
==== Front
Cell Rep Med
Cell Rep Med
Cell Reports Medicine
2666-3791
Elsevier

S2666-3791(24)00379-3
10.1016/j.xcrm.2024.101665
101665
Article
Targeting miR-29 mitigates skeletal senescence and bolsters therapeutic potential of mesenchymal stromal cells
Ding Zhen 12
Ma Guixing maguixing@live.com
12∗
Zhou Bo 1
Cheng Siyuan 1
Tang Wanze 1
Han Yingying 1
Chen Litong 1
Pang Wei 1
Chen Yangshan 1
Yang Dazhi 1
Cao Huiling caohl@sustech.edu.cn
13∗∗
1 Department of Biochemistry, School of Medicine, Guangdong Provincial Key Laboratory of Cell Microenvironment and Disease Research, Key University Laboratory of Metabolism and Health of Guangdong, Southern University of Science and Technology, Shenzhen 518055, China
∗ Corresponding author maguixing@live.com
∗∗ Corresponding author caohl@sustech.edu.cn
2 These authors contributed equally

3 Lead contact

20 8 2024
20 8 2024
20 8 2024
5 8 1016655 3 2024
7 6 2024
8 7 2024
© 2024 The Author(s)
2024
https://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).
Summary

Mesenchymal stromal cell (MSC) senescence is a key factor in skeletal aging, affecting the potential of MSC applications. Identifying targets to prevent MSC and skeletal senescence is crucial. Here, we report increased miR-29 expression in bone tissues of aged mice, osteoporotic patients, and senescent MSCs. Genetic overexpression of miR-29 in Prx1-positive MSCs significantly accelerates skeletal senescence, reducing cortical bone thickness and trabecular bone mass, while increasing femur cross-sectional area, bone marrow adiposity, p53, and senescence-associated secretory phenotype (SASP) levels. Mechanistically, miR-29 promotes senescence by upregulating p53 via targeting Kindlin-2 mRNA. miR-29 knockdown in BMSCs impedes skeletal senescence, enhances bone mass, and accelerates calvarial defect regeneration, also reducing lipopolysaccharide (LPS)-induced organ injuries and mortality. Thus, our findings underscore miR-29 as a promising therapeutic target for senescence-related skeletal diseases and acute inflammation-induced organ damage.

Graphical abstract

Highlights

• miR-29 expression increases with aging in bone tissues and in senescent MSCs

• miR-29 overexpression in Prx1-positive MSCs accelerates MSCs and skeletal senescence

• miR-29 promotes cellular senescence through Kindlin-2/p53 axis

• miR-29 knockdown MSCs alleviate skeletal senescence and systemic inflammation

Ding et al. report that miR-29 expression increases in bone tissues in aged mice and OP patients, as well as in senescent MSCs, promoting skeletal senescence by upregulating p53 via targeting Kindlin-2. miR-29 knockdown in BMSCs impedes senescence, enhances bone mass and bone regeneration, and reduces LPS-induced inflammation and mortality.

Keywords

skeletal senescence
miR-29
p53
Kindlin-2
MSC therapy
bone
osteoporosis
aging
bone repair
Published: August 20, 2024
==== Body
pmcIntroduction

The musculoskeletal system provides mechanical support, protects organs, enables movement, and stores calcium and bone marrow. Senescence alters bone mass and geometry, causing bone degeneration and age-related osteoporosis (OP), characterized by decreased trabecular and cortical bone thickness and volume and increased cortical bone porosity.1,2,3,4 Senescence expands bone marrow volume,5 diminishes bone mechanical properties and physiological functions, and leads to the onset of OP and fractures. Senescent bones create inflammatory niches, exacerbating inflammatory response and cellular senescence induced by factors like lipopolysaccharide (LPS).6,7,8 Despite these evident impacts, the mechanisms governing skeletal senescence are unclear, and no drugs exist for its prevention, necessitating the identification of potential targets.

Cellular senescence is a fundamental mechanism in skeletal senescence.9 Mesenchymal stromal cells (MSCs) can differentiate into osteoblasts, adipocytes, and chondrocytes.10 Aging reduces the quantity and functionality of bone marrow MSCs (BMSCs) in both mice and humans.11,12 Senescent BMSCs exhibit increased adipogenic13,14,15 and decreased osteogenic differentiation, leading to higher bone marrow adipose tissue and inflammation, hallmarks of skeletal senescence.6,16,17,18,19,20 The senescence-associated secretory phenotype (SASP) in BMSCs drives skeletal senescence.21 BMSCs are widely used to treat various diseases such as degenerative conditions, autoimmune disorders, and inflammatory diseases,22,23,24 but their senescence poses significant challenges for transplantation due to donor-related factors or in vitro culture.25,26 Identification of potential targets to prevent BMSC senescence is crucial to enhance their therapeutic efficacy.

MicroRNAs (miRNAs) are small noncoding RNAs that modulate post-transcriptional gene expression in processes such as cell proliferation, tumorigenesis, apoptosis, immunity, and the differentiation of BMSCs.27,28,29,30,31 Their concise sequences, robust stability, and potent biological functions make miRNAs promising therapeutic targets. miRNA mimics and inhibitors are in clinical trials for diseases like cardiovascular disease, hepatitis, and cancer.32,33,34,35,36 Nevertheless, the role of miRNAs in regulating MSCs and skeletal senescence in vivo remains largely unexplored. The miR-29 family, comprising miR-29a, miR-29b, miR-29c, encoded by two gene loci, miR-29a/b1 on chromosome 6 and miR-29b2/c on chromosome 1,37 targets the same mRNA due to their identical seed sequence and is critical in various biological processes such as skeletal muscle development, cardiovascular homeostasis, immunoregulation, and tumorigenesis.37,38,39,40,41,42 While there are several in vitro studies on the involvement of miR-29 in MSCs senescence,43,44,45 the findings are controversial, highlighting the need for systematic in vivo studies.

Considering the superior clinical application prospects of miRNAs and MSCs, identification of potential miRNAs governing MSCs and skeletal senescence, along with the elucidation of underlying mechanisms, holds substantial theoretical and clinical implications for the management of bone degenerative disorders that are closely related to skeletal senescence, such as OP. In this study, we establish that miR-29 expression increases in bone tissues in mice with aging or in people with OP, as well as in senescent MSCs. We demonstrate that overexpression of miR-29 in Prx1-positive MSCs significantly accelerates MSCs and skeletal senescence through the miR-29/Kindlin-2/p53 axis. Knocking down miR-29 by lentivirus with miRNA sponge technology significantly rejuvenates BMSCs. Administration of BMSCKD significantly impedes skeletal senescence, augments bone mass in aged or ovariectomized (OVX) mice and accelerates the regeneration of calvarial defects. This intervention notably mitigates LPS-induced acute organ injuries, including those affecting the lung, kidney, and liver injury, with a particularly noteworthy reduction in the elevated mortality rate.

Results

miR-29 plays a key role in the senescence of MSCs

To identify key miRNAs related to skeletal senescence, we re-analyzed the expression profiles of skeletal miRNAs in aged mice (Sequence Read Archive [SRA] datasets: SRA074415) and OVX mice (GEO datasets: GSE109705). This analysis revealed 13 miRNAs significantly upregulated in bone tissues from both aged (22-month-old) and OVX mice (C57BL/6 background, 9-month-old, with surgery conducted at 3 months of age).46,47 Among these, the miR-29 family members—miR-29a, miR-29b, and miR-29c—were prominently upregulated (Figure 1A), suggesting a crucial role for miR-29 in skeletal senescence.Figure 1 miR-29 promotes the senescence of MSCs

(A) The upregulated miRNAs in both bone tissues of aged mice (22 months) and OVX mice.

(B) FISH staining for miR-29a expression on tibial sections.

(C) Quantification of (B).

(D) RT-qPCR analyses for miR-29a expression in bone tissues. N = 5 mice per group for (C) and (D).

(E) FISH staining for miR-29a expression on bone sections of normal control (Con) and patients with OP.

(F) Quantification of (E). N = 8 samples in control group and N = 10 samples in OP group.

(G–I) Primary BMSCs were used for SA-β-Gal staining (G and H) and RT-qPCR analyses for miR-29a expression (I). (G) Representative images of SA-β-Gal. (H) Quantification of (G).

(J–L) Primary BMSCs infected with miR-29a mimic or negative control RNA (NC) were used for SA-β-Gal staining (K and L) and RT-qPCR analyses for miR-29a overexpression (J). (K) Representative images of SA-β-Gal. (L) Quantification of (K).

(M–O) Primary BMSCs infected with miR-29a inhibitor or negative control RNA (NC) were used for SA-β-Gal staining (N and O) and RT-qPCR analyses for miR-29a expression (M). (N) Representative images of SA-β-Gal. (O) Quantification of (N). n = 5 biologically independent experiments for (G–O). For RT-qPCR analyses, U6 expression was used for internal reference. Results are presented as mean ± standard deviation (S`D). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, versus controls. Two-tailed Student’s t test.

Next, we examined miR-29 expression in bone tissues. Both fluorescence in situ hybridization (FISH) staining of tibial sections and quantitative reverse-transcription PCR (RT-qPCR) analyses revealed significant increases in miR-29a expression in the bone tissues of aged mice (18-month-old) compared to young mice (5-month-old) (Figures 1B–1D). Importantly, FISH staining of human bone sections also demonstrated a drastic increase in miR-29a expression in bone tissues from patients with OP compared to normal controls (Figures 1E and 1F). Collectively, these findings imply a potential association between miR-29 expression and skeletal senescence.

Given that MSC senescence is a key contributor to skeletal senescence, we investigated whether miR-29 regulates the senescence of BMSCs. Primary BMSCs were isolated from young (2-month-old) and aged (18-month-old) mice for senescence-associated β-galactosidase (SA-β-Gal) staining and RT-qPCR analysis. The results indicated significant upregulation of both SA-β-Gal (Figures 1G and 1H) and miR-29 expression (Figure 1I) in BMSCs from aged mice compared to young mice. Next, we examined the impact of miR-29 on BMSC senescence through loss-and-gain experiments in vitro. To streamline the experiments, we synthesized only an miR-29a mimic, as all three miR-29 family members share the same seed sequence. BMSCs isolated from 2-month-old male C57BL/6 mice and cultured to passage 4 were transfected with either an miR-29a mimic or an inhibitor and cultured for 72 h. RT-qPCR analysis confirmed high transfection efficiency of the miR-29a mimic in primary BMSCs (Figure 1J). SA-β-gal staining revealed a significant increase in SA-β-gal-positive BMSCs in the miR-29a mimic-transfected group compared to the negative control group (Figures 1K and 1L). Conversely, SA-β-Gal-positive BMSCs were notably reduced (Figures 1N and 1O) when miR-29a was knocked down (Figure 1M).

These findings collectively suggest that miR-29 dramatically accelerates the senescence of BMSCs.

Targeted overexpression of miR-29 in the limb and head Prx1-positive MSCs has no marked effect on skeletal development in mice

BMSCs are vital for skeletal homeostasis. Various markers, such as Nestin, LeptinR, and Prx1, are used to label BMSCs.48 Prx1 and LeptinR are expressed in adult MSCs,49 while Nestin expression is reduced.49 Nestin-Cre is also utilized in the nervous system.50 LeptinR marks BMSCs but not periosteal cells.49 Prx1 is highly expressed in limb and calvarial MSCs,51 identifying stem cells essential for bone maintenance and repair.52 Therefore, to explore the role of miR-29 in skeletal senescence in vivo, we crossed Prx1-Cre transgenic mice with floxed miR-29a/b1 mice (miR-29a/b1fl/fl), resulting in mice with miR-29 overexpression specifically in the limb and head MSCs (Prx1-Cre; miR-29a/b1fl/+, referred to as miR-29Prx1). Prx1-Cre-negative littermates (miR-29a/b1fl/+) served as control mice (referred to as Con) (Figure S1A). The birth rate of miR-29Prx1 mice followed Mendelian frequency patterns. FISH staining and RT-qPCR analysis revealed significantly increased miR-29a levels in limbs of miR-29Prx1 mice compared to control mice (Figure S1B–S1D). At post-natal day 0 (P0), no discernible abnormalities in the gross appearance or body weight were observed between control and miR-29Prx1 mice (Figures S1E and S1F). Alizarin red and Alcian blue double staining of whole-mount skeletons demonstrated no obvious abnormalities in the skull vault, clavicle, rib cage, sternum, forelimbs, and hindlimbs of miR-29Prx1 mice (Figures S1G and S1H). Additionally, no significant differences were found in the mineralized parts (stained red by alizarin red) of long bones (humerus, ulna, radius, femur, and tibia) (Figure S1I) or the length of the primary ossification center between the two groups (Figures S1J and S1K). Hematoxylin and eosin (H&E) staining of humeral sections from P0 mice revealed no differences in chondrocyte arrangement in the proliferation zone or cell size in the hypertrophic zone of the humeral growth plate (Figures S1L and S1M). Furthermore, the development of secondary ossification center was similar between the two genotypes, as revealed by Alcian blue staining of tibial sections from P16.5 mice (Figures S1N and S1O). Collectively, these results indicate that miR-29 overexpression in Prx1+ MSCs has no obvious effect on early skeletal development.

miR-29 overexpression in MSCs markedly accelerates skeletal senescence in mice

Next, we analyzed the bone mass of both genotypes of mice. Microcomputed tomography (μCT) analyses of the distal femur of 2-month-old mice revealed a significant reduction in both trabecular and cortical bone mass in miR-29Prx1 mice compared to control littermates (Figure 2A). Specifically, in female miR-29Prx1 mice, there was a 25.8% decrease in bone mineral density (BMD) (Figure 2B), a 71.6% decrease in bone volume/tissue volume fraction (BV/TV) (Figure 2C), a 56.8% decrease in trabecular number (Tb.N) (Figure 2E), and a 138.0% increase in trabecular separation (Tb.Sp) (Figure 2D). Male miR-29Prx−1 mice exhibited similar bone mass loss. Cortical thickness (Cort.Th) of miR-29Prx1 mice decreased by 28.3% in females and 24.7% in males (Figure 2F). Additionally, the cross-sectional area of femur bone marrow increased by 42.0% in female and 43.5% in male miR-29Prx1 mice (Figure 2G), indicating significant bone marrow volume expansion. The femur length of miR-29Prx1 mice was slightly increased compared to controls (Figure 2H). In addition, the calvarial bone mass of miR-29Prx1 mice was also slightly but significantly decreased (Figure 2I), as evidenced by reduced BMD and BV/TV (Figures 2J and 2K). The vertebral bone mass (lumber spine [L4]), where Prx1-Cre is not expressed, did not show significant changes in miR-29Prx1 mice (Figures 2L–2N). The decrease in cortical bone thickness and the increase in bone marrow volume are distinctive morphological features of aging bone.1,2 Another typical feature of bone aging is the increase of bone marrow adipose tissue.53 H&E staining of tibal sections revealed significant increases in bone marrow adipose tissue, with a 473.1% increase in adipose tissue proportion and a 305.7% increase in adipocyte number in miR-29Prx1 mice relative to control mice (Figures 2O–2Q).Figure 2 Targeted genetic overexpression of miR-29 in MSCs markedly accelerates skeletal senescence in mice

Two-month-old miR-29Prx1 mice and control mice (Con) were used for the following analyses.

(A) Three-dimensional (3D) reconstruction from μCT scans of the distal femurs with the indicated sexes.

(B–H) Quantitative analyses of BMD, BV/TV, Tb.Sp, Tb.N, and Cort.Th of distal femurs, cross-sectional area of bone marrow of midsection of femurs, and length of femurs. N = 6 mice in male miR-29Prx1 group, N = 5 mice in the remaining groups.

(I) Three-dimensional (3D) reconstruction from μCT scans of the calvarial of female mice.

(J and K) Quantitative analyses of BMD and BV/TV of calvaria. N = 5 mice per group.

(L) Three-dimensional (3D) reconstruction from μCT scans of the vertebrae (lumber spine [L4]) of female mice.

(M and N) Quantitative analyses of BMD and BV/TV of vertebrae. N = 5 mice in miR-29Prx1 group and N = 6 mice in control group.

(O) Representative images of H&E staining of tibial sections of female mice.

(P and Q) Adipocyte number and bone marrow adipose proportion were calculated.

(R) RT-qPCR analyses. RNA samples were isolated from bone tissues. The expression level of mRNA was normalized to Gapdh.

(S) IF and IHC staining.

(T–V) Quantification of p53 (T), p16ink4a (U), and p21 (V).

(W and X) WB analyses of bone tissue (W). (X) Quantification of (W). N = 5 mice per group for (O–X). Results are presented as mean ± standard deviation (SD). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, versus controls. Two-tailed Student’s t test.

Next, we evaluated the expression of p53, a marker of cellular senescence, through immunofluorescence (IF) staining of tibial sections. The results revealed a significant increase in p53 expression in miR-29Prx1 mice (Figures 2S and 2T). Although p53 is widely recognized as a marker of cellular senescence, it also plays roles in regulating cell proliferation, apoptosis, etc.54,55 To further confirm cellular senescence in the bone tissues of miR-29Prx1 mice, we examined p16ink4a, a specific biomarker of senescence,56,57 and p21, which is a specific downstream senescence marker associated with p53.58 Immunohistochemistry (IHC) staining of tibial sections (Figures 2S, 2U, and 2V) and western blot (WB) analyses (Figures 2W and 2X) of protein samples from bone tissues revealed significantly higher levels of p16ink4a and p21 in miR-29Prx1 mice. Moreover, RT-qPCR analyses demonstrated a marked upregulation of SASP-associated genes, including IL-1α, IL-6, IL-8, Mmp-3, Cxcl1, and Cxcl3, in bone tissues of miR-29Prx1 mice (Figure 2R). The composition and intensity of SASP are highly dynamic and largely dependent on the cell type. However, core components of SASP are consistently elevated across different types, including chemokines such as Cxcl1, Cxcl2, and Cxcl3.59 The SASP factors selected for our study are widely recognized and utilized as indicators of senescence.6,60,61,62,63

Collectively, the aforementioned findings indicate that miR-29 overexpression dramatically accelerates skeletal senescence, resulting in substantial bone mass loss and other aging-related skeletal changes.

miR-29 overexpression in MSCs severely impairs osteoblast formation without affecting osteoclast formation

Given that bone mass is intricately regulated by the balance between osteoblast-mediated bone formation and osteoclast-mediated bone resorption, we first investigated the impact of miR-29 overexpression in MSCs on osteoblast formation. Calcein-blue double-labeling experiments revealed a substantial compromise in the osteoblast activity in miR-29Prx1 mice (Figure S2A). This was evidenced by dramatical decreases in mineral apposition rate (MAR) by 52.3% and 72.4%, mineralizing surface per bone surface (MS/BS) by 20.4% and 41.6%, and bone-formation rate (BFR) by 61.3% and 83.9% in both femoral metaphyseal cancellous and cortical bones (Figures S2B–S2G). Von Kossa staining of undecalcified femoral sections further illustrated significant reductions in osteoid volume/tissue volume (OV/TV) (Figures S2H and S2I). IHC staining of tibial sections revealed notable decreases in Osteocalcin (Ocn) and Runx2 levels on trabecular surfaces in miR-29Prx1 mice (Figures S2J–S2L). WB analysis of protein samples derived from bone tissues confirmed significant reductions in bone formation markers, including Runx2, Osterix (Osx), and alkaline phosphatase (Alp) (Figures S2M and S2N).

Subsequently, we determined whether miR-29 overexpression in MSCs affects osteoclast formation. Tartrate-resistant acid phosphatase (TRAP) staining of tibial sections, marking osteoclasts in vivo, revealed that miR-29 overexpression had no significant influence on osteoclast formation in 2-month-old mice (Figure S2O). Osteoclast number/bone perimeter (Oc.Nb/BPm) and osteoclast surface/bone surface (Oc.S/BS) in spongiosa bones were comparable between control and miR-29Prx1 mice (Figures S2P and S2Q). Furthermore, an in vitro osteoclast differentiation assay using primary bone marrow monocytes showed similar numbers of multinucleated TRAP-positive osteoclasts in both the groups (Figures S2R and S2S).

These findings demonstrate that miR-29 overexpression in MSCs significantly impairs osteoblast-mediated bone formation but has no obvious effect on osteoclast formation. Therefore, the observed bone mass loss in miR-29Prx1 mice is primarily due to compromised osteoblast formation and activity.

miR-29 overexpression in Dmp1-positive osteocytes expedites skeletal senescence and aged-related bone mass loss

Osteocytes, which reside within the bone matrix, are terminally differentiated osteoblasts originating from MSCs and constitute 90%–95% of the total bone cells in adult skeleton.64 To investigate the potential role of miR-29 in osteocytes, we generated mice with miR-29 overexpression specifically in osteocytes (Dmp1-Cre; miR-29a/b1fl/+, denoted as miR-29Dmp1) using Dmp1-Cre transgenic mice.65 miR-29a/b1fl/+ littermates were used as control mice (referred to as Con). RT-qPCR analyses confirmed a significant increase in miR-29a in bone tissues of miR-29Dmp1 mice compared to controls (Figure S3A). μCT analysis revealed a significant reduction in bone mass in 7-month-old miR-29Dmp1 mice (Figure S3B). Specifically, trabecular BMD decreased by 46.6% and 43.6%, and BV/TV decreased by 42.1% and 47.9% in female and male miR-29Dmp1 mice, respectively (Figure S3C and S3D). The Tb.N (Figure S3E) and Cort.Th (Figures S3A and S3F) in male miR-29Dmp1 mice also showed marked decreases. No significant difference in bone mass was observed between male 5-month-old miR-29Dmp1 mice and control mice (Figures S3A–S3E), indicating that the bone mass loss phenotype in miR-29Dmp1 mice aggravates with aging. The skeletal morphology of miR-29Dmp1 mice indicated a senescence phenotype similar to miR-29Prx1 mice. IF and IHC staining of tibial sections from 7-month-old female mice revealed the notably elevated expression of senescence markers p53, p16ink4a, and p21 (Figures S3G–S3J), and reduced expression of osteogenic markers Runx2 and Ocn (Figures S3G, S3K, and S3L) in miR-29Dmp1 mice. TRAP staining showed no significant effect on osteoclast formation in miR-29Dmp1 mice (Figures S3M–S3O). Taken together, these findings suggest that miR-29 accelerates skeletal senescence at both the MSC and terminally differentiated osteocyte stages.

miR-29 overexpression attenuates osteogenic while enhancing adipogenic differentiation of MSCs via accelerated cellular senescence

To determine the possible mechanisms underlying decreased osteoblast activity caused by miR-29 overexpression, we performed colony-forming unit-fibroblast (CFU-F) assays and colony-forming unit-osteoblast (CFU-OB) assays on primary bone marrow cells from 2-month-old control and miR-29Prx1 mice.66 The results revealed a significant decrease in both CFU-Fs (Figures S4A and B) and CFU-OBs (Figures S4A and S4C) in miR-29Prx1 mice compared to controls. Next, osteogenic induction of primary BMSCs from miR-29Prx1 mice showed a drastic reduction in osteoblastic marker genes (Alp, Runx2, Osx), as demonstrated by Alp staining and WB analyses (Figures S4D–S4G). In contrast, senescence marker genes (p53, p16ink4a, p21) were significantly upregulated (Figures S4D and S4E). SA-β-Gal staining revealed an increased proportion of senescent BMSCs in miR-29Prx1 mice (Figures S4H and S4I). Additionally, BMSCs from miR-29Prx1 mice exhibited substantially elevated mRNA levels of SASP-associated genes (Figure S4J), enhanced adipogenic differentiation (shown by oil red O staining) (Figures S4K and L), and increased expression of adipogenic marker genes (Fatty acid synthase [Fas], Peroxisome proliferator activated receptor gamma [PPARγ]) (shown by WB analyses) (Figures S4M and N), compared to controls. Collectively, these findings demonstrate that miR-29 overexpression impedes osteoblast bone formation by accelerating BMSCs senescence, inhibiting osteogenic differentiation, and promoting adipogenic differentiation, alongside increased expression of senescence indicators such as p53, p16ink4a, p21, SA-β-Gal, and SASP.67,68

miR-29 promotes MSCs senescence via modulating the Kindlin-2/p53 axis

To elucidate the mechanisms through which miR-29 modulates MSCs differentiation and senescence, RNA sequencing and Gene Ontology (GO) pathway analysis were performed using primary BMSCs from 2-month-old miR-29Prx1 and control mice. Results revealed significant alterations in cell adhesion pathways due to miR-29 overexpression (Figure 3A). Focal adhesion proteins, known for regulating cell adhesion, growth, survival, migration, and senescence,69,70 were notably affected. Utilizing miRNA databases (miRDB database and TargetScan database), we predicted the top 350 target molecules of miR-29a. The intersection of the GO term “focal adhesion” and the set of target molecules predicted by miR-29 from miRDB and TargetScan highlighted Kindlin-2 as the sole gene significantly regulated by miR-29 (Figure 3B). Kindlin-2, a critical focal adhesion protein, plays crucial roles in cellular senescence and bone homeostasis.71,72,73 Our prior studies demonstrated that Kindlin-2 deficiency promotes inflammatory responses and bone disorders71,74,75 and impairs osteoblastic differentiation of BMSCs, leading to drastic bone loss.73 Additionally, Sossey-Alaoui et al. reported in a recent study that the loss of Kindlin-2 induces cellular senescence by promoting p53 and downstream signaling pathways.72 Thus, findings from our group and others collectively suggest that Kindlin-2 serves as a potential target for regulating MSCs and skeletal senescence through the regulation of p53 expression.Figure 3 miR-29 promotes MSCs senescence via Kindlin-2/p53 axis

(A) GO analysis of changed pathways identified through RNA-seq of the primary BMSCs from 2-month-old female miR-29Prx1 mice and control mice.

(B) Venn map analyzing the factors of GO: Focal adhesion overlap with predicted miR-29 target genes.

(C) IHC staining of Kindlin-2 on tibial sections from 2-month-old mice.

(D) Quantification of (C).

(E) WB analyses using protein extracts isolated from bone tissues of 2-month-old female mice.

(F) Quantification of (E). N = 5 mice per group for (C–F).

(G) WB analyses using protein extracts of primary BMSCs derived from 2-month-old female mice.

(H) Quantification of (G). n = 5 biologically independent experiments.

(I) RT-qPCR analysis of RNA samples isolated from bone tissues of 2-month-old female mice. Kindlin-2 mRNA was normalized to Gapdh mRNA. N = 5 mice per group.

(J and K) Double luciferase assay. 3T3-E1 cells were transfected with plasmids harboring the wild-type KINDLIN-2 3′-UTR (WT) or the 3′-UTR with a mutation (Mut) in the miR-29 binding site downstream of the luciferase coding region together with miR-29 mimic or non-specific control (NC). Lysates were analyzed for luciferase activity.

(L–N) 3T3-E1 cells transfected with miR-29a mimic and negative control RNA (NC) were used for RT-qPCR analyses (L) and WB analyses (M and N).

(O–Q) 3T3-E1 cells transfected with miR-29 sponge and negative control plasmid (NC) were used for RT-qPCR analyses (O) and WB analyses (P and Q).

(R–V) 3T3-E1 cells were initially transfected with a miR-29a mimic, followed by transfection with a Kindlin-2-Flag plasmid (K2-Flag) 24 h later. After 72 h, cells were subjected to RT-qPCR analyses (V) and WB analyses (R and S) and SA-β-Gal staining (T and U). n = 5 biologically independent experiments for (J–V). For RT-qPCR analyses, miR-29a was normalized to U6. Results are presented as mean ± standard deviation (SD). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, versus controls. Two-tailed Student’s t test.

To investigate whether miR-29 modulates MSCs senescence via Kindlin-2, we assessed Kindlin-2 expression in bone tissues. IHC staining and WB analyses demonstrated significant reductions in Kindlin-2 protein levels in bone tissues and primary BMSCs of miR-29Prx1 mice compared to controls (Figures 3C–3H). Similarly, Kindlin-2 protein levels were markedly decreased in bone tissues of miR-29Dmp1 mice (Figures S3P and S3Q). RT-qPCR analysis using RNA samples extracted from bone tissues confirmed the downregulation of Kindlin-2 mRNA expression in miR-29Prx1 mice (Figure 3I). A double fluorescein reporter assay using the wild-type (WT) 3′-UTR region of KINDLIN-2 (WT) and the mutant region (Mut) demonstrated that miR-29a directly accelerates the degradation of Kindlin-2 mRNA (Figures 3J and 3K). Additionally, WB and RT-qPCR analyses demonstrated that miR-29a mimic transfection led to significant downregulation of Kindlin-2 and upregulation of p53 (Figures 3L–3N), while miR-29a knockdown using miRNA sponge technology (miR-29 sponge) increased Kindlin-2 expression and decreased p53 expression (Figures 3O–3Q). To further confirm that miR-29 overexpression promotes cellular senescence via inhibiting Kindlin-2, we overexpressed Kindlin-2 in 3T3-E1 cells 24 h post-transfection with miR-29a mimic and assessed cellular senescence. RT-qPCR analyses confirmed the overexpression of miR-29 (Figure 3V). Results showed that Kindlin-2 overexpression significantly reversed the increased expression of senescence markers (p53, p16ink4a, and p21) as revealed by WB analyses (Figures 3R and 3S) and mitigated the increased cellular senescence induced by miR-29 overexpression as indicated by SA-β-Gal staining (Figures 3T and 3U). Collectively, the aforementioned findings establish Kindlin-2 as a direct target of miR-29, with miR-29 promoting MSCs and skeletal senescence through the Kindlin-2/p53 axis.

Further investigations in various OP models, including aged mice (18-month-old), obese mice induced by a high-fat diet, and patients with OP, showed significant decreases in Kindlin-2 levels and increases in p53 levels in bone tissues (Figures S5A–S5G). Collectively, these findings suggest that the miR-29-regulated Kindlin-2/p53 axis plays a key role in the OP pathogenesis.

BMSCs with miR-29 knockdown (BMSCKD) accelerate calvarial bone regeneration more effectively than WT BMSCs (BMSCNC)

The sub-culturing process for expanding primary BMSCs in vitro often leads to proliferative senescence, posing challenges for MSC-based applications.76,77,78 Our in vitro experiments confirmed that BMSCs are highly susceptible to cellular senescence, especially during prolonged subculture (Figures 4B and 4C). To assess whether miR-29 knockdown could rejuvenate MSCs, we used lentiviruses with miR-29 sponge (Lv-KD) to knock down miR-29. RT-qPCR analysis confirmed efficient knockdown (Figure 4A), and SA-β-gal staining revealed that miR-29 knockdown markedly delayed cellular senescence of BMSCs (BMSCKD) during continuous subculture (Figures 4B and 4C). CCK-8 assay results demonstrated greater proliferative capacity in BMSCKD, especially after continuous subculture (Figure 4D). While the mRNA expression of hallmark SASP genes was elevated by approximately 1.3- to 8.7-fold in primary BMSCs derived from miR-29Prx1 mice compared to controls (Figure S4J), miR-29 knockdown significantly inhibited the mRNA expression of these genes (Figure 4E) and reduced the expression levels of p53, p16ink4a, and p21, while increasing Kindlin-2 expression (Figures 4F and 4G). Furthermore, osteoblastic marker genes, including Osx, Alp, and Runx2, were markedly increased in BMSCKD upon induction for osteoblastic differentiation (Figures 4F and 4G).Figure 4 Treatment of BMSCKD accelerates calvarial bone regeneration

BMSCs infected with lentivirus expressing miR-29 sponge plasmid (BMSCKD) or negative control (BMSCNC) were used for the following experiments.

(A) RT-qPCR analyses for miR-29a expression.

(B) Representative images of SA-β-Gal staining.

(C) Quantification of (B).

(D) CCK-8 assay for cell proliferation at P7.

(E) RT-qPCR analyses using indicated primers. mRNAs were normalized to Gapdh mRNA.

(F and G) WB analyses. n = 5 biologically independent experiments for (A–G).

(H) Three-dimensional (3D) reconstruction from μCT scans of the calvariae of mice. A critical-sized calvarial bone defect model (3-mm diameter) was created in 4-month-old C57BL/6 mice; then BMSCNC and BMSCKD were applied to the wound of the calvarial bone defects. After 3 weeks, the mice were sacrificed for μCT analysis.

(I–K) Quantitative analyses of healing area proportion, BMD, and BV/TV.

(L) Representative images of H&E staining of calvarial sections at injured area. The defect areas were marked with black lines.

(M) Quantitative analyses of length of injured area.

(N) IF and IHC staining of calvarial sections.

(O–Q) Quantification of p53 (O), Kindlin-2 (P), and Runx2 (Q).

(R) TRAP staining of calvarial sections.

(S and T) Oc.S/BS (S) and Oc.N/BPm (T) were measured using ImageJ. N = 5 mice per group for (H–T). Results are presented as mean ± standard deviation (SD). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, versus controls. Two-tailed Student’s t test.

To assess the potential application value of BMSCKD compared to BMSCNC, we initially established a mouse model of calvarial defects. μCT analysis revealed that BMSCKD treatment significantly accelerated the healing of calvarial bone defect compared to the BMSCNC treatment (Figure 4H), with 25.6% of the bone defect filled in the BMSCKD-treated group versus 11.1% in the BMSCNC-treated group (Figure 4I). BMD and BV/TV of the injured area were significantly increased with BMSCKD treatment (Figures 4J and 4K). H&E staining of calvarial bone sections revealed more regenerated new bone in the BMSCKD-treated group (Figures 4L and 4M), and IF staining indicated a marked decrease in p53 expression in the BMSCKD-treated group (Figures 4N and 4O). IHC staining revealed significant upregulations of Kindlin-2 and Runx2 in the BMSCKD-treated group (Figures 4N, 4P, and 4Q). There was no significant difference in osteoclast activity between the two groups (Figures 4R–4T).

Collectively, BMSCKD exhibit enhanced osteoblastic differentiation capacity and bone-forming activity compared to BMSCNC, both in vitro and in vivo, suggesting a higher application value for BMSCKD in promoting bone regeneration after injury or fracture.

BMSCKD treatment mitigates age-related phenotypes in mice more efficiently than BMSCNC

Our aforementioned findings underscore the pivotal role of miR-29 in MSCs senescence, positioning miR-29 as a potential target for addressing skeletal senescence. To further explore the anti-aging function of BMSCKD in an aging microenvironment, we evaluated aged mice (24-month-old). IF and IHC staining of tibial sections validated the notably elevated expression of senescence markers p53, p16ink4a, and p21 in aged mice compared to young mice (5-month-old) (Figures S6A–S6D). BMSCNC and BMSCKD were administrated to 24-month-old female C57BL/6 mice. μCT analysis revealed a significant increase in trabecular bone mass in the BMSCKD-treated group compared to the BMSCNC-treated group (Figures 5A–5D). BMSCKD treatment increased BV/TV and Tb.Th by 112.1% and 33.9%, respectively, and reduced Tb.Sp by 32.8% (Figures 5B–5D), although BMSCKD treatment did not significantly increase the cortical bone thickness (Figures 5A and 5E), likely due to insufficient cell dosage or treatment duration. The BMSCKD-treated group exhibited decreased expression of p53, p16ink4a, and p21 (Figures 5F–5I), and increased expression of Kindlin-2 and the osteogenic markers, including Runx2 and Ocn (Figures 5F, and 5J–5L), indicating enhanced osteoblast differentiation and bone formation. TRAP staining showed similar osteoclast formation in the two groups (Figure 5M–5O), while H&E staining revealed reduced bone marrow adipose tissue in the BMSCKD-treated group (Figures 5P and 5Q). Collectively, these results suggest that BMSCKD treatment increases bone mass by delaying skeletal senescence.Figure 5 Treatment of BMSCKD mitigates age-related phenotypes in mice

(A) Three-dimensional (3D) reconstruction from μCT scans of the distal femurs. Female 24-month-old mice were administrated with BMSCKD or BMSCNC via tail vein injection, and mice were sacrificed 40 days post-injection for μCT analysis of the distal femurs for bone mass analysis.

(B–E) Quantitative analyses of BV/TV, Tb.N, Tb.Sp, and Cort.Th.

(F–Q) Tibial sections were subjected to IF staining for p53 expression (F and G), IHC staining for p16ink4a (F and H), p21 (F and I), Kindlin-2 (F and J), Runx2 (F and K), and Ocn (F and L), TRAP staining for osteoclast formation (M–O), and H&E staining (P and Q). Oc.S/BS (N) and Oc.N/BPm (O) were measured using ImageJ.

(R) H&E staining and Masson staining of lung sections.

(S) Quantifications of lung pathological score.

(T) Quantifications of lung collagen proportion.

(U) Representative images of H&E staining of liver, kidney, and pancreas sections.

(V and W) ELISA assay of serum levels of IL-1β (V) and TNF-α (W). N = 5 mice per group. Results are presented as mean ± standard deviation (SD). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, versus controls. Two-tailed Student’s t test.

Histomorphological analyses by H&E staining and Masson trichrome staining of the lung revealed a marked reduction in fibrosis and a more intact pulmonary alveolar structure in the BMSCKD-treated group (Figures 5R–5T), with lower pathological score79 and collagen proportion (Figures 5S and 5T), indicating improved lung structure and function. No significant histological differences were observed in the liver, kidney, and pancreas between the two groups (Figure 5U). BMSCs initially accumulate in the lungs post-injection,80,81 followed by subsequent accumulation in the liver and other organs,80 which may enhance therapeutic efficacy for lung conditions. Chronic inflammation is a hallmark of aging, and, therefore, we detected the serum levels of representative inflammatory factors tumor necrosis factor alpha (TNF-α) and interleukin (IL)-1β. BMSCKD administration significantly reduced levels of these inflammatory factors compared to BMSCNC treatment (Figures 5V and 5W), suggesting that BMSCKD transplantation alleviates chronic inflammation in aged mice. The anti-inflammatory function of BMSCKD may contribute to mitigate age-related phenotypes in aged mice. Collectively, miR-29 knockdown in BMSCs offers a superior approach to mitigating skeletal senescence, age-related OP, and lung damage associated with aging. These findings highlight miR-29 as a therapeutic target for senescence and propose BMSCKD administration as a potential strategy for treating age-related diseases.

BMSCKD treatment protects against OP induced by OVX more efficiently than BMSCNC

Estrogen deficiency in postmenopausal women is a significant factor promoting skeletal senescence and OP progression,82 with approximately 1 in 3 women aged over 50 experiencing an osteoporotic fracture.83 While the OVX mouse model is commonly utilized to simulate OP induced by estrogen deficiency,84,85 studies indicate that estrogen deficiency can also accelerate cellular senescence, characterized by increased SASP, elevated levels of reactive oxygen species, and mitochondria dysfunction.84,85 Therefore, to assess whether BMSCKD can better mitigate skeletal senescence and OP under estrogen-deficient conditions, we utilized an OVX mouse model. μCT analysis revealed a significant decrease in bone mass in the OVX group compared to the sham group. While BMSCNC treatment markedly increased bone mass in OVX mice, BMSCKD treatment further enhanced this bone-forming capacity (Figures 6A–6C). IF and IHC staining of tibial sections demonstrated enhanced p53, p16ink4a, and p21 expression in OVX group, which decreased following BMSCNC treatment and further decreased with BMSCKD treatment (Figures 6D–6G). The expression levels of Kindlin-2, Runx2, and Ocn mirrored the bone mass trends across the groups (Figures 6D, and 6H–6J). By the way, the downregulated Kindlin-2 level and upregulated p53 level were also observed in the bone tissue of OVX mice as an OP mouse model (Figures 6D-6G), similar with the results in other OP model. BMSC treatment significantly inhibited osteoclast overactivation caused by OVX, with no significant differences between BMSCNC- and BMSCKD-treated groups (Figures 6K–6M). Collectively, these findings suggest that primary BMSCKD exhibit superior efficacy than BMSCNC in combating skeletal senescence and OP caused by estrogen deficiency, positioning BMSCKD administration as a potential strategy for the treatment of OP in postmenopausal women.Figure 6 Treatment of BMSCKD protects against OP induced by OVX

(A) Three-dimensional (3D) reconstruction from μCT scans of the distal femurs. Female 2-month-old mice were subjected to OVX surgery or sham, and one-week post-OVX, BMSCNC and BMSCKD were administrated to the OVX mice via tail vein injection. Mice were sacrificed 2-month post cell injection for μCT analysis of the distal femurs for bone mass analysis.

(B and C) Quantitative analyses of BMD and BV/TV.

(D–J) Tibial sections were subjected to IF and IHC staining with the indicated antibodies.

(K–M) TRAP staining of tibial sections. Oc.S/BS (L) and Oc.N/BPm (M) were measured using ImageJ. N = 5 mice per group. Results are presented as mean ± standard deviation (SD). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, versus controls. One-way ANOVA.

BMSCKD treatment protects against organ damage caused by LPS more efficiently than BMSCNC

MSC transplantation has been shown to safeguard against organ injury from acute inflammation. To ascertain whether BMSCKD possess a more potent protective ability, we utilized an LPS-induced acute inflammation mouse model. All untreated mice died within 36 h post-LPS injection. BMSCNC treatment significantly increased the survival rate to 30% at 60 h, while BMSCKD treatment further elevated it to 80% (Figure 7A). μCT analysis indicated no significant differences in bone mass among the groups (Figures 7B–7E). However, IHC staining unveiled higher levels of Runx2 and Kindlin-2 in the BMSCNC-treated groups, with the BMSCKD-treated group showing the greatest increase (Figures 7F–7H). TRAP staining demonstrated that BMSCs treatment mitigated LPS-induced osteoclast formation equally well in both BMSCNC- and BMSCKD-treated groups (Figures 7I–7K).Figure 7 Treatment of BMSCKD significantly alleviates LPS-induced organ damage and mortality rate

3-month-old female mice were injected with LPS intraperitoneally (25 mg/kg) followed by BMSCNC or BMSCKD or normal saline administration via tail vein injection and were then used for the following experiments.

(A) Survival curve. N = 10 mice per group. Log rank test was used.

(B) Three-dimensional (3D) reconstruction from μCT scans of the distal femurs.

(C–E) Quantitative analyses of BMD, BV/TV, and Cort.Th.

(F–K) Tibial sections were subjected to IHC staining and TRAP staining. Oc.S/BS (J) and Oc.N/BPm (K) were measured using ImageJ.

(L) Representative images of H&E staining of lung sections, kidney sections, spleen sections, and liver sections.

(M) Quantification of pathological score of lung tissue.

(N) Quantification of kidney tubular damage proportion. N = 5 mice per group. Results are presented as mean ± standard deviation (SD). ∗p < 0.05, ∗∗p < 0.01, ∗∗∗p < 0.001, ∗∗∗∗p < 0.0001, versus controls. One-way ANOVA.

LPS, commonly referred to as endotoxin, causes acute inflammatory organ injury, affecting the lung, kidney, spleen, liver, and skeleton. H&E staining revealed that BMSCNC treatment significantly alleviated lung injury, reducing alveolar wall thickening, alveolar congestion, and interstitial inflammatory cell infiltration, with BMSCKD treatment showing even greater improvement (Figures 7L and 7M). Similarly, BMSCKD treatment more effectively reduced renal injury markers, including tubular dilation, vacuolar degeneration edema, and interstitial damage with inflammatory cell infiltration (Figures 7L and 7N). The spleen injury caused by LPS, evidenced by the indistinct red-white marrow demarcation in spleen tissue, an enlarged gap between cells, and loosened interstitium, was further alleviated by BMSCKD treatment compared to BMSCNC treatment (Figure 7L). Interestingly, in the liver, BMSCNC treatment provided limited relief from severe hepatocyte swelling, but BMSCKD treatment significantly improved liver condition (Figure 7L). IHC staining for TNF-α and IL-1β in organ tissues showed that BMSCNC treatment reduced these inflammatory markers compared to saline treatment, with BMSCKD treatment achieving further reductions (Figures S7A–S7C). Specifically, BMSCKD treatment reduced TNF-α and IL-1β expression by 67.6% and 73.1% in the lung and by 67.3% and 71.5% in the liver, respectively, compared to BMSCNC treatment. These findings may explain the superior protective effect of BMSCKD treatment in the liver and lung.

Collectively, these findings demonstrate that BMSCKD have a markedly stronger protective effect than BMSCNC in reducing mortality and organ damage caused by LPS-induced acute systemic inflammation, suggesting BMSCKD administration as a potential strategy to counteract organ damage and improve survival rates in acute inflammation.

Discussion

This study identifies miRNA-29 as a key regulator of MSCs and skeletal senescence via p53 induction by suppressing Kindlin-2. Targeting miRNA-29 offers a strategy to rejuvenate MSCs and delay skeletal senescence. Currently, approximately 200 million individuals worldwide suffer from OP.86 Given the limitations of current OP treatments, BMSCKD display substantially superior efficacy over BMSCNC in mitigating skeletal senescence, enhancing bone injury regeneration, increasing bone mass in aged or estrogen-deficient mice, safeguarding against acute organ damage, and improving survival rates from systemic inflammation, highlighting the clinical potential of BMSCKD.

Cumulative evidence highlights p53’s pivotal role in cellular senescence and DNA damage response elicited by cellular stressors. Activation of p53 initiates pathways like p53/p21 and/or p16ink4a/Rb to regulate cellular senescence.87,88,89,90,91,92 Studies indicate increased miR-29 expression in aging tissues (brain, liver, heart, muscle, etc.), suggesting its role in tissue senescence through multiple signaling pathways.93,94,95,96,97 Our findings reveal significantly upregulated miR-29 in aged bone tissues in mice and in the bone tissues of patients with OP. The enforced expression of miR-29 significantly enhances cellular senescence with elevated p53 and SA-β-Gal, inhibiting BMSC osteogenesis but promoting adipogenesis. Inhibiting miR-29 rejuvenates BMSCs, reducing p53 and SA-β-Gal, enhancing osteogenesis, and inhibiting adipogenesis. Genetic overexpression of miR-29a in Prx1-positive MSCs or Dmp1-positive osteocytes increases p53 expression, accelerating skeletal senescence, reducing trabecular/cortical bone mass, enlarging bone marrow cavity, and increasing bone marrow adiposity, compromising osteoblast bone-forming activity.

Studies from our group and others consistently emphasize the pivotal role of Kindlin-2 in regulating skeletal senescence and modulating the p53 signaling pathway.72,73,98,99 This study identifies Kindlin-2 as a direct target of miR-29, mediating p53 regulation. Firstly, altered miR-29 expression correlates positively with p53 levels but negatively with Kindlin-2 levels. Secondly, the genetic miR-29 overexpression in Prx1-positive MSCs or Dmp1-positive osteocytes reduces Kindlin-2 and increases p53 expression. Finally, a double fluorescein report assay confirms miR-29’s direct targeting of Kindlin-2 mRNA through its WT 3′-UTR region.

Skeletal senescence significantly contributes to age-related OP. Estrogen deficiency in postmenopausal women is another major factor fostering skeletal senescence and OP progression.84,100 Direct transplantation of BMSCs has demonstrated efficacy in age-related OP and OVX mouse models, protecting the skeletal system.101,102,103,104,105,106 Genetic modification of BMSCs enhances osteogenic or angiogenic capabilities pre-transplantation, involving genes like Pum2, Fabp3, and Bone morphogenic protein (BMP) family members.107,108,109,110,111 However, cellular senescence from aged donors or prolonged in vitro culture remains a challenge.112,113 Transplanted MSCs are also susceptible to influences leading to functional decline in an aging microenvironment in vivo.114 MiRNAs offer promising therapeutic targets for various diseases,115 with undergoing clinical evaluation.116,117,118 Specifically, this study highlights the successful miR-29 knockdown in BMSCs rejuvenating osteogenic capacity and reducing senescence markers in aged mice or OVX mice, suggesting potential for enhancing MSC therapy efficacy.

People with OP face a high fracture risk, with approximately 1 in 3 women over the age of 50 experiencing osteoporotic fractures.83 Mortality rates are notably elevated, especially in femoral neck fractures, reaching up to 30%, with hip fractures associated with a 15%–20% increased mortality rate within a year.119 This study employs a calvarial defect model to assess the osteogenic potential and bone regeneration capacity of BMSCKD. Results show that BMSCKD exhibit significantly enhanced osteogenic potentials and bone regeneration capacities compared to BMSCNC. Composite scaffolds combining MSCs and biomaterial scaffolds have shown efficacy in calvarial defect repair.120,121,122 BMSCKD show promise for bone defect repair in this study, potentially enhancing outcomes when integrated with biomaterial scaffolds, warranting further investigation of this synergy.

Acute lung injury, often progressing to acute respiratory distress syndrome, carries mortality rates of 35%–55% when triggered by sepsis or other factors.123,124 Acute kidney injury, linked to sepsis, exhibit hospital mortality rates up to 60.3%,125,126 while acute liver injury poses a significant health concern with hospital mortality rates of admitted patients up to 10%.127,128 MSCs are recognized for their potent immunomodulatory capabilities,129 offering therapeutic potential across various inflammatory conditions such as kidney injury, lung injury, osteoarthritis, rheumatoid arthritis, and inflammatory bowel disease.130,131,132 MSCs regulate immune responses by influencing diverse cell types including macrophages, neutrophils, mast cells, dendritic cells, natural killer cells, T cells, and B cells through secretion of soluble factors, crucial for balancing inflammatory processes.133 Senescent MSCs exhibit an enhanced SASP.134 Persistent chronic inflammation stands as a pivotal factor in the aging process. In this study, we illustrate that BMSCKD administration substantially alleviates lung injury and inflammatory cell infiltration in aged mice, surpassing BMSCNC effects. In LPS-induced acute inflammation, BMSCKD enhance osteogenic activity compared to BMSCNC or saline, indicating potential for bone mass protection under extended inflammatory conditions. Moreover, BMSCKD treatment markedly reduces inflammatory factors IL-1β and TNF-α and enhances survival in LPS-induced models compared to BMSCNC (Figures S7A–S7C), aligning with MSCs’ ability to modulate cytokine production and nuclear factor κB/NLRP3 signaling pathways to mitigate inflammation.135,136,137 These findings underscore a compelling strategy to augment the immune-regulatory capabilities of MSCs in combating organ injury and inflammation.

In conclusion, we establish the central role of miR-29 in accelerating MSCs and skeletal senescence through modulating the Kindlin-2/p53 axis. Targeting miR-29 emerges as a promising strategy to rejuvenate MSCs. Administration of MSCs with miR-29 knockdown presents a potential therapeutic avenue for age-related disorders, including OP, acute inflammation, etc.

STAR★Methods

Key resources table

REAGENT or RESOURCE	SOURCE	IDENTIFIER	
Antibodies	
	
Kindlin-2	Proteintech	Cat#11453-1-AP; RRID:AB_2262660	
GAPDH	ZSGB-BIO	Cat#TA-08; RRID:AB_2747414	
Tubulin	ZSGB-BIO	Cat#TA-10; RRID:AB_3095964	
Runx2	HUABIO	Cat#ET1612-47; RRID:AB_2924311	
Osterix (SP7)	HUABIO	Cat#ER1914-47	
Alkaline phosphatase (Alp)	Abclonal	Cat#A0514; RRID:AB_2861462	
Osteocalcin (Ocn)	Bioss Antibodies	Cat#bs-4917R; RRID:AB_2916189	
p53	ABClonal	Cat#A19585; RRID:AB_2862683	
Fas	ABClonal	Cat#A19050; RRID:AB_2862543	
PPARγ	ABClonal	Cat#A0270; RRID:AB_2757083	
p16ink4a	ABClonal	Cat#A0262; RRID:AB_2757075	
p21	ABClonal	Cat#A19094; RRID:AB_2862586	
	
Chemicals, peptides, and recombinant proteins	
	
Ascorbic acid	Sigma	Cat#A4403	
Calcein	Sigma	Cat#C0875	
Adipogenic medium	Procell	Cat#PD-004	
Lipofectamine RNAiMAX	Invitrogen	Cat#13778150	
MesenCult Proliferation Medium	STEMCELL	Cat#05513	
Giemsa	Sigma	Cat#GS500	
LPS	Sigma	Cat#L4524	
SYBR	Beyotime	Cat#D7260	
	
Critical commercial assays	
	
Osteo-Bed Bone Embedding kit	Sigma	Cat#EM0200	
MicroRNA FISH kit	GenePharma	N/A	
Total RNA isolating kit	Transgen	Cat#ER601-01	
First-standard cDNA synthesis kit	GeneCopeia	Cat#QP114	
miRNA RT-qPCR detection kit	GeneCopeia	Cat#QP011	
EnVision+System-HRP (DAB) kit	Dako	Cat#K401111-2	
	
Deposited data	
	
RNA-seq data	This paper	Mendeley Data: https://doi.org/10.17632/zn2hxj6yz3.1	
Western blot original images	This paper	Mendeley Data: https://doi.org/10.17632/zn2hxj6yz3.1	
miRNA expression profile of young and aged bone of mice	He X. et al.47	SRA datasets: SRA074415	
MicroRNA expression in sham and ovariectomy (OVX) mice femur tissue	Zhao J. et al.48	DEO datasets: GSE109705	
	
Experimental models: Cell lines	
	
3T3-E1 cell line	Servicebio	Cat#STCC20026P	
	
Experimental models: Organisms/strains	
	
Mouse: C57BL/6	Laboratory animal center of southern university of science and technology	N/A	
Mouse: miR-29flox/+	This study	N/A	
	
Oligonucleotides	
	
RT-qPCR Primer	Sangon Biotech	See Table S1	
miRNA mimic and inhibitor	GenePharm	See Table S2	
	
Software and algorithms	
	
ImageJ	National Institutes of Health	Version 8.0	
GraphPad Prism 9	GraphPad software	Version 9.5.0	

Resource availability

Lead contact

Further information and requests for resources should be directed to the lead contact, Dr. Huiling Cao (caohl@sustech.edu.cn).

Materials availability

This study did not generate new unique reagents.

Data and code availability

• RNA-seq data have been deposited at the Mendeley Data and are publicly available as of the date of publication. DOI is listed in the key resources table. Accession numbers are listed in the key resources table. This paper analyzes existing, publicly available data. These accession numbers for the datasets are listed in the key resources table. Original WB images have been deposited at Mendeley and are publicly available as of the date of publication. The DOI is listed in the key resources table.

• This study does not report original code

• Any additional information required to reanalyze the data reported in this paper is available from the lead contact upon request.

Experimental model and study participant details

Animal study

The miR-29a/b1 gene loci were cloned, and a long stop element with LoxP was added in front of the gene loci. Subsequently, it was inserted at the Rosa2.6 loci of mice. To achieve miR-29 overexpress in MSCs, miR-29a/b1fl/fl mice were bred with Prx1-cre mice, resulting in the generation of Prx1-Cre; miR-29a/b1fl/+ mice (referred to as miR-29Prx1). The miR-29a/b1fl/+ mice were used as control (referred to as Con). For miR-29 overexpression in osteocytes, Dmp1-cre mice were bred with miR-29a/b1fl/fl mice, generating Dmp1-Cre; miR-29a/b1fl/+ mice (referred to as miR-29Dmp1). All animals used in this study were maintained on a C57/BL6 background through successive crosses with normal C57BL/6 mice for more than 10 generations. Mice were maintained in a controlled environment with a humidity level of 22 ± 2°C and a 12-h dark/light cycle, provided with ample food and water. All protocols for animal studies were approved by the Institutional Animal Care and Use Committee of Southern University of Science and Technology.

For critical sized calvarial bone defect model,138 anesthetized 4-month-old male C57BL/6 mice underwent a midline sagittal incision to expose the calvarium by separating the scalp skin, subcutaneous tissue, and periosteum tissue. A cranial drill was used to create a 3 mm diameter bone defect in the middle of parietal bone along the median sagittal section, with the defect edge positioned 1 mm away from the sagittal sutures. BMSCs at Passage 4 (1 × 105 cells, harvested at 80% confluence) were suspended in 1% alginic acid in saline solution and applied to the the calvarial bone defects.139 Subsequently, 10 μl of 150 mM CaCl2 solution was added to the alginic acid at the defect site to facilitate hydrogel cross-linking. The wound was sutured after 30 s. Mice were scarified 3 weeks post-surgery, and the entire calvarium was fixed in 4% paraformaldehyde for subsequent histological analysis. Micro-CT scanning parameters were set at 60 kV, 100 μA, and 926 ms, with a resolution of 10 μm. The defect area was measured using micro-CT reconstructed images analyzed with ImageJ software. For histology analysis, the calvaria was sectioned sagittally at the center of the defect site and embedded in paraffin. The first three rounded sections were used for histological examination, and defect diameter measurements were performed using ImageJ software.

For aged mice, BMSCs (1 × 106 cells/mouse) were administered to 24-month-old female mice through tail vein injection. After 40 days, the mice were euthanized, and the tissue were collected and subjected to subsequent assays.

For OVX mice model, the OVX surgery were conduct at the age of 2 months. One week after surgery, BMSCs (1 × 106 cells/mouse) were administered to mice through tail vein injection. The mice were euthanized, after 2 weeks and the tissue were collected and subjected to subsequent assays.

For LPS induced acute inflammation mouse model, BMSCNC or BMSCKD or saline was administrated to 10-week-old female mice through tail vein injection immediately after LPS administration. Mice were randomly divided into 3 groups and were injected intraperitoneally with LPS (25 mg/kg, Sigma, L4524). Immediately after that, BMSCs (1 × 106 cells/mouse) were injected intravenously. Mice were administrated every 12 h.

Human samples

Human bone tissue samples were obtained from the hospital. All the protocols were approved by the ethics committee of the hospital (IRB No: 2021 -192). The information of healthy control and people with OP were listed in Table. S3. Bone mineral density of all participates was measured and the participates whose T scores ≤ −2.5 were assigned to the OP group, and whose T scores ≥ −1 were assigned to the control group.140

Cell culture

For primary BMSCs,141 primary BMSCs were isolated and maintained in culture medium (α-MEM supplemented with 10% FBS (fetal bovine serum, Gibco, A5669401), 100 U/mL penicillin and 100 μg/mL streptomycin) at 37°C in a 5% CO2 incubator. For osteogenic differentiation, 2 × 105 BMSCs/well were seeded in a 6-well plate and cultured in osteogenic medium (α-MEM containing 10% FBS and 50 μg/mL ascorbic acid) for 7 days. Adipogenic differentiation was induced by seeding 2 × 105 BMSCs/well in a 6-well plate and culturing them in adipogenic medium (PD-004, Procell)142 for 8 days, followed by Oil Red O staining. MSCs from passages 3 to 7 were used in this study.

3T3-E1 cells were cultured in α-MEM medium with 10% FBS, 100 U/mL penicillin and 100 μg/mL streptomycin at 37°C in a 5% CO2 incubator. Transfection of miR-29a mimic and miR-29a inhibitor was performed as the construction using Lipofectamine RNAiMAX (Invitrogen, 13778100) regent. The information of miR-29a mimic and miR-29a inhibitor were listed in Table S2.

Method details

Micro-computerized tomography (μCT) analysis

Femurs obtained from mice were fixed in 4% paraformaldehyde solution and analyzed for bone mass using μCT (SkyScan 1172 Micro-CT, Bruker MicroCT).66 The guidelines for techniques and terminology adhered to were set by the American Society for Bone and Mineral Research. The scanning parameters were configured at 60 kV, 100 μA, and 926 ms with a resolution of 10 μm. A region of interest (ROI) measuring 1.5 mm region in length and positioned 1/30 of the total femur length proximal to the distal growth plate was selected for trabecular measurement analysis. For cortical bone analysis, the ROI encompassed the midsection of the femur, measuring 1 mm in length. The key parameters were calculated, including BMD, BV/TV, Tb.N, Tb.Th, Tb.Sp and Cort.Th.

Calcein double labeling experiment and MAR, MS/BS, and BFR analysis

For, calcein double-labelling experiment,66 intraperitoneal injection of calcein solution (20 mg/kg, Sigma, C0875) was performed on mice at the 1st and 5th days, with sacrifice taking place on the 7th day. Femurs obtained from mice were fixed in 70% ethanol and embedded using Osteo-Bed Bone Embedding kit (Sigma, EM0200). Sections of 5-μm thickness were photographed using a fluorescence microscope (Olympus-BX53) under excitation light at a wavelength of 488 nm. MAR, MS/BS, and BFR was calculated.

Lentivirus package

Lentivirus was produced using HEK293T cell line.108 15 μg lentiviral vectors (pLv-NC-GFP or pLv-miR-29 scope-GFP) with Paspx2 vector and PMD2.G vector were transfected in HEK293T cells in a 10 cm dish. Medium after culturing cells for 48 h and 72 h was subjected to centrifugation at 80000 × g for 2 h to purify lentivirus. The resulting lentiviruses were resuspended in 100 μL PBS (Solarbio life science, P1022), and the lentiviral titer was estimated to be approximately 2 × 109 PFU/mL.

CFU-F and CFU-OB assay

Bone marrow nucleated cells isolated from 2-month-old male mice were seeded in 6-well plate at a density of 2 × 106 cells/well. For CFU-F assay, the cells were cultured in the Mouse MesenCult Proliferation Medium (STEMCELL Technologies, 05513) for 14 days, then Giemsa staining (Sigma, GS500) was performed according to the manufacturer’s protocol. For CFU-OB assay, the cells were cultured in differentiation medium (α-MEM containing 10% FBS, 50 mg/mL L-ascorbic acid and 2 mmol/L β-glycerophosphate) for 21 days, then alizarin red staining was performed.66

Fluorescence in situ hybridization (FISH)

5-μm-thick mice tibal sections or human bone sections underwent fluorescence in situ hybridization (FISH) using the microRNA FISH Kit (GenePharma, Suzhou) following the manufacturer’s instructions. Initially, sections were deparaffinized and permeabilized with proteinase K for 20 min at 37°C, followed by treatment with blocking solution to inhibit endogenous peroxidase activity. After dehydration, sections were exposed to a microRNA probe mix for miR-29a (1 μM biotin-probe: 1 μM SA-Cy3: PBS = 2: 1: 7) and subjected to hybridization for 18 h at 37°C. Subsequent to serial washes with washing buffer, sections were incubated with DAPI and sealed with anti-fluorescence quench sealant. Image acquisition was performed by Zessis LSM980 confocal microscope. FISH quantification was conducted using ImageJ software. Specifically, each group comprised 5 mice, with 3 sections selected from each mouse. The section depth of different samples was standardized. The average fluorescence intensity or gray value of 5 fields was determined for every section, and all data were normalized to the control group. For human samples, 8 samples from normal controls and 10 samples from patients with OP were utilized for analysis.

RT-qPCR analysis

Total RNA, encompassing mRNA and miRNA, was extracted using Trizol buffer and purified with a total RNA isolating kit (Transgen, ER601-01). Nanodrop 3000 assessed RNA quality via the 260/280 ratio. Subsequently, 1 μg of mRNA underwent reverse transcription into complementary DNA using an RT regent kit (Beyotime, D7190). SYBR Green (Beyotime, D7260) was used to quantify the PCR amplification.143 Expression levels were calculated using the ΔCt-method. The mRNA primers were synthesized by Sangon and the primer pairs used in this study are described in Table S1. For miRNAs, addition of a poly-A tail preceded reserve transcription into complementary DNA using a first-standard cDNA synthesis kit (GeneCopeia, QP114), and detection occurred through a miRNA RT-qPCR Detection kit (GeneCopeia, QP011). The miRNA primers were synthesized by GeneCopeia.

Western blot analysis

Cells were lysed in RIPA buffer (Sigma, R0278) and bone tissue was ground to powder in liquid nitrogen and subsequently lysed in RIPA buffer. The protein concentration was measured using BCA kit (Cwbio, CW0014). Protein lysates were separated using SDS-PAGE and transferred onto a PVDF membrane. Proteins were immunoblotted with the primary antibodies (Kindlin-2 antibody (rabbit, Proteintech, 11453-1-AP, 0.6 μg/mL), Gapdh antibody (mouse, ZSJB-BIO, TA-08, 1 μg/mL), Runx2 antibody (rabbit, HUABIO, ET1612-47, 1 μg/mL), Tubulin antibody (mouse, ZSJB-BIO, TA-10, 1 μg/mL), Osterx (rabbit, HUABIO, ER1914-47, 1 μg/mL), Alkaline phosphatase (rabbit, Abclonal, A0514, 0.33 μg/mL), p53, (rabbit, Abclonal, A19585, 1.2 μg/mL), Fas, (rabbit, Abclonal, A19050, 0.6 μg/mL), PPARγ (rabbit, Abclonal, A0270, 1.68 μg/mL), p16ink4a (rabbit, Abclonal, A0270, 2.92 μg/mL), p21,(rabbit, Abclonal, A0270, 1.1 μg/mL)) at 4°C for 12 h, followed by incubation with their conjugated secondary antibodies with HRP. Information of antibodies used in this study was presented in Table S2. Blots were developed using chemiluminescent HRP substrate. ImageJ software was used for the quantification of WB.144 Briefly, blots were analyzed using ImageJ software to measure the gray value, with the ratio of the target protein relative to the internal control defined as the quantification parameter.

Histological analysis

Tissue samples were fixed in 4% paraformaldehyde solution for 24–48 h. Bone tissue was firstly decalcified using an EDTA (BBI, A60017) solution, followed by dehydration and embedding with paraffin. Mice tibial or calvarial sections of 5-μm thickness were then deparaffinized, rehydrated, and utilized for H/E, TRAP or Alcian blue staining.145 The quantification of TRAP staining was conducted using ImageJ software. Each group comprised 5 mice, with 3 sections selected from each mouse. The depth of sections from different samples was matched accordingly. The average gray value was calculated for every section across 5 fields. Specifically, the ROI was delineated based on the margin of cancellous bone, and the the number and proportion of TRAP positive cells were measured. For quantifying the number and proportion of adipocytes, 3 sections from each mouse were analyzed, with 5 mice per group. The average adipocyte count and adipose proportion across 5 fields of one section were computed for analysis.

Pathology scoring methodology

For pathology scoring methodology,79 the injury severity was graded from 0 to 4, considering five independent variables: hemorrhage, neutrophils in the alveolar space, hyaline membranes, pertinacious debris filling the airspaces, and septal thickening. The scoring system ranged from 0 (no damage) to 4 (>75% damage), with intermediate scores indicating varying degrees of damage. The inflammation score was determined independently by three researchers who were blinded to the experimental conditions, and the average of their scores was computed to obtain the final score. For sampling protocol, 5 mice were included in each group, with 3 sections selected from each mouse. The average fluorescence intensity or gray value was calculated for every section across 5 fields.

Immunohistochemistry (IHC) and immunofluorescence (IF) staining

Tissues were dehydrated and embedded with paraffin. Sections of 5-μm thickness underwent deparaffinization and antigen retrieval in a citric acid solution at 58°C for 16 h. For IHC staining, mice tibial or calvarial sections of 5-μm thickness were incubated with indicated antibodies (Kindlin-2 antibody (rabbit, Proteintech, 11453-1-AP, 3 μg/mL), Runx2 antibody (rabbit, HUABIO, ET1612-47, 5 μg/mL), OCN (rabbit, Bioss, bs-4917R, 5 μg/mL), p16ink4a (rabbit, Abclonal, A0270, 16.8 μg/mL), p21 (rabbit, Abclonal, A0270, 22 μg/mL) or control IgG using EnVision+System-HRP (DAB) kit (Dako North America Inc, K401111-2) according to the manufacturer’s instruction.146 Images were captured by Lecia Aperio VERSA scanner. The quantification of Runx2 and Ocn was calculated by ImageJ software. Briefly, 5 mice were included in each group, and 3 sections from each mouse were selected. The depth of sections from different samples was matched accordingly. The average fluorescence intensity or gray value of 5 fields was calculated for every section. All data were normalized to the control group. For IF staining, mice tibial or calvarial sections of 5-μm thickness were incubated with primary antibodies (p53, rabbit, Abclonal, A19585, 6 μg/mL) at 4°C for 8 h followed by incubation with fluorescent labeled secondary antibodies for 1 h at room temperature. Images were captured by Zessis LSM980 confocal microscope. The quantification of p53 was calculated by ImageJ software. Briefly, it included 5 animals in one group and 3 sections from every animal were selected. The section depth of different samples was corresponded. The average fluorescence intensity or gray value was calculated for every section across 5 fields. All data were normalized to the control group.

Quantification and statistical analysis

The sample size for mouse experiments in this study was determined based on our prior experience. Mice were randomly assigned to groups in the experiments conducted in this study. IF, IHC, and histology analyses were carried out and assessed in a double-blinded manner. GraphPad Prism 9 (Version 9.5.0) was used for statistical analyses. All data were expressed as the mean ± standard deviations (s.d.) and analyzed for statistical significance using two-tailed Student’s t test or one-way ANOVA. Log rank test was used in survival analysis. p < 0.05 was considered statistically significant.

Supplemental information

Document S1. Figures S1–S7 and Tables S1 and S2

Document S2. Article plus supplemental information

Acknowledgments

We acknowledge the assistance of Core Research Facilities of Southern University of Science and Technology. This work was supported, in part, by the 10.13039/501100001809 National Natural Science Foundation of China grants (82372476 and 82350710800 ), the 10.13039/501100012166 National Key Research and Development Program of China grant (2019YFA0906001 ), and the Guangdong Provincial Science and Technology Innovation Council grant (2017B030301018 ).

Author contributions

Study design: Z.D., G.M., and H.C. Study conducts, data collection, and analysis: Z.D., G.M., B.Z., S.C., W.T., Y.H., L.C., W.P., Y.C., D.Y., and H.C. Data interpretation: H.C., G.M., and Z.D. Drafting the manuscript: H.C. and Z.D. Z.D., G.M., and H.C. take the responsibility for the integrity of the data analysis.

Declaration of interests

The authors declare no competing interests.

Supplemental information can be found online at https://doi.org/10.1016/j.xcrm.2024.101665.
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