3.1 Proliferation and osteogenic capacity was decreased in senescent BMSCs
Young and senescent BMSCs were derived from 4-week- and 18-month-old SD rats, respectively. Flow cytometric plots (Supplementary Figure S1) indicated the percentages of CD29+/CD45–/CD90+ stem cells in Y-BMSCs and O-BMSCs were ~ 98.7% and ~ 99.3%, respectively, which could meet the requirements of the following experiments. Despite of the similar quantity of stem cells, O-BMSCs exhibited high level SA-β-gal activity, the most widely used biomarker for senescent and aging cells (Fig. 1A). Proliferation curves of Y-BMSCs and O-BMSCs cultured in complete culture medium was assessed by CCK8 assay, which revealed that the proliferation ability of O-BMSCs was significantly reduced compared to Y-BMSCs (Fig. 1B).
The osteogenic differentiation capacity of O-BMSCs was also significantly impaired, manifested as remarkably reduced ALP activity (Fig. 1C). The expressions of osteogenic-related genes (ALP, Col-1, Runx2) and aging-related genes (SATB2, p53, p21, p16) were further analyzed by qRT-PCR (Fig. 1D). The expressions of ALP, Col-1, Runx2 and SATB2 of O-BMSCs were significantly decreased compared to Y-BMSCs, whereas p53, p21 and p16 were significantly increased. Of note, tumor-suppressor proteins p53, p21, and p16 are major regulators of G1/S cell–cycle checkpoint upregulated by senescence-indued stimuli [40–42], while SATB2 is a suppressor of cellular senescence that inhibits p16 [43]. Immunofluorescence staining of osteogenic proteins (BMP-2, Col-1, OSX) and aging-related SATB2 (Fig. 1F) presented a consistent trend with PCR results. These results demonstrated the degeneration of proliferation and osteogenic differentiation capacity of senescent BMSCs.
BMSCs senescence is commonly acknowledged as a pre-death state in which cell cycling and self-renewal capacity are extinguished [44], which was manifested as reduced proliferation of O-BMSCs in this study. Osteogenic differentiation of O-BMSCs was simultaneously impaired under possible underlying mechanisms including decreased expression of BMSCs-specific surface antigens, changing proportions of subpopulations within a heterogeneous BMSCs population, telomere loss and other senescence-related cellular defects [45]. Therefore, it is important to monitor and rejuvenate the proliferative and osteogenic functions of O-BMSCs for promoting aging bone regeneration. Current strategies to maintain BMSCs self-renewal and differentiation potential include utilization of growth factors, drugs, extracellular vesicles etc. to alter the cell culture microenvironment [46–48]. Herein, a previously optimized osteoinductive extracellular vesicles derived from young BMSCs was introduced and its effect on O-BMSCs was explored in the following experiments.
3.2 OI-exos from Y-BMSCs enhanced proliferation and osteogenesis of O-BMSCs
In an attempt to rejuvenate the senescence and restore the osteogenicity of BMSCs, osteoinductive exosomes (OI-exos) were extracted from Y-BMSCs as previously reported [21] and applied to treat O-BMSCs (Fig. 2A). The morphology of OI-exos observed by TEM (Fig. 2B) presented round, membrane-bound vesicles with diameters of 30–150 nm. The particle size distribution of OI-exos in aqueous phase measured by NTA (Fig. 2C) ranged from 100 to 250 nm with the peak at ~ 160 nm. The western blot assay (Fig. 2D) identified the expression of extracellular vesicles markers including TSG101, Alix and CD9, which were enriched in exosomes rather than cell lysates. The above results confirmed the successful preparation of OI-exos for following experiments. In the following in vitro experiments, an optimized exosome concentration of 2 × 1010 particles/mL according to previous study [21].
To investigate the internalization of Y-BMSC-derived OI-exos into O-BMSCs, the extracellular vesicles were labeled with DiI and incubated with O-BMSCs for 1h, 3h, 6h and 12h followed by fluorescence observation. The results revealed that O-BMSCs exhibited exosome uptake from 1 h, which gradually increased time-dependently (Fig. 2E-2F). Quantification of fluorescence intensity showed that the difference of exosome uptake from 6h to 12h remained statistically significant, indicating the continuous uptake of extracellular vesicles consistently proceeded after 12 h. The internalization of OI-exos into O-BMSCs was the prerequisite for further application.
To further explore the effect of OI-exos on O-BMSCs, proliferation and osteogenic differentiation of exosome-treated O-BMSCs were evaluated with untreated O-BMSCs as control and Y-BMSCs as positive control. After OI-exos treatment, O-BMSCs exhibited reduced aging-related SA-β-gal activity, though still higher than Y-BMSCs (Fig. 3A). Proliferation of O-BMSCs was significantly enhanced on day 4 and 7 (Fig. 3B). Similar trends were also observed in ALP activity (Fig. 3C-3D), osteogenic-related genes (Fig. 3E) and osteogenic proteins (Fig. 3F), showing enhanced osteogenic differentiation of O-BMSCs incubated with OI-exos. Though there was still a certain gap between the exosome-treated O-BMSCs and Y-BMSC in osteogenic- and aging-related phenotypes, the expression of related genes (osteogenic ALP, BMP-2 and OCN, aging-related p53 and p21) of extracellular vesicles-treated O-BMSCs exhibited comparable levels to that of Y-BMSCs. Collectively, our results indicated that OI-exos alleviated senescence of O-BMSCs and enhanced its proliferation and osteogenic differentiation capacity from a level of gene expression, though the related cellular phonotype, which was affected by more complexities, was only partially restored by the intervention of OI-exos. These results provided an effective approach for delaying aging process and improving osteogenic differentiation ability of senescent BMSCs.
Recently it has been suggested that the cargo of extracellular vesicles is significantly altered with age and with age-related diseases, and that “younger” extracellular vesicles could reverse some degenerative, age-related diseases [49, 50]. For instance, young extracellular vesicles derived from human exfoliated deciduous teeth are reported to exert abundant anti-senescent effects and potently rescue senescent tendon stem/progenitor cells into regenerative status at physiological and epigenetic levels [51]; extracellular vesicles from young MSCs are proven to ameliorate senescent phenotype of aged MSCs and enhance their function for myocardial repair by transferring exosomal miR-136 and downregulating Apaf1 [52]; serum extracellular vesicles from young rats with high expression of miRNA-19b-3p rescued the decreased osteogenic differentiation ability of aged BMSCs by inhibiting PTEN expression, providing a new strategy for treatment of bone microdamage and prevention of fractures [53]. The alleviated senescence, enhanced proliferation and osteogenesis of O-BMSC treated by Y-BMSC-derived OI-exos demonstrated in this study added to existing proofs that exosomes are potential therapeutics for senescence rejuvenation.
3.3 Exosomal lncRNA-ENSRNOG00000056625 potentially compensated senescence-impaired osteogenesis in O-BMSCs
As extracellular vesicles exert functions via delivery of nucleic acids (mRNA, microRNA, lncRNAs) into recipient cells [54, 55], following investigations on the underlying mechanism of young exosome-mediated osteogenesis of O-BMSCs focused on exosomal transcriptomics. Our previous study has indicated that multicomponent miRNA (let-7a-5p, let-7c-5p, miR-328a-5p and miR-31a-5p) in OI-exos enhanced osteogenesis via regulating Bmpr2/Acvr2b competitive receptor towards Bmpr-activated Smad pathway [21]. Though many previous studies have uncovered the mechanisms regarding exosomal mRNAs and miRNAs, the exosomal lncRNAs and their effects on osteogenesis remain largely unexplored. The abundant differential lncRNAs in OI-exos compared to control exosomes (con-exos) detected by microarray (Fig. 4A) prompted us to further explore the potential functions of exosomal lncRNAs in osteogenesis. Cellular RNA-seq was performed to identify the transcriptomic differences between O-BMSCs and Y-BMSCs (Fig. 4B). As depicted in Fig. 4C, considering that down-regulated lncRNAs in O-BMSCs could be compensated by exosomal lncRNAs from OI-exos, 204 up-regulated lncRNAs in OI-exos and 56 down-regulated lncRNAs in O-BMSCs were intersected. Two lncRNAs (lncRNA-ENSRNOG00000056625 and lncRNA-ENSRNOG00000056685) were obtained by intersection. To excavate the potential lncRNA-miRNA-mRNA interaction networks, 17 miRNAs that base-paired with the two intersected lncRNA sequences were predicted using miRanda database, and intersected with the 201 differentially expressed miRNAs in O-BMSCs to screen out pivotal miRNAs. 169 target genes of the 4 intersected miRNAs were predicated using miRWalk3.0 and miRDB databases, and intersected with the 3791 lncRNA-correlated mRNAs in O-BMSCs, which were screened by the Pearson’s correlation coefficients. The above-intersected 2 lncRNAs, 4 miRNAs and 74 mRNAs were analyzed according to ceRNA network theory to screen out the lncRNA-miRNA-mRNA relationship where the lncRNA and mRNA were linked by the same miRNA, competitively inhibited and negatively correlate with the miRNA. Finally, potential ceRNA networks consisting of 2 lncRNAs, 3 miRNAs and 12 mRNAs were constructed for further validation (Fig. 4D).
The expressions of the two lncRNAs were determined in Y-BMSCs and O-BMSCs by qRT-PCR. Significant down-regulation of lncRNA-ENSRNOG00000056625 was validated in O-BMSCs compared to Y-BMSCs, whereas lncRNA-ENSRNOG00000056685 expression showed no significant changes between Y-BMSCs and O-BMSCs (Fig. 5A). The remarkably enhanced expression of lncRNA-ENSRNOG00000056625 in OI-exo compared to con-exo was also confirmed (Fig. 5B). After treatment with OI-exo, the lncRNA-ENSRNOG00000056625 level in O-BMSCs was significantly increased compared with untreated O-BMSCs, though still lower than Y-BMSCs (Fig. 5C). These data demonstrated that the downregulated lncRNA-ENSRNOG00000056625 in O-BMSCs was compensated by OI-exo.
LncRNA-ENSRNOG00000056625-overexpressing (lnc-OE) plasmids were transfected into O-BMSCs with negative control lncRNA sequence (lnc-NC) to further indentify the effect of lncRNA-ENSRNOG00000056625 on osteogenic differentiation. Successful transfection was confirmed by the significantly increased expression of lncRNA-ENSRNOG00000056625 in O-BMSCs (Fig. 5D). Significantly decreased SA-β-gal activity and increased ALP activity of O-BMSCs were observed after lnc-OE transfection (Fig. 5E-5F). The expression of osteogenic-related genes including ALP, BMP-2, Col-1 and OCN were significantly up-regulated in lnc-OE-transfected O-BMSCs, while the expression of aging-related genes, p53 and p21, were significantly down-regulated (Fig. 5G). Immunofluorescence staining also demonstrated that lncRNA-ENSRNOG00000056625 overexpression promoted the expression of BMP-2 and Col-1 in protein level (Fig. 5H-5I). Taken together, these results demonstrate that OI-exos transferred osteogenic-promoting lncRNA-ENSRNOG00000056625 into O-BMSCs and enhanced osteogenic differentiation capacity of O-BMSCs.
Bone degenerative diseases or aging often involve the dysregulation of transcriptional networks and signaling pathways [56, 57]. Functions of lncRNAs in bone biology and bone diseases have been uncovered in recent years, for example, lncRNA-OG was reported to promote the osteogenic differentiation of BMSCs by regulating hnRNPK [58]; lncRNA-DANCR was shown to recruit enhancer of EZH2 by interacting with 305-nt transcript and enhancer of zestehomolog2, and then inhibit transcription of the target gene Runx2 and osteogenic differentiation [59]. In addition, some lncRNAs played dual roles in the differentiation of osteoblasts, showing both positive and negative regulation by targeting different downstream genes. LncRNA H19 was reported to negatively regulate osteogenic differentiation via reducing the expression of Dkk4, which encodes a protein of the Dickkopf family [60]; another research indicated that lncRNA H19 promoted osteoblast differentiation by functioning as a competing endogenous RNA [61]. Only a few studies revealed the functions of exosomal lncRNAs, e.g. exosomal lncRNA H19 acts as "sponges" to miR-106 and regulates the expression of angiogenic factor, Angpt1 that activates lnc-H19/Tie2-NO signaling in mesenchymal and endothelial cells [62]; Nonetheless, the effects of exosomal lncRNAs on bone-related biological process, especially in aging microenvironment remained unexplored until this study.
3.4 lncRNA-ENSRNOG00000056625 sponged miR-1843a-5p and enhanced osteogenesis of O-BMSCs
After validating the osteogenic-enhanceing effect of lncRNA-ENSRNOG00000056625, the miRNAs in its potential CeRNA networks, miR-1843a-5p and miR-150-5p, were further determined in Y-BMSCs and O-BMSCs by qRT-PCR. Both candidate miRNAs contained binding sites with lncRNA-ENSRNOG00000056625. As shown in Fig. 6A, a significantly increased expression of miR-1843a-5p was detected in O-BMSCs compared to Y-BMSCs, while the expression of miR-150-5p exhibited no significant difference. The expression of lncRNA-ENSRNOG00000056625 and miR-1843a-5p showed opposite trends in O-BMSCs, according with the general pattern of lncRNA sponging miRNA in ceRNA mechanisms. The interaction between miR-1843a-5p and lncRNA-ENSRNOG00000056625 was further investigated according to their bioinformatically predicted binding site (Fig. 6B). The luciferase assay indicated the miR-1843a-5p mimics transfection repressed the luciferase activity of the lncRNA-WT rather than lncRNA-MT reporters (Fig. 6C). To verify whether lncRNA-ENSRNOG00000056625 regulated osteogenic differentiation of O-BMSCs through sponging miR-1843a-5p, lncRNA-OE and miR-1843a-5p overexpression (miR-OE) plasmids were co-transfected in O-BMSCs. Opposite to the suppressed senescent and enhanced osteogenic prototype of O-BMSCs after lnc-OE transfection, miR-OE alone significantly decreased ALP activity and increased SA-β-gal activity of O-BMSCs, and co-transfection of miR-OE compromised the positive effects of lnc-OE (Fig. 6D-6E). qRT-PCR results demonstrated that lnc-OE-upregulated osteogenic-related genes (ALP, BMP-2, Col-1, OCN) and lnc-OE-downregulated aging-related genes (p53, p21) regressed to an insignificant level when the cells were co-transfected with miR-OE plasmids (Fig. 6F). The lnc-OE-upregulated BMP-2 and Col-1 protein expression level (Fig. 6G) were also reversed by miR-OE co-transfection. In summary, these data demonstrated that lncRNA-ENSRNOG00000056625 alleviated aging and elevated osteogenic capacity of O-BMSCs through serving as a sponge for miR-1843a-5p, which promoted senescence and negatively regulated osteogenesis.
Mounting evidences have proved that CeRNA networks of lncRNAs was one of the classic regulatory mechnaisms that mediate the process of bone regeneration [63, 64]. For instance, LncRNA NORAD promotes BMSC differentiation and proliferation by targeting miR-26a-5p in steroid-induced osteonecrosis of the femoral head [65]; lncRNA MALAT1 promotes the osteogenic differentiation of BMSCs and inhibited osteoclastic differentiation of macrophages in osteoporosis by upregulating IGF2BP1 expression via competitively binding to miR-124-3p [66]. The results in this study firstly annotated the functions of lncRNA-ENSRNOG00000056625 and miR-1843a-5p in osteogenic- and aging-related process, and demonstrated that lncRNA-ENSRNOG00000056625 served as a sponage for miR-1843a-5p to alleviate senescence and promote osteogenesis. In the following study, the target genes that regulated by miR-1843a-5p and correated with lncRNA-ENSRNOG00000056625 were screened to explore their functions for osteogensis of O-BMSCs.
3.5 miR-1843a-5p targeted Mob3a and negatively regulated osteogenesis of O-BMSCs
It is well-recognized that miRNAs play regulatory roles through inhibition of their targeted genes in bone regeneration [67, 68]. The bioinformatically predicted downstream target genes of miR-1843a-5p were further determined by qRT-PCR (Fig. 7A), and the results validated three significantly down-regulated genes in O-BMSCs compared to Y-BMSCs, Mob3a, Zim1, and Sms. Mob3a, belonging to Mob family genes, which activate the Hippo pathyways kinases (MST/LATS) or NDR kinases [69], has been reported to bypass BRAF and RAS-induced senescence via Hippo pathway. A relative of Mob3a, Mob1, has been reported to be inhibited by exosomal miR-186 derived from BMSCs to promote osteogenesis via upregulating YAP, a member of hippo signaling pathway, in postmenopausal osteoporosis [70]. Another previous study indicates the interaction between Mob1b and miR-135b-5p regulates osteogenesis via Hippo signaling pathway [71]. By contrast, few literatues have been reported regarding Zim1 and Sms in osteogenic- or aging-related process. Therefore, Mob3a was considered as the most potential target in osteogenic- and aging-related process, and hence selected for further investigation.
The predicted binding sites of Mob3a complementary to miR-1843a-5p (Fig. 7B) exhibited an identical sequence (UACCUCC) to that of lncRNA-ENSRNOG00000056625, indicating a potential competing relationship. Luciferase assay revealed that miR-1843a-5p mimics transfection repressed the luciferase activity of the Mob3a-WT rather than Mob3a-MT reporters (Fig. 7C). To further verify whether miR-1843a-5p regulate osteogenic differentiation of O-BMSCs through functional targeting Mob3a, Mob3a overexpression (Mob3a-OE) plasmids was constructed and co-transfected with miR-OE plasmids. The miR-OE-enhanced SA-β-gal activity were significantly decreased after co-transfection with Mob3a-OE plasmids (Fig. 7D). In contrast, ALP staining demonstrated that the miR-OE-repressed osteogenic activity was rescued by Mob3a-OE plasmids (Fig. 7E). Mob3a-OE transfection alone induced reduced senescence and enhanced osteogenic differentiation (Fig. 7D-7E). qRT-PCR results demonstrated that miR-OE-downregulated osteogenic-related genes (ALP, BMP-2, Col-1, OCN) and miR-OE-upregulated aging-related genes (p53, p21) returned to an insignificant level when the cells were co-transfected with Mob3a-OE plasmids (Fig. 7F). The miR-OE-downregulated BMP-2 and Col-1 protein expression level (Fig. 7G) were also reversed by Mob3a-OE plasmids co-transfection. Collectively, these results suggest that that the osteogenic differentiation of O-BMSCs was regulated by miR-1843a-5p through targeting Mob3a in O-BMSCs.
3.6 OI-exo induced Mob3a upregulation, YAP dephosphorylation and nuclear translocation
Previous study has reported that Mob3a inhibits Hippo/MST/LATS signaling, elevates YAP expression, and then bypasses oncogene-induced senescence [69]. However, the relationship between miR-1843a-5p with Mob3a and the downstream YAP-involving pathway in O-BMSCs remains unknown. It has been reported that extracellular vesicles derived from HUCMSCs effectively inhibits BMSC apoptosis and prevents rat disuse osteoporosis via miR-1263/Mob1/Hippo signaling pathway, where exosomal miR-1263 binds to the 3' UTR of Mob1 and exerts its function by directly targeting Mob1 in recipient cells to activate YAP [72]. Herein, the above data demonstrated that Mob3a is a direct target of exosomal miR-1843a-5p in O-BMSCs. However, whether OI-exos regulated osteigenic differentiation of O-BMSCs through YAP pathway and the association between Mob3a and YAP remain unknown.
As shown in Fig. 10A, Mob3a protein expression in O-BMSCs was significantly inferior to Y-BMSCs, and was elevated by OI-exos though still lower than Y-BMSCs (Fig. 8A, a1 and a2), which was in line with the senescent and osteogenic trends in Fig. 3. The protein expression of p-YAP (ser127 and ser397), which was downstream of Mob3a and upregulated in O-BMSCs, was significantly suppressed by OI-exos, though lowest p-YAP expression was observed in Y-BMSCs (Fig. 8A, a3 and a4). The trend of nuclear YAP protein levels was opposite to p-YAP and similar to Mob3a (Fig. 8A, a5). Phosphorylation of YAP is commonly known as a state of degradation which is an opposite procedure to nuclear translocation. Our results also demonstrated that OI-exos suppressed the phosphorylation and degradation of YAP, and promoted its nuclear translocation.
To validate the function of exosome-transfered lncRNA-ENSRNOG00000056625 and its relationship with miR-1843a-5p, co-transfection of lnc-OE and/or miR-OE was performed (Fig. 8B). The results shown that Mob3a protein level significantly increased after lncRNA-OE transfection, and the upregulated Mob3a was decreased after co-transfection of miR-OE (Fig. 8B, b1 and b2). Moreover, the expression of p-YAP (ser127 and ser397) in O-BMSCs was decreased by lncRNA-OE transfection and increased by miR-OE (Fig. 8B, b3 and b4). In contrast, the nuclear YAP level showed an opposite trend to p-YAP results (Fig. 8B). These data indicated that lncRNA-ENSRNOG00000056625 reduced the phosphorylation of YAP and promoted YAP transfer into nucleus, which could be blocked by miR-1843a-5p. Combined with the bioinformatic analysis (Fig. 4) and exosomal lncRNA component verification (Fig. 5B), it is implied that exosomal lncRNA-ENSRNOG00000056625 contributed to the YAP signaling
To further verify the interaction between miR-1843a-5p and Mob3a in YAP signaling, co-transfection of miR-OE and/or mRNA-OE was performed (Fig. 8C). P-YAP (ser127 and ser397) was significantly upregulated by miR-OE plasmids, while the upregulation was suppressed by Mob3a overexpression plasmids (Fig. 8C, c1-c3). Correspondngly, nuclear YAP expression was positively regulated by Mob3a overexpression and impeded by miR-1843a-5p overexpression (Fig. 8C, c4).
YAP is the downstream effector of the Hippo pathway that regulates tissue growth and cell plasticity during animal development and regeneration [73–76]. Remarkably, YAP promotes regeneration in tissues or organs with poor or compromised regenerative capacity especially in the old or diseased individuals [77, 78]. Our previous studies have demonstrated that concentrated growth factor promotes gingival regeneration through AKT/Wnt/β-catenin and YAP signaling pathways [79]. Also, YAP plays an important roles in promoting osteogenesis, suppressing adipogenesis, and thus maintaining bone homeostasis in bone regeneration [80]. Collectively, it was suggested that lncRNA-ENSRNOG00000056625, which was differentially high expressed in OI-exo, functioned as a promoter of YAP translocation into nucleus via sponging miR-1843a-5p and upregulating Mob3a expression, increasing proliferation and osteogenic differentiation and alleviating senescence of O-BMSCs.
3.7 OI-exo-loaded MBG scaffold promoted bone regeneration in aged rats
Biomaterial scaffolds have been increasingly designed to deliver exosomes for in vivo bone regeneration in recent years [81–84]. In our previous study, MBG scaffold with hierarchical macro-/micro-/meso-porous structure and inherent osteoinductivity has been developed as an effective carrier for lyophilized delivery of OI-exo and demonstrated to promote bone regeneration of young rats in vivo [21]. Herein, on the bases that OI-exos alleviated senescence and enhanced proliferation and osteogenesis of O-BMSC, OI-exo-loaded MBG scaffold was further applied in calvarial defect of aged rats to evaluate its effect on enhancing aging bone regeneration and alleviating age-related bone resorption in vivo.
As observed by SEM, OI-exo-loaded MBG scaffold (MBG + exo) exhibited interconnected macropores (≥ 200 µm, Fig. 9A, a1) and surface micropores (0.5-2 µm, Fig. 9A, a2) for lyophilized delivery of extracellular vesicles (30–150 nm, Fig. 9A, a3). Wide-angle XRD spectrum (Fig. 9B) and EDS spectrum (Fig. 9C) of MBG indicated a typical amorphous bioglass composition of trinary Si/Ca/P oxide, which was the basis for excellent cytocompatibility and degradability. TEM image of MBG (Fig. 9D) showed ordered mesoporous structure with an average pore size of ~ 7.8 nm according to N2 adsorption-desorption analysis (Fig. 9E). The mesopores of MBG provide high surface aria for biomineralization and potential growth factor delivery in bone repair applications.
The in vivo bone regenerative effect of MBG + exo was evaluated in calvarial defects of aged rats (Fig. 4F) with unloaded MBG scaffold (MBG) applied in aged and young rats for comparison. 12 weeks after surgery, micro-CT analysis (Fig. 9G-9H) showed considerable new bone formation (20 ± 1% BV/TV) fused with residual material in young rats, reconfirming the inherent osteoconductivity and osteoinductivity of MBG. However, in aged rats, MBG scaffolds unexpectedly exhibited complete degradation with inferior bone formation (16 ± 2% BV/TV). In comparison, MBG + exo induced significantly enhanced bone formation (9.5 ± 2% BV/TV), though did not alleviate the precipitate degradation of scaffold. Literature has reported that senescent individuals are in a state of chronic hypoinflammation [85–87] and show excess activity of osteoclasts [88–90], which might lead to accelerated degradation of MBG scaffold.
Bone formation at different stages (Week 3, 6 and 9) were evaluated by sequential fluorescent labeling (Fig. 10A-10C). The results showed that bone deposition amount and rate in aged rats treated by MBG + exo was significantly higher than MBG in all time, though still lower than that in young rats treated by MBG. Histological analyses by van Gieson (VG), haematoxylin and eosin (HE), and Masson’s trichrome staining provided more details of the regenerative outcomes. As shown in Fig. 10D, VG staining, a universal identification method for hard tissue sections, showed significantly more newly formed bone in MBG + exo group than MBG group. HE and Masson’s trichrome staining indicated that residual material was only observed in young rats, while tremendous fibrous connective tissue occupied the non-osseous areas in the defect area of aged rats. Newly formed bone partially bridged the bottom defect area of aged rats treated with MBG + exo. The trends of new bone area observed histologically were in accordance with micro-CT quantitive data.
The in vivo results demonstrated that though excess activity of bone resorption in senescent individuals appeared to be a tremendous challenge in aged bone regeneration, young BMSCs-derived OI-exo showed promising efficacy in alleviating senescence and enhancing aged osteogenesis in animal experiments. The underlying mechanism of young extracellular vesicles-enhanced osteogenesis of O-BMSCs was further investigated in the following of this research.