Activation of the Hh Pathway in Periosteum-Derived Mesenchymal Stem Cells Induces Bone Formation in Vivo
2010; Elsevier BV; Volume: 177; Issue: 6 Linguagem: Inglês
10.2353/ajpath.2010.100060
ISSN1525-2191
AutoresQun Wang, Chunlan Huang, Fanjie Zeng, Ming Xue, Xinping Zhang,
Tópico(s)Genetic Syndromes and Imprinting
ResumoWhile the essential role of periosteum in cortical bone repair and regeneration is well established, the molecular pathways that control the early osteogenic and chondrogenic differentiation of periosteal stem/progenitor cells during repair processes are unclear. Using a murine segmental bone graft transplantation model, we isolated a population of early periosteum-callus-derived mesenchymal stem cells (PCDSCs) from the healing autograft periosteum. These cells express typical mesenchymal stem cell markers and are capable of differentiating into osteoblasts, adipocytes, and chondrocytes. Characterization of these cells demonstrated that activation of the hedgehog (Hh) pathway effectively promoted osteogenic and chondrogenic differentiation of PCDSCs in vitro and induced bone formation in vivo. To determine the role of the Hh pathway in adult bone repair, we deleted Smoothened (Smo), the receptor that transduces all Hh signals at the onset of bone autograft repair via a tamoxifen-inducible RosaCreER mouse model. We found that deletion of Smo markedly reduced osteogenic differentiation of isolated PCDSCs and further resulted in a near 50% reduction in periosteal bone callus formation at the cortical bone junction as determined by MicroCT and histomorphometric analyses. These data strongly suggest that the Hh pathway plays an important role in adult bone repair via enhancing differentiation of periosteal progenitors and that activation of the Hh pathway at the onset of healing could be beneficial for repair and regeneration. While the essential role of periosteum in cortical bone repair and regeneration is well established, the molecular pathways that control the early osteogenic and chondrogenic differentiation of periosteal stem/progenitor cells during repair processes are unclear. Using a murine segmental bone graft transplantation model, we isolated a population of early periosteum-callus-derived mesenchymal stem cells (PCDSCs) from the healing autograft periosteum. These cells express typical mesenchymal stem cell markers and are capable of differentiating into osteoblasts, adipocytes, and chondrocytes. Characterization of these cells demonstrated that activation of the hedgehog (Hh) pathway effectively promoted osteogenic and chondrogenic differentiation of PCDSCs in vitro and induced bone formation in vivo. To determine the role of the Hh pathway in adult bone repair, we deleted Smoothened (Smo), the receptor that transduces all Hh signals at the onset of bone autograft repair via a tamoxifen-inducible RosaCreER mouse model. We found that deletion of Smo markedly reduced osteogenic differentiation of isolated PCDSCs and further resulted in a near 50% reduction in periosteal bone callus formation at the cortical bone junction as determined by MicroCT and histomorphometric analyses. These data strongly suggest that the Hh pathway plays an important role in adult bone repair via enhancing differentiation of periosteal progenitors and that activation of the Hh pathway at the onset of healing could be beneficial for repair and regeneration. Periosteum plays a key role in repair and regeneration via activation of multipotent mesenchymal stem cells (MSCs).1Allen MR Hock JM Burr DB Periosteum: biology, regulation, and response to osteoporosis therapies.Bone. 2004; 35: 1003-1012Abstract Full Text Full Text PDF PubMed Scopus (295) Google Scholar, 2Wlodarski KH Normal and heterotopic periosteum.Clin Orthop Relat Res. 1989; : 265-277PubMed Google Scholar After fracture or osteotomy, periosteum undergoes a unique morphogenic process that, to some extent, recapitulates the fundamental features of bone and cartilage differentiation in the developing limb.3Ferguson C Alpern E Miclau T Helms JA Does adult fracture repair recapitulate embryonic skeletal formation?.Mech Dev. 1999; 87: 57-66Crossref PubMed Scopus (443) Google Scholar, 4Sandell LJ Adler P Developmental patterns of cartilage.Front Biosci. 1999; 4: D731-D742Crossref PubMed Google Scholar Within a few days after injury, periosteum becomes thickened due to activation and proliferation of the residing mesenchymal stem/progenitor cells. These stem/progenitor cells proceed to differentiate into osteoblasts or chondrocytes and eventually form bone via both intramembranous and endochondral pathways. Endochondral bone formation takes place at the cortical bone junctions where the blood supply is poor. 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Because the amino acid sequence is highly conserved, Hh ligands are functionally interchangeable. 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Ptc represses the activity of Smo, which regulates proteolytic processing of downstream zinc-finger transcription factors Gli1, 2, and 3. 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A comprehensive knowledge of these pathways in bone repair will be important to the optimization of future pathway-targeted strategies for treatment of bony defects. The lack of a defined phenotype and cellular markers for mesenchymal progenitor cells significantly hampers the efforts to track cell fate and further understand the molecular pathways underlying the expansion and differentiation of periosteal progenitor cells during repair processes. We previously established a segmental bone graft transplantation model in mice that permits molecular and cellular analyses of periosteum's contribution to bone graft healing and incorporation.31Tiyapatanaputi P Rubery PT Carmouche J Schwarz EM O'Keefe RJ Zhang X A novel murine segmental femoral graft model.J Orthop Res. 2004; 22: 1254-1260Crossref PubMed Scopus (74) Google Scholar Using this model, we tracked periosteal cell fate and examined the unique periosteum tissue morphogenesis during bone graft healing.32Xie C Reynolds D Awad H Rubery PT Pelled G Gazit D Guldberg RE Schwarz EM O'Keefe RJ Zhang X Structural bone allograft combined with genetically engineered mesenchymal stem cells as a novel platform for bone tissue engineering.Tissue Eng. 2007; 13: 435-445Crossref PubMed Scopus (103) Google Scholar, 33Zhang X Xie C Lin AS Ito H Awad H Lieberman JR Rubery PT Schwarz EM O'Keefe RJ Guldberg RE Periosteal progenitor cell fate in segmental cortical bone graft transplantations: implications for functional tissue engineering.J Bone Miner Res. 2005; 20: 2124-2137Crossref PubMed Scopus (270) Google Scholar, 34Zhang X Awad HA O'Keefe RJ Guldberg RE Schwarz EM A perspective: engineering periosteum for structural bone graft healing.Clin Orthop Relat Res. 2008; 466: 1777-1787Crossref PubMed Scopus (176) Google Scholar We found that the expansion and further differentiation of the progenitor cells accounted for about 70% of bone and cartilage formation during the initiation stage of healing. Although these cells were eventually remodeled and replaced by host bone, the formation of donor cell–derived callus was essential for the initiation of bone graft healing. The removal of the donor periosteum resulted in the elimination of bone formation and marked impairment of bone graft healing.33Zhang X Xie C Lin AS Ito H Awad H Lieberman JR Rubery PT Schwarz EM O'Keefe RJ Guldberg RE Periosteal progenitor cell fate in segmental cortical bone graft transplantations: implications for functional tissue engineering.J Bone Miner Res. 2005; 20: 2124-2137Crossref PubMed Scopus (270) Google Scholar Further engraftment of multipotent mesenchymal stem cells on devitalized bone allografts improves healing and graft incorporation.32Xie C Reynolds D Awad H Rubery PT Pelled G Gazit D Guldberg RE Schwarz EM O'Keefe RJ Zhang X Structural bone allograft combined with genetically engineered mesenchymal stem cells as a novel platform for bone tissue engineering.Tissue Eng. 2007; 13: 435-445Crossref PubMed Scopus (103) Google Scholar, 33Zhang X Xie C Lin AS Ito H Awad H Lieberman JR Rubery PT Schwarz EM O'Keefe RJ Guldberg RE Periosteal progenitor cell fate in segmental cortical bone graft transplantations: implications for functional tissue engineering.J Bone Miner Res. 2005; 20: 2124-2137Crossref PubMed Scopus (270) Google Scholar With the goal of understanding the molecular pathways that regulate periosteal mesenchymal cell differentiation during cortical repair, in this study we isolated a population of early periosteum-derived mesenchymal stem cells (PCDSCs) from the healing bone autografts. These isolated cells express the typical mesenchymal stem cell markers (Sca-I, CD105, SSEA-4, CD29, and CD140) and are capable of differentiating into osteoblasts, adipocytes, and chondrocytes. Further characterization of these cells demonstrated that activation of hedgehog signaling was extremely effective in driving bone formation in vivo. Furthermore, using a Tamoxifen (TM)–inducible RosaCreER mouse model that allows efficient deletion of genes during initiation of adult repair,35Badea TC Wang Y Nathans J A noninvasive genetic/pharmacologic strategy for visualizing cell morphology and clonal relationships in the mouse.J Neurosci. 2003; 23: 2314-2322PubMed Google Scholar, 36Xie C Xue M Wang Q Schwarz EM O'Keefe RJ Zhang X Tamoxifen-inducible CreER-mediated gene targeting in periosteum via bone-graft transplantation.J Bone Joint Surg Am. 2008; 90: 9-13Crossref PubMed Scopus (11) Google Scholar we demonstrated that disruption of Hh signaling by postnatal deletion of Smo significantly reduced periosteal bone callus formation. Smof/f, Ptch1-LacZ heterozygotes, RosaCreER, GFP transgenic mice were purchased from the Jackson Laboratory (Bar Harbor, ME). Immunocompromised mice (bg-nu/nu-xid) were purchased from Harlan Sprague Dawley Inc (Indianapolis, IN). All animal surgeries were performed on 8- to 10-week-old adult mice. All procedures were approved by the Institutional Committee of Animal Resources. The Hh agonist Purmorphamine (Cayman Chemicals, Ann Arbor, MI) was used at 2 μmol/L in all in vitro experiments. Hh inhibitor Cyclopamine was used at indicated doses according to the manufacturer (Toronto Research Chemical Inc., North York, Ontario). Adenovirus encoding green fluorescence protein (Ad-GFP) and N-terminal peptide of human Shh (ShhN)37Lou H Crystal RG Leopold PL Enhanced efficacy of cholesterol-minus sonic hedgehog in postnatal skin.Mol Ther. 2005; 12: 575-578Abstract Full Text Full Text PDF PubMed Scopus (7) Google Scholar were kindly provided by Dr. Philip Leopold (Weill Medical College of Cornell University, New York, NY). Adenovirus encoding recombinant human BMP-2 was purified as previously described.33Zhang X Xie C Lin AS Ito H Awad H Lieberman JR Rubery PT Schwarz EM O'Keefe RJ Guldberg RE Periosteal progenitor cell fate in segmental cortical bone graft transplantations: implications for functional tissue engineering.J Bone Miner Res. 2005; 20: 2124-2137Crossref PubMed Scopus (270) Google Scholar Tamoxifen (TM) (Sigma-Aldrich, St. Louis, MO) was administered via peritoneal injection at a dose of 1 mg/10 g of body weight. Tamoxifen (TM) was injected every other day before and after surgery for a maximum of 5 doses.36Xie C Xue M Wang Q Schwarz EM O'Keefe RJ Zhang X Tamoxifen-inducible CreER-mediated gene targeting in periosteum via bone-graft transplantation.J Bone Joint Surg Am. 2008; 90: 9-13Crossref PubMed Scopus (11) Google Scholar, 38Chen M Lichtler AC Sheu TJ Xie C Zhang X O'Keefe RJ Chen D Generation of a transgenic mouse model with chondrocyte-specific and tamoxifen-inducible expression of Cre recombinase.Genesis. 2007; 45: 44-50Crossref PubMed Scopus (112) Google Scholar The femoral bone graft transplantation was performed as previously described.33Zhang X Xie C Lin AS Ito H Awad H Lieberman JR Rubery PT Schwarz EM O'Keefe RJ Guldberg RE Periosteal progenitor cell fate in segmental cortical bone graft transplantations: implications for functional tissue engineering.J Bone Miner Res. 2005; 20: 2124-2137Crossref PubMed Scopus (270) Google Scholar, 39Ito H Koefoed M Tiyapatanaputi P Gromov K Goater JJ Carmouche J Zhang X Rubery PT Rabinowitz J Samulski RJ Nakamura T Soballe K O'Keefe RJ Boyce BF Schwarz EM Remodeling of cortical bone allografts mediated by adherent rAAV-RANKL and VEGF gene therapy.Nat Med. 2005; 11: 291-297Crossref PubMed Scopus (239) Google Scholar, 40Xie C Ming X Wang Q Schwarz EM Guldberg RE O'Keefe RJ Zhang X COX-2 from the injury milieu is critical for the initiation of periosteal progenitor cell mediated bone healing.Bone. 2008; 43: 1075-1083Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar Briefly, 10-week-old recipient mice were anesthetized by peritoneal injection of a mix of 10 mg/ml Ketamine and 1 mg/ml Xylazine. A 4-mm mid-diaphyseal segment was removed from the femur of the donor mice using a sharp diamond-cutting wheel attached to a cordless Dremel (Dremel Minimite model 750). Grafts were carefully dissected to remove the muscles without compromising the periosteum and immediately used to reconstruct a 4-mm segmental defect in the same mouse (autograft) or mice of the same strain (isograft). In the indicated experiments, we used live bone grafts from GFP transgenic mice as donor and the wild-type littermates as recipients. The bone graft was stabilized using a 25G stainless pin placed through intramedullary marrow cavity. To isolate mesenchymal progenitor cells from autograft periosteum, live bone isograft transplantation was performed between GFP transgenic mice and their wild-type littermates. Donor GFP+ grafts were collected at day 5 posttransplantation. Bone marrow was removed by repeated flushing of the marrow cavities with serum-free α-MEM medium. Tissues attached to the periosteal surface of the donor grafts were scraped off and pooled in a Petri dish. After digestion with Collagenase D (Roche Applied Science, Indianapolis, IN) at a concentration of 1 mg/ml for 1 hour, all cells were pooled and suspended in α-MEM medium containing 1% penicillin and streptomycin, 1% glutamine, and 20% fetal bovine serum (FBS). Cells were allowed to adhere to the culture dish and grow to confluence. Cells collected from the second and third passage were used for differentiation assay and fluorescence activated cell sorting (FACS) analyses for mesenchymal stem cell surface markers. In other indicated experiments, autograft transplantations were performed in various mouse models including Smof/f and Smof/f;CreER mice. Periosteal progenitors were subsequently isolated from the donor graft periosteum on day 5 postgrafting. Cells were suspended in phosphate-buffered saline containing 4% FBS. Phycoerythrin-coupled antibodies against CD34, CD45, CD133, CD105, SSEA-4, CD29, CD140 were used to stain cell surface markers. FACS analyses were performed using FACS Calibur at the Flow Cytometry Core facility. In some indicated experiments, GFP+ cells were sorted to 99% purity and used for differentiation analyses. For adipogenesis assay, cells were cultured with αMEM medium containing horse serum (12.5%), FBS (12.5%), penicillin/streptomycin (50 U/ml), and hydrocortisone (10−4 mol/L). Cultures were harvested on day 10 for Oil Red O staining and RNA analyses to examine PPARγ2 and CEBPβ gene expression. For osteogenesis assay, cells were cultured with fresh osteogenic differentiation media containing 10 mmol/L β-glycerolphosphate and 50 μmol/L ascorbic acid. Cells were harvested at indicated times for alkaline phosphatase (ALP) staining and gene analyses. For chondrogenesis assay, a total of 2 × 105 cells in 10 μl of media were placed as micromass in the center of a 24-well plate and incubated in 5% CO2 at 37°C for 1 hour. Basic chondrogenic differentiation medium containing high glucose DMEM supplemented with 1% ITS-Premix, L-ascorbic acid-2-phosphate (0.1 mmol/L), dexamethasone (1 × 107 mol/L), proline (400 μg/ml) and TGFβ (10 ng/ml) was added. Pellets were harvested on day 7 for Alcian Blue staining. A total of four pellets from each group were pooled for RNA analyses. In indicated experiments, adenovirus expressing human BMP-2 or ShhN was used to infect isolated cells before the micromass culture. PCDSCs were isolated from the donor autografts of GFP transgenic mice. Cells were infected with control adenovirus Ad-GFP or Ad-ShhN at a multiplicity of infection (MOI) of 10 or 100. A total of 1 × 106 cells were seeded onto a collagen scaffold (BD Bioscience, Franklin Lakes, NJ) and implanted subcutaneously into athymic nude mice as previously described.41Krebsbach PH Kuznetsov SA Satomura K Emmons RV Rowe DW Robey PG Bone formation in vivo: comparison of osteogenesis by transplanted mouse and human marrow stromal fibroblasts.Transplantation. 1997; 63: 1059-1069Crossref PubMed Scopus (442) Google Scholar, 42Shi S Gronthos S Chen S Reddi A Counter CM Robey PG Wang CY Bone formation by human postnatal bone marrow stromal stem cells is enhanced by telomerase expression.Nature Biotechnol. 2002; 20: 587-591Crossref PubMed Scopus (317) Google Scholar Implants were harvested 8 weeks after surgery, and bone formation was determined by microCT and histology. To determine the incorporation of donor GFP+ progenitors into bone tissue, implants were fixed in 4% paraformaldehyde followed by decalcification in 10% EDTA for 2 weeks. Cryosections were prepared and GFP+ cells were visualized and photographed using a Zeiss Axio Imager. 35S-UTP–labeled sense and antisense riboprobes against murine Shh or Ihh genes were synthesized from a plasmid (kindly provided by Dr. Matthew Hilton) as previously described.43Zhang X Schwarz EM Young DA Puzas JE Rosier RN O'Keefe RJ Cyclooxygenase-2 regulates mesenchymal cell differentiation into the osteoblast lineage and is critically involved in bone repair.J Clin Invest. 2002; 109: 1405-1415Crossref PubMed Scopus (598) Google Scholar, 44Xie C Liang B Xue M Lin AS Loiselle A Schwarz EM Guldberg RE O'Keefe RJ Zhang X Rescue of impaired fracture healing in COX-2-/- mice via activation of prostaglandin E2 receptor subtype 4.Am J Pathol. 2009; 175: 772-785Abstract Full Text Full Text PDF PubMed Scopus (79) Google Scholar The specific activity of the probes was determined by radioactivity. The sections were incubated in hybridization buffer (50% formamide, 0.3 M NaCl, 20 mmol/L Tris-HCl, 5 mmol/L EDTA, 10% dextran sulfate, 0.02% Ficoll, 0.02% BSA, 0.02% polyvinyl pyrrolidone, and 0.5 mg/ml yeast RNA) containing riboprobe at 10,000 cpm/μl. Hybridization was performed at 55°C overnight. Emulsion-dipped slides were exposed for about 7–14 days depending on the intensity of the signals. Anti-sense probes of Ihh were further used as controls for the experiment. Immunohistochemical staining for the nuclear antigen PCNA protein was performed using a staining kit purchased from Zymed (S. San Francisco, CA). The staining procedures were followed as instructed by the manufacturer. To determine the number of PCNA+ cells, three sections of each sample were used to count the positive cells in periosteal callus. The mean of the three sections represented each sample. A group of three samples was included for each time point. The mean from three samples divided by the average of the area of the periosteal callus was used in statistical analyses. Total RNA was prepared using a Qiagen RNA extraction kit. Exactly 0.5 μg of mRNA per callus was pooled and used in reverse transcription to make single-strand cDNA. Single-strand cDNA was synthesized using a commercial first strand cDNA synthesis kit (Invitrogen). Quantitative RT-PCR reaction was performed using SyberGreen (ABgene, Rochester, NY) in a RotorGene real time PCR machine (Corbett Research, Carlsbad, CA). All genes were compared to a standard β-actin control. Data were assessed quantitatively using analysis of variance comparing relative levels of transcript expression as a function of time. All primers used for the assessment can be found in the Table 1.Table 1PrimersGenesPrimer forwardPrimer reverseMurine actin5′-AGATGTGGATCAGCAAGCAG-3′5′-GCGCAAGTTAGGTTTTGTCA-3′ Smo5′-GACCACTCCCATAAGGGCTA-3′5′-GAAGAGGTTGGCCTAGTGGA-3′ Col X5′-CTTTGTGTGCCTTTCAATCG-3′5′-GTGAGGTACAGCCTACCAGTTTT-3′ Col II5′-CCACACCAAATTCCTGTTCA-3′5′-ACTGGTAAGTGGGGCAAGAC-3′ PPARr5′-AGGCCGAGAAGGAGAAGCTGTTG-3′5′-TGGCCACCTCTTTGCTCTGCTC-3′ C/EBP a5′-GAGCGCCATCGACTTCAG-3′5′-CTTGCGCACAGCGATGTT-3′ Osx5′-ACTGGCTAGGTGGTGGTCAG-3′5′-GGTAGGGAGCTGGGTTAAGG-3′ Bmp-25′-TGGAAGTGGCCCATTTAGAG-3′5′-GCTTTTCTCGTTTGTGGAGC-3′ OC5′-AGGGAGGATCAAGTCCCG-3′5′-GAACAGACTCCGGCGCTA-3′ ALP5′-TTGTGCGAGAGAAAGAGAGAGA-3′5′-GTTTCAGGGCATTTTTCAAGGT-3′ Ptc15′-TTCTGCTGCCTGTCCTCTTA-3′5′-GCAAACCGGACGACACTT-3′ Gli15′-TTGTCCAGCTTGGATGAAGG-3′5′-CCCAGACGGCGAGACAC-3′ SFRP-15′-CCCTCCAAGGCTTGAGTAAAAG-3′5′-AGCACATGCATAGGCGGTGTA-3′ SFRP-25′-ATCCGCAAGCTGCAATGCTA-3′5′-TGTGCTTGGGAAACCGGAAA-3′Human Bmp25′-CCTCAGCAGAGCTTCAGGTT-3′5′-TGCTTGCATTCTGATTCACC-3′ Shh5′-ACTCCGAGCGATTTAAGGAACT-3′5′-CAGACGTGGTGATGTCCACTG-3′ Open table in a new tab Femurs were harvested at day 17 postgrafting and scanned using a Viva microCT system (Scanco Medical, Bassersdorf, Switzerland) at a voxel size of 10.5 μm to image bone. New bone formation was measured as previously described.33Zhang X Xie C Lin AS Ito H Awad H Lieberman JR Rubery PT Schwarz EM O'Keefe RJ Guldberg RE Periosteal progenitor cell fate in segmental cortical bone graft transplantations: implications for functional tissue engineering.J Bone Miner Res. 2005; 20: 2124-2137Crossref PubMed Scopus (270) Google Scholar, 40Xie C Ming X Wang Q Schwarz EM Guldberg RE O'Keefe RJ Zhang X COX-2 from the injury milieu is critical for the initiation of periosteal progenitor cell mediated bone healing.Bone. 2008; 43: 1075-1083Abstract Full Text Full Text PDF PubMed Scopus (56) Google Scholar The threshold was chosen using 2D evaluation of several slices in the transverse anatomical plane so that mineralized callus were identified while surrounding soft tissue was excluded. An average threshold of 220 was optimal and used uniformly for all samples. Each sample was contoured around the external callus and along the edge of the cortical bone, excluding the marrow cavity. New bone volume was measured on the surface of the host and donor side in bone autografted samples as previously described.33Zhang X Xie C Lin AS Ito H Awad H Lieberman JR Rubery PT Schwarz EM O'Keefe RJ Guldberg RE Periosteal progenitor cell fate in segmental
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