<i>Mmp13</i> expression during non-stabilized fracture healing.

<p>(Left column) Safranin-O/Fast Green (SO) and Trichrome (TC) stained sagittal sections through the WT callus at 6 (A), 10 (E) and 14 (I) days post-fracture. Cartilage (red) develops during the soft callus phase of healing (A), is resorbed during the hard callus phase (E) and is replaced by bone (blue, I). (Middle/Right column) In situ hybridization analyses of <i>Mmp13</i> expression and osteoblast/chondrocyte differentiation markers. (B–D) At 6 days post-fracture, <i>Mmp13</i> is expressed in the callus and overlaps with <i>Col1</i>-expressing cells (early osteoblasts) but not <i>Mmp9</i>-expressing cells (osteoclasts and inflammatory cells). (F–H) At day 10, <i>Mmp13</i> mRNA is detected in hypertrophic chondrocytes also expressing <i>Col10</i> and <i>Vegf</i>. (J–L) At day 14, <i>Mmp13</i> is expressed in mature osteoblasts co-expressing <i>Col1</i> and/or <i>Oc</i>. Scale bars: A, E, I = 1mm; B-D, F-H, J-L-200 µm.</p

MMP13 is required for normal healing by intramembranous ossification.

<p>(A, Left column) SO stain of stabilized fracture calluses at day 10 post-fracture show that unlike <i>Mmp9</i>^−/−mice, no cartilage is formed during stabilized fracture healing in <i>Mmp13</i>^−/−mice (<i>n</i> = 14) compared to WT mice (<i>n</i> = 3). (A, Right column) At day 28, stabilized fracture calluses in <i>Mmp13</i>^−/− mice (<i>n</i> = 12) appear to have increased bone volume as compared to WT (<i>n</i> = 14) by histology. (B) Masson's Trichrome staining of cortical defect samples at 21 and 28 days post-surgery suggests an increased amount of bone in <i>Mmp13</i>^−/−mice. Labels designate compact (Co) and spongy (S) regions of defect. Histomorphometric analyses (within the boxed area) of WT (d21 <i>n</i> = 5, d28 <i>n</i> = 6) and <i>Mmp13</i>^−/− (d21 <i>n</i> = 6, d28 <i>n</i> = 6) cortical defect samples confirm that there is an increase in spongy bone volume (SV/DV) but not compact volume (CoV/DV) in the defect area measured at day 21 and 28 (*p<0.05) in <i>Mmp13</i>^−/− as compared to WT. Bonferroni corrected t-test, bars represent means ± SD. Scale bars: A = 1 mm, B = 500 µM.</p

Transplant of WT bone marrow does not rescue the <i>Mmp13</i>^−/−non-stabilized fracture healing phenotype.

<p>(A) Immunostaining for GFP on callus tissues from <i>Mmp13^−/−</i> mice transplanted with bone marrow from <i>β-actin GFP</i> mice (GFP→<i>Mmp13^−/−</i> mice) . (Left panel) Bone marrow cells (bm) are positive for GFP (black staining) showing they are donor-derived but the adjacent cortex (c) is negative. (Middle panel) Chondrocytes at day 14 and (Right panel) osteocytes embedded in the new bone (arrows) at day 28 do not stain for GFP, showing they are host-derived. (B, Left column) SO and (Right column) Masson's Trichrome staining of non-stabilized fracture calluses from <i>Mmp13</i>^−/− mice transplanted with WT bone marrow (WT → <i>Mmp13</i>^−/−) and <i>Mmp13</i>^−/−mice transplanted with <i>Mmp13</i>^−/−bone marrow (<i>Mmp13</i>^−/−→ <i>Mmp13</i>^−/−) show no difference in the amount of cartilage volume at 14 days post-fracture (WT → <i>Mmp13^−/− n</i> = 6, <i>Mmp13^−/−</i>→ <i>Mmp13^−/− n</i> = 5) and no difference in the amount bone at day 28 (WT → <i>Mmp13^−/− n</i> = 7, <i>Mmp13^−/−</i>→ <i>Mmp13^−/− n</i> = 4). (C) Histomorphometric analyses of total cartilage volume as a proportion of total callus volume (CV/TV; day 14) and total bone volume as a proportion of total callus volume (BV/TV; day 28) demonstrate no significant difference between WT → <i>Mmp13</i>^−/− and <i>Mmp13</i>^−/− → <i>Mmp13</i>^−/− animals, suggesting that bone marrow transplant does not rescue the <i>Mmp13</i>^−/non-stabilized fracture healing phenotype. Bonferroni corrected t-test, bars represent means ± SD. Scale bars: (A, left and middle) = 50 µm, (A, right) = 25 µm, B = 1 mm.</p

<i>Mmp13</i>^−/− mice display an accumulation of cartilage during non-stabilized fracture healing.

<p>(A) SO staining of WT and <i>Mmp13</i>^−/−fracture callus at 7, 10, 14, and 21 days post-fracture shows that cartilage persists in the <i>Mmp13</i>^−/− callus from 14 through 21 days post-fracture. Scale bar = 1 mm (B) Histomorphometric measurements of total callus volume (TV), total cartilage volume (CV) and total cartilage volume as a proportion of total callus volume (CV/TV) in WT and <i>Mmp13</i>^−/− mice at day 7 (WT <i>n</i> = 6, <i>Mmp13</i>^−/−<i>n</i> = 6), 10 (WT <i>n</i> = 8, <i>Mmp13</i>^−/−<i>n</i> = 6), 14 (WT <i>n</i> = 8, <i>Mmp13</i>^−/−<i>n</i> = 6), 21 (WT <i>n</i> = 6, <i>Mmp13</i>^−/−<i>n</i> = 6) and 28 (WT <i>n</i> = 6, <i>Mmp13</i>^−/−<i>n</i> = 6). There is a statistically significant increase in total cartilage volume in <i>Mmp13</i>^−/− calluses compared with WT at day 7 (**p<0.01), 14 (*p<0.05) and 21 (*p<0.05). There is a statistically significant increase in total cartilage volume as a proportion of total callus volume in <i>Mmp13</i>^−/− calluses compared with WT at day 7 (**p<0.01), 14 (*p<0.05) and 21 (*p<0.05). Wilcoxon test, bars represent means ± S.D. At 21 days post-fracture, all <i>Mmp13^−/−</i> calluses contained cartilage as compared to 1/3 of WT. At 28 days post-fracture, only <i>Mmp13^−/−</i> calluses (1/3) still contained cartilage.</p

Mechanical properties of intact tibiae and unstabilized fracture calluses.

<p>Results are given as mean ± SD of sample group (intact WT <i>n</i> = 10, <i>Mmp13^−/− n</i> = 10; 14 days post fracture WT <i>n</i> = 7, <i>Mmp13^−/− n</i> = 5; 21 days WT <i>n</i> = 2, <i>Mmp13^−/− n</i> = 7; 28 days WT <i>n</i> = 6, <i>Mmp13^−/− n</i> = 2).</p

Ultra-Structural Analysis (Transmission Electron Microscopy) of GBM from Control and MMP-Inhibitor–Treated Mice

<div><p>(A) Control C57Bl/6 mice at 14 wk of age. The 5-wk-old normal mice also exhibited normal GBM architecture (unpublished data).</p> <p>(B) Kidneys from 5-wk-old <i>α3(IV)^−/−</i> mice before the onset of proteinuria, displaying the beginning of splitting of the GBM and podocyte effacement (arrow). </p> <p>(C). Kidneys from <i>α3(IV)^−/−</i> mice at 14 wk of age, which were treated from 5 wk of age with MMP-inhibitors for 9 wk, displayed moderate GBM-splitting and podocyte effacement (arrows). </p> <p>(D) Kidneys from 12-wk-old <i>α3(IV)^−/−</i> mice without treatment showed severe lesions of the GBM associated with podocyte effacement (arrows). Original magnification: ×12,250. </p></div

Integral steps in endochondral ossification are unperturbed during non-stabilized fracture repair in <i>Mmp13^−/−</i> mice.

<p>(A) Overlay of SO stained sections with in situ hybridization for <i>Col2</i> (red) indicate no difference in the early differentiation of chondrocytes in WT and <i>Mmp13</i>^−/− calluses at day 5. Overlay of SO stained sections with in situ hybridization for <i>Col10</i> (yellow) shows a delay in hypertrophic chondrocyte removal in the <i>Mmp13</i>^−/− callus at day 14. Scale bar = 1 mm (B) Cellular analyses of WT and <i>Mmp13</i>^−/− calluses at day 14 show that blood vessels (PECAM) and osteoclasts (TRAP) are present in the <i>Mmp13</i>^−/− callus while aggrecan cleavage by MMPs (DIPEN epitope) is reduced in the <i>Mmp13</i>^−/− callus. Scale bar = 1 mm</p

<i>Mmp13</i>^−/− mice display increased bone volume during non-stabilized fracture healing.

<p>(A) Trichrome staining of WT and <i>Mmp13</i>^−/−non-stabilized fracture calluses shows an increase in the amount of bone in the <i>Mmp13</i>^−/− callus compared to WT at days 28 and 56 post-fracture. Scale bar = 1 mm (B) Histomorphometric measurements of total bone volume (BV) and total bone volume as a proportion of total callus volume (BV/TV) in WT and <i>Mmp13</i>^−/− mice at days 7 (WT <i>n</i> = 6, <i>Mmp13</i>^−/−<i>n</i> = 6), 10 (WT <i>n</i> = 8, <i>Mmp13</i>^−/−<i>n</i> = 6), 14 (WT <i>n</i> = 8, <i>Mmp13</i>^−/−<i>n</i> = 6), 21 (WT <i>n</i> = 6, <i>Mmp13</i>^−/−<i>n</i> = 6), 28 (WT <i>n</i> = 6, <i>Mmp13</i>^−/−<i>n</i> = 6) and 56 (WT <i>n</i> = 5, <i>Mmp13</i>^−/−<i>n</i> = 4) confirm this observation There is a statistically significant decrease in BV in <i>Mmp13</i>^−/− calluses compared with WT at day 7 (*p<0.05), but a statistically significant increase in BV and BV/TV in <i>Mmp13</i>^−/− calluses compared with WT at days 28 (**p<0.01 and p<0.05 respectively) and 56 (*p<0.05). Wilcoxon test, bars represent means ± S.D. (C) Histomorphometric measurements indicate a statistically significant difference in total spongy bone volume in <i>Mmp13</i>^−/− calluses compared with WT at day 28 (**p<0.01) but no difference is detected in total compact bone volume. (D) Micro-CT analyses show that bone mineral density is significantly increased in the <i>Mmp13^−/−</i> callus compared to WT at 21 (**p<0.01) and 28 (*p<0.05) days post-fracture. Bonferroni corrected t-test, bars represent means ± SD.</p

A model for MMP13 action in the cartilage and bone compartments of the non-stabilized fracture callus.

<p>MMP13 secreted from hypertrophic chondrocytes (HC) and osteoblasts (OB) acts upon the ECM to produce a pre-processed ECM in both compartments. This pre-processed ECM is then invaded by blood vessels and further modified by osteoclasts (OC) secreting MMP9, leading to the production of a processed ECM. This processed ECM then promotes further steps of callus maturation including hypertrophic chondrocyte apoptosis, replacement of cartilage by bone and new bone remodeling.</p

Effect of Combined MMP-Inhibition on the Progression of Renal Disease in <i>α3(IV) ^−/−</i> Mice

<div><p>(A–D) In situ zymography. In situ zymography displayed gelatin-degrading activity in normal kidneys (A), which was substantially increased in kidneys of 14-wk-old <i>COL4A3^−/−</i> mice (B). Incubation with the MMP-2, MMP-3, or MMP-9–inhibitor cocktail significantly blocked MMP activities in kidneys from wild-type mice (C) and kidneys from <i>COL4A3^−/−</i> mice (D). </p> <p>(E and F) Renal function in <i>α3(IV)^−/−</i> mice. Urinary protein excretion (E) was measured in urine, which was collected over a 24-h period in metabolic cages. Serum creatinine (F) was determined in serum samples that were obtained bi-weekly from mice. The graphs display the progression of urinary protein excretion and serum creatinine levels from 4 to 14 wk of age. In each panel, disease progression is shown in untreated control mice (black), in mice that received MMP-inhibitors starting at 5 wk of age (red), and in mice that received MMP-inhibitors starting at 8 wk of age (green). </p> <p>(G–N) Kidney histology from <i>α3(IV)^−/−</i> mice. The images display representative periodic acid-Schiff (PAS)–stained kidney sections from <i>α3(IV)^−/−</i> mice, together with images displaying representative kidneys from untreated α <i>3(IV)^−/−</i> mice to illustrate disease progression (G–L). At the age of 3 wk, the kidneys appear completely normal (G). At 4 wk of age, a few glomeruli appear hypercellular, representative of inflammatory cells (H). At the age of 6 wk, a few tubules filled with protein casts appear (I). At 8 wk of age, a few sclerotic glomeruli are present, and atrophic tubules containing protein casts become more abundant (J). At the age of 10 wk, increased numbers of mononuclear cells associated with widening of the interstitial space indicate the onset of interstitial fibrosis (K). At 14 wk of age, the kidneys display severe tubular atrophy, glomerulosclerosis, and interstitial fibrosis, reflecting ESRD (L). A kidney section is displayed of a 14-wk-old <i>α3(IV)^−/−</i> mouse that received MMP-inhibitors from 4 wk of age (M). Initiation of treatment after week 4 (compare with H) led to substantially ameliorated disease after 14 wk (M, compare with L). A representative kidney section of a <i>α3(IV)^−/−</i> mice at week 11 that received MMP-2–inhibitors, MMP-9–inhibitors, and MMP-3–inhibitors, starting at week 8 and displaying enhanced progression of disease, is displayed (N, compare with K). (Original magnification of PAS-stained histologies: ×200). </p> <p>(O and P) Representative kidney sections from wild-type control mice at 4 wk of age (O) and 14 wk of age (P) are displayed. (Original magnification of PAS-stained histologies: ×200).</p></div