeq R package (1.10.1). The P values were adjusted USA) following the manufacturer’s recommendations, using Benjamini and Hochberg’s approach. Genes with an and library quality was assessed on the Agilent Bioanaly- adjusted P value less than 0.05 found by DESeq were con- zer 2100 system. Clustering of the index-coded samples sidered to be differentially expressed. For the LC-MS/MS was performed on a cBot Cluster Generation System analysis, proteins with an LFQ intensity ratio between the using the TruSeq PE Cluster Kit v3-cBot-HS (Illumina) two groups (old and young) larger than 2 and overlapped according to the manufacturer’s instructions. The library between two replicants were selected for Gene Ontology preparations were sequenced on an Illumina HiSeq (GO) and Kyoto Encyclopedia of Genes and Genomes 2000/2500 platform, and 100-bp/50-bp single-end reads (KEGG) enrichment analysis performed by the clusterPro- were generated. Raw data in fastq format were first proc- filer R package in Bioconductor [32]. GO terms and essed through in-house Perl scripts. Clean data were ob- KEGG pathways with corrected P values less than 0.05 tained by removing reads containing adapters, reads were considered significantly enriched. containing poly-N, and low-quality reads from the raw data. At the same time, the Q20, Q30, and GC content Statistical analyses of the clean data were calculated. All downstream ana- All statistical analyses were performed with SPSS soft- lyses were based on high-quality clean data. Bowtie ware, version 20.0. When data sets adhered to a normal v0.12.9 was used to align the single-end clean reads to distribution, Student’s t test was used to evaluate the UniGene sequences. HTSeq v0.6.1 was used to count statistical differences between the two groups. The the read numbers mapped to each gene. The reads per Mann-Whitney U test was used for data that were not kilobase transcriptome per million reads (RPKM) of each normally distributed. P < 0.05 was considered significant. gene was calculated based on the length of the gene and All numerical data are expressed as the mean ± s.d. the read count mapped to the gene. Results Exosome identification The morphological changes in aged bone The morphology of OLCS exosomes was observed by Compared with young bones, aged bones displayed sig- transmission electron microscopy (Hitachi, Japan). Laser nificant decreases in bone mass indicated by the reduced scattering microscopy (ZetaView®, Germany) was used to micro-CT parameters of BV/TV, Tb.N, and CBT measure the particle size of the exosomes (Fig. 6b). The (Fig. 1a–g). Histological analysis showed a decreased expression of exosome markers (CD9 and CD81) was con- number of osteocytes and an increased number of empty firmed by Western blotting (Fig. 6c). Anti-CD9 and CD81 lacunae in aged relative to young cortical bone (Fig. 1l). antibodies were purchased from Abcam (Cambridge, UK). Compared with young bone, the inner surface of aged cortical bone was less smooth with more visible resorp- Liquid chromatography-mass spectrometry analysis tion pits, below which empty lacunae were easily ob- Exosomes were lysed in SDT buffer and ultrasonicated, served (Fig. 1h–k). To further investigate the condition and protein concentrations were subjected to SDS-PAGE. of the extracellular matrix, Toluidine Blue, Masson, and Proteins were then digested using the FASP protocol [30]. Sirius Red staining were performed. The results showed Two micrograms of enzymatic hydrolysis lysate (obtained abundant dendrites connecting into a network around by filter-aided sample preparation) were analysed using young osteocytes in young bone (Fig. 1m), while con- 150 μm × 20 mm Thermo EASY column SC001 traps tinuous dendrites were rare in aged cortical bone (Thermo, USA). Liquid chromatography-mass spectrom- (Fig. 1n). Masson staining revealed predominantly blue etry (LC-MS/MS) was performed using the QExactive staining in young cortical bone (Fig. 1o); in aged bone, platform in positive ion mode with a scanning range of however, bone matrix was mainly stained in red (Fig. 1p), 300-1800 m/z. Twenty fragment spectra (MS2 scan) were indicating a change in collagen composition in bone collected after each full scan. The resolution for MS1 and matrix during ageing. Sirius Red staining demonstrated MS2 at m/z 200 was set to 70000 and 17500, respectively. poor densification and loose tissue structure of collagen The raw data were analysed using MaxQuant software fibres in aged bone matrix (Fig. 1q, r). Further, morpho- (1.3.0.5). Label-free quantitation (LFQ) analysis [31] was logical abnormalities in the shapes of cell bodies, den- performed, and the proteins with a certain LFQ intensity drites, and nuclei were shown by TEM (Fig. 2). The Zhang et al. Journal of Orthopaedic Surgery and Research (2019) 14:129 Page 4 of 17 Fig. 1 Ageing-related changes of bone and bone matrix. a–f Representative micro CT images: overall views (a, b), trabecular views (c, d), and cross section views (e, f) of young and aged mouse femurs. g Micro CT measurements for bone volume to total volume ratio (BV/TV), trabecular number (Tb.N), and cortical bone thickness (CBT) in mouse femurs. h–k Representative images of H&E staining of mouse femurs: low magnification (h, i) and high magnification of cortical bone area (j, k), young and aged. White arrows (k) indicate the empty lacunae, and black arrows indicate the resorption pits (i, k). l The osteocyte and empty lacuna counts per unit area in the young and aged cortical bone. m, n Representative images of Toluidine Blue staining of cortical bone, young and aged. o–r Distribution of collagen in the bone indicated by Masson’s trichrome (o, p) and Sirius Red staining (q, r), young and aged. Data in all bar plots are shown as means ± SD, *P < 0.05 osteocytes of aged mice showed many morphological ab- Exosome identification and exosomal proteins identified normalities, such as cytoplasmic lysis, empty cytoplasm, by LC-MS/MS and abnormal nuclei (Fig 2d–l). Moreover, tethering ele- Given that secretory substances of the bone can regulate ments between osteocyte dendrites and the canalicular the functions of adjacent bone cells (OCs, OBs, MSCs) or wall were visible in young bone (Fig. 2c) but were absent even distant organs, exosomes, small membrane-bound in aged bone (Fig. 2i). vesicles that carry biological macromolecules to target sites, might be an effective mediator of bone secretion and GO and KEGG pathway enrichment analysis of the DEGs could show functional alterations from the perspective of between aged and young cortical bone cells bone secretion. In this context, OLCS exosomal proteins Differential expression analysis of the RNA-seq results of both groups were isolated for LC-MS/MS analysis. identified 271 upregulated and 477 downregulated differen- Three conventional experiments were performed to con- tially expressed genes (DEGs) between aged and young cor- firm the exosomal nature of isolated vesicles: visualization of tical bone cells (Fig. 3). The most enriched GO terms and the isolated vesicles by electron microscopy revealed the pres- KEGG pathways are listed in Figs. 4 and 5. With reference ence of spherical structures approximately 100 nm in size to the given functions of osteocytes, the key molecules in (Fig. 6a); particle size measurement indicated a size range be- our DEGs that can perform and represent these functions tween80and 200nm,withapeak size of 120.9nmin diam- are highlighted, as changes in the expression levels of these eter (Fig. 6b), and the expression of the general exosome functional molecules can partially reflect the characteristics markers CD9 and CD81 was validated by Western blotting of bone cell senescence. showing MWs of 25 and 20 kDa, respectively (Fig. 6c). Zhang et al. Journal of Orthopaedic Surgery and Research (2019) 14:129 Page 5 of 17 Fig. 2 Ageing-related morphological changes in osteocytes. a–c Micromorphology of osteocytes in young mouse cortical bone, shown by TEM. Osteocyte shape (a) and the ultrastructure of osteocyte processes (b, c). The connecting fibers between cell processes and pericanalicular matrix, indicated by a black arrow (c). In the aged bone, osteocyte dendrites lack fibers anchoring to the canalicular wall (i). d–l TEM images of osteocytes in aged mouse cortical bone. Aged osteocytes show a variety of abnormal cytomorphological features: cytoplasmic lysis (d, e), indicated by a white star; empty cytoplasm (f–h), indicated by a black star; and (j–l) abnormal forms of the nucleus, shrinkage (j), and swelling (k, l) Further, in subsequent LC-MS/MS analysis, a total of were enriched in the GO cellular component term “extra- 1019 proteins in young and 700 proteins in aged OLCS cellular exosomes” (data not shown), which was at the top exosomes were identified. A large number of of the list sorted by P value, and the following GO terms: exosome-associated proteins were found as evidence that focal adhesion, membrane, vesicles, extracellular matrix, authentic exosomes were obtained: generally, more than and membrane raft (data not shown), which are all 58% (592 out of 1019 in the young group) and 63% (444 exosome-related cellular components. Furthermore, many out of 700 in the aged group) of the identified proteins exosome markers were found both in young and aged Zhang et al. Journal of Orthopaedic Surgery and Research (2019) 14:129 Page 6 of 17 Fig. 3 Transcriptome analysis of aged and young mouse cortical bone cells by RNA-seq. a Heat map of differential gene expression between the young (Y) and aged (O) groups. b Volcano plots for the differentially expressed genes (DEGs), aged vs young. Numbers are given for the upregulated (red plots) and downregulated (green plots) DEGs OLCS exosomes, including the commonly studied exosome and young groups (the same GO terms between two markers tetraspanins (CD9, CD63, CD81), flotillin and cav- groups were eliminated) were preserved and are listed in eolin (FLOT1, FLOT2, CAV1), major histocompatibility Table 1. LFQ analysis identified 236 downregulated and complex protein (RT1.Alu, RT1-CE14, RT1-Bu alpha, 177 upregulated differentially expressed proteins (DEPs) RT1-Bu beta, RT1.Alu, RT1.Ab), and integrins (ITGA1, 2, 5 in OLCS exosomes of the aged group relative to that of and ITGB1,2,3,6). Moreover,manyother potential the young control. The top 50 DEPs (sorted by log2 fold markers were also included: annexins (ANXA1, 2, 3, 4, 5, 6, change in LFQ intensity) are listed by heatmap depiction 7, and 11), transcription factors (EF1A and EF2), heat shock in Figs. 7 a and b. The enriched GO terms among down- proteins (HSPA8, HSP90AA1, HSP90AB1, and HSPD1), regulated and upregulated DEPs are listed in Figs. 7c phosphatidylserine-binding protein (MFGE8/lactadherin), and d. Ageing characteristics of bone indicated by the and growth factor receptor (EGFR [only in the young results of this study were summarized in Fig. 8. group]). Last but not least, several proteins that are consid- ered to be absent or underrepresented in exosomes have Discussion not been identified, for example, Argonaute/RISC complex The morphological changes in aged bone and osteocytes (AGO) and golgin (GM130). Based on the morphological evaluation and observation of young and aged long bones (Figs. 1 and 2), the main GO analysis of DEPs in exosomes derived from young and phenotypes of aged cortical bone can be generally sum- aged cortical bone marized as (1) loss of bone mass, (2) changes in bone The top GO terms (BP) for the young group (with an in- matrix (structure and composition), and (3) poor osteo- put of 1019 proteins) are listed in Fig. 6d. Among them, cyte status. We assume that poor osteocyte status may the ageing- and bone remodelling-related GO terms and be the root cause of bone ageing phenotypes, and mo- associated proteins are displayed in Figs. 6 e and f, re- lecular evidence that can link the senescence of osteo- spectively. The differing GO terms between the aged cyte network to bone ageing urgently needs clarification. Zhang et al. Journal of Orthopaedic Surgery and Research (2019) 14:129 Page 7 of 17 Fig. 4 Top GO terms and KEGG pathways among downregulated DEGs. Enriched KEGG pathways (a) and GO terms (b) for downregulated DEGs, aged vs young. The abscissa represents DEG numbers. c Network of the most enriched GO terms (BP) and associated DEGs, downregulated, aged vs young. The sizes of the yellow nodes are proportional to the numbers of DEGs related to a given GO term Acquisition of biological information from bone cells and differentiated morphology with abundant dendrite con- its limitation nectivity. Heart perfusion can effectively remove the In this study, the biological information was obtained blood cells from bone tissue. The pretreatment of the directly from the cortical bone. So, the key issue is en- cortical bone surface by a quick flush with TRIzol re- suring that osteocytes are the most abundant cells in the agent before RNA extraction and by a short digestion sampling site. In this context, we took a small segment with trypsin and collagenase before exosome extraction of cortical bone where the osteocytes showed highly can reduce the influence of adherent cells, including Zhang et al. Journal of Orthopaedic Surgery and Research (2019) 14:129 Page 8 of 17 Fig. 5 Top GO terms and KEGG pathways among upregulated DEGs. Enriched KEGG pathways (a) and GO terms (b) for upregulated DEGs, aged vs young. The abscissa represents DEG numbers. c Network of the most enriched GO terms (BP) and associated DEGs, upregulated, aged vs young. The sizes of the yellow nodes are proportional to the numbers of DEGs related to a given GO term osteoblasts (OBs), lining cells, and osteoclasts (OCs). murine femoral mid-diaphysis [33]. Additionally, histo- Nevertheless, it is difficult to remove the vascular endo- logical observations of serial sections in our preliminary thelial cells (VECs) in the bone matrix. Research on the experiment suggested that the proportion of osteocytes quantitative morphometry of the bone vasculature has was more than 90% (90.49%) of the total number of cells shown that the vascular volume density accounts for in the middle 3-mm segments of the cortical bone 00.13% of the cortical total volume in the region of the (Additional file 1: Figure S1 and S2). Although the Zhang et al. Journal of Orthopaedic Surgery and Research (2019) 14:129 Page 9 of 17 Fig. 6 Exosome characterization and proteomics analysis of exosomal proteins. (a) Representative TEM image of exosomes isolated from young rat cortical bone (scale bar = 100 nm). b Particle size measurement of exosomes by dynamic light scattering. c Western blot analysis of CD9 and CD81 in exosomes. d The most enriched GO terms among the proteins identified in young rat OLCS exosomes. The abscissa represents protein numbers. e Network of the ageing-related GO terms and associated proteins identified in young rat OLCS exosomes. f Network of the bone remodelling-related GO terms and associated proteins identified in young rat OLCS exosomes. The sizes of the yellow nodes are proportional to the numbers of DEGs related to a given GO term osteocytes are the majority of cells in our sampling site, of this study. More accurate sampling methods and the information we obtained was not from pure osteo- cutting-edge sequencing methods, such as laser micro- cytes but a mixture, which we consider as the limitation dissection based on hard tissue and single-cell Zhang et al. Journal of Orthopaedic Surgery and Research (2019) 14:129 Page 10 of 17 Table 1 The most enriched GO terms (BP) among proteins identified in exosomes of both groups GO terms among proteins identified in GO terms among proteins identified in exosomes (aged) exosomes (young) Protein Establishment of protein localization to Protein depolymerization (11) membrane (35) Cellular protein complex disassembly (13) Protein peptidyl-prolyl isomerization (10) Positive regulation of protein localization to nucleus (14) Chaperone-mediated protein complex assembly Protein nitrosylation (4) (4) Positive regulation of protein secretion (24) DNA DNA conformation change (23/217) None Telomere maintenance (15/113) Cell appendage Lamellipodium organization (16) None morphogenesis Ruffle organization (10) Membrane raft assembly (4) Podosome assembly (6) Regulation of cell junction assembly (12) Metabolism Nitric oxide metabolic process (12) None Regulation of nitric oxide biosynthetic process (12) Tricarboxylic acid metabolic process (8) Cellular transportation Golgi vesicle transport (25) None Energy coupled proton transmembrane transport (12) Positive regulation of protein transport(45) Cell clearance Lysosome organization (5) None Protein targeting to lysosome (5) Cell homeostasis Regulation of pH (13) Negative regulation of homeostatic process (18) Cellular response Cellular response to peptide hormone stimulus Acute-phase response (10) (34) Response to fungus (7) Response to parathyroid hormone (6) Response to immobilization stress (7) Response to angiotensin (7) Response to lipopolysaccharide (34) Response to mineralocorticoid (10) Killing by host of symbiont cells (4) Cell stress Response to endoplasmic reticulum stress (23) Respiratory burst (5) Cellular response to oxidative stress (23) Cell death Negative regulation of apoptotic signaling Regulation of intrinsic apoptotic signaling pathway in response to pathway (15) DNA damage (6) Cytolysis (6) Membrane invagination (9) Immunologic process I-kappaB kinase/NF-kappaB signaling (24) Activation of immune response (27) Positive regulation of leukocyte-mediated immunity (13) B cell-mediated immunity (16) Positive regulation of inflammatory response (13) Neutrophil migration (11) Granulocyte chemotaxis (12) Positive regulation of T cell activation (16) Leukocyte migration (29) Cell-cell recognition (9) Complement activation, lectin pathway (4) Cell-matrix adhesion Regulation of cell-matrix adhesion (16) None Skeletal system Positive regulation of ossification (14) None Osteoclast differentiation (5) Nervous system Axon ensheathment (17) Negative regulation of nervous system development (23) Peripheral nervous system axon regeneration (5) Glial cell differentiation (25) Other systems/tissue Myeloid cell development (12) Fibroblast proliferation (12) Regulation of muscle contraction (18) Renal system development (25) Regulation of coagulation (16) Smooth muscle cell proliferation (14) The numbers in parentheses are the counts of proteins annotated for each GO term Zhang et al. Journal of Orthopaedic Surgery and Research (2019) 14:129 Page 11 of 17 Fig. 7 GO analysis of the differentially expressed exosomal proteins. a, b Heatmap depiction of the top 50 overlapping downregulated (a, green) and upregulated (B, red) proteins in OLCS exosomes, aged vs young. c, d The most enriched GO terms among downregulated (c) and upregulated (d) exosomal proteins, aged vs young. The numbers are DEP counts for each GO term sequencing, may be helpful to optimize the methods of downregulated (Fig. 4a). Taken together, these lines of this study. evidence tend to show the attenuation of the mechanical sensing of aged bone cells. DEGs involved in bone mechanosensation The expression of many key factors involved in the bone Hormone receptors mechanosensation is downregulated in the aged cortical Here, the gene expression of some hormone receptors, bone. First, the critical structural molecules that allow including Pth1r, VDR, Fgf1r, and Fgf2r, was downregu- osteocytes to bear mechanical force are downregulated lated in aged cortical bone. Parathyroid hormone (PTH), (Fig. 4): integrins and related factors (e.g., Itga10, Itgb4, secreted by the parathyroid glands, plays a central role Chad, Fap), components of focal adhesions (e.g., Lamb2, in maintaining bone metabolic homeostasis by binding Thbs2/4, Tnc, Tnn, Pdgfra), cytoskeleton proteins (e.g., to the PTHR, a G protein-coupled membrane-spanning Actn2, Cav1, Flnc, Myl2, Mylk4), and the main calnexin receptor. Activation of PTH1R increases both bone for- (Cx43/Gja1) that composes the gap junction between os- mation and resorption by inhibiting Sost expression and teocytes. Second, key genes whose products involve in elevating the RANKL/OPG ratio, respectively [13]. Vita- mechanical stimulation induced second messenger pro- min D binds to vitamin D receptor (VDR), as is the case duction: key enzymes involved in NO, PGE2, and ATP with nuclear receptors. Its main function is to mediate synthesis (e.g., NOS1, COX1-3, ND1-5), factors involved calcium and phosphorus metabolism by regulating the in the regulation of homeostasis and the intracellular expression of the molecules (e.g., DMP1, MEPE, PHEX) flow of calcium ions (Casq1, Jph2, Trdn, Jsrp1, Hrc, involved in the synthesis and secretion of inorganic Capn3, Ryr1), and Sost, an osteocyte-specific marker pyrophosphate and the regulation of bone matrix that integrates osteocyte mechanotransduction and bone mineralization [15]. The expression of FGF23, the core mass by antagonizing Wnt/beta-catenin signalling [34, regulator of body calcium and phosphorus [14], has been 35]. Additionally, downstream signalling elicited by demonstrated to be influenced by both PTH/PTHR and mechanical force-induced messengers, such as vitamin D/VDR signalling through the fibroblast growth cGMP-PKG, PI3K-AKT, and WNT signalling, is factor receptor (FGFR) [13, 36]. Given their important Zhang et al. Journal of Orthopaedic Surgery and Research (2019) 14:129 Page 12 of 17 Fig. 8 Ageing characteristics of bone indicated by transcriptomic and exosomal proteomic analysis of cortical bone cells. The scheme enumerates the main findings of this study. Up-and down-regulations of bone cell functions and signaling pathways are shown in pink and light green columns, respectively role in mediating the calcium phosphate homeostasis be- these molecules could affect the mineralization and tween the bone and the circulatory system, downregula- mechanical properties of the extracellular matrix. tion of these receptors may alter how cells perceive and It has been demonstrated that the mediation of osteo- respond to hormones during ageing. clastic bone resorption depends mainly on the receptor activator of nuclear factor kappa-Β ligand (RANKL)/oste- DEGs involved in bone formation and resorption oprotegerin (OPG) ratio. RANKL, produced by osteocytes, Many key osteogenic molecules and pathways are down- is the major osteoclast differentiation factor [37, 38]. Al- regulated in aged group, including members of the BMP though our data showed no significant difference in and WNT signalling pathways (Tgfb2, Bmp2, Wnt5b, RANKL expression, downregulation of Opg/Tnfrsf11b Wnt16, Lrp4, Lrp5) and their target genes (Gremlin, may increase the RANKL/OPG ratio, which could be the Twist, Ocn, Postn, Gja1, Opg, Cdh2/11/15, and Alp1) reason for the excessive bone resorption in the aged bone. (Fig. 4b). Moreover, many regulators of these two path- In addition, the upregulation of several key genes of osteo- ways are also downregulated; some are positive regula- clast stimulating factors, such as Adam8, Tyrobp, Sbno2, tors, e.g., Col1a1, Bambi, Cav1, Fgfr2, and Kank1, while and Ccr1, could promote osteoclast formation. Further- some are inhibitors, including Twist, Dkk1, Nbl1, and more, higher expression levels of genes involved in regu- Cdh2. Downregulation of these regulatory factors indi- lating proinflammatory cytokine responses are positively cates that the dominant role of bone cells in regulating associated with osteoclast activation in the aged bone. bone formation weakens during ageing. Regarding the formation and maintenance of bone DEGs related to immunity matrix, gene expression levels are downregulated for a From our data, it is obvious that the proinflammatory variety of collagens (e.g., COL1, COL2, COL3, COL5, state is the most significant feature of bone aging, as the COL8, COL11), molecules involved in collagen fibril upregulated genes were enriched in many inflammatory organization (e.g., LUM, P3H1, GREM1, LOX), and GO terms involved in both innate and adaptive immun- many non-collagenous bone matrix proteins (OCN, ON, ity. In innate immunity, pattern recognition receptors POSTN, DMP1, MEPE, PHEX) (Fig. 4). The lack of (PRRs), a group of receptors that activate the formation Zhang et al. Journal of Orthopaedic Surgery and Research (2019) 14:129 Page 13 of 17 of inflammasomes by identifying pathogen-associated DEGs involved in cellular energy metabolism molecular patterns (PAMPs) and damage-associated mo- Mitochondrial and endoplasmic reticulum (ER) insuffi- lecular patterns (DAMPs) in the environment, play im- ciencies are widely considered to be putative hallmarks portant roles in regulating pathogen recognition and of cell senescence [41]. Here, signs of decline in energy activation of the intracellular inflammatory response metabolism in the aged bone cells were evident: first, in [39]. In our data, pattern recognition-related inflamma- the aged group, of the 69 downregulated transcripts tory molecules and pathways were upregulated in the whose products serve mitochondrial functions, many are aged group (Fig. 5), including (1) two important pattern involved in ATP production (tricarboxylic acid cycle, re- recognition receptor (PRR) pathways—the Toll-like re- spiratory chain, and oxidative phosphorylation). Down- ceptor and NOD-like receptor signalling pathways regulation of these key genes may directly reduce the (Fig. 5a); (2) molecules and signalling pathways down- cellular energy supply. In addition, several downregu- stream of PRRs that trigger innate immunity, including lated molecules are key components of complex I NF-κB signalling (Fig. 5a) and genes related to interferon (ND1-4) and complex IV (COX1-3) in the respiratory production (Irf1, Ptpn22, Myd88, Cd14, Pycard); (3) chain, whose dysfunction could cause electron leakage NLRP inflammasome components (Nlrp3, Pycard, and increase the production of reactive oxygen species Card11); and (4) molecules that are regulated in re- (ROS), one of the most important factors for cell senes- sponse to viruses, fungi, and lipopolysaccharide (Fig. 5a). cence. Finally, the gene expression of several key en- These lines of evidence suggest that pathogen infections zymes that respond to cellular stress was downregulated: may be the cause of the enhanced proinflammatory state for example, LONP, which can protect cells from various in aged cortical bone. In addition, a large number of up- stress conditions by regulating mitochondrial metabol- regulated DEGs were enriched in GO terms involved in ism and repairing mitochondrial DNA [42], and PINK, adaptive immunity, such as regulation of cytokine pro- which plays an irreplaceable role in mediating mitochon- duction and secretion, tumour necrosis factor (TNF) drial autophagy [43]. In addition, the products of some production, lymphocyte proliferation, differentiation, DEGs located in the ER were downregulated. FKBP9, and migration (Fig. 5b). It is worth mentioning that CRTAP, and FKBP10 function as protein-folding factors some known regulators of adaptive immunity were mediated by chaperones [44]. MNF2 and VDAC are typ- found in OLCS exosomes (see below in the “Immune-as- ical key factors on the ER–mitochondria interface [45, sociated proteins” section), and most of them were up- 46]. These changes in energy metabolism could be the regulated in the aged group, which might be further root causes of bone ageing. evidence for the involvement of aged bone cells in the Changes in some cellular metabolism pathways are regulation of adaptive immunity. also important clues for determining the underlying On the other hand, there are many receptors of mechanisms of bone ageing. The FoxO signalling path- proinflammatory cytokines whose gene expression way is regulated by and counteracts external changes levels were increased in the aged group: some of that disturb homeostasis, including metabolic stress, oxi- them are involved in the mediation of immunosup- dative stress, and growth factor deprivation [47]. Ac- pressive signals, such as Lilrb4, Pilra, Il10ra, and cording to the KEGG analysis, the FoxO signalling Il1r2; some play pathogenic roles in inflammatory and pathway was upregulated in the aged group, which may autoimmune diseases such as rheumatoid arthritis, for be further corroboration of the severe cellular stress in example, Il17ra; and Il18rap and Il21r, whose prod- aged bone cells. Furthermore, FoxO signalling may have ucts are involved in the activation of signalling path- special cellular functions in the bone: it has been dem- ways associated with inflammation, including NF-κB, onstrated that activation of FoxO in osteoblasts can hin- MAPK8, and JAK-STAT. Changes in the expression der bone formation, as it can competitively inhibit the levels of these molecules could affect the responses of WNT pathway by diverting β-catenin from TCF- to aged bone cells to inflammatory factors. Notably, FoxO-mediated transcription [48]. Moreover, the FoxO molecules negatively regulating JAK-STAT signalling pathway is normally negatively regulated through phos- were downregulated (Fig. 4b), while molecules posi- phorylation mediated by the PI3K-AKT pathway in di- tively regulating the JAK-STAT cascade were upregu- verse organisms [49]. Meanwhile, PI3K-AKT is lated in the aged group (Fig. 5b). Given that responsible for mechanical force-induced osteogenesis in JAK-STAT signalling is considered to have the cap- the bone [8]. Consistently, in our data, FoxO signalling was acity to mediate the responses of target cells to in- upregulated (Fig. 5a) and accompanied by the downregula- flammatory cytokines and plays an important role in tion of PI3K-AKT and WNT signalling (Fig. 4a, b). Thus, regulating cell apoptosis [40], the active regulation of the crosstalk among these three pathways could be the mo- JAK-STAT signalling might be the link between cell lecular explanation and underlying link between the bone responses to inflammatory cytokines and cell death. loss and decline in cell function that occur during ageing. Zhang et al. Journal of Orthopaedic Surgery and Research (2019) 14:129 Page 14 of 17 The loss- and gain-of-function of OLCS exosomes with transcription factors and stress kinases that can mediate age indicated by GO analysis among exosomal proteins inflammatory and tumourigenic signalling, e.g., NF-κB The results revealed that the skeleton could secrete large and AMPK [55]. Thus, the increased expression of these amounts of regulatory proteins involved in regulating a SASP proteins in senescent OLCS exosomes may explain wide range of cellular activities, from regulating energy how aged bone cells contribute to the environmental ef- metabolism and protein transportation to cell differenti- fects of adjacent and distal cells and systems. ation, morphogenesis, and movement (Fig. 6d). Add- itionally, biological processes involving the maintenance Immune-associated proteins of cell homeostasis and anti-senescence were included, Our analyses of the immune-associated proteins in such as molecules involved in the regulation of telomere OLCS exosomes have shown interesting findings regard- maintenance, protein folding and stabilization, tissue re- ing the cross-regulation between the skeleton and the generation, lysosome organization, and proteins in re- immune system: First, approximately 10% (85 out of sponse to ROS and hormones (Fig. 6d, e). 1019 proteins identified in the young group, and 76 out In clear contrast to young controls, the aged OLCS of 700 proteins in the aged group) of the identified pro- exosomes lacked functional proteins for cellular activ- teins found in OLCS exosomes were enriched in ities, including maintenance of the morphological prop- immune-related GO terms, and approximately 80% (67 erties of cells, maintenance of cell homeostasis, and out of 85/76 in young/aged group) of them overlapped regulation of cellular metabolism and transportation between the young and aged groups. Although most of (Table 1). Moreover, the results revealed a functional de- them were foundtohaveahigherLFQ intensityinaged cline in the regulation of DNA conformation, telomere OLCS exosomes than in young ones, their overall compos- maintenance, protein complex assembly, and localization ition in exosomes was relatively constant across age. To (Table 1). In addition, proteins involved in lysosome some extent, these results indicate that there is a certain organization and cellular responses to hormones (para- group of molecules in OLCS exosomes, both aged and thyroid, angiotensin, and mineralocorticoid) were absent young, that may serve an immune regulatory function. Sec- in the aged group (Table 1). On the other hand, the ond, among these overlapping immune-associated proteins enriched proteins in the aged group were mainly associ- were some key factors that have been shown in existing ated with activation of immune responses, cell damage, studies to regulate the activities of both immune cells and and cell death (Table 1). Most of the features mentioned bone cells, including CD44, CD47, CD59, TGF-β2, and above mirrored the findings at the RNA level; some of GSN. Among these, CD44 is a novel marker of osteocytic them have been demonstrated or categorized in existing differentiation in bone [56]; meanwhile, it is also widely studies linked to cell senescence [41] and could elucidate expressed in many kinds of immune cells (T cells, granulo- both the causes and consequences of bone ageing. cytes, monocytes) and is critical for their maturation [57, 58]. CD47, known as a “don’t eat me” signal to macro- Proteins identified in OLCS exosomes phages, plays a critical role in immune cell activation [59, Senescence-associated secretory phenotype 60], and it also has a profound effect on skeletal remodel- Studies have demonstrated that aged cells can secrete ling and bone maintenance through its actions on both os- proinflammatory factors, growth factors, chemokines teoblasts and osteoclasts [61]. For CD59, this molecule was and proteases to mediate age-related inflammatory re- recently demonstrated to be a regulator of bone growth sponses, wound healing, and even tumour progression; and homeostasis by interfering with the complement sys- this condition is now referred to as the tem in innate immunity [62]. TGF-β, a multifunctional senescence-associated secretory phenotype (SASP) [50, cytokine, plays integral roles in the regulation of adaptive 51]. Here, several classical SASP proteins, including immunity, mainly through activating T cells and effector TGF-β2, OPG, MMP9, TIMP1, MIF1, PRDXs, and and regulatory T cells [63]; in the bone, it is a critical regu- IGFBP3, were found to be upregulated in aged OLCS lator coupling of bone formation and resorption [64]. All exosomes. Among them, TGF-β2 and OPG are import- these proteins, except CD44, showed increased expression ant mediators of bone metabolism; serum levels of in aged OLCS exosomes. Similarly, many other immuno- TGF-β2 and OPG have shown significant positive corre- regulatory molecules were upregulated in aged OLCS exo- lations with bone turnover markers in Chinese women somes, including several complement molecules and [52]. TIMP1, a tissue inhibitor of metalloproteinases regulators: C3, C1QA, C1QB, C1QC, C4A, C8B, MBL1, (MMPs), is expressed in both osteocytes and osteoblasts MASP2, FCNB; positive regulators of T cell/B cell activa- and maintains the balance of bone matrix degradation tion: SLC4A1, SPN, TFRC, ANXA1, CORO1A, FLOT, by regulating MMP levels [53]. Moreover, TIMP/MMPs PNP, PTPRC; and regulators of leukocyte proliferation, mi- can also accommodate the immune influx in the extra- gration, and aggregation: S100A9, S100A8, PNP, PLG, MIF, cellular matrix [54]. MIF1 is known to activate several ECM1, SERPINE1, SLC4A1, GSTP1, ELANE. Zhang et al. Journal of Orthopaedic Surgery and Research (2019) 14:129 Page 15 of 17 On the other hand, only a small number of Conclusion immune-associated proteins were found to have higher A global view of changes in the gene expression profiles of intensities in the young group relative to the aged group, bone cells and secretory proteins in OLCS exosomes sug- including M-CSF (CSF-1), CD1d, Galectin1 (LGALS1), gests key characteristics of bone ageing. Some changes con- CD90 (THY1), and RHOA. Among them, M-CSF sidered hallmarks of cell ageing have been reviewed (macrophage colony-stimulating factor 1) is a haemato- previously, e.g., reductions in energy metabolism, cellular poietic growth factor involved in the proliferation, differ- responses to hormones, DNA conformation, telomere entiation, and survival of monocytes and macrophages; maintenance, and an increased level of the proinflamma- in the bone, it is one the most important cytokines for tory state. Moreover, newly identified changes are indicative the maturation and function of osteoclasts. CD1d is a of reductions in bone-specific functions, e.g., mechanical key molecule necessary to activate CD1d-dependent sensation and bone remodelling regulation. Further, our natural killer T cells that regulate certain tissue-specific data suggest that exosomes of young OLCS have multiple immune activities [65]. Galectin1, a homodimeric molecules that could play roles in maintaining cellular galactose-binding lectin, is able to selectively bind with a homeostasis; however, these functions are weakened during T cell surface receptor (NP-1) and inhibit T cell prolifer- ageing. Nevertheless, the upregulated proteins in aged ation [66, 67]. CD90, also known as the T lymphocyte OLCS exosomes provide clues that these aged OLCS exo- differentiation antigen, is a conserved cell surface pro- somes could have additional functions, such as promoting tein with a single V-like immunoglobulin domain; CD90 wound healing and scavenging free radicals, that help the can activate the T cell receptor [68] and is expressed as a surrounding cells resist senescence. On the other hand, surface marker for bone marrow stem cells [69, 70] and aged cells are considered the principal promoters of sys- osteoblasts [71]. temic and local inflammation that characterize ageing and Collectively, a large number of immune regulatory promote age-related diseases. Herein, we first found that a factors for both innate and adaptive immunity were large number of immune-related DEGs were upregulated found in OLCS exosomes. These findings may provide in aged bone cells, and then, we determined that many clues for exploring the mutual regulation between the regulatory factors both for innate and adaptive immunity skeleton and immune system and the mechanism of are found in OLCS exosomes, indicating that exosomes bone ageing. may act as shuttles propagating inflammatory states from the aged cells to the immune cells. Taken together, this study provides new insights into a further understanding of Bone remodelling-associated proteins in OLCS exosomes bone ageing. Proteins that are involved in osteoblast differentiation (e.g., COL-1/5, LRP, MMP-2, DCN, CX43, β-CATENIN, Additional files TGF-β, ALP) and osteoclast differentiation (e.g., M-CSF, ANXA, FBN1, SLC9, SRC, ATP6AP) were found to be Additional file 1: Figure S1 and S2. Description of the upregulated in the young group relative to the aged sampling method of cortical bone cell in this study. (Fig. S1. Cell group (Fig. 7a, c), indicating that exosomes may be one composition of mouse cortical bone shown by H&E staining. Fig. S2. The sampling method of this study). (DOCX 21222 kb) of the possible mechanisms of bone secretion in the regulation of bone remodelling and decrease with age. Abbreviations BV/TV: Bone volume to total volume ratio; CBT: Cortical bone thickness; Antioxidative proteins in OLCS exosomes CKD: Chronic kidney disease; CVD: Cardiovascular disease; DAMPs: Damage- associated molecular patterns; DEGs: Differentially expressed genes; Since cell ageing is identified as a decline in various DEPs: Differentially expressed proteins; ER: Endoplasmic reticulum; GO: Gene functions of the cell, almost all evidence that supports Ontology; KEGG: Kyoto Encyclopedia of Genes and Genomes; LC-MS/ an increase in harmful effects or a loss of beneficial MS: Liquid chromatography-mass spectrometry; LCS: Lacunar-canalicular system; LFQ: Label-free quantitation; MSCs: Mesenchymal stem cells; effects in the aged cell is reasonable. However, there OLCS: Osteocytic lacunar-canalicular system; PAMPs: Pathogen-associated is an exception: some exosomal proteins involved in molecular patterns; ROS: Reactive oxygen species; RPKM: Reads per kilobase maintaining cell redox homeostasis and removal of transcriptome per million reads; SASP: Senescence-associated secretory phenotype; Tb.N: Trabecular number; VECs: Vascular endothelial cells ROS are upregulated in aged OLCS exosomes relative to young ones (Fig. 7b, d), including several key mol- Acknowledgements ecules, such as peroxiredoxins (PRDXs), superoxide Not applicable. dismutase 2 (SOD2), thioredoxin 1 (TRX1), glutathi- one S-transferase P1 (GSTP1), and haptoglobin, which Funding This study was funded by the National Science Foundation of China (81470715 manifests in aged OLCS may exert a positive effect and 81771043), Y. Sun Key Project of Chinese National Programs for Research on surrounding or even distant cells to resist oxida- and Development (2016YFC1102705, Y. 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