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Impaired remodeling phase of fracture repair in the absence of matrix metalloproteinase-2.

Lieu S, Hansen E, Dedini R, Behonick D, Werb Z, Miclau T, Marcucio R, Colnot C - Dis Model Mech (2010)

Bottom Line: In humans and mice, MMP2 deficiency causes a musculoskeletal phenotype.Unlike Mmp9- and Mmp13- mutations, which affect both cartilage and bone in the callus, the Mmp2- mutation delayed bone remodeling but not cartilage remodeling.However, we did not detect changes in expression of Mmp9, Mmp13 or Mt1-Mmp (Mmp14) in the calluses of Mmp2- mice compared with wild type by in situ hybridization, but we observed decreased expression of Timp2 in the calluses of Mmp2-, Mmp9- and Mmp13- mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, University of California at San Francisco, San Francisco, CA 94143, USA.

ABSTRACT
The matrix metalloproteinase (MMP) family of extracellular proteases performs crucial roles in development and repair of the skeleton owing to their ability to remodel the extracellular matrix (ECM) and release bioactive molecules. Most MMP- skeletal phenotypes that have been previously described are mild, thus permitting the assessment of their functions during bone repair in the adult. In humans and mice, MMP2 deficiency causes a musculoskeletal phenotype. In this study, we assessed the role of MMP2 during mouse fracture repair and compared it with the roles of MMP9 and MMP13. Mmp2 was expressed at low levels in the normal skeleton and was broadly expressed in the fracture callus. Treatment of wild-type mice with a general MMP inhibitor, GM6001, caused delayed cartilage remodeling and bone formation during fracture repair, which resembles the defect observed in Mmp9(-/-) mice. Unlike Mmp9- and Mmp13- mutations, which affect both cartilage and bone in the callus, the Mmp2- mutation delayed bone remodeling but not cartilage remodeling. This remodeling defect occurred without changes in either osteoclast recruitment or vascular invasion of the fracture callus compared with wild type. However, we did not detect changes in expression of Mmp9, Mmp13 or Mt1-Mmp (Mmp14) in the calluses of Mmp2- mice compared with wild type by in situ hybridization, but we observed decreased expression of Timp2 in the calluses of Mmp2-, Mmp9- and Mmp13- mice. In keeping with the skeletal phenotype of Mmp2- mice, MMP2 plays a role in the remodeling of new bone within the fracture callus and impacts later stages of bone repair compared with MMP9 and MMP13. Taken together, our results indicate that MMPs play unique and distinct roles in regulating skeletal tissue deposition and remodeling during fracture repair.

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Mmp2 expression relative to other MMPs at day 10 post-fracture. (A) Safranin-O (SO) staining of wild-type callus tissues. Boxed area shows the transition between hypertrophic cartilage and bone, and is shown at higher magnification in B–L. (B–K) In situ hybridization on adjacent sections using (B) osteocalcin (Oc), (C) collagen type 10 (Col10), (D) Mmp2, (E) Mmp9, (F) Mmp13, (G) Mt1-Mmp, (H) Bsg and (I) Timp2 antisense mRNA probes, (J) Mmp2 sense mRNA probe, and (K) Vegf antisense mRNA probe. (L) PECAM immunostaining on an adjacent section illustrates the chondro-vascular junction. Scale bars: 1 mm (A); 200 μm (B–L).
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f3-0040203: Mmp2 expression relative to other MMPs at day 10 post-fracture. (A) Safranin-O (SO) staining of wild-type callus tissues. Boxed area shows the transition between hypertrophic cartilage and bone, and is shown at higher magnification in B–L. (B–K) In situ hybridization on adjacent sections using (B) osteocalcin (Oc), (C) collagen type 10 (Col10), (D) Mmp2, (E) Mmp9, (F) Mmp13, (G) Mt1-Mmp, (H) Bsg and (I) Timp2 antisense mRNA probes, (J) Mmp2 sense mRNA probe, and (K) Vegf antisense mRNA probe. (L) PECAM immunostaining on an adjacent section illustrates the chondro-vascular junction. Scale bars: 1 mm (A); 200 μm (B–L).

Mentions: MMPs play important roles during bone formation via endochondral ossification. This process requires the deposition of a cartilage matrix, which is remodeled and replaced by bone in part via the action of MMPs. Because non-stabilized tibial fractures heal via endochondral ossification, we chose this model to compare expression patterns of MMPs. Mmp2 is expressed at low levels in uninjured bone, including in osteocytes (data not shown) (Inoue et al., 2006; Mosig et al., 2007). Low levels of Mmp2 expression were detected as early as day 3 post-fracture at the fracture site and in the surrounding soft tissues immediately adjacent to the fracture site (Fig. 1C). Mmp2 expression was low and diffuse compared with the highly localized expression of Mmp9 in osteoclasts (Fig. 1D) (Colnot et al., 2003), Mmp13 in the activated periosteum (Fig. 1E) (Behonick et al., 2007), and Mt1-Mmp signal in the activated periosteum and surrounding soft tissues (Fig. 1F). By day 6, Mmp2 expression was still diffuse (Fig. 2D) but, compared with day 3, was stronger in areas of the callus in which cartilage and bone form, as shown by adjacent sections stained with collagen type 1 and collagen type 2 in situ probes (Fig. 2A–C). This pattern differed from the Mmp9 expression pattern in osteoclasts (Fig. 2E,F), and Mmp13 expression in cartilage and bone (Fig. 2G). Diffuse expression of Mt1-Mmp (Fig. 2H) and the MMP activator basigin [Bsg; also known as extracellular matrix metalloproteinase inducer (EMMPRIN); Fig. 2I] were also found in areas that overlapped with Mmp2 expression. At day 10, when the callus comprises a large amount of cartilage tissue surrounded by areas of new bone, Mmp2 expression was still detected at low levels throughout the callus (Fig. 3D). On adjacent sections, Mmp9 expression was confined to the chondro-vascular junction (Fig. 3E), Mmp13 expression was high in hypertrophic cartilage and bone (Fig. 3F), Mt1-Mmp expression was high at the chondro-vascular junction but low in cartilage and bone (Fig. 3G), and Bsg expression was low throughout the callus (Fig. 3H). Interestingly, the MMP inhibitor Timp2 was also expressed in most areas of the callus, with a stronger signal observed at the junction of cartilage and bone, at which several MMPs are highly expressed (Fig. 3I).


Impaired remodeling phase of fracture repair in the absence of matrix metalloproteinase-2.

Lieu S, Hansen E, Dedini R, Behonick D, Werb Z, Miclau T, Marcucio R, Colnot C - Dis Model Mech (2010)

Mmp2 expression relative to other MMPs at day 10 post-fracture. (A) Safranin-O (SO) staining of wild-type callus tissues. Boxed area shows the transition between hypertrophic cartilage and bone, and is shown at higher magnification in B–L. (B–K) In situ hybridization on adjacent sections using (B) osteocalcin (Oc), (C) collagen type 10 (Col10), (D) Mmp2, (E) Mmp9, (F) Mmp13, (G) Mt1-Mmp, (H) Bsg and (I) Timp2 antisense mRNA probes, (J) Mmp2 sense mRNA probe, and (K) Vegf antisense mRNA probe. (L) PECAM immunostaining on an adjacent section illustrates the chondro-vascular junction. Scale bars: 1 mm (A); 200 μm (B–L).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3046093&req=5

f3-0040203: Mmp2 expression relative to other MMPs at day 10 post-fracture. (A) Safranin-O (SO) staining of wild-type callus tissues. Boxed area shows the transition between hypertrophic cartilage and bone, and is shown at higher magnification in B–L. (B–K) In situ hybridization on adjacent sections using (B) osteocalcin (Oc), (C) collagen type 10 (Col10), (D) Mmp2, (E) Mmp9, (F) Mmp13, (G) Mt1-Mmp, (H) Bsg and (I) Timp2 antisense mRNA probes, (J) Mmp2 sense mRNA probe, and (K) Vegf antisense mRNA probe. (L) PECAM immunostaining on an adjacent section illustrates the chondro-vascular junction. Scale bars: 1 mm (A); 200 μm (B–L).
Mentions: MMPs play important roles during bone formation via endochondral ossification. This process requires the deposition of a cartilage matrix, which is remodeled and replaced by bone in part via the action of MMPs. Because non-stabilized tibial fractures heal via endochondral ossification, we chose this model to compare expression patterns of MMPs. Mmp2 is expressed at low levels in uninjured bone, including in osteocytes (data not shown) (Inoue et al., 2006; Mosig et al., 2007). Low levels of Mmp2 expression were detected as early as day 3 post-fracture at the fracture site and in the surrounding soft tissues immediately adjacent to the fracture site (Fig. 1C). Mmp2 expression was low and diffuse compared with the highly localized expression of Mmp9 in osteoclasts (Fig. 1D) (Colnot et al., 2003), Mmp13 in the activated periosteum (Fig. 1E) (Behonick et al., 2007), and Mt1-Mmp signal in the activated periosteum and surrounding soft tissues (Fig. 1F). By day 6, Mmp2 expression was still diffuse (Fig. 2D) but, compared with day 3, was stronger in areas of the callus in which cartilage and bone form, as shown by adjacent sections stained with collagen type 1 and collagen type 2 in situ probes (Fig. 2A–C). This pattern differed from the Mmp9 expression pattern in osteoclasts (Fig. 2E,F), and Mmp13 expression in cartilage and bone (Fig. 2G). Diffuse expression of Mt1-Mmp (Fig. 2H) and the MMP activator basigin [Bsg; also known as extracellular matrix metalloproteinase inducer (EMMPRIN); Fig. 2I] were also found in areas that overlapped with Mmp2 expression. At day 10, when the callus comprises a large amount of cartilage tissue surrounded by areas of new bone, Mmp2 expression was still detected at low levels throughout the callus (Fig. 3D). On adjacent sections, Mmp9 expression was confined to the chondro-vascular junction (Fig. 3E), Mmp13 expression was high in hypertrophic cartilage and bone (Fig. 3F), Mt1-Mmp expression was high at the chondro-vascular junction but low in cartilage and bone (Fig. 3G), and Bsg expression was low throughout the callus (Fig. 3H). Interestingly, the MMP inhibitor Timp2 was also expressed in most areas of the callus, with a stronger signal observed at the junction of cartilage and bone, at which several MMPs are highly expressed (Fig. 3I).

Bottom Line: In humans and mice, MMP2 deficiency causes a musculoskeletal phenotype.Unlike Mmp9- and Mmp13- mutations, which affect both cartilage and bone in the callus, the Mmp2- mutation delayed bone remodeling but not cartilage remodeling.However, we did not detect changes in expression of Mmp9, Mmp13 or Mt1-Mmp (Mmp14) in the calluses of Mmp2- mice compared with wild type by in situ hybridization, but we observed decreased expression of Timp2 in the calluses of Mmp2-, Mmp9- and Mmp13- mice.

View Article: PubMed Central - PubMed

Affiliation: Department of Orthopaedic Surgery, University of California at San Francisco, San Francisco, CA 94143, USA.

ABSTRACT
The matrix metalloproteinase (MMP) family of extracellular proteases performs crucial roles in development and repair of the skeleton owing to their ability to remodel the extracellular matrix (ECM) and release bioactive molecules. Most MMP- skeletal phenotypes that have been previously described are mild, thus permitting the assessment of their functions during bone repair in the adult. In humans and mice, MMP2 deficiency causes a musculoskeletal phenotype. In this study, we assessed the role of MMP2 during mouse fracture repair and compared it with the roles of MMP9 and MMP13. Mmp2 was expressed at low levels in the normal skeleton and was broadly expressed in the fracture callus. Treatment of wild-type mice with a general MMP inhibitor, GM6001, caused delayed cartilage remodeling and bone formation during fracture repair, which resembles the defect observed in Mmp9(-/-) mice. Unlike Mmp9- and Mmp13- mutations, which affect both cartilage and bone in the callus, the Mmp2- mutation delayed bone remodeling but not cartilage remodeling. This remodeling defect occurred without changes in either osteoclast recruitment or vascular invasion of the fracture callus compared with wild type. However, we did not detect changes in expression of Mmp9, Mmp13 or Mt1-Mmp (Mmp14) in the calluses of Mmp2- mice compared with wild type by in situ hybridization, but we observed decreased expression of Timp2 in the calluses of Mmp2-, Mmp9- and Mmp13- mice. In keeping with the skeletal phenotype of Mmp2- mice, MMP2 plays a role in the remodeling of new bone within the fracture callus and impacts later stages of bone repair compared with MMP9 and MMP13. Taken together, our results indicate that MMPs play unique and distinct roles in regulating skeletal tissue deposition and remodeling during fracture repair.

Show MeSH
Related in: MedlinePlus