Expression of Mmp2, Mmp9, Mmp13 and Mt1-Mmp during fracture repair
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 (). Mmp2
expression was low and diffuse compared with the highly localized expression of Mmp9
in osteoclasts () (Colnot et al., 2003
in the activated periosteum () (Behonick et al., 2007
), and Mt1-Mmp
signal in the activated periosteum and surrounding soft tissues (). By day 6, Mmp2
expression was still diffuse () 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 (). This pattern differed from the Mmp9
expression pattern in osteoclasts (), and Mmp13
expression in cartilage and bone (). Diffuse expression of Mt1-Mmp
() and the MMP activator basigin [Bsg
; also known as extracellular matrix metalloproteinase inducer (EMMPRIN); ] 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 (). On adjacent sections, Mmp9
expression was confined to the chondro-vascular junction (), Mmp13
expression was high in hypertrophic cartilage and bone (), Mt1-Mmp
expression was high at the chondro-vascular junction but low in cartilage and bone (), and Bsg
expression was low throughout the callus (). 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. 1. Mmp2 expression relative to that of other MMPs at day 3 post-fracture. (A–F) Safranin-O (SO) staining of wild-type callus tissues (A) and in situ hybridization on adjacent sections near the fracture (boxed area in A) using (B) collagen type 1 (more ...)
Fig. 2. Mmp2 expression relative to other MMPs at day 6 post-fracture. (A) Safranin-O (SO) staining of wild-type callus tissues. (B–I) On adjacent sections (boxed area in A) are shown in situ hybridization signals for (B) collagen type 1 (Col1), (C) collagen (more ...)
Fig. 3. 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) (more ...)
Effects of the general MMP inhibitor GM6001 on fracture repair
Given the presence of multiple MMPs in the fracture callus, we first assessed the total contribution of MMPs on bone repair by applying the MMP inhibitor GM6001 directly into the fracture callus of wild-type mice. The treatment was applied during the early soft callus phase of repair (days 6–9). By day 10, the proportions of cartilage and bone were not significantly different between treated and control calluses (). GM6001 affected the initial hard callus phase of repair. Healing was delayed and marked by a significant increase in the proportion of cartilage within the callus at day 14 and a significant decrease in the proportion of bone ().
Fig. 4. The MMP inhibitor GM6001 delays cartilage remodeling and bone formation during fracture repair. Histomorphometric analyses at days 10 (d10) and 14 (d14) post-fracture of cartilage volume as a proportion of total callus volume (CV/TV) and bone volume as (more ...)
Delayed bone remodeling in Mmp2-null mutant fracture calluses
When we created non-stabilized tibial fractures in Mmp2–/– mice, we observed a delay in bone remodeling as shown by representative histological stains () and histomorphometric analyses (). The callus volume was increased at day 10 but not at days 14 and 21 in Mmp2–/– mice compared with wild-type counterparts (). The total cartilage volume and the proportions of cartilage in the callus were also unchanged in Mmp2–/– compared with wild-type mice (). At day 10, the total bone volume and proportion of bone in the callus were not significantly different between Mmp2–/– and wild-type mice. By days 14 and 21, we observed an increase in the total bone volume and the proportion of bone in Mmp2–/– compared with wild-type mice, indicating a delay in bone remodeling in Mmp2–/– mice.
Fig. 5. Lack of MMP2 delays bone remodeling during fracture repair. (A) Trichrome staining on sections of (top) wild-type (WT) and (bottom) Mmp2–/–fracture calluses at days 10 (d10), 14 (d14) and 21 (d21) post-fracture. (B) Quantification via (more ...)
To investigate the cellular bases of this phenotype, we performed cellular and molecular analyses on representative sections through the fracture calluses at various time points. We observed normal osteoclast recruitment as indicated by tartrate-resistant acid phosphatase (TRAP) staining at day 10 post-fracture (). Degradation of the cartilage matrix, as shown by antibody staining for the DIPEN epitope (an epitope within aggrecan that is exposed specifically upon MMP cleavage), revealed no changes in the state of proteoglycan cleavage in Mmp2–/– calluses compared with those in wild type (). Similarly, blood vessel invasion assessed via platelet endothelial cell adhesion molecule (PECAM) immunostaining was normal in Mmp2–/– fracture calluses () and expression of the angiogenic factor Vegf in hypertrophic cartilage was similar in Mmp2–/– calluses compared with wild type. Given that the absence of MMP2 affected mostly the bone compartment of the callus, we concentrated our expression analyses on bone but found no changes in the expression pattern of osteopontin (), collagen type 1 () and osteocalcin () at day 14 post-fracture.
Fig. 6. Lack of MMP2 does not affect recruitment of osteoclasts, cartilage matrix remodeling, angiogenesis or expression of bone markers in the fracture callus. (A–H) TRAP staining, DIPEN immunostaining, PECAM immunostaining and Vegf in situ hybridization (more ...)
Analysis of functional redundancy among MMP family members
The Mmp2–/– fracture repair phenotype differed from the more severe bone repair phenotypes that we observed previously in Mmp9–/– and Mmp13–/– mice. We thus investigated whether MMP2 loss was compensated for by other MMPs that are expressed in the callus. When we compared Mmp2–/– and wild-type calluses, we did not observe qualitative differences in the expression profiles or expression levels of Mmp9, Mmp13 or Mt1-Mmp (). However, we observed a decrease in Timp2 expression in the callus of Mmp2–/– mice compared with wild type (). Likewise, Mmp2, Mmp13 and Mt1-Mmp expressions were unchanged in Mmp9–/– calluses compared with wild-type calluses, but Timp2 exhibited a decrease in the intensity of in situ hybridization signal (). The expression of Timp2 was also decreased in Mmp13–/– calluses compared with wild-type calluses, whereas the expressions of Mmp2, Mmp9 and Mt1-Mmp were similar to wild type (). These results thus indicate that the absence of Mmp2 might not impact the expression of other MMPs in cartilage and bone, suggesting that normal expression levels might be sufficient to partially compensate for the lack of Mmp2.
Fig. 7. MMP and Timp2 expression patterns in wild-type, Mmp13–/–, Mmp9–/– and Mmp13–/– mice during fracture repair. (A–L) Lack of MMP2 does not affect expression of Mmp9, Mmp13 or Mt1-Mmp but decreases (more ...)