Articular cartilage regeneration by activated skeletal stem cells
AbstractOsteoarthritis (OA) is a degenerative disease resulting in irreversible, progressive destruction of articular cartilage1. The etiology of OA is complex and involves a variety of factors, including genetic predisposition, acute injury and chronic inflammation2,3,4. Here we investigate the ability of resident skeletal stem-cell (SSC) populations to regenerate cartilage in relation to age, a possible contributor to the development of osteoarthritis5,6,7. We demonstrate that aging is associated with progressive loss of SSCs and diminished chondrogenesis in the joints of both mice and humans. However, a local expansion of SSCs could still be triggered in the chondral surface of adult limb joints in mice by stimulating a regenerative response using microfracture (MF) surgery. Although MF-activated SSCs tended to form fibrous tissues, localized co-delivery of BMP2 and soluble VEGFR1 (sVEGFR1), a VEGF receptor antagonist, in a hydrogel skewed differentiation of MF-activated SSCs toward articular cartilage. These data indicate that following MF, a resident stem-cell population can be induced to generate cartilage for treatment of localized chondral disease in OA.
Data availabilityMicroarray data described in this study was deposited with the gene expression omnibus with the accession number GSE151303. There are no restrictions on sharing the data. All data are available from the corresponding author upon reasonable request.References1.Robinson, W. H. et al. Low-grade inflammation as a key mediator of the pathogenesis of osteoarthritis. Nat. Rev. Rheumatol. 12, 580–592 (2016).CAS
PubMed
PubMed Central
Google Scholar
2.Rahmati, M., Mobasheri, A. & Mozafari, M. Inflammatory mediators in osteoarthritis: a critical review of the state-of-the-art, current prospects, and future challenges. Bone 85, 81–90 (2016).CAS
PubMed
Google Scholar
3.Marques-Rocha, J. L. et al. Noncoding RNAs, cytokines, and inflammation-related diseases. FASEB J. 29, 3595–3611 (2015).CAS
PubMed
Google Scholar
4.Liu-Bryan, R. & Terkeltaub, R. Emerging regulators of the inflammatory process in osteoarthritis. Nat. Rev. Rheumatol. 11, 35–44 (2015).CAS
PubMed
Google Scholar
5.Hoemann, C. D. et al. Chondroinduction is the main cartilage repair response to microfracture and microfracture with BST-CarGel: results as shown by ICRS-II histological scoring and a novel zonal collagen type scoring method of human clinical biopsy specimens. Am. J. Sports Med. 43, 2469–2480 (2015).PubMed
Google Scholar
6.Mithoefer, K., McAdams, T., Williams, R. J., Kreuz, P. C. & Mandelbaum, B. R. Clinical efficacy of the microfracture technique for articular cartilage repair in the knee: an evidence-based systematic analysis. Am. J. Sports Med. 37, 2053–2063 (2009).PubMed
Google Scholar
7.Goldberg, A., Mitchell, K., Soans, J., Kim, L. & Zaidi, R. The use of mesenchymal stem cells for cartilage repair and regeneration: a systematic review. J. Orthop. Surg. Res. 12, 39 (2017).PubMed
PubMed Central
Google Scholar
8.Vos, T. et al. Years lived with disability (YLDs) for 1160 sequelae of 289 diseases and injuries 1990–2010: a systematic analysis for the global burden of disease study 2010. Lancet 380, 2163–2196 (2012).PubMed
PubMed Central
Google Scholar
9.Murray, C. J. et al. The state of US health, 1990–2010: burden of diseases, injuries, and risk factors. JAMA 310, 591–608 (2013).CAS
PubMed
PubMed Central
Google Scholar
10.Hootman, J. M., Helmick, C. G., Barbour, K. E., Theis, K. A. & Boring, M. A. Updated projected prevalence of self-reported doctor-diagnosed arthritis and arthritis-attributable activity limitation among US adults, 2015–2040. Arthritis Rheumatol. 68, 1582–1587 (2016).PubMed
PubMed Central
Google Scholar
11.Johnson, V. L. & Hunter, D. J. The epidemiology of osteoarthritis. Best. Pr. Res Clin. Rheumatol. 28, 5–15 (2014).
Google Scholar
12.Marshall, M., Watt, F. E., Vincent, T. L. & Dziedzic, K. Hand osteoarthritis: clinical phenotypes, molecular mechanisms and disease management. Nat. Rev. Rheumatol. 14, 641–656 (2018).PubMed
Google Scholar
13.Bijlsma, J. W., Berenbaum, F. & Lafeber, F. P. Osteoarthritis: an update with relevance for clinical practice. Lancet 377, 2115–2126 (2011).PubMed
Google Scholar
14.Steadman, J. R., Rodkey, W. G., Briggs, K. K. & Rodrigo, J. J. [The microfracture technic in the management of complete cartilage defects in the knee joint]. Orthopade 28, 26–32 (1999).CAS
PubMed
Google Scholar
15.Makris, E. A., Gomoll, A. H., Malizos, K. N., Hu, J. C. & Athanasiou, K. A. Repair and tissue engineering techniques for articular cartilage. Nat. Rev. Rheumatol. 11, 21–34 (2015).CAS
PubMed
Google Scholar
16.Knutsen, G. et al. Autologous chondrocyte implantation compared with microfracture in the knee. A randomized trial. J. Bone Jt. Surg. Am. 86-A, 455–464 (2004).
Google Scholar
17.Devitt, B. M., Bell, S. W., Webster, K. E., Feller, J. A. & Whitehead, T. S. Surgical treatments of cartilage defects of the knee: systematic review of randomised controlled trials. Knee 24, 508–517 (2017).PubMed
Google Scholar
18.Vanlauwe, J. et al. Five-year outcome of characterized chondrocyte implantation versus microfracture for symptomatic cartilage defects of the knee: early treatment matters. Am. J. Sports Med. 39, 2566–2574 (2011).PubMed
Google Scholar
19.Piuzzi, N. S. et al. Accelerated growth of cellular therapy trials in musculoskeletal disorders: an analysis of the NIH Clinical Trials Data Bank. Orthopedics 42, e144–e150 (2019).20.Sipp, D., Robey, P. G. & Turner, L. Clear up this stem-cell mess. Nature 561, 455–457 (2018).CAS
PubMed
Google Scholar
21.Jo, C. H. et al. Intra-articular injection of mesenchymal stem cells for the treatment of osteoarthritis of the knee: a proof-of-concept clinical trial. Stem Cells 32, 1254–1266 (2014).CAS
PubMed
Google Scholar
22.Vega, A. et al. Treatment of knee osteoarthritis with allogeneic bone marrow mesenchymal stem cells: a randomized controlled trial. Transplantation 99, 1681–1690 (2015).CAS
PubMed
Google Scholar
23.Pers, Y. M. et al. Adipose mesenchymal stromal cell-based therapy for severe osteoarthritis of the knee: a phase I dose–escalation trial. Stem Cells Transl. Med. 5, 847–856 (2016).PubMed
PubMed Central
Google Scholar
24.Worthley, D. L. et al. Gremlin 1 identifies a skeletal stem cell with bone, cartilage, and reticular stromal potential. Cell 160, 269–284 (2015).CAS
PubMed
PubMed Central
Google Scholar
25.Sacchetti, B. et al. Self-renewing osteoprogenitors in bone marrow sinusoids can organize a hematopoietic microenvironment. Cell 131, 324–336 (2007).CAS
PubMed
Google Scholar
26.Chan, C. K. et al. Identification and specification of the mouse skeletal stem cell. Cell 160, 285–298 (2015).CAS
PubMed
PubMed Central
Google Scholar
27.Chan, C. K. F. et al. Identification of the human skeletal stem cell. Cell 175, 43–56 e21 (2018).CAS
PubMed
PubMed Central
Google Scholar
28.Ambrosi, T. H., Longaker, M. T. & Chan, C. K. F. A revised perspective of skeletal stem cell biology. Front Cell Dev. Biol. 7, 189 (2019).PubMed
PubMed Central
Google Scholar
29.Luria, E. A., Owen, M. E., Friedenstein, A. J., Morris, J. F. & Kuznetsow, S. A. Bone formation in organ cultures of bone marrow. Cell Tissue Res. 248, 449–454 (1987).CAS
PubMed
Google Scholar
30.Gronthos, S., Simmons, P. J., Graves, S. E. & Robey, P. G. Integrin-mediated interactions between human bone marrow stromal precursor cells and the extracellular matrix. Bone 28, 174–181 (2001).CAS
PubMed
Google Scholar
31.Kratchmarova, I., Blagoev, B., Haack-Sorensen, M., Kassem, M. & Mann, M. Mechanism of divergent growth factor effects in mesenchymal stem cell differentiation. Science 308, 1472–1477 (2005).CAS
PubMed
Google Scholar
32.Marecic, O. et al. Identification and characterization of an injury-induced skeletal progenitor. Proc. Natl Acad. Sci. USA 112, 9920–9925 (2015).CAS
PubMed
Google Scholar
33.Jiang, Y. & Tuan, R. S. Origin and function of cartilage stem/progenitor cells in osteoarthritis. Nat. Rev. Rheumatol. 11, 206–212 (2015).PubMed
Google Scholar
34.Ransom, R. C. et al. Genetic dissection of clonal lineage relationships with hydroxytamoxifen liposomes. Nat. Commun. 9, 2971 (2018).PubMed
PubMed Central
Google Scholar
35.Loeser, R. F., Collins, J. A. & Diekman, B. O. Ageing and the pathogenesis of osteoarthritis. Nat. Rev. Rheumatol. 12, 412–420 (2016).CAS
PubMed
PubMed Central
Google Scholar
36.Gulati, G. S. et al. Isolation and functional assessment of mouse skeletal stem cell lineage. Nat. Protoc. 13, 1294–1309 (2018).CAS
PubMed
PubMed Central
Google Scholar
37.Eldracher, M., Orth, P., Cucchiarini, M., Pape, D. & Madry, H. Small subchondral drill holes improve marrow stimulation of articular cartilage defects. Am. J. Sports Med. 42, 2741–2750 (2014).PubMed
Google Scholar
38.Rinkevich, Y., Lindau, P., Ueno, H., Longaker, M. T. & Weissman, I. L. Germ-layer and lineage-restricted stem/progenitors regenerate the mouse digit tip. Nature 476, 409–413 (2011).CAS
PubMed
Google Scholar
39.Fang, H. & Beier, F. Mouse models of osteoarthritis: modelling risk factors and assessing outcomes. Nat. Rev. Rheumatol. 10, 413–421 (2014).PubMed
Google Scholar
40.Loeser, R. F. et al. Microarray analysis reveals age-related differences in gene expression during of harpagoside by upregulation of the BMP2 and Wnt signaling pathways in osteoblasts and suppression of differentiation in osteoclasts. J. Nat. Prod. 80, 434–442 (2017).
Google Scholar
41.Ransom, R. C. et al. Mechanoresponsive stem cells acquire neural crest fate in jaw regeneration. Nature 563, 514–521 (2018).CAS
PubMed
PubMed Central
Google Scholar
42.Ray, A., Singh, P. N., Sohaskey, M. L., Harland, R. M. & Bandyopadhyay, A. Precise spatial restriction of BMP signaling is essential for articular cartilage differentiation. Development 142, 1169–1179 (2015).CAS
PubMed
PubMed Central
Google Scholar
43.Bragdon, B. et al. Earliest phases of chondrogenesis are dependent upon angiogenesis during ectopic bone formation in mice. Bone 101, 49–61 (2017).CAS
PubMed
PubMed Central
Google Scholar
44.Urist, M. R. Bone: formation by autoinduction. Science 150, 893–899 (1965).CAS
PubMed
Google Scholar
45.Keeney, M. et al. Scaffold-mediated BMP-2 minicircle DNA delivery accelerated bone repair in a mouse critical-size calvarial defect model. J. Biomed. Mater. Res. A 104, 2099–2107 (2016).CAS
PubMed
PubMed Central
Google Scholar
46.Chung, H. J. et al. Anti-osteoporotic activity of harpagoside by upregulation of the BMP2 and Wnt signaling pathways in osteoblasts and suppression of differentiation in osteoclasts. J. Nat. Prod. 80, 434–442 (2017).CAS
PubMed
Google Scholar
47.Gonzalez-Fernandez, T., Tierney, E. G., Cunniffe, G. M., O’Brien, F. J. & Kelly, D. J. Gene delivery of TGF-beta3 and BMP2 in an MSC-laden alginate hydrogel for articular cartilage and endochondral bone tissue engineering. Tissue Eng. Part A 22, 776–787 (2016).CAS
PubMed
Google Scholar
48.Hamilton, J. L. et al. Targeting VEGF and its receptors for the treatment of osteoarthritis and associated pain. J. Bone Miner. Res. 31, 911–924 (2016).PubMed
PubMed Central
Google Scholar
49.Nagai, T. et al. Bevacizumab, an anti-vascular endothelial growth factor antibody, inhibits osteoarthritis. Arthritis Res Ther. 16, 427 (2014).PubMed
PubMed Central
Google Scholar
50.Tevlin, R. et al. Pharmacological rescue of diabetic skeletal stem cell niches. Sci. Transl. Med. 9, eaag2809 (2017).51.Adouni, M. & Dhaher, Y. Y. A multi-scale elasto-plastic model of articular cartilage. J. Biomech. 49, 2891–2898 (2016).PubMed
Google Scholar
52.Chandran, P. L., Dimitriadis, E. K., Mertz, E. L. & Horkay, F. Microscale mapping of extracellular matrix elasticity of mouse joint cartilage: an approach to extracting bulk elasticity of soft matter with surface roughness. Soft Matter 14, 2879–2892 (2018).CAS
PubMed
PubMed Central
Google Scholar
53.Kretzschmar, K. et al. Profiling proliferative cells and their progeny in damaged murine hearts. Proc. Natl Acad. Sci. USA 115, E12245–E12254 (2018).CAS
PubMed
Google Scholar
54.Nagao, M. et al. Vascular endothelial growth factor in cartilage development and osteoarthritis. Sci. Rep. 7, 13027 (2017).PubMed
PubMed Central
Google Scholar
55.Steinberg, J. & Zeggini, E. Functional genomics in osteoarthritis: past, present, and future. J. Orthop. Res. 34, 1105–1110 (2016).PubMed
PubMed Central
Google Scholar
56.Prockop, D. J. et al. Defining the risks of mesenchymal stromal cell therapy. Cytotherapy 12, 576–578 (2010).PubMed
Google Scholar
57.Jones, I. A., Togashi, R., Wilson, M. L., Heckmann, N. & Vangsness, C. T. Jr. Intra-articular treatment options for knee osteoarthritis. Nat. Rev. Rheumatol. 15, 77–90 (2019).PubMed
PubMed Central
Google Scholar
58.Conrad, B., Han, L. H. & Yang, F. Gelatin-based microribbon hydrogels accelerate cartilage formation by mesenchymal stem cells in three dimensions. Tissue Eng. Part A 24, 1631–1640 (2018).CAS
PubMed
PubMed Central
Google Scholar
59.Taylor, S. E. et al. Identification of human juvenile chondrocyte-specific factors that stimulate stem cell growth. Tissue Eng. Part A 22, 645–653 (2016).CAS
PubMed
Google Scholar
60.Glyn-Jones, S. et al. Osteoarthritis. Lancet 386, 376–387 (2015).CAS
PubMed
Google Scholar
61.Seita, J. et al. Gene Expression Commons: an open platform for absolute gene expression profiling. PLoS ONE 7, e40321 (2012).CAS
PubMed
PubMed Central
Google Scholar
Download referencesAcknowledgementsThe authors thank N. Williams, C. Queen, C. Crumpton, E. Kang, M. Butcher, L. Quinn, P. Perira and R. Mann for advice and assistance, and A. McCarty, M. Alvarez, and VSC Staff for animal model support. The authors also thank M. Ekhart, and the PAN facility at Stanford for help in gene-expression analysis. In addition, the authors thank S. Goodman and the Orthopedic Department of Stanford for donation of human adult specimens. The study was supported by NIH (R01 DE027323, R56 DE025597, R01 DE026730, R01 DE021683, R21 DE024230, U01HL099776, U24DE026914 and R21 DE019274), CIRMTR1-01249, Oak Foundation, Hagey Laboratory, Pitch Johnson Fund and Gunn/Olivier Research Fund to M.T. Longaker; Siebel Fellowship, PCF YI Award, Stinehart/Reed, and NIHNIAK99AG049958-01A1, American Federation of Aging Research and Arthritis National Research Foundation to C.K.F.C.; HHMI Fellowship to G.S.G.; the German Research Foundation (DFG) Fellowship 399915929 to T.H.A.; NIH, NIGMS R01GM123069, NIH1R01AR071379, ACS Clowes Award, Department of Defense CDMRP W81XWH-18-1-0653, OR170174 to B.L; PSRF National Endowment and Stanford TTE fellowship to M.P.M.; NIHS10 RR02933801 to Stanford Stem Cell FACS core. The project was supported, in part, by Award Number 1S10OD021514-01 from the National Center for Research Resources (NCRR). Its contents are solely the responsibility of the authors and do not necessarily represent the official views of the NCRR or the National Institutes of Health.Author informationAffiliationsInstitute for Stem Cell Biology and Regenerative Medicine, Stanford University School of Medicine, Stanford, CA, USAMatthew P. Murphy, Gunsagar S. Gulati, Owen Marecic, Ryan C. Ransom, Malachia Y. Hoover, Irving L. Weissman, Michael T. Longaker & Charles K. F. ChanDepartment of Surgery, Stanford University School of Medicine, Stanford, CA, USAMatthew P. Murphy, Lauren S. Koepke, Michael T. Lopez, Thomas H. Ambrosi, Yuting Wang, Ryan C. Ransom, Holly Steininger, Liming Zhao, Natalina Quarto, Derrick C. Wan, Michael T. Longaker & Charles K. F. ChanBlond McIndoe Laboratories, Division of Cell Matrix Biology and Regenerative Medicine, School of Biological Sciences, Faculty of Biology, Medicine and Health, The University of Manchester, Manchester Academic Health Science Centre, Manchester, UKMatthew P. MurphyDepartment of Bioengineering, Stanford University, Palo Alto, CA, USAXinming Tong, Stuart B. Goodman & Fan YangDepartment of Orthopaedic Surgery, Tongji Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, ChinaYuting Wang & Liming ZhaoDepartment of Stanford Nano Shared Facilities, Stanford University, Palo Alto, CA, USAMarcin P. WalkiewiczDepartment of Surgery, University of Michigan, Ann Arbor, MI, USABenjamin LeviDepartment of Orthopaedic Surgery, Stanford University, Palo Alto, CA, USAStuart B. Goodman & Fan YangContributionsConception and design was done by M.P.M., I.L.W., M.T. Longaker and C.K.F.C. Experiments, data analysis and data interpretation were carried out by M.P.M., L.S.K., M.T. Lopez, T.H.A., G.S.G., O.M., Y.W., R.C.R., M.Y.H., H.S., and L.Z. Synthesis of hydrogels was carried out by X.T. and F.Y. Atomic Force Microscopy training and assistance was provided by M.P.W. Drafting and revision of the manuscript was carried out by M.P.M., L.S.K., N.Q., B.L., D.C.W., S.B.G., M.T. Longaker and C.F.K.C. All authors received and approved the final document.Corresponding authorsCorrespondence to
Michael T. Longaker or Charles K. F. Chan.Ethics declarations
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The authors declare no competing interests.
Additional informationPeer review information Michael Basson and Brett Benedetti were the primary editors on this article and managed its editorial process and peer review in collaboration with the rest of the editorial team.Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.Extended dataExtended Data Fig. 1 Mouse microarray data analysis.a, Pathways significantly enriched in Ad/MF and P3 mSSC populations. Top, (from left-right) Protein secretion, MYC targets, E2F targets. Bottom, (from left-right) G2M checkpoint, oxidative phosphorylation and TGFb signaling. b-c, mSSC expression (b) and bulk tissue expression (c) of key genes involved in cartilage development, cell cycle and inflammation. d, mSSC gene expression levels after in vitro treatment of PBS, BMP2, or sVEGFR1.Extended Data Fig. 2 In vitro assays for groups (N, MF).a, Alizarin red staining of uninjured/normal adult (N) and adult MF mSSCs (left) and mBCSPs (right), and quantification of the alizarin red stain after extraction and photometric measurement at OD450. (n = 3 mice per group). * 0.032; ** 0.0014. b, Alcian blue staining of N and MF mSSCs (left) and mBCSPs (right), and quantification of the Alcian blue stain after extraction and photometric measurement at OD595. (n = 3 mice per group). ** 0.0010; *** 0.0004. c, Oil red O staining of Ad and Ad/MF mSSCs (Left) and mBCSPs (Right). Scale bar 100μm. (n = 3 mice per group). Graphs show mean + /- s.e.m. Two-tailed Student’s t-test. Exact P values to 2 significant figures.Extended Data Fig. 3 Microdissection before and after MF surgery.a, Schematic depicting the distal femur of a mouse and its major tissue layers before and after MF surgery. b, Representative flow cytometry analysis of the articular surface (top), the periosteum (middle), and bone (bottom) with relative populations of mOP, mCP, mBCSP, and mSSC. c, Quantification of mSSC/million events in bone, cartilage, and periosteum. Graph shows mean + /- s.e.m. Ordinary one-way ANOVA test with post-hoc analysis using Šídák method to compare between specific means. Exact P values to 2 significant figures. (n = 5 mice per group). d, Quantification of mSSC/million events comparing MF joints and joints with articular cartilage and periosteum removed prior to MF surgery (mMF). (n = 5 mice per group). e, Alcian blue staining of mSSC following chondrogenic differentiation with treatment of PBS or BMP2 + sVEGFR1. Scale bars 100μm. Representative of three independent experiments. f, Quantification of the Alcian blue stain after extraction and photometric measurement at OD595. Unless otherwise stated, graphs show mean + /- s.e.m. Two-tailed Student’s t-test. Exact P values to 2 significant figures.Extended Data Fig. 4 OA progression.a, Schematic of the experimental outline. Destabilization of the medial meniscus (DMM) was performed on adult mice, and the distal femurs were harvested after 8 weeks. Analysis was performed by IF, pentachrome, and Safranin-O Fast Green staining. b, Left, representative IF of indicated markers in sham and DMM joints. Scale bars 100μm. Right, Safranin-O Fast Green and pentachrome stains of sham and joints 8 weeks after DMM. Scale bars 500μm. (n = 3 per group). c, Quantification of IF stains in sham versus DMM. Graphs show mean + /- s.e.m. Two-tailed Student’s t-test. Exact P values to 2 significant figures. (n = 3 per group).Extended Data Fig. 5 IHC and quantification of OA/MF with factors at 4 weeks.a, Representative IF of (from left-right) ACAN, COL 1, COL 2, COL 10 and MMP 13 in 3 treatment conditions (top: PBS, middle: BMP2, bottom: BMP2 + sVEGFR1) at 4 weeks after MF. Scale bars 250μm. (n = 8 per group). b, Quantification of IF stains for indicated markers of MF + factors at 4 weeks. Graphs show mean + /- s.e.m. Ordinary one-way ANOVA test with post-hoc analysis using Šídák method to compare between specific means. Exact P values to 2 significant figures. c, Representative IF isotype and positive controls. (n = 3 per group). d, Repeat examples of pentachrome staining of BMP2 + sVEGFR1 sections at low (scale bars 500μm) and high (scale bars 100μm) magnification. Representatives of eight independent experiments.Extended Data Figure 6 Histology of OA/MF at 2 weeks and 8 weeks.a, Representative pentachrome stains and respective IF of (from left-right) ACAN, COL 1, COL 2, COL 10 and MMP 13 in 3 treatment conditions (top: PBS; middle: BMP2; bottom: BMP2 + sVEGFR1) at 2 weeks after MF. Scale bars 500μm. (n = 4 per group). b, Representative pentachrome stains and respective IF stains for the indicated markers in 3 treatment conditions (top row: PBS; middle: BMP2; bottom: BMP2 + sVEGFR1) at 8 weeks after MF. Scale bars 500μm. (n = 4 per group). c, Quantification of IF stains for the indicated markers at 2 weeks (top) and 8 weeks (bottom) after MF. (n = 3 per group per timepoint). Graphs show mean + /- s.e.m. Ordinary one-way ANOVA test with post-hoc analysis using Šídák method to compare between specific means. Exact P values to 2 significant figures.Extended Data Fig. 7 Week 16 timepoint.a, Representative pentachrome and respective IF of (from left-right) ACAN, COL 1, COL 2, COL 10 and MMP 13 in 3 treatment conditions (top: PBS, middle: BMP2, bottom: BMP2 + sVEGFR1). Scale bars 500μm. (n = 4 per group). b, Quantification of IF stains for indicated markers of MF + factors. Graphs show Mean + /- SEM. Ordinary one-way ANOVA test with post-hoc analysis using Šídák method to compare between specific means. Exact P values to 2 significant figures. (n = 3 per group). c, From left-right, gross images of distal femur (scale bars 1 mm); respective pentachrome (scale bars 500μm); 3D Peak Force Error; 3D Deformation; Force Volume in 4 conditions (PBS, BMP2, BMP2 + sVEGFR1, Uninjured). (n = 4 per group).Extended Data Fig. 8 Effects of aged and irradiated niches.a, Quantification of mSSC/million events in normal and MF joints of adult (9wks) and aged (1 yr) mice. Graph shows mean + /- s.e.m. Two-tailed Student’s t-test. Exact P values to 2 significant figures. (n = 10 mice per group). b, Representative pentachrome stains of 2-weeks post MF surgery in aged joints with (from left-right) PBS, BMP2, and BMP2 + sVEGFR1. Scale bar 500μm. (n = 3 per group). c, Schematic of the experimental outline. Microfracture surgery is performed on the joints of GFP + adult mice (Ad/MF). GFP + mSSCs are sorted by flow cytometry and cotransplanted with BMP2 + sVEGFR1 into MF defects of irradiated mice. Analysis is performed by brightfield microscopy (BF), immunofluorescence (IF), and pentachrome staining. d, Top, Representative BF (left) and fluorescence microscopy (right) of GFP + Ad/MF mSSC 4 weeks after they were transplanted into the MF defect of an irradiated joint. Scale bars 500μm. (n = 5 mice). Middle, Representative pentachrome stain (scale bar 500μm) shown with higher magnification (scale bar 100μm) and respective IF of GFP + Ad/MF mSSC 4 weeks after they were transplanted into the MF defect of an irradiated joint. Positive staining marked by white arrows. Representatives of three separate experiments. Bottom right, a negative control IF stain was performed for each experiment. Scale bars 500μm.Extended Data Fig. 9 Atomic Force Microscopy at 8 weeks post MF.a, Left and middle, schematic of Rheology experimental outline. Right, Quantification for the modulus of the regenerate (PBS, BMP2, BMP2 + sVEGFR1.) Graph shows mean + /- s.e.m. Ordinary one-way ANOVA test (p =
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