Defective bone repair in mast cell-deficient <i>Cpa3^Cre/+</i> mice

<div><p>In the adult skeleton, cells of the immune system interact with those of the skeleton during all phases of bone repair to influence the outcome. Mast cells are immune cells best known for their pathologic role in allergy, and may be involved in chronic inflammatory and fibrotic disorders. Potential roles for mast cells in tissue homeostasis, vascularization and repair remain enigmatic. Previous studies in combined mast cell- and Kit-deficient <i>Kit</i>^{<i>W-sh/W-sh</i>} mice <i>(Kit</i>^<i>W-sh</i>) implicated mast cells in bone repair but <i>Kit</i>^<i>W-sh</i> mice suffer from additional Kit-dependent hematopoietic and non- hematopoietic deficiencies that could have confounded the outcome. The goal of the current study was to compare bone repair in normal wild type (<i>WT</i>) and <i>Cpa3</i>^<i>Cre/+</i> mice, which lack mast cells in the absence of any other hematopoietic or non- hematopoietic deficiencies. Repair of a femoral window defect was characterized using micro CT imaging and histological analyses from the early inflammatory phase, through soft and hard callus formation, and finally the remodeling phase. The data indicate 1) mast cells appear in healing bone of <i>WT</i> mice but not <i>Cpa3</i>^<i>Cre/+</i> mice, beginning 14 days after surgery; 2) re-vascularization of repair tissue and deposition of mineralized bone was delayed and dis-organised in <i>Cpa3</i>^<i>Cre/+</i> mice compared with <i>WT</i> mice; 3) the defects in <i>Cpa3</i>^<i>Cre/+</i> mice were associated with little change in anabolic activity and biphasic alterations in osteoclast and macrophage activity. The outcome at 56 days postoperative was complete bridging of the defect in most <i>WT</i> mice and fibrous mal-union in most <i>Cpa3</i>^<i>Cre/+</i> mice. The results indicate that mast cells promote bone healing, possibly by recruiting vascular endothelial cells during the inflammatory phase and coordinating anabolic and catabolic activity during tissue remodeling. Taken together the data indicate that mast cells have a positive impact on bone repair.</p></div

Quantitative micro CT analysis of bone vasculature.

<p>Quantitative micro CT analysis of bone vasculature.</p

Macroscopic evaluation of bone repair over time.

<p>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 <i>WT</i> (B) and <i>Cpa3</i>^<i>Cre/+</i> (F) femora. At 28 days PO, the defect is bridged with primary bone in many <i>WT</i> (C), but not <i>Cpa3</i>^<i>Cre/+</i> (G asterix) mice. By 56d PO the majority of <i>WT</i> femora have assumed their pre-operative anatomy (D), whereas most of those from <i>Cpa3</i>^<i>Cre/+</i> 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 <i>WT</i> and N = 6 <i>Cpa3</i>^<i>Cre/+</i> at 5d PO; N = 16 <i>WT</i> and N = 11 <i>Cpa3</i>^<i>Cre/+</i> at 14d PO; N = 8 <i>WT</i> and N = 10 <i>Cpa3</i>^<i>Cre/+</i> at 28d PO and N = 6 <i>WT</i> and N = 8 <i>Cpa3</i>^<i>Cre/+</i> at 56d PO.</p

Micro CT analysis of bone and vessel regeneration.

<p>Femora harvested from <i>WT</i> and <i>Cpa3</i>^<i>Cre/+</i> 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 <i>WT</i> femur whereas mal-union is evident in the <i>Cpa3</i>^<i>Cre/+</i> 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 <i>Cpa3</i>^<i>Cre/+</i> mice (F1 asterix), and is restricted to cortical bone by 56d PO (D1, H1).</p

Histochemical analysis of bone in contralateral cortex.

<p>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 <i>Cpa3</i>^<i>Cre/+</i> 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 <i>WT</i> (B) and <i>Cpa3</i>^<i>Cre/+</i> (F) bones, accompanied by high ALP activity. Active bone formation is sustained at 28d (G) and 56d (H) PO in <i>Cpa3</i>^<i>Cre/+</i> mice, but is less apparent in <i>WT</i> mice (C, D). Images are representative of N = 7 <i>WT</i> and N = 6 <i>Cpa3</i>^<i>Cre/+</i> at 5d PO; N = 16 <i>WT</i> and N = 11 <i>Cpa3</i>^<i>Cre/+</i> at 14d PO; N = 8 <i>WT</i> and N = 10 <i>Cpa3</i>^<i>Cre/+</i> at 28d PO and N = 6 <i>WT</i> and N = 8 <i>Cpa3</i>^<i>Cre/+</i> at 56d PO.</p

CD34 immunohistochemistry in regenerating bone.

<p>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 <i>WT</i> bone compared with weak, disorganised staining in <i>Cpa3</i>^<i>Cre/+</i> bone. By 28 days PO CD34 immunoreactivity is restricted primarily to the periosteum in <i>WT</i> bone, but persists in the remaining fibrous tissue in <i>Cpa3</i>^<i>Cre/+</i> bone (asterix). Images are representative of N = 7 <i>WT</i> and N = 6 <i>Cpa3</i>^<i>Cre/+</i> at 5d PO; N = 11 <i>WT</i> and N = 9 <i>Cpa3</i>^<i>Cre/+</i> at 14d PO and N = 8 <i>WT</i> and N = 6 <i>Cpa3</i>^<i>Cre/+</i> at 28d PO.</p

Quantitative staining analysis of cellular activity.

<p>Quantitative staining analysis of cellular activity.</p

Identification of osteoclasts and macrophages in regenerating bone.

<p>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 <i>WT</i> than in <i>Cpa3</i>^<i>Cre/+</i> 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 <i>WT</i> (A1) and <i>Cpa3</i>^<i>Cre/+</i> (B1) bones. In <i>WT</i> 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 <i>Cpa3</i>^<i>Cre/+</i> bone (F1). Images are representative of N = 7 <i>WT</i> and N = 6 <i>Cpa3</i>^<i>Cre/+</i> at 5d PO; N = 16 <i>WT</i> and N = 11 <i>Cpa3</i>^<i>Cre/+</i> at 14d PO; N = 8 <i>WT</i> and N = 10 <i>Cpa3</i>^<i>Cre/+</i> at 28d PO and N = 6 <i>WT</i> and N = 8 <i>Cpa3</i>^<i>Cre/+</i> at 56d PO.</p

Generation of Sam68-Deficient Mice

<div><p>(A) The genomic organizations of the wild-type and targeted <i>sam68</i> alleles after homologous recombination are depicted. The location of the DNA fragment used as a probe for the Southern blot analysis is shown, as well as the sizes of the two BglII fragments detected for wild-type and targeted <i>sam68</i> alleles. The targeted allele replaces exon 4 and part of exon 5 of <i>sam68</i> with a PGK-neomycin cassette.</p><p>(B) Southern-blot analysis of genomic DNA from wild-type (+/+), heterozygous (+/−), and homozygous (−/−) mice. DNA fragments corresponding to wild-type (4.5 kb) and the targeted (5.5 kb) alleles are illustrated.</p><p>(C) Western blot analysis of Sam68 expression. Protein extracts from wild-type, heterozygous, and homozygous cells subjected to immunoblot analyses using normal rabbit serum, anti-Sam68 AD1 antibody, the peptide antibody AD1 preabsorbed with the immunogenic peptide corresponding to amino acids 330–348 of mouse Sam68, anti-Sam68 Sc333 antibody that recognizes the C-terminal 20 amino acids of Sam68, and anti-actin antibodies as loading control. The migration of the molecular mass markers is known on the left in kDa.</p></div

Histologic Analysis of Undecalcified Bone from Sam68<b>^+/+</b> and Sam68^−/− Mice

<p>Sections of tibia fixed in 4% paraformaldehyde and embedded in plastic were stained for ALP (A–C, E–G) activity to identify osteoblasts or for TRAP (B–D, F–H) activity to identify osteoclasts. Staining patterns were similar in 4-month-old Sam68^+/+ (A and B), 4-month-old Sam68^−/− (C and D), and 12-month-old Sam68^−/− (G and H) mice compared with 12-month-old Sam68^+/+ mice (E and F). Magnification at source, left panels ×10 and right panels ×40. Micrographs are representative of those taken from five to seven sections in each group of animals.</p