microcomputed tomography (microct) scanner vivact 80  (SCANCO USA INC)

Journal: bioRxiv

Article Title: Proliferation of Early and Mature Osteoblasts Is Required for Bone Fracture Healing in Mice

doi: 10.1101/2025.05.27.656371

Figure Lengend Snippet: (A) Femur fractures were created in 12-week-old wildtype control (CTL) and Col1-TK experimental (EXPT) mice, followed by ganciclovir (GCV) dosing for 3, 7, or 14 days (n=7-9). (B) (Left) Representative radiographs at 21 days post-fracture (DPF). (Right) Quantification of radiographic healing. (C) 3D microCT images at 21 DPF (scale bar, 1 mm). (D) Sagittal callus sections were stained with picrosirius red (PSR) to visualize collagen-rich bone and alcian blue (AB) for proteoglycan-rich cartilage, and callus composition was determined (scale bar=1 mm). (Graphs depict mean ± SD; individual data points shown [pink=female; blue=male]. Statistics: Chi-square test (B) or two-Way ANOVA with Tukey Post Hoc test (C,D); *p<0.05, **<0.01, ****<0.0001; ns: p>0.05)

Article Snippet: Femurs were scanned ex vivo using microCT (VivaCT 40, Scanco, 10.5μm voxel size, 55kV, 145μA, 300ms integration time).

Techniques: Control, Staining

Journal: bioRxiv

Article Title: Proliferation of Early and Mature Osteoblasts Is Required for Bone Fracture Healing in Mice

doi: 10.1101/2025.05.27.656371

Figure Lengend Snippet: (A) Radiographic scoring by dosing duration at 7, 14 and 21 days post-fracture (DPF) (n=7-9). (Score 2=neither cortex bridged; 6=both cortices bridged). (B) microCT parameters from the callus region at 21 DPF (includes callus + cortical bone). (Dashed lines indicate avg. value of contralateral intact femurs; shading denotes +/- SD). (C) Histological callus composition at 21 DPF was determined from sections stained by picrosirius red and alcian blue (PSRAB). Image is representative section from Col1-TK mouse treated for 7 days with GCV. (Cg: cartilage, Wb: woven bone, F: fibrous tissue, Ps: periosteum.) (Graphs B, C depict mean ± SD; statistical differences determined by Fisher’s Exact Test)

Article Snippet: Femurs were scanned ex vivo using microCT (VivaCT 40, Scanco, 10.5μm voxel size, 55kV, 145μA, 300ms integration time).

Techniques: Staining

Journal: bioRxiv

Article Title: Proliferation of Early and Mature Osteoblasts Is Required for Bone Fracture Healing in Mice

doi: 10.1101/2025.05.27.656371

Figure Lengend Snippet: (A) After femur fracture Col1-TK mice were treated with water (Control, n=3) or GCV (Exptl, n=3) for 10 days. Cells isolated from whole-fracture callus were subjected to scRNAseq. (B) UMAP plot of total callus cells with annotated cluster identities. (C) The number of cells from immune clusters on Day 5 (Clusters 2, 6, 7, 10, 11, 12, 14, 16) and Day 10 (Clusters 7, 10, 13, 15) normalized to total cell numbers. (D) Mesenchymal cells were selected in silico and re-clustered. (E) Expression of canonical genes for osteoblasts, chondrocytes, and hypertrophic chondrocytes displayed as violin plots and projected on UMAPS. (F) Mesenchymal cells were identified by treatment group, and the percent of cells in each cluster was quantified (n=3). (G) Callus bone volume was quantified by microCT (n=5-8). (H) Histological sections of callus at 10 DPF were analyzed to determine total callus area and callus tissue composition. (I) Representative images of TK IHC of control (Col1-TK + water) and exptl (Col1-TK + GCV). (Statistics: two-Way ANOVA with Holm-Sidak Post Hoc test (C, F) or two-tailed t-test (G, H). Scale bars: PSRAB = 1 mm, TK=1 mm.)

Article Snippet: Femurs were scanned ex vivo using microCT (VivaCT 40, Scanco, 10.5μm voxel size, 55kV, 145μA, 300ms integration time).

Techniques: Control, Isolation, In Silico, Expressing, Two Tailed Test

Journal: bioRxiv

Article Title: Proliferation of Early and Mature Osteoblasts Is Required for Bone Fracture Healing in Mice

doi: 10.1101/2025.05.27.656371

Figure Lengend Snippet: (A) Overlay of Prrx1 (mesenchymal cells), Pecam1 (endothelial cells), or Ptprc /CD45 (immune cells) of all cells 10 days post-fracture. (B) Top 5 DEGs for each cluster of all cells from 10 DPF. (C) UMAP colored by group (Control, Exptl). The percent of cells in each cluster by group was quantified (number of cells in cluster/total number of cells from that callus) for each replicate callus and plotted (n=3). (D) Number of Skeletal Stem and Progenitor Cells (SSPCs, defined as cells expressing Acta2 , Ly6a , Itgav , Thy1 , and Ctsk ) normalized to the total number of cells from that replicate callus. (E) Expression of myofibroblast genes ( Acta2 , Tagln2 , Actg1 , and Tpm2 ), nonspecific fibroblast genes ( Col1a1 , Col3a1 , and Postn ), and innate immune genes ( Isg15 , Ifit1 , Irf7 ) used to annotate mesenchymal cell clusters. (F) MicroCT analysis of fracture callus. (G) Histological analysis of callus composition. (Graphs depict mean±SD; statistical differences were determined by a two-Way ANOVA with Holm-Sidak Post-Hoc test.)

Article Snippet: Femurs were scanned ex vivo using microCT (VivaCT 40, Scanco, 10.5μm voxel size, 55kV, 145μA, 300ms integration time).

Techniques: Control, Expressing

Journal: bioRxiv

Article Title: Proliferation of Early and Mature Osteoblasts Is Required for Bone Fracture Healing in Mice

doi: 10.1101/2025.05.27.656371

Figure Lengend Snippet: (A) Mice were treated with TMX starting at 10-wks age, followed by femur fracture at 12-wks, followed by 2 weeks GCV treatment. Healing was assessed 14 days post-fracture. Genotype controls were mice lacking either Osx-CreERT2 (Cre-) or ROSA-TK (TK-) alleles; experimental mice had both alleles (Cre+/TK+) (n=6). ( B) TK+ mice were treated with water for TK IHC, which shows robust TK expression in callus woven bone only in Cre+ mice (green dashed line=callus periphery; WoB=Woven Bone; Mu=Muscle). (C) (Left) Representative radiographs. (Right) Quantification of radiographic healing. ( D) Histological analysis of PSRAB stained-sections to assess callus size and composition. (E) Callus bone volume was quantified by microCT. (Statistical differences determined by Chi-Square test (C) or by two-way ANOVA with Holm-Sidak Post-Hoc test (D, E); *p<0.05, **p<0.01. Scale bars: TK=0.25 mm, PSRAB=1 mm, microCT=1 mm.)

Article Snippet: Femurs were scanned ex vivo using microCT (VivaCT 40, Scanco, 10.5μm voxel size, 55kV, 145μA, 300ms integration time).

Techniques: Expressing, Staining

Journal: bioRxiv

Article Title: Proliferation of Early and Mature Osteoblasts Is Required for Bone Fracture Healing in Mice

doi: 10.1101/2025.05.27.656371

Figure Lengend Snippet: (A) Histological analysis of callus. Ablation of proliferating Osterix-expressing cells for 14 days post-fracture significantly reduces the amount of woven bone and significantly increases the amount of fibrous tissue within the fracture callus, without affecting %cartilage. These data are summarized in the stacked bar graph in . (B) microCT analysis of the callus region (includes callus + cortical bone). Ablation of proliferating Osterix-expressing cells for 14 days post-fracture significantly reduces Total Volume (TV) and Bone Volume (BV) at the site of fracture callus. (Dashed lines indicate avg. value of contralateral intact femurs; shading denotes +/- SD). (C) microCT analysis of non-fractured bones. Treatment with GCV does not significantly change TV, BV, or femur length in the contralateral, non-fractured femurs. (All mice in A-C were dosed with GCV.) (D) Impairments in fracture callus size and bone volume are only seen in mice that are Cre+;TK+ and treated with GCV, even if all received TMX treatment. (E) Impairments in fracture callus size and bone volume are only seen in mice that are Cre+;TK+ and are treated with TMX, even if all received GCV treatment. (The third bar of graphs in panels D and E show identical data, also shown in the fourth bar of panel B.) (Graphs depict mean±SD and statistical differences were determined by (A-C) a Two-Way ANOVA with Holm-Sidak Post-Hoc test or (D & E) Kruskal-Wallis test.)

Article Snippet: Femurs were scanned ex vivo using microCT (VivaCT 40, Scanco, 10.5μm voxel size, 55kV, 145μA, 300ms integration time).

Techniques: Expressing

Journal: bioRxiv

Article Title: Proliferation of Early and Mature Osteoblasts Is Required for Bone Fracture Healing in Mice

doi: 10.1101/2025.05.27.656371

Figure Lengend Snippet: (A) Femur fracture was performed at 12-wks age, followed by 2 weeks GCV treatment. Healing was assessed 14 days after fracture. Genotype controls included mice lacking either Ocn-Cre or RosaTK alleles; experimental mice were Cre+/TK+ (n=5-10). (B) TK+ mice were treated with water for HSV-TK IHC, which shows TK expression in callus woven bone only in Cre+ mice. (C) (Left) Representative radiographs. (Right) Quantification of radiographic healing. (D) Histological analysis of fracture callus composition on PSRAB-stained slides showed that experimental mice have altered composition, with less woven bone and more fibrous tissue compared to control. (E) Callus bone volume quantified by microCT. (Statistical differences determined by Chi-Square test (C) or two-way ANOVA with Holm-Sidak Post-Hoc test (D, E). Scale bars: TK=0.25 mm, PSRAB=1 mm, microCT=1 mm.)

Article Snippet: Femurs were scanned ex vivo using microCT (VivaCT 40, Scanco, 10.5μm voxel size, 55kV, 145μA, 300ms integration time).

Techniques: Expressing, Staining, Control

Journal: bioRxiv

Article Title: Proliferation of Early and Mature Osteoblasts Is Required for Bone Fracture Healing in Mice

doi: 10.1101/2025.05.27.656371

Figure Lengend Snippet: (A) Fracture callus composition was determined from histology. TK+/Ocn-Cre+ mice have reduced woven bone and increased fibrous tissue within the fracture callus without affecting cartilage composition. These data are summarized in the stacked bar graph in . (B) MicroCT analysis of callus region (includes callus + cortical bone). TK+/OcnCre+ mice have reduced Total Volume (TV) and Bone Volume (BV). (Dashed lines indicate avg. value of contralateral intact femurs; shading denotes +/- SD). (C) MicroCT of intact femurs. Genotype does not alter TV, BV, or Femur Length in the contralateral, non-fractured femurs from the same cohort of mice. (All mice in A-C were dosed with GCV.) (D) Impairments in fracture callus morphology are only seen in mice that are Cre+;TK+ and treated with GCV. (Graphs depict mean±SD and statistical differences were determined by a two-Way ANOVA with Holm-Sidak Post-Hoc test.)

Article Snippet: Femurs were scanned ex vivo using microCT (VivaCT 40, Scanco, 10.5μm voxel size, 55kV, 145μA, 300ms integration time).

Techniques:

Journal: bioRxiv

Article Title: Proliferation of Early and Mature Osteoblasts Is Required for Bone Fracture Healing in Mice

doi: 10.1101/2025.05.27.656371

Figure Lengend Snippet: (A) Ocn-Cre;ROSA-TK mice were treated with GCV for 2 weeks post-fracture (as depicted in ), then GCV was withdrawn for the following 10 weeks. X-ray scoring shows that Ocn-Cre-mice fully bridge and remodel by 12 weeks post-fracture, whereas Ocn-Cre+ mice have significantly impaired bridging at 2 weeks post-fracture that does not fully recover by 12 weeks. (B) PSRAB staining of representative samples shows that Ocn-Cre-mice 12 weeks post-fracture have bony healing at the fracture site, whereas Ocn-Cre+ mice still have cartilaginous and fibrotic calluses 12 weeks post-fracture. (C) Ocn-Cre;ROSA-TK mice were treated with GCV for 3-, 5-, or 10-days post-fracture and euthanized on the last day of treatment indicated. PSRAB staining and analysis shows early changes in callus composition. (D) microCT analysis of Ocn-Cre;RosaTK mice treated with GCV for 3-, 5-, or 10-days post-fracture and euthanized on the last day of treatment indicated shows that control Cre-mice increase callus mineralization over time, whereas Cre+ experimental mice do not create a mineralized callus. (Graphs depict mean±SD; statistical differences were determined by (A) Chi-Square test, or (C-D) two-Way ANOVA with Holm-Sidak Post-Hoc test.)

Article Snippet: Femurs were scanned ex vivo using microCT (VivaCT 40, Scanco, 10.5μm voxel size, 55kV, 145μA, 300ms integration time).

Techniques: Staining, Control

Journal: bioRxiv

Article Title: Proliferation of Early and Mature Osteoblasts Is Required for Bone Fracture Healing in Mice

doi: 10.1101/2025.05.27.656371

Figure Lengend Snippet: (A) Mice were treated with TMX starting at 10-wks age, followed by femur fracture at 12-wks, followed by 2 weeks GCV treatment. Healing was assessed 14 days after fracture. Control mice were Cre-;TK+ while experimental mice were Cre+;TK+. (B) TK+ mice were treated with water for HSV-TK IHC, which shows robust TK expression in callus woven bone only in Cre+ mice. (C) Fracture callus bridging was quantified based on radiographic images. (D) Histological analysis of PSRAB stained-sections was used to assess callus size and composition. (E) Callus bone volume was quantified using microCT. (Statistical differences were determined by Chi-Square test (B) or by two-tailed t-test (C, D). Scale bars: PSRAB=1 mm, TK IHC=0.25 mm, microCT=1 mm.)

Article Snippet: Femurs were scanned ex vivo using microCT (VivaCT 40, Scanco, 10.5μm voxel size, 55kV, 145μA, 300ms integration time).

Techniques: Control, Expressing, Staining, Two Tailed Test

Journal: bioRxiv

Article Title: Proliferation of Early and Mature Osteoblasts Is Required for Bone Fracture Healing in Mice

doi: 10.1101/2025.05.27.656371

Figure Lengend Snippet: (A) Cre;ROSA-TK mice were treated with GCV and also treated with carbenoxolone (CBX) to block gap junction intercellular communication. In three lines of mice receiving CBX, callus bone volume measured by microCT was significantly less in Cre+ experimental vs. Cre-control. Callus bone volume in Cre+/CBX+ mice was not different from experimental Cre+ mice that did not receive CBX (the latter data also shown in , , and ). (B) When data from each Cre line were aggregated to overcome low sample size, Cre+ mice had impaired callus mineralization compared to Cre-mice, both treated with CBX. (Graphs depict mean±SD; statistical differences were determined by One-Way ANOVA with Fisher’s LSD Post-Hoc (A) or Mann-Whitney U test (B).)

Article Snippet: Femurs were scanned ex vivo using microCT (VivaCT 40, Scanco, 10.5μm voxel size, 55kV, 145μA, 300ms integration time).

Techniques: Blocking Assay, Control, MANN-WHITNEY

Journal: bioRxiv

Article Title: Proliferation of Early and Mature Osteoblasts Is Required for Bone Fracture Healing in Mice

doi: 10.1101/2025.05.27.656371

Figure Lengend Snippet: (A) Mice were treated with TMX starting at 10-wks age, followed by femur fracture at 12-wks, followed by 2 weeks GCV treatment. Healing was assessed 14 days after fracture. Control mice were Cre-;TK+ while experimental mice were Cre+;TK+. (B) Radiographs showed less mineralized callus in Cre+ mice, resulting in modestly poorer callus bridging scores than in Cre-controls. (C) Histological analysis shows a marginally smaller callus area in Cre+ mice, but normal percent cartilage and woven bone compared to Cre-controls. (D) Fractured bones were assessed by microCT; there were no significant differences in callus bone volume or BMD between Cre+ mice and control mice. (Statistical differences were determined by Chi-Square test (B) or by two-tailed t-test (C,D). Scale bars: PSRAB=1 mm, TK IHC=0.5 mm, microCT=1 mm.)

Article Snippet: Femurs were scanned ex vivo using microCT (VivaCT 40, Scanco, 10.5μm voxel size, 55kV, 145μA, 300ms integration time).

Techniques: Control, Two Tailed Test