Fig 1.
Identification of mature and immature mast cells.
Mice heterozygous for knock-in of Cre recombinase in the first exon of the Cpa3 gene were generated by backcross of founder Cpa3Cre/+ mice, shipped from the colony in Heidelberg, Germany, to C57Bl6 mice. PCR genotyping (A) of DNA isolated from ear punch specimens of 4-week-old mice yields 320bp and 450bp products for heterozygous Cpa3Cre/+ mice. Back skin biopsies were harvested from N = 3 wild type (WT, B, D) and N = 3 Cpa3Cre/+ (C, E) mice were embedded in paraffin and adjacent sections stained with acidified toluidine blue (aTB) or immunochemically with mast cell (MC) tryptase to identify mast cells. Representative images show numerous purple stained granular mast cells in WT (B) but not in Cpa3Cre/+ (C) skin. MC tryptase immunopositive cells (D arrows) are also seen in WT but not in Cpa3Cre/+ skin (E). Bilateral drill hole defects were generated in the femora of adult N = 37 WT and N = 35 Cpa3Cre/+ mice and cohorts of animals euthanized from 5d to 56d post-operative (PO). Sections of bone stained with aTB show mature mast cells (arrows) in marrow adjacent to the defect in WT (B2-B4) but not in Cpa3Cre/+ (C1-C4) mice. MC tryptase positive cells (arrows) are seen in WT tissue starting at 14d PO (D2-D4 but not in Cpa3Cre/+ mice (E2-E4). Images are representative of N = 7 WT and N = 6 Cpa3Cre/+ at 5d PO; N = 13 WT and N = 10 Cpa3Cre/+ at 14d PO; N = 8 WT and N = 10 Cpa3Cre/+ at 28d PO and N = 5 WT and N = 8 Cpa3Cre/+ at 56d PO.
Table 1.
Quantitative staining analysis of cellular activity.
Fig 2.
Micro CT analysis of bone and vessel regeneration.
Femora harvested from WT and Cpa3Cre/+ mice at the indicated time points were scanned at a resolution of 5μm on a Skyscan 1172 instrument. Representative 3D models (A-H), reconstructed from 2D images were tilted at a 45° angle to show healing of the defect over time. At 14d PO (B, F) significant new bone (white) is seen in the medullary canal and on the periosteal surface at the level of the defect (B, F arrows). By 56d PO, bone regeneration and remodelling have effectively closed the defect in the WT femur whereas mal-union is evident in the Cpa3Cre/+ femurs, with holes penetrating the new cortical bone (H arrow). 3D models of hemi-femora (A1-H1) show the distribution of blood vessels (white) at the same time points. Revascularization of bone reaches a peak at 14d postoperative, with a skewed distribution in Cpa3Cre/+ mice (F1 asterix), and is restricted to cortical bone by 56d PO (D1, H1).
Table 2.
Quantitative micro CT analysis of bone architecture.
Table 3.
Quantitative micro CT analysis of bone vasculature.
Fig 3.
CD34 immunohistochemistry in regenerating bone.
Bones were decalcified, embedded in paraffin and 5μm sections stained immunochemically for CD34 expression. Representative images of the defect, medulla and contralateral cortex show robust staining of cells lining vessels at 5d PO in WT bone compared with weak, disorganised staining in Cpa3Cre/+ bone. By 28 days PO CD34 immunoreactivity is restricted primarily to the periosteum in WT bone, but persists in the remaining fibrous tissue in Cpa3Cre/+ bone (asterix). Images are representative of N = 7 WT and N = 6 Cpa3Cre/+ at 5d PO; N = 11 WT and N = 9 Cpa3Cre/+ at 14d PO and N = 8 WT and N = 6 Cpa3Cre/+ at 28d PO.
Fig 4.
Macroscopic evaluation of bone repair over time.
2D micro CT images (A-H) were compared with 5 μm histological sections of un-decalcified bone stained with von Kossa and toluidine blue (VK/TB) to distinguish mineralised (black) from un-mineralized tissue (blue). Representative mid-sagittal images show shards of old bone (A, E arrows) remaining in the defect at 5d PO and significant new bone at 14d PO in the medullary canal and on the periosteal surface opposite the defect in both WT (B) and Cpa3Cre/+ (F) femora. At 28 days PO, the defect is bridged with primary bone in many WT (C), but not Cpa3Cre/+ (G asterix) mice. By 56d PO the majority of WT femora have assumed their pre-operative anatomy (D), whereas most of those from Cpa3Cre/+ mice exhibit mal-union on the defect side (H asterix) and large channels separating old from new bone on the contralateral cortex (H arrows). Images are representative of N = 7 WT and N = 6 Cpa3Cre/+ at 5d PO; N = 16 WT and N = 11 Cpa3Cre/+ at 14d PO; N = 8 WT and N = 10 Cpa3Cre/+ at 28d PO and N = 6 WT and N = 8 Cpa3Cre/+ at 56d PO.
Fig 5.
Histochemical analysis of bone in defect/medulla.
Representative images of 5 μm sections of un-decalcified bone stained with von Kossa and toluidine blue (A-H) were compared with the equivalent region of 5μm sections of decalcified bone stained with alkaline phosphatase (ALP). Prominent osteoblasts against osteoid seams can be seen at 14d (F) and 28d (G) PO in Cpa3Cre/+ bones compared with WT bones (B-C). ALP activity peaks at 14 days PO in both WT (B) and Cpa3Cre/+ (F) bones and declines thereafter. Images are representative of N = 7 WT and N = 6 Cpa3Cre/+ at 5d PO; N = 16 WT and N = 11 Cpa3Cre/+ at 14d PO; N = 8 WT and N = 10 Cpa3Cre/+ at 28d PO and N = 6 WT and N = 8 Cpa3Cre/+ at 56d PO.
Fig 6.
Histochemical analysis of bone in contralateral cortex.
Representative images of 5 μm sections of von Kossa stained un-decalcified bone (A-H) were compared with 5μm sections of decalcified bone stained with ALP. A thick, fibrous periosteum is apparent in Cpa3Cre/+ bones (E, G asterix) in the absence of any significant difference in ALP activity. Bone formation with large osteoblasts adjacent to osteoid is apparent at 14d PO in WT (B) and Cpa3Cre/+ (F) bones, accompanied by high ALP activity. Active bone formation is sustained at 28d (G) and 56d (H) PO in Cpa3Cre/+ mice, but is less apparent in WT mice (C, D). Images are representative of N = 7 WT and N = 6 Cpa3Cre/+ at 5d PO; N = 16 WT and N = 11 Cpa3Cre/+ at 14d PO; N = 8 WT and N = 10 Cpa3Cre/+ at 28d PO and N = 6 WT and N = 8 Cpa3Cre/+ at 56d PO.
Fig 7.
Identification of osteoclasts and macrophages in regenerating bone.
5 μm sections of decalcified bone were stained with tartrate resistant acid phosphatase (TRAP) or immunochemically with the macrophage marker F4/80. Representative images show more TRAP activity in WT than in Cpa3Cre/+ bones at 5d (A vs B) and 14d (C vs D) PO, and less at 28d (E vs F) PO. F4/80 positive macrophages were seen in condensed mesenchyme filling the defect/medulla at 5d PO in both WT (A1) and Cpa3Cre/+ (B1) bones. In WT bone, F4/80 positive cells can be seen lining vessels at 14d (C1) PO and scattered throughout bone marrow at 28d (E1) PO, whereas they were embedded in fibrous tissue in Cpa3Cre/+ bone (F1). Images are representative of N = 7 WT and N = 6 Cpa3Cre/+ at 5d PO; N = 16 WT and N = 11 Cpa3Cre/+ at 14d PO; N = 8 WT and N = 10 Cpa3Cre/+ at 28d PO and N = 6 WT and N = 8 Cpa3Cre/+ at 56d PO.