SPARC from Young CSF Rejuvenates aged Skull Bone Marrow by targeting neutrophil-derived MMP25

SPARC from Young CSF Rejuvenates aged Skull Bone Marrow by targeting neutrophil-derived MMP25 | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article SPARC from Young CSF Rejuvenates aged Skull Bone Marrow by targeting neutrophil-derived MMP25 Ping Zhang, Yang Li, Huixin Lv, Weibiao Lin, Xiaoxing Huang, Zechuan Li, and 10 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-8242292/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Skeletal aging remains poorly understood, particularly in craniofacial bones that interface closely with the central nervous system. Here, we identify a unique aging microenvironment in skull bone marrow (BM) characterized by neutrophil accumulation and elevated MMP25, which drives skull skeletal stem cell (SSC) dysfunction via NF-κB-mediated inflammation. Neutrophil-specific deletion of Mmp25 alleviates age-associated bone loss and preserves SSC regenerative capacity. We further demonstrate that cerebrospinal fluid from young mice (yCSF) rejuvenates aged SSCs, with Secreted Protein Acidic and Rich in Cysteine (SPARC) identified as a key youth-associated factor diminished in aged CSF. Mechanistically, SPARC suppresses PU.1-dependent transcription of MMP25, thereby reducing inflammation and restoring osteogenic potential in aged SSCs. These findings define a CSF-skull BM axis linking neuroimmune signaling to skeletal aging and identify the SPARC-PU.1-MMP25 pathway as a therapeutic target for craniofacial bone regeneration. Biological sciences/Cell biology/Cell signalling Health sciences/Molecular medicine Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Full Text Additional Declarations There is NO Competing Interest. Supplementary Files SupplementalMaterial.pdf Supplementary information ExtendedDataVedio1.mp4 Extended Data Vedio 1 Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-8242292","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Article","associatedPublications":[],"authors":[{"id":563952883,"identity":"0e38a3bd-79f4-4a4f-b4b9-13fa493637ad","order_by":0,"name":"Ping 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Tang","email":"","orcid":"https://orcid.org/0000-0002-8625-7717","institution":"Peking University","correspondingAuthor":false,"prefix":"","firstName":"Fuchou","middleName":"","lastName":"Tang","suffix":""},{"id":563952897,"identity":"c7891ce7-a156-4b4a-9c82-cb04ae852d43","order_by":14,"name":"Cun-Yu Wang","email":"","orcid":"","institution":"Peking University School and Hospital of Stomatology","correspondingAuthor":false,"prefix":"","firstName":"Cun-Yu","middleName":"","lastName":"Wang","suffix":""},{"id":563952898,"identity":"d420076c-8dff-40b2-ab2d-e87218dc4b6a","order_by":15,"name":"Yongsheng Zhou","email":"","orcid":"https://orcid.org/0000-0002-4332-0878","institution":"Peking University School and Hospital of Stomatology","correspondingAuthor":false,"prefix":"","firstName":"Yongsheng","middleName":"","lastName":"Zhou","suffix":""}],"badges":[],"createdAt":"2025-11-30 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03:00:39","extension":"pdf","order_by":9,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":5356039,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalMaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8242292/v1/63bb961442c38f48b6b085e4.pdf"},{"id":100744547,"identity":"8e15abbb-8059-4602-9f4c-b95d0e3478f2","added_by":"auto","created_at":"2026-01-21 02:59:51","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":38710737,"visible":true,"origin":"","legend":"\u003cp\u003eAge-associated decline of cranial skeletal stem cell function (a) Representative micro-computed tomography (µCT) images of coronal parietal bone sections from mice aged 1, 6, 12, and 18 months (M) (upper), along with quantification of bone volume/tissue volume ratio (BV/TV), trabecular number (Tb. N) and trabecular separation (Tb. Sp) (bottom). n = 6 mice for the 1M, 6M and 12M groups; n = 5 mice for the 18Mgroup. Scale bar, 1 mm. (b) Representative H\u0026amp;E staining of parietal bone sections from mice aged 1M, 6M, 12M, and 18M.Yellow arrows indicate marrow adipose tissue. n = 3. Scale bar, 100 µm. (c) µCT assessment and quantitative analysis of bone regeneration in calvarial defects from mice aged 1M, 6M, 12M, and 18M at 8 weeks post-surgery. n = 6. Scale bar, 1 mm. (d) Subcutaneous transplantation of PDGFRA⁺ and PDGFRA⁻ cells from young (1M) mice, with evaluation of their ectopic osteogenic potential after 4 weeks. H\u0026amp;E staining with a β-TCP scaffold (left) and three-dimensional µCT reconstruction with a Matrigel scaffold (middle), along with quantification of bone volume (right). n = 3. Scale bars, 100 µm (H\u0026amp;E) and 1 mm(µCT). (e) Schematic of in vivo subcutaneous serial transplantation. Created in BioRender. Yang, L. (2025) BioRender.com/ph6ozw9. (f) FACS analysis of ZsGreen+ (PDGFRA+) and mCherry+ (PDGFRA-) cells after the first transplantation. (g) Representative H\u0026amp;E staining of bone formation from ZsGreen+ (PDGFRA+) and mCherry+ (PDGFRA-) cells with β-TCPscaffold. Scale bar, 200 µm. (h) Flow cytometry analysis showing PDGFRA+ SSCs in the skull BM from young (1M) and aged (12M) mice (left), with quantification of PDGFRA+ SSC frequency (right). n = 7. (i) SA-β-Gal staining of senescent PDGFRA+ SSCs isolated from young (1M) and aged (12M) mice. n = 3. Scale bar, 100 µm. (j) qPCR analysis of P16 and P21 mRNA expression in PDGFRA+ SSCs isolated from young (1M) and aged (12M) mice. n = 5. (k) H\u0026amp;E staining (left) and µCT analysis (middle) of ectopic bone formation derived from PDGFRA⁺ cells in young (1M) and aged (12M) mice, with quantification of bone volume (right). n = 3. Scale bars, 100 µm (H\u0026amp;E) and 1 mm (µCT). (l) Representative immunofluorescence images showing tdTomato+ SSCs in the skull BM of 1M, 3M, 6M, and 12M Pdgfra-CreERT2tdTomato mice, analyzed 30 days after tamoxifen induction. Scale bar, 25 µm. (m) tdTomato-labeled PDGFRA+ SSCs give rise to COL1A1+ osteoblasts in young (1M) and aged (12M) mice, 30 days after tamoxifen induction. White arrows indicate tdTomato+ osteoblasts. Quantification of COL1A1+tdTomato+ cells is shown on the right. n = 3. Scale bars, 25 µm.\u003c/p\u003e","description":"","filename":"Figure1.png","url":"https://assets-eu.researchsquare.com/files/rs-8242292/v1/df6f9160898f0ca5baec8952.png"},{"id":100744559,"identity":"443b14b1-e073-4d0d-ac23-f1d7e923c818","added_by":"auto","created_at":"2026-01-21 03:00:00","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":24478793,"visible":true,"origin":"","legend":"\u003cp\u003eNeutrophil-derived MMP25 accumulates with age and drives skull SSC dysfunction (a) qPCR analysis of Mmp25 mRNA expression in neutrophils (Neu), non-hematopoietic cells (NHC), lymphocytes (Lym), and bone marrow-derived macrophages (BMM) in 12M aged mice. n = 5. (b) Western blot analysis and quantification of MMP25 expression in Neu, NHC, Lym, and BMMin12Maged mice. n = 4. (c) qPCR analysis of Mmp25 mRNA expression in neutrophils from 1M, 6M, 12M, and 18M mice. n = 5. (d) Western blot analysis and quantification of MMP25 expression in neutrophils from 1M, 6M, 12M, and 18M mice. n = 3. (e) Schematic depicting the treatment of PDGFRA⁺ SSCs with neutrophil-derived conditioned media (CM). Created in BioRender. Yang, L. (2025) BioRender.com/3o92gcm. (f) Representative SA-β-Gal staining of PDGFRA+ SSCs treated with control medium, neutrophil-CM (Neu-CM), CM from negative control-treated neutrophils (Neu + NC-CM), or CM from siRNA-Mmp25-treated neutrophils (Neu + si-Mmp25 CM). n = 3. Scale bars, 100 µm. (g) qPCR analysis of P16 and P21 mRNA expression in PDGFRA+ SSCs cultured with different CM as depicted in f. n = 4. (h) ARS staining of osteogenic differentiation from PDGFRA+ SSCs cultured with different CM as depicted in f. (i) Oil Red O staining and quantification of adipogenic differentiation of PDGFRA+ SSCs cultured in different CM as depicted in f. n = 4. Scale bars, 100 µm. (j) Flow cytometry analysis showing increased CXCR2+CD177+ neutrophils and elevated MMP25 expression in aged (12M) mice compared to young (1M) mice, along with quantification of CXCR2+CD177+ neutrophils, MMP25+ cells, and MMP25mean fluorescence intensity (MFI). n = 5. (k) Schematic illustration of neutrophil-specific Mmp25 deletion strategy (upper panel), with 10M male Mmp25fl/fl mice treated with AAV9-Cxcr2-eGFP (Mmp25Neu+/+) or AAV9-Cxcr2-cre-eGFP (Mmp25NeuΔ) via tail vein injection for 2 months. Representative µCT images (lower panel) along with quantification of BV/TV, Tb.N, and Tb.Sp (right). n = 6. (l) H\u0026amp;E staining of parietal bone sections from Mmp25Neu+/+ and Mmp25NeuΔ mice. Yellow arrows indicate marrow adipose tissue. n = 3. Scale bar, 20 µm. (m) Representative images of calcein double labeling and analysis of mineral apposition rate (MAR) and bone formation rate (BFR) in parietal bone of 12M Mmp25Neu+/+ andMmp25NeuΔ mice. n = 5. Scale bar, 20 µm. (n) Flow cytometry analysis and quantification of PDGFRA+ SSCs in skull BM from Mmp25Neu+/+ andMmp25NeuΔ mice. n =5. (o) Representative images of SA-β-Gal staining and quantification of senescent PDGFRA+ SSCsfrom Mmp25Neu+/+ andMmp25NeuΔ mice. n = 3. Scale bar, 100 µm. (p) qPCR analysis of P16 and P21 mRNA expression in PDGFRA+ SSCs from Mmp25Neu+/+ andMmp25NeuΔ mice. n = 5. (q) Representative images of ARS staining of PDGFRA+ SSCs from Mmp25Neu+/+ and Mmp25NeuΔ mice. (r) Representative images of Oil Red O staining and quantification of adipogenic differentiation in PDGFRA+ SSCs from Mmp25Neu+/+ and Mmp25NeuΔ mice. n = 4. Scale bar, 100 µm.\u003c/p\u003e","description":"","filename":"Figure2.png","url":"https://assets-eu.researchsquare.com/files/rs-8242292/v1/c0d15292865fd34d9bc09957.png"},{"id":100744543,"identity":"36cbff05-0d63-46b1-b34e-e1c6ecdf432e","added_by":"auto","created_at":"2026-01-21 02:59:49","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":4043672,"visible":true,"origin":"","legend":"\u003cp\u003eMMP25 amplifies inflammatory signaling via NF-κB activation (a) scRNA-seq analysis of skull BM cells from Mmp25Neu+/+ and Mmp25NeuΔ mice. t-SNE visualization shows distinct cell populations color-coded by cell type (left) and quantification of cell proportions (right). (b) Dotplots showing expression of cell-type specific marker genes in skull BM cells. (c) Dotplot of inflammatory marker genes comparing in Mmp25Neu+/+ andMmp25NeuΔ mice. (d) Gene expression patterns in Stromal cells from Mmp25Neu+/+ and Mmp25NeuΔ mice. (e) t-SNE plot of neutrophil subsets isolated from Mmp25Neu+/+ and Mmp25NeuΔ mice. (f) Dotplot analysis of subset-specific marker genes defining neutrophil subsets. (g) Gene Ontology (GO) enrichment analysis comparing Mmp25Neu+/+ and Mmp25NeuΔ mice. (h) KEGG pathway enrichment analysis of down regulated genes in Mmp25Neu+/+ and Mmp25NeuΔ mice. (i) qPCR analysis of Mmp25 expression in neutrophils from 12-month-old mice transfected with NC or Mmp25 siRNA(si-2) for 48hours. n = 5. (j) Western blot analysis of MMP25 expression in in neutrophils after transfection with NC or Mmp25 siRNA(si-2) for 48hours. (k) p-P65, P65, p-IκBα, and IκBα protein levels in neutrophils after transfection with NC or Mmp25 siRNA (si-2) for 48hours (left). Quantification of p-P65/P65 and p-IκBα/IκBα ratios is shown on the right. n = 4. (l) ELISA quantification of IL1β and TNF in the CM 48 hours after NC or si-2 transfection. n = 5. (m) qPCR analysis of Mmp25 expression in neutrophils from one-month-old mice transfected with vectors or MMP25 overexpression plasmid. n = 5. (n) Western blot analysis of MMP25 expression in neutrophils from 1-month-old mice after transfection with vectors or MMP25 overexpression plasmid for 48 hours. (o) Western blot analysis of p-P65, P65, p-IκBα, and IκBα protein levels in neutrophils from 1-month-old mice after transfection with vectors or MMP25 overexpression plasmid for 48 hours (left). Quantification of p-P65/P65 and p-IκBα/IκBα ratios is shown on the right. n = 4. (p) ELISA quantification of IL1β and TNF in CM collected 48 hours after transfection with either a control vector or an MMP25 overexpression plasmid. n = 5.\u003c/p\u003e","description":"","filename":"Figure3.png","url":"https://assets-eu.researchsquare.com/files/rs-8242292/v1/363d8a290fe73ef02875bf66.png"},{"id":100744703,"identity":"a9031a93-0c6c-4791-a7f2-dd7f6575557b","added_by":"auto","created_at":"2026-01-21 03:00:20","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":8116270,"visible":true,"origin":"","legend":"\u003cp\u003eYoung cerebrospinal fluid rejuvenates aged SSCs and restores cranial bone regeneration (a) Ex vivo two-photon imaging of skull BM at 30 min, 50 min, and 7 days after intracisternal (IC) injection of AF488-OVA-labeled yCSF (right, created in BioRender. Yang, L. (2025) BioRender.com/699whad). Intravenous (IV) injection of CD31 labeled vasculature. Data are representative of three independent experiments. Scale bar, 100 µm. (b) Timeline of EdU administration following artificial cerebrospinal fluid (aCSF) or yCSF treatment (left, created in BioRender. Yang, L. (2025) BioRender.com/iuf4lqr). Quantification of tdTomato+ and EdU+tdTomato+ cells in 12-month-old Pdgfra-CreERT2tdTomato mice after 72 hours (right). n = 5. (c) Schematic illustration of the experimental design showing the in vivo treatment of 12-month-old mice with aCSF or yCSF for 7 days (7 d). Created in BioRender. Yang, L. (2025) BioRender.com/qns7y70. (d) Representative flow cytometry plots showing P16 expression in tdTomato+ cells from 12-month-old Pdgfra-CreERT2tdTomato mice, 7 days after treatment with aCSF or yCSF (left). Quantification of P16+ cells within the tdTomato+ population (right). n = 6. (e) Representative SA-β-Gal staining and quantification of PDGFRA+ SSCs isolated from aged (12M) mice 7 days after aCSF or yCSF treatment. n = 3. Scale bar, 100 µm. (f) qPCR analysis of P16 and P21 mRNA expression in PDGFRA+ SSCs isolated from aged (12M) mice 7 days after aCSF or yCSF treatment. n = 6. (g) H\u0026amp;E staining (left) and corresponding µCT images (right) of ectopic bone formation by PDGFRA+ SSCs isolated from aged (12M) mice following 7-day aCSF or yCSF treatment, and quantification of bone volume (right). n = 3. Scale bars, 100 µm(H\u0026amp;E) and 1 mm(µCT). (h) ARS staining of osteogenic differentiation of PDGFRA+ SSCs isolated from aCSF- or yCSF-treated (7 d) aged (12M) mice. (i) Oil Red O staining and quantification of adipogenic differentiation of PDGFRA+ SSCs derived from aged (12M) mice 7 days post-aCSF or yCSF treatment. n = 4. Scale bar, 100 µm. (j) Representative immunofluorescence images of skull BM sections from aCSF- or yCSF-treated aged (12M) Pdgfra-CreERT2tdTomato mice showing COL1A1 expression (green) in tdTomato+ cells (red). Higher magnification of the boxed regions (II-V) with merged images demonstrating tdTomato+ osteoblasts (white arrows). Quantification of COL1A1+tdTomato+ cells is shown on the right. n = 3. Scale bar, 25 µm. (k) Representative images of calcein double labelling and analysis of MAR and BFR in the parietal bone from aged (12M) mice 7 days post-aCSF or yCSF administration. n =5. Scale bar, 20 µm. (l) Representative µCT images of calvarial bone defect healing in aged (12M) mice 8 weeks after surgery, with defects created following 7 days of aCSF or yCSF treatment, and quantification of BV/TV and Tb.N (right). n = 5. Scale bar, 1 mm. (m) Schematic illustration of experimental design (upper, created in BioRender. Yang, L. (2025) BioRender.com/4omxzkc). Representative µCT images of skulls from aged (12M) mice after 4-week continuous aCSF or yCSF administration (lower), and quantification of BV/TV, Tb.N, and Tb.Sp (right). n = 6. Scale bar, 1 mm. (n) qPCR analysis of Mmp25 mRNA expression in neutrophils isolated from aged (12M) mice 7 days after aCSF or yCSF treatment. n = 5. (o) Western blot analysis and quantification of MMP25 expression in neutrophils isolated from aged (12M) mice 7 days after aCSF or yCSF treatment. n = 4. (p) Flow cytometry analysis of CXCR2+CD177+ neutrophils and MMP25 expression 7 days after aCSF or yCSF treatment in aged (12M) mice (left), with quantification shown on the right. n = 5.\u003c/p\u003e","description":"","filename":"Figure4.png","url":"https://assets-eu.researchsquare.com/files/rs-8242292/v1/cb08ecc5f86aa91e5afae81c.png"},{"id":100744705,"identity":"f5c402d6-fa67-4b12-9923-4642497b4955","added_by":"auto","created_at":"2026-01-21 03:00:21","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":4911282,"visible":true,"origin":"","legend":"\u003cp\u003eSPARC mediates the rejuvenating effect of yCSF by suppressing MMP25 (a) Heatmap showing differentially expressed proteins (|log2FC| \u0026gt; 1, p \u0026lt; 0.01) in the CSF of young (1M) and aged (12M) mice identified via mass spectrometry. (b) ELISA quantification of SPARC in the CSF of young (1M) and aged (12M) mice. n =6. (c) EdU administration after PBS or SPARC treatment (left panel, created in BioRender. Yang, L. (2025) BioRender.com/dyec38n). Quantification of tdTomato+ and EdU+tdTomato+ cells in Pdgfra-CreERT2tdTomato mice after 72 hours (right). n = 3. (d) Schematic illustration of the 7-day SPARC treatment strategy in aged mice. Created in BioRender. Yang, L. (2025) BioRender.com/xgmaff6. (upper); Representative SA-β-Gal staining of PDGFRA+ SSCs from aged (12M) mice following 7-day SPARC treatment (lower). n = 3. Scale bar, 100 µm. (e) qPCR analysis of P16 and P21 mRNA expression in PDGFRA+ SSCs isolated from aged (12M) mice 7 days after PBS or SPARC treatment. n = 5. (f) ARS staining of the osteogenic differentiation of PDGFRA+ SSCs from aged (12M) mice treated with PBS or SPARC for 7 days. (g) Oil Red O staining and quantification of adipogenic differentiation of PDGFRA+ SSCs from aged (12M) mice after 7-day PBS or SPARC treatment. n = 4. Scale bar, 100 µm. (h) Representative immunofluorescence images showing COL1A1 expression (green) and tdTomato+ cells (red) in skull BM from aged (12M) Pdgfra-CreERT2tdTomato mice treated with PBS or SPARC for 7 days. Quantification of COL1A1+tdTomato+ cells is shown on the right. n = 3. Scale bar, 25 µm. (i) Representative images of calcein double labeling in the parietal bone from aged (12M) mice treated with PBS or SPARC for 7 days, with quantification of MAR and BFR. n = 5. Scale bar, 20 µm. (j) Representative µCT images of skull defect healing in aged (12M) mice 8 weeks after surgery. Defects were created after 7-day PBS or SPARC treatment. Quantification of BV/TV and Tb.N is shown on the right. n = 5. Scale bar, 1 mm. (k) Long-term effects of SPARC on bone quality analyzed by µCT following 4-week continuous administration in aged (12M) mice. Quantification of structural parameters is shown on the right. n = 6. Scale bar, 1 mm. (l) Flow cytometry analysis of CXCR2+CD177+ neutrophils and MMP25 expression 7 days after PBS or SPARC treatment in aged (12M) mice (left), with quantification shown on the right. n = 5. (m) qPCR analysis of Mmp25 mRNA expression in neutrophils from 12-month-old mice treated with SPARC (2 µg/mL) or PBS for 48 hours in vitro. n = 5. (n) Western blot analysis and quantification of MMP25 expression in CXCR2+CD177+ neutrophils from 12-month-old mice treated with SPARC (2 µg/mL) or PBS for 48 hours in vitro. n = 4. (o) qPCR analysis of Mmp25 mRNA expression in neutrophils isolated from 12-month-old mice 7 days after SPARC (20 µg/mL) or PBS treatment in vivo. n = 6. (p) Western blot analysis and quantification of MMP25 expression in CXCR2+CD177+ neutrophils isolated from 12-month-old mice 7 days after SPARC (20 µg/mL) or PBS treatment in vivo. n = 4. (q) Schematic illustration of the experimental design showing AAV9-Mmp25 or AAV9-CTRL injections followed by IC SPARC administration (upper panel, created in BioRender). Representative µCT images of skull from aged (12M) mice receiving indicated treatments (lower), and quantification of BV/TV, Tb.N, and Tb.Sp. n = 6. Scale bar, 1 mm. (r) Representative images of calcein double labeling in calvaria from aged (12M) mice receiving AAV9-CTRL or AAV9-Mmp25 with SPARC or PBS treatment (left), and quantification of BFR and MAR (right). n = 5. Scale bar, 20 µm.\u003c/p\u003e","description":"","filename":"Figure5.png","url":"https://assets-eu.researchsquare.com/files/rs-8242292/v1/7d1cb0e5a1710a0c8623168b.png"},{"id":100744610,"identity":"89575c4f-d320-475b-9e8c-0d801ffecc76","added_by":"auto","created_at":"2026-01-21 03:00:17","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":4534532,"visible":true,"origin":"","legend":"\u003cp\u003eSPARC represses PU.1-dependent chromatin accessibility at the Mmp25 locus (a) Genome-wide ATAC-seq signal profiles and heatmaps around transcription start sites (TSS, ± 3 kb) of skull neutrophils from 1-, 12-, and 18-month-old mice, illustrating age-associated alterations in chromatin accessibility.. (b) Representative ATAC-seq tracks of the Mmp25 locus in skull neutrophils from 1M, 12M, and 18M mice. The highlighted (2 kb) region shows increased chromatin accessibility at the promoter with aging. (c) Aggregate ATAC-seq signal and heatmaps around TSS (±3 kb) in aged mice treated with PBS or SPARC for 7 days, showing SPARC-induced chromatin closure at age-associated accessible regions. (d) Representative ATAC-seq tracks of the Mmp25 locus in PBS- or SPARC-treated aged skull neutrophils. The highlighted (2 kb) region shows reduced promoter accessibility following SPARC treatment. (e) Motif enrichment analysis revealed that PU.1-binding motifs were highly enriched in regions with increased accessibility during aging, whereas these motifs were depleted in regions showing decreased accessibility after SPARC treatment, indicating that SPARC reverses PU.1-associated chromatin activation in aged skull bone marrow. (f) ChIP-qPCR analysis showing PU.1 binding to the Mmp25 promoter. Left, schematic of two predicted PU.1-binding sites (P1 and P2) upstream of the Mmp25 TSS. Right, PU.1 enrichment at P1 and P2 regions in control and PU.1-knockdown (si-PU.1) cells. n = 3. (g) qPCR analysis of PU.1 mRNA expression in neutrophils from young and aged mice. n = 5. (h) Western blot analysis and quantification of PU.1 protein expression in neutrophils from young and aged mice. n = 3. (i) qPCR analysis of PU.1 mRNAexpression in neutrophils after transfection with NC or PU.1 siRNAfor 48 hours in vitro. n = 5. (j) Western blot analysis and quantification of PU.1 protein expression in neutrophils after transfection with NC or PU.1 siRNAin vitro. n = 3. (k) qPCR analysis of PU.1 mRNA expression in neutrophils after transfection with vectors or PU.1 overexpression plasmid in vitro. n = 6. (l) Western blot analysis and quantification of PU.1 protein expression in neutrophils following transfection with a control vector or a PU.1 overexpression plasmid in vitro. n =3. (m) qPCR analysis of Mmp25 mRNA expression in neutrophils. Left, PU.1 knockdown (si-PU.1); right, PU.1 overexpression. n = 6. (n) Western blot analysis of MMP25, p-P65, P65, p-IκBα, and IκBα protein levels in neutrophils from 12-month-old mice after transfection with NC or PU.1 siRNA for 48 hours in vitro (left). Quantification of MMP25/β-actin, p-P65/P65 and p-IκBα/IκBα ratios is shown on the right. n = 3. (o) Western blot analysis of MMP25, p-P65, P65, p-IκBα, and IκBα protein levels in neutrophils from 1-month-old mice after transfection with vectors or PU.1 overexpression plasmid for 48 hours in vitro (left). Quantification of MMP25/β-actin, p-P65/P65 and p-IκBα/IκBα ratios is shown on the right. n = 3. (p) qPCR analysis of PU.1 mRNA expression in neutrophils isolated from 12-month-old mice treated with SPARC (2 µg/mL) or PBS for 48 hours in vitro. n = 5. (q) Western blot analysis and quantification of PU.1 expression in neutrophils isolated from 12-month-old mice treated with SPARC (2 µg/mL) or PBS for 48 hours in vitro. n =5. (r) qPCR analysis of PU.1 mRNA expression in neutrophils isolated from 12-month-old mice 7 days after SPARC (20 µg/mL) or PBS treatment in vivo. n = 4 for SPARC, n = 5 for PBS. (s) Western blot analysis and quantification of PU.1 expression in neutrophils isolated from 12-month-old mice 7 days after SPARC (20 µg/mL) or PBS treatment in vivo. n = 4. (t) qPCR analysis of PU.1 mRNA expression in neutrophils from 1-month-old mice following transfection with an empty vector or a PU.1 overexpression plasmid and subsequent treatment with or without SPARC (2 µg/mL) for 48 hours in vitro. n = 3. (u) qPCR analysis of Mmp25 mRNA expression in neutrophils from 1-month-old mice following transfection with an empty vector or a PU.1 overexpression plasmid and subsequent treatment with or without SPARC (2 µg/mL) for 48 hours in vitro. n = 3. (v) Western blot analysis of p-P65, P65, p-IκBα, and IκBα protein levels in neutrophils from 1-month-old mice 7 days after SPARC (20 µg/mL) or PBS treatment in vivo (left). Quantification of p-P65/P65 and p-IκBα/IκBα ratios is shown on the right. n = 3. (w) Western blot analysis of p-P65, P65, p-IκBα, and IκBα protein levels in neutrophils from 1-month-old mice following transfection with an empty vector or a PU.1 overexpression plasmid and subsequent treatment with or without SPARC (2 µg/mL) for 48 hours in vitro. n = 3.\u003c/p\u003e","description":"","filename":"Figure6.png","url":"https://assets-eu.researchsquare.com/files/rs-8242292/v1/cdc7618c86ed9ff71bcd7d52.png"},{"id":100744537,"identity":"ea7bab18-cf31-403a-876a-b147221c16cc","added_by":"auto","created_at":"2026-01-21 02:59:42","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":7714576,"visible":true,"origin":"","legend":"\u003cp\u003eSPARC in human yCSFrejuvenates aged cranial bone marrow (a) Representative SA-β-Gal staining of skull BM stromal cells from young and aged individuals. n = 3. Scale bar, 100 µm. (b) ARS staining of osteogenic differentiation in skull BM stromal cells from young and aged individuals. (c) Representative H\u0026amp;E staining of ectopic bone formation by skull BM stromal cells from young and aged individuals 4 weeks after subcutaneous transplantation with β-TCP scaffold. Scale bar, 100 µm. (d) Oil Red O staining and quantification of adipogenic differentiation in skull BM stromal cells of young and aged individuals. n = 3. Scale bar, 100 µm. (e-f) scRNA-seq analysis of the human skull BM cells (e, created in BioRender. Yang, L. (2025) BioRender.com/u22nwqh). t-SNE plot showing distinct cell populations and proportions between young and aged individuals (f). (g-h) Violin plots showing the expression of CDKN1A, IL1B, and IL6 in skull BM cells and the expression of SPP1, RUNX2, LPL, and CEBPD in stromal cells from young and aged individuals. (i) t-SNE plot of human neutrophil subsets. (j) Violin plots showing the expression of MMP25 across neutrophil subsets. (k) Violin plots showing the expression of MMP25 in neutrophil cells from young and aged individuals. (l) qPCR analysis of MMP25 mRNA expression in neutrophils from young and aged individuals. n = 3. (m) Western blot analysis and quantification of MMP25 protein levels in neutrophils from young and aged individuals. n = 3. (n) Violin plots showing the expression of PU.1 in BM cells from young and aged individuals (left). qPCR analysis of PU.1 mRNA expression in neutrophils from young and aged individuals (right). n = 6. (o) Quantification of SPARC in the CSF of young and aged individuals using ELISA. n =3. (p) qPCR analysis of MMP25 mRNAexpression in neutrophils from aged individuals after 48 hours treatment with 20% yCSF. n = 3. (q) Western blot analysis of MMP25 expression in neutrophils from aged individuals after 48 hours treatment with 20% yCSF. (r) qPCR analysis of MMP25 mRNA expression in aged neutrophils from aged individuals after 48 hours treatment with SPARC (2 µg/mL). n = 3. (s) Western blot analysis and quantification of MMP25 expression in neutrophils from aged individuals after 48 hours treatment with SPARC (2 µg/mL). n = 3. (t) Schematic diagram showing the collection of CM from young neutrophils overexpressing either the vector or MMP25 (OE-MMP25), followed by treatment with yCSF or SPARC, and subsequent functional analysis of young skull BM stromal cells. Created in BioRender. Yang, L. (2025) BioRender.com/tmw273x. (u) Representative SA-β-Gal staining of senescent cells in young skull BM stromal cells treated with CM from the indicated groups. n = 3. Scale bar, 100 µm. (v) qPCR analysis of P16 and P21 mRNAexpression in young skull BM stromal cells treated with different CM. n = 3. (w) ARS staining for osteogenic differentiation in young skull BM stromal cells treated with CM from the indicated groups. (x) Oil Red O staining and quantification of adipogenic differentiation in young skull BM stromal cells treated with CM from the indicated groups. n = 3. Scale bar, 100 µm.\u003c/p\u003e","description":"","filename":"Figure7.png","url":"https://assets-eu.researchsquare.com/files/rs-8242292/v1/cd59a8b43f8a2087744bd93f.png"},{"id":103505765,"identity":"eadefe6b-0b4c-4891-9652-4954ed624e01","added_by":"auto","created_at":"2026-02-26 13:32:56","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":10141680,"visible":true,"origin":"","legend":"Article File","description":"","filename":"Manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8242292/v1_covered_b381bacf-41dd-4763-952a-934775101b8c.pdf"},{"id":100744704,"identity":"84c1e041-51a5-4980-b95e-c89d85ab2dfe","added_by":"auto","created_at":"2026-01-21 03:00:20","extension":"pdf","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":5356039,"visible":true,"origin":"","legend":"Supplementary information","description":"","filename":"SupplementalMaterial.pdf","url":"https://assets-eu.researchsquare.com/files/rs-8242292/v1/f550b10edab6a994968fcc0e.pdf"},{"id":100744540,"identity":"dc769057-32be-4fdb-b5e6-e6c43a09bd63","added_by":"auto","created_at":"2026-01-21 02:59:45","extension":"mp4","order_by":2,"title":"","display":"","copyAsset":false,"role":"supplement","size":29571609,"visible":true,"origin":"","legend":"Extended Data Vedio 1","description":"","filename":"ExtendedDataVedio1.mp4","url":"https://assets-eu.researchsquare.com/files/rs-8242292/v1/f06f67a84db16c8c3ab8fd08.mp4"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"SPARC from Young CSF Rejuvenates aged Skull Bone Marrow by targeting neutrophil-derived MMP25","fulltext":[],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":false,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":true,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":true,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":true,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-8242292/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-8242292/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"Skeletal aging remains poorly understood, particularly in craniofacial bones that interface closely with the central nervous system. Here, we identify a unique aging microenvironment in skull bone marrow (BM) characterized by neutrophil accumulation and elevated MMP25, which drives skull skeletal stem cell (SSC) dysfunction via NF-κB-mediated inflammation. Neutrophil-specific deletion of Mmp25 alleviates age-associated bone loss and preserves SSC regenerative capacity. We further demonstrate that cerebrospinal fluid from young mice (yCSF) rejuvenates aged SSCs, with Secreted Protein Acidic and Rich in Cysteine (SPARC) identified as a key youth-associated factor diminished in aged CSF. Mechanistically, SPARC suppresses PU.1-dependent transcription of MMP25, thereby reducing inflammation and restoring osteogenic potential in aged SSCs. These findings define a CSF-skull BM axis linking neuroimmune signaling to skeletal aging and identify the SPARC-PU.1-MMP25 pathway as a therapeutic target for craniofacial bone regeneration.","manuscriptTitle":"SPARC from Young CSF Rejuvenates aged Skull Bone Marrow by targeting neutrophil-derived MMP25","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2026-01-21 02:56:47","doi":"10.21203/rs.3.rs-8242292/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"e6d22230-3452-4be9-bdc2-8798eca5d774","owner":[],"postedDate":"January 21st, 2026","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[{"id":60049673,"name":"Biological sciences/Cell biology/Cell signalling"},{"id":60049674,"name":"Health sciences/Molecular medicine"}],"tags":[],"updatedAt":"2026-02-23T15:12:09+00:00","versionOfRecord":[],"versionCreatedAt":"2026-01-21 02:56:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-8242292","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-8242292","identity":"rs-8242292","version":["v1"]},"buildId":"XKTyCvWXoU3ODBz1xrDgd","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}