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• W1973104883 abstract "Protease-activated receptor (PAR)-1, a G-protein-coupled receptor activated by thrombin, mediates thrombin-induced proliferation of osteoblasts. The current study was undertaken to define the role of PAR-1 in bone repair. Holes were drilled transversely through the diaphysis of both tibiae of PAR-1-null and wild-type mice. Three days later, fewer cells had invaded the drill site from adjacent bone marrow in PAR-1-null mice than in wild-type mice, and a lower percentage of cells were labeled with [3H]thymidine in PAR-1-null drill sites. More osteoclasts were also observed in the drill site of PAR-1-null mice than in wild-type mice 7 days after drilling. New mineralized bone area was less in the drill site and on the adjacent periosteal surface in PAR-1-null mice than in wild-type mice at day 9. From day 14, no obvious differences could be seen between PAR-1-null and wild-type tibiae. In vitro thrombin caused a dose-dependent increase in proliferation of bone marrow stromal cells isolated from wild-type mice but not PAR-1-null mice. Thrombin stimulated survival of bone marrow stromal cells from both wild-type and PAR-1-null mice, but it did not affect bone marrow stromal cell migration in either wild-type or PAR-1-null cells. The results indicate that PAR-1 plays an early role in bone repair. Protease-activated receptor (PAR)-1, a G-protein-coupled receptor activated by thrombin, mediates thrombin-induced proliferation of osteoblasts. The current study was undertaken to define the role of PAR-1 in bone repair. Holes were drilled transversely through the diaphysis of both tibiae of PAR-1-null and wild-type mice. Three days later, fewer cells had invaded the drill site from adjacent bone marrow in PAR-1-null mice than in wild-type mice, and a lower percentage of cells were labeled with [3H]thymidine in PAR-1-null drill sites. More osteoclasts were also observed in the drill site of PAR-1-null mice than in wild-type mice 7 days after drilling. New mineralized bone area was less in the drill site and on the adjacent periosteal surface in PAR-1-null mice than in wild-type mice at day 9. From day 14, no obvious differences could be seen between PAR-1-null and wild-type tibiae. In vitro thrombin caused a dose-dependent increase in proliferation of bone marrow stromal cells isolated from wild-type mice but not PAR-1-null mice. Thrombin stimulated survival of bone marrow stromal cells from both wild-type and PAR-1-null mice, but it did not affect bone marrow stromal cell migration in either wild-type or PAR-1-null cells. The results indicate that PAR-1 plays an early role in bone repair. Thrombin is a serine protease, which plays a central role in blood coagulation through its cleavage of fibrinogen, but also exerts specific receptor-mediated effects on cell function. Three thrombin receptors have been identified, protease-activated receptors (PARs)-1, -3, and -4, which are members of the seven transmembrane domain G-protein-coupled receptor family.1Vu TK Hung DT Wheaton VI Coughlin SR Molecular cloning of a functional thrombin receptor reveals a novel proteolytic mechanism of receptor activation.Cell. 1991; 64: 1057-1068Abstract Full Text PDF PubMed Scopus (2650) Google Scholar, 2Mackie EJ Pagel CN Smith R de Niese MR Song SJ Pike RN Protease-activated receptors: a means of converting extracellular proteolysis into intracellular signals.IUBMB Life. 2002; 53: 277-281Crossref PubMed Scopus (55) Google Scholar Protease-activated receptor-1 is expressed by osteoblasts (bone-forming cells), and mediates thrombin-induced proliferation of these cells.3Abraham LA Jenkins AL Stone SR Mackie EJ Expression of the thrombin receptor in developing bone and associated tissues.J Bone Miner Res. 1998; 13: 818-827Crossref PubMed Scopus (34) Google Scholar, 4Abraham LA Mackie EJ Modulation of osteoblast-like cell behavior by activation of protease-activated receptor-1.J Bone Miner Res. 1999; 14: 1320-1329Crossref PubMed Scopus (46) Google Scholar, 5Song S-J Pagel CN Pike RN Mackie EJ Studies on the receptors mediating responses of osteoblasts to thrombin.Int J Biochem Cell Biol. 2005; 371: 206-213Crossref Scopus (27) Google Scholar Thrombin also stimulates osteoclastic bone resorption in vitro.6Gustafson GT Lerner U Thrombin, a stimulator of bone resorption.Biosci Rep. 1983; 3: 255-261Crossref PubMed Scopus (62) Google Scholar, 7Hoffmann O Klaushofer K Koller K Peterlik M Mavreas T Stern P Indomethacin inhibits thrombin-, but not thyroxin-stimulated resorption of fetal rat limb bones.Prostaglandins. 1986; 31: 601-608Crossref PubMed Scopus (55) Google Scholar Thrombin is generated during tissue injury, and PAR-1 activation appears to be involved in pathological processes including inflammation and wound healing.8Strukova SM Thrombin as a regulator of inflammation and reparative processes in tissues.Biochemistry (Mosc). 2001; 66: 8-18Crossref PubMed Scopus (78) Google Scholar, 9Cocks TM Moffatt JD Protease-activated receptors: sentries for inflammation?.Trends Pharmacol Sci. 2000; 21: 103-108Abstract Full Text Full Text PDF PubMed Scopus (200) Google Scholar, 10Cirino G Cicala C Bucci MR Sorrentino L Maraganore JM Stone SR Thrombin functions as an inflammatory mediator through activation of its receptor.J Exp Med. 1996; 183: 821-827Crossref PubMed Scopus (233) Google Scholar For example, when thrombin or a PAR-1-activating peptide is applied to incisional skin wounds in rats, wound healing is accelerated.11Carney DH Mann R Redin WR Pernia SD Berry D Heggers JP Hayward PG Robson MC Christie J Annable C Fenton JW Glenn KC Enhancement of incisional wound healing and neovascularization in normal rats by thrombin and synthetic thrombin receptor-activating peptides.J Clin Invest. 1992; 89: 1469-1477Crossref PubMed Scopus (132) Google Scholar Because both osteoblast proliferation and bone resorption are important components of the process of bone repair, we hypothesized that thrombin, through PAR-1, may participate in this process. The current study was undertaken to investigate this hypothesis, using mice with a targeted disruption of the PAR-1 gene.12Connolly AJ Ishihara H Kahn ML Farese Jr, RV Coughlin SR Role of the thrombin receptor in development and evidence for a second receptor.Nature. 1996; 381: 516-519Crossref PubMed Scopus (440) Google Scholar Fifty percent of these mice die at about day 9.5 of gestation, and the remainder survive to become apparently normal adults. Embryonic death of PAR-1-null mice can be prevented by targeted expression of PAR-1 in endothelial cells.13Griffin CT Srinivasan Y Zheng YW Huang W Coughlin SR A role for thrombin receptor signaling in endothelial cells during embryonic development.Science. 2001; 293: 1666-1670Crossref PubMed Scopus (239) Google Scholar Although PAR-1 is expressed by platelets and mediates platelet aggregation in humans, PAR-1 is not expressed by mouse platelets, and therefore is not required for normal blood coagulation in this species.14Kahn ML Zheng YW Huang W Bigornia V Zeng D Moff S Farese Jr, RV Tam C Coughlin SR A dual thrombin receptor system for platelet activation.Nature. 1998; 394: 690-694Crossref PubMed Scopus (864) Google ScholarA number of models of bone repair have been established for use in mice, including an unstabilized fracture model, stabilized fracture models involving internal or external fixation, and models involving bone defects without fracture.15Bourque WT Gross M Hall BK A reproducible method for producing and quantifying the stages of fracture repair.Lab Anim Sci. 1992; 42: 369-374PubMed Google Scholar, 16Hiltunen A Vuorio E Aro HT A standardized experimental fracture in the mouse tibia.J Orthop Res. 1993; 11: 305-312Crossref PubMed Scopus (171) Google Scholar, 17Paccione MF Warren SM Spector JA Greenwald JA Bouletreau PJ Longaker MT A mouse model of mandibular osteotomy healing.J Craniofac Surg. 2001; 12: 444-450Crossref PubMed Scopus (13) Google Scholar, 18Li G White G Connolly C Marsh D Cell proliferation and apoptosis during fracture healing.J Bone Miner Res. 2002; 17: 791-799Crossref PubMed Scopus (55) Google Scholar, 19Li X Gu W Masinde G Hamilton-Ulland M Rundle CH Mohan S Baylink DJ Genetic variation in bone-regenerative capacity among inbred strains of mice.Bone. 2001; 29: 134-140Abstract Full Text Full Text PDF PubMed Scopus (37) Google Scholar, 20Uusitalo H Rantakokko J Ahonen M Jamsa T Tuukkanen J KaHari V Vuorio E Aro HT A metaphyseal defect model of the femur for studies of murine bone healing.Bone. 2001; 28: 423-429Abstract Full Text Full Text PDF PubMed Scopus (80) Google Scholar, 21Campbell TM Wong WT Mackie EJ Establishment of a model of cortical bone repair in mice.Calcif Tissue Int. 2003; 8: 8Google Scholar Similar responses to bone injury are seen in all of these models, with an initial phase of cellular invasion into the defect, followed by formation of callus tissue composed of woven bone with varying amounts of cartilage. The callus is then modeled over time through the actions of osteoclasts and osteoblasts, to restore lamellar bone and normal architecture.In the current study, we have used PAR-1-null and the corresponding wild-type (WT) mice in a model of bone repair recently established in our laboratory.21Campbell TM Wong WT Mackie EJ Establishment of a model of cortical bone repair in mice.Calcif Tissue Int. 2003; 8: 8Google Scholar In this model, a hole is drilled transversely through the full diameter of the tibia. The drill site fills rapidly with a hematoma, and over the ensuing days the hematoma is invaded by cells from the adjacent bone marrow. These cells produce trabecular woven bone which fills the drill site by day 7. The woven bone is modeled from about day 9, to restore normal bone architecture within about four weeks.21Campbell TM Wong WT Mackie EJ Establishment of a model of cortical bone repair in mice.Calcif Tissue Int. 2003; 8: 8Google Scholar We observed a number of differences between the drill sites of PAR-1-null and WT mice during the early stages of repair. With the aim of elucidating the functional basis for some of these differences, in vitro studies of bone marrow cells isolated from PAR-1-null and WT mice have also been conducted.Materials and MethodsMaterialsPurified human α-thrombin was prepared as described.22Stone SR Hofsteenge J Kinetics of the inhibition of thrombin by hirudin.Biochemistry. 1986; 25: 4622-4628Crossref PubMed Scopus (511) Google Scholar Cell culture media and additives were obtained from Invitrogen (Melbourne, Australia). All other chemicals and reagents were purchased from Sigma-Aldrich (St. Louis, MO) unless otherwise stated.Mice in which the protease activated-receptor-1 gene had been disrupted by homologous recombination12Connolly AJ Ishihara H Kahn ML Farese Jr, RV Coughlin SR Role of the thrombin receptor in development and evidence for a second receptor.Nature. 1996; 381: 516-519Crossref PubMed Scopus (440) Google Scholar (PAR-1-null mice) were kindly provided by Dr. S. R. Coughlin (University of California, San Francisco). These mice have been extensively back-crossed on the C57BL/6J background. Age-matched C57BL/6J mice were used as WT controls. All work involving animals was approved by the Animal Experimentation Ethics Committee of the School of Veterinary Science, University of Melbourne.SurgerySurgery was performed on 12- to 13-week-old PAR-1-null and wild-type male mice as described.21Campbell TM Wong WT Mackie EJ Establishment of a model of cortical bone repair in mice.Calcif Tissue Int. 2003; 8: 8Google Scholar Briefly, a single hole, 0.5 mm in diameter, was drilled with a 25-gauge needle. The defect passed through the entire diameter of the tibia from the medial to the lateral aspect just proximal to the distal end of the tibial crest. Surgery was carried out on both hind limbs. At various times postdrilling (0, 1, 2, 3, 5, 7, 9, 14, 28, and 42 days), mice were weighed, killed by cervical dislocation and the tibia and fibula along with the associated muscle were excised.HistologyThe left tibia was fixed for 1.5 hours in 4% paraformaldehyde in PBS and processed for preparation of cryosections. The right tibia was fixed in 70% ethanol and processed for embedding in LR-White-Hard (London Resin Company, Reading, UK). Some tissues were then demineralized in 0.33 mol/L ethylenediaminetetraacetic acid disodium salt (pH 7.4), whereas others were processed undemineralized. Transverse cryosections (10 μm thick) were prepared and stained for the presence of tartrate-resistant acid phosphatase (TRAP) to identify osteoclasts, as described.21Campbell TM Wong WT Mackie EJ Establishment of a model of cortical bone repair in mice.Calcif Tissue Int. 2003; 8: 8Google Scholar Tissues were embedded in LR-White according to the manufacturer's instructions.Transverse sections (5 μm) were cut using a slab microtome with a tungsten knife (Polycut E, Leica, Bannockburn, IL). Double-sided adhesive tape was used to attach undemineralized sections to glass slides. Demineralized sections were spread using 40% acetone and 1% benzoyl alcohol, collected on 3-aminopropyltriethoxysilane-coated glass slides, then stained with Epoxy Stain (ProSciTech, Thuringowa Central, QLD, Australia). Undemineralized sections were stained with Von Kossa.Histomorphometric AnalysisHistomorphometric analyses were conducted on images of sections captured with a digital camera (Spot, Diagnostic Instrument Inc, Sterling Heights MI) linked to an Olympus BX 60 microscope. All cell counts and area and length measurements were made with Image-Pro Plus (Media Cybernetics, Silver Spring, MD) image analysis software.The tibial cortical bone width and total cortical bone area were measured on Epoxy-stained demineralized plastic sections of undrilled tibiae of 12-week old mice. The sections used for these measurements were at the same level of the tibia as used for drilling in the bone repair model. The sections of fibula present in the same sections were used for measurement of osteoblast surface and eroded surface as a percentage of total endosteal and periosteal surface. In TRAP-stained cryosections from 12-week-old mice, osteoclasts on endosteal and periosteal surfaces of the fibula were counted manually as TRAP-positive cells with more than two nuclei; osteoclast counts were expressed as cells/mm bone surface. Three to four sections from each animal (4 WT and 4 PAR-1-null mice) were used for histomorphometric analyses of uninjured bone.For cell counts and new bone area measurement in sections of drilled tibiae, images were captured at different regions within the drill site and on the periosteal surface, as defined in Figure 1. Two to three sections from each animal (5 WT and 5 PAR-1-null mice for each time point) were used for cell counts and bone measurement. Demineralized epoxy-stained plastic sections through the drill site were used. Total cell number in each field at different regions within the drill site (defined in Figure 1) was counted manually using a 40x objective. Osteoclasts were counted manually as TRAP-positive cells, with more than two nuclei, adherent to bone surfaces, in demineralized TRAP-stained transverse cryosections through the drill site.For autoradiographic studies in vivo, three 12- to 13-week-old PAR-1-null and WT male mice were drilled; [3H]thymidine (1 μCi/g; Amersham, Buckinghamshire, UK) was injected intraperitoneally 42 hours after drilling and then mice were killed at day 3 (30 hours after [3H]thymidine injection). Both tibiae were collected and used for production of demineralized plastic sections as described above. After being dipped in Ilford nuclear research emulsion K.5 (Knutsford, Cheshire, UK), sections were stored in the dark for 5 weeks at 4°C, developed in Kodak GBX (Coburg, Victoria, Australia), then stained with 1:100 epoxy stain. Three or four sections from each animal were used for cell counts. Total cell number and labeled nuclei (nuclei containing more than 10 grains) were counted. Results for labeled cells were expressed as the percentage of total cells in each region.For bone area measurement, undemineralized Von Kossa-stained plastic sections through the drill sites were used. Total new trabecular bone area (Von Kossa-positive) was measured in each field. Results for new trabecular bone area are expressed as a percentage of total tissue area.Bone Marrow Stromal Cell IsolationSingle cell suspensions of bone marrow stromal cells (BMSCs) were prepared from left and right femurs and tibiae of 4–5-week-old mice, as described.23Hankenson KD Bain SD Kyriakides TR Smith EA Goldstein SA Bornstein P Increased marrow-derived osteoprogenitor cells and endosteal bone formation in mice lacking thrombospondin 2.J Bone Miner Res. 2000; 15: 851-862Crossref PubMed Scopus (75) Google Scholar Cells were pelleted, resuspended, and counted using the trypan blue exclusion method in a hemocytometer to determine the total number of viable marrow cells obtained from both femurs and tibiae. Cells were seeded into 75 cm2 flasks for growth. To separate adherent stromal cells from nonadherent hemopoietic cells, medium was changed after 48 hours, removing all nonadherent cells together with the culture medium; adherent stromal cells were washed twice with PBS. Cells were fed every two or three days until confluent, then trypsinized and plated in appropriate vessels for further experiments. Cells were cultured in minimal α-essential medium containing 10% fetal calf serum, gentamicin (50 μg/ml), amphotericin B (2.5 μg/ml), and sodium ascorbate (50 μg/ml) at 37°C under 5% CO2 in air.Colony-Forming Unit-Fibroblast AssayFreshly isolated single cell suspensions of bone marrow were plated in 60-mm dishes at a density of 105Song S-J Pagel CN Pike RN Mackie EJ Studies on the receptors mediating responses of osteoblasts to thrombin.Int J Biochem Cell Biol. 2005; 371: 206-213Crossref Scopus (27) Google Scholar cells/cm2. To maintain the influence of nonadherent hemopoietic cells, only one third of the volume of medium was removed and replaced on days 3 and 6. On day 9, the dishes were washed with PBS, fixed with 4% paraformaldehyde in phosphate-buffered saline (PBS) for 10 minutes, and stained for alkaline phosphatase (ALP) activity using an ALP staining kit (Sigma) following the manufacturer's instructions. Colonies of cells containing more than 20 cells were counted manually as colony-forming units-fibroblast, to be referred to as CFU-Fs. The number of ALP-positive CFU-Fs (≥20% ALP-positive cells) was also counted.Apoptosis AssayApoptotic cells were identified on the basis of nuclear morphology with 4,6-diamidino-2 phenylindole (DAPI) staining. Cells were plated in eight-chamber slides, grown to confluence and then deprived of serum for 24 hours before treatment with thrombin for 72 hours. Cell monolayers were fixed, permeabilized and mounted in Gelvatol (25% polyvinyl alcohol, 30% glycerol, and 0.1% sodium azide) containing DAPI (1 μg/ml). Fluorescent images were captured and cell counts conducted with Image Pro Plus software; total and apoptotic nuclei were counted in two or three representative fields per well using a 20× objective. Total cells were counted automatically, and apoptotic cells in the same field were counted by eye, identified on the basis of morphology (nuclear condensation and fragmentation). Values for apoptotic cells were expressed as the percentage (±SEM; n = 4) of total cells.Cell Proliferation AssayCell proliferation was assayed by 5-bromo-2′-deoxyuridine (BrdU) incorporation using a kit (Roche, Nutley, NJ USA). Proliferation assays for WT and PAR-1-null BMSCs were always carried out at the same time, and initial plating cell densities were the same in WT and PAR-1-null mice. BMSCs were cultured in 96–well plates until approximately 80% confluent. Cells were washed and deprived of serum for 24 hours and then treated with thrombin in serum-free medium for an additional 24 hours; BrdU labeling solution was added simultaneously with thrombin. Cells were fixed, and incorporated BrdU was detected by a direct immunoperoxidase method and tetramethylbenzidine substrate, according to the instructions supplied with the kit. The absorbance was read at 450 nm and 690 nm (in dual wavelength mode) in a Labsystems (Helsinki, Finland) Multiskan MS plate reader.Migration AssayThe migration assays were performed in 24-well Transwell cell culture plates (Corning Inc., Corning, NY) with polycarbonate filters (6.5 mm in diameter with 8 μm pores). Red blood cells were removed from freshly isolated bone marrow cells using 0.84% NH4Cl, then cells were washed with PBS and suspended at 2 × 106/ml in serum-free medium. The lower chambers of Transwell plates were filled with 0.6 ml of serum-free medium with or without 100 nmol/L thrombin, and the upper chambers with 0.1 ml of cell suspension. The cells were allowed to migrate for 5 hours at 37°C in a humidified atmosphere with 5% CO2 in air. The inserts were carefully removed. Nonadherent cells within the lower chambers were collected with their medium, and viable cells were counted using the trypan blue exclusion method in a hemocytometer. Adherent cells were fixed with 4% paraformaldehyde in PBS and stained with Mayer's hematoxylin. Adherent cells in five representative fields/well were counted under the microscope.RNA IsolationRNA was isolated from first passage BMSCs, long bone and bone marrow using the following method. RNA was isolated from the diaphyseal region of tibiae and femurs which had been stripped of all attendant soft tissue, and from which the metaphyses had been removed and bone marrow expelled with PBS. The diaphyseal regions of tibiae and femurs were washed thoroughly with PBS flushed from a 21-gauge needle. Bones were snap frozen in liquid nitrogen and ground to a fine powder before the addition of Tri-Reagent; first passage BMSCs and bone marrow were lysed directly in Tri-Reagent. RNA was extracted from the samples using Tri-Reagent (Sigma-Aldrich) according to the manufacturer's instructions.Reverse Transcriptase Polymerase Chain Reaction (RT-PCR)Polymerase chain reaction was used to investigate the expression of thrombin receptors and prothrombin. The first strand cDNA synthesis was carried out on total RNA using M-MLV reverse transcriptase according to the manufacturer's instructions (Promega, Madison, WI). Primers for the mouse PAR and glyceraldehyde-3-phosphate (GAPDH) genes have been described elsewhere.24Pagel CN de Niese MR Abraham LA Chinni C Song SJ Pike RN Mackie EJ Inhibition of osteoblast apoptosis by thrombin.Bone. 2003; 33: 733-743Abstract Full Text Full Text PDF PubMed Scopus (61) Google Scholar The sequence for the prothrombin primers was as follows: forward, CTA TGT CGC ACG TCC GC; reverse, GCA CTG GAT ACC GGT ATG G (product size 392 bp). The PCR reactions were carried out using the following thermal profile: 95°C for 30 seconds, annealing for 30 seconds at the appropriate temperature (55°C for PAR-1 and PAR-4; 51°C for PAR-3 and prothrombin) extension at 72°C for 30 seconds, for 30 cycles followed by a final extension step for 5 minutes at 72°C. Mouse liver cDNA was used as a positive control to confirm that the primers could detect expression of mouse prothrombin.Real-time PCR was carried out to investigate the relative expression of PAR-3 and PAR-4 in bone marrow isolated from WT and PAR-1 null mice (3 mice of each genotype). The sequence for the real-time PAR-3 and PAR-4 primers was as follows: GAPDH: forward GAT GCC CCC ATG TTT GTG AT; reverse TTG CTG ACA ATC TTG AGT GAG TTG T; PAR-3: forward GCT CCA TTT GTC AGC TCC TC; reverse GGA GGG AAG GGG ACA TGT AT; PAR-4: forward GCT ACA GCC ATG CAC TCA GA; reverse AGG GCT CGG GTT TGA ATA GT. Real-time PCR was carried out on an MX3000p real-time PCR machine (Stratagene, La Jolla, CA) using 0.25 μmol/L forward and reverse primers and Platinum Sybr Green qPCR Supermix-UDG containing ROX reference dye (Invitrogen, Carlsbad, CA). Reactions were run on the following thermal profile; hot start at 95°C for 3 minutes, then denaturation at 95°C for 30 seconds, annealing at 58°C for 30 seconds and extension at 72°C for 30 seconds, all for 40 cycles. Data were collected to the Sybr and ROX channels at the end of each extension step, and the final cycle was followed by a thermal melt step. Data collected for each sample were then analyzed using the 2−ΔΔCT method25Livak KJ Schmittgen TD Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔ CT method.Methods. 2001; 25: 402-408Crossref PubMed Scopus (119535) Google Scholar to give expression of PAR-3 and PAR-4 (normalized to GAPDH expression in each sample) in PAR-1 null bone marrow relative to normalized PAR-3 and PAR-4 expression in WT bone marrow.Statistical AnalysesData were analyzed using one-tailed unpaired Student's t-test assuming unequal variances; P values <0.05 were considered significant.ResultsHistomorphometry of Uninjured Bone in PAR-1-Null and WT MiceTo determine whether there were differences in cortical bone size between PAR-1-null and WT mice at the site of surgery, cortical bone width and area were determined using transverse sections of undrilled tibiae of 12-week-old animals. The width (178 ± 8 μm for WT mice and 174 ± 7 μm for PAR-1-null mice) and area (0.945 ± 0.033 mm2 for WT mice and 0.875 ± 0.032 mm2 for PAR-1-null mice) of cortical bone showed no significant difference between PAR-1-null and WT mice. In addition, transverse sections through the fibula from 12-week-old animals were used to investigate parameters related to formation and resorption in uninjured bone. Osteoblast surface, eroded surface and osteoclast number were quantitated (Table 1); there were no significant differences for any of these parameters between WT and PAR-1-null animals.Table 1Histomorphometry of Cortical Bone in the Fibula of PAR-1-Null and WT MiceWTPAR-1-nullOsteoblast surface (%)*All values represent mean ± SEM (n = 4).20.0 ± 1.118.9 ± 0.4Eroded surface (%)*All values represent mean ± SEM (n = 4).25.0 ± 0.726.5 ± 1.2Osteoclast number/mm bone surface*All values represent mean ± SEM (n = 4).3.7 ± 0.14.5 ± 0.4* All values represent mean ± SEM (n = 4). Open table in a new tab Bone Repair in PAR-1-Null and WT MiceIn general, bone repair proceeded at a similar rate in PAR-1-null and WT mice, but differences were observed between the two genotypes at specific times and locations.A hematoma rapidly filled the bone defect in both WT and PAR-1-null mice soon after drilling. Cells of hemopoietic appearance were present in the drill site at day 1 (not shown), but very few cells remained at day 2 (Figure 2, A and B). There were no apparent differences in blood clot formation or cellularity during the first two days between the two genotypes. In the tibiae of WT mice, a new wave of cells was observed in the drill site three days postdrilling and most of these cells were of mesenchymal appearance. At this stage, it appeared that there were fewer cells in the drill site in PAR-1-null mice than in WT mice (Figure 2, C and D). The cell density was counted in three different regions of the drill site (see Figure 1 for an illustration of the regions used for cell counts). There were about 50% fewer cells in all three areas of the drill sites of PAR-1-null mice when compared with those of WT mice (Figure 2G).Figure 2Invasion of the drill site by cells during the early stages of bone repair in PAR-1-null and WT mice. Plastic demineralized transverse Epoxy-stained sections through 2-day (A, B), 3-day (C, D), and 5-day (E, F) drill sites of WT (A, C, E) and PAR-1-null (B, D, F) mice. Arrows indicate bone fragments resulting from drilling. Scale bar = 100 μm. G: Cell counts for three different regions of the drill site at day 3: lateral, medial and bone marrow (BM; see Figure 1). Data represent cell number in each field (mean ± SEM, n = 5). H: [3H]thymidine-labeled cells were counted in the same regions of the drill site at day 3. Values for [3H]thymidine-labeled cells are presented as a percentage of total cells (mean ± SEM, n = 3). Significant differences between values for PAR-1-null and WT mice are indicated as *(P < 0.05) or ***(P < 0.001).View Large Image Figure ViewerDownload Hi-res image Download (PPT)To investigate whether this difference in cell number could be attributed to differential rates of proliferation, mice were injected with [3H]thymidine 42 hours after drilling, then killed 30 hours later (ie, 3 days after drilling). Autoradiographic studies of sections from these mice indicated that the percentage of [3H]thymidine-labeled cells in the drill site was two- to fourfold lower in PAR-1-null than in WT mice (Figure 2H).A periosteal reaction was observed adjacent to the drill site three days after drilling. The flat resting periosteal cells present before drilling became cuboidal. The periosteum was expanded dramatically from a single cell layer to multiple cell layers. However, there was no obvious difference in the periosteal reaction between WT and PAR-1-null mice at day 3 (data not shown).By day 5, the drill site was filled with randomly arranged cells surrounded by fibrillar extracellular matrix, with no obvious difference between WT and PAR-1-null animals (Figure 2, E and F). New mineralized trabecular woven bone was present at day 7 in the drill site in both PAR-1-null and WT mice, as visualized using sections of undemineralized tissue, stained with Von Kossa stain (Figure 3, A and C). At this stage, there appeared to be no difference in the amount of new bone between the two genotypes. Two days later, however, it appeared that there was less new mineralized bone in the drill site in PAR-1-nu" @default.
• W1973104883 created "2016-06-24" @default.
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• W1973104883 date "2005-03-01" @default.
• W1973104883 modified "2023-10-10" @default.
• W1973104883 title "The Role of Protease-Activated Receptor-1 in Bone Healing" @default.
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