The assembly and degradation of extracellular matrix (ECM) molecules are crucial processes during bone development. In this study, we show that ECM remodeling is a critical rate-limiting step in endochondral bone formation. Matrix metalloproteinase (MMP) 13 (collagenase 3) is poised to play a crucial role in bone formation and remodeling because of its expression both in terminal hypertrophic chondrocytes in the growth plate and in osteoblasts. Moreover, a mutation in the human MMP13 gene causes the Missouri variant of spondyloepimetaphyseal dysplasia. Inactivation of Mmp13 in mice through homologous recombination led to abnormal skeletal growth plate development. Chondrocytes differentiated normally but their exit from the growth plate was delayed. The severity of the Mmp13- null growth plate phenotype increased until about 5 weeks and completely resolved by 12 weeks of age. Mmp13-null mice had increased trabecular bone, which persisted for months. Conditional inactivation of Mmp13 in chondrocytes and osteoblasts showed that increases in trabecular bone occur independently of the improper cartilage ECM degradation caused by Mmp13 deficiency in late hypertrophic chondrocytes. Our studies identified the two major components of the cartilage ECM, collagen type II and aggrecan, as in vivo substrates for MMP13. We found that degradation of cartilage collagen and aggrecan is a coordinated process in which MMP13 works synergistically with MMP9. Mice lacking both MMP13 and MMP9 had severely impaired endochondral bone, characterized by diminished ECM remodeling,prolonged chondrocyte survival, delayed vascular recruitment and defective trabecular bone formation (resulting in drastically shortened bones). These data support the hypothesis that proper ECM remodeling is the dominant rate-limiting process for programmed cell death, angiogenesis and osteoblast recruitment during normal skeletal morphogenesis.

Endochondral ossification, the process by which most of the skeleton is formed, is a powerful system for studying various aspects of the biological response to degraded extracellular matrix (ECM). In addition, the dependence of endochondral ossification upon neovascularization and continuous ECM remodeling provides a good model for studying the role of the matrix metalloproteases (MMPs) not only as simple effectors of ECM degradation but also as regulators of active signal-inducers for the initiation of endochondral ossification. The daunting task of elucidating their specific role during endochondral ossification has been facilitated by the development of mice deficient for various members of this family. Here, we discuss the ECM and its remodeling as one level of molecular regulation for the process of endochondral ossification, with special attention to the MMPs.In the past decade, multiple discoveries have facilitated our understanding of skeletal development. Transcription factors and/or growth factors that are involved in the genetic and molecular control of OSTEOGENESIS (see glossary box), CHONDROGENESIS and joint formation have been identified and their pathways partially elucidated [1]. The attention now turns to a different level of molecular regulation -that provided by the extracellular matrix (ECM) of the developing bone and the proteases that remodel it. Recent studies attest to the importance of unique composition of distinct ECMs within the developing skeleton, as well as their influence on cell differentiation and function [2][3][4].Bone develops in two different ways. Mesenchymal cells can directly differentiate into bone by the process of INTRAMEMBRANOUS OSSIFICATION, which is responsible for the formation of most of the craniofacial skeleton. Alternatively, these cells can differentiate into cartilage, which then provides a template for bone morphogenesis by the process of ENDOCHONDRAL OSSIFICATION; this process is responsible for the formation of most of the vertebrate appendicular and axial skeleton ( Figure 1a). Endochondral ossification occurs at two distinct sites in the vertebrate long bone -the primary (diaphyseal) and the secondary (epiphyseal) sites of ossification. Bone development initiates at the primary site. The secondary (epiphyseal) site is under independent control and is ossified later (Figure 1b). During this process, a new structure, the GROWTH PLATE, is formed between the DIAPHYSIS and the EPIPHYSIS by the segregation of CHONDROCYTES at different stages of differentiation. Under the control of signaling through Indian hedgehog (Ihh), bone morphogenetic proteins (BMPs) and fibroblast growth factor 18, a region of resting chondrocytes feeds into a zone of proliferating chondrocytes that then undergo hypertrophy and subsequently apoptosis. The ECM produced by and surrounding the terminally differentiated hypertrophic chondrocytes is calcified, partially degraded by the hypertrophic chondrocytes themselves [5], as well as by CHONDROCLASTS and/or preosteoclasts, and be...

Extracellular matrix (ECM) remodeling is important during bone development and repair. Because matrix metalloproteinase 13 (MMP13, collagenase-3) plays a role in long bone development, we have examined its role during adult skeletal repair. In this study we find that MMP13 is expressed by hypertrophic chondrocytes and osteoblasts in the fracture callus. We demonstrate that MMP13 is required for proper resorption of hypertrophic cartilage and for normal bone remodeling during non-stabilized fracture healing, which occurs via endochondral ossification. However, no difference in callus strength was detected in the absence of MMP13. Transplant of wild-type bone marrow, which reconstitutes cells only of the hematopoietic lineage, did not rescue the endochondral repair defect, indicating that impaired healing in Mmp13−/− mice is intrinsic to cartilage and bone. Mmp13−/− mice also exhibited altered bone remodeling during healing of stabilized fractures and cortical defects via intramembranous ossification. This indicates that the bone phenotype occurs independently from the cartilage phenotype. Taken together, our findings demonstrate that MMP13 is involved in normal remodeling of bone and cartilage during adult skeletal repair, and that MMP13 may act directly in the initial stages of ECM degradation in these tissues prior to invasion of blood vessels and osteoclasts.

Matrix metalloproteinases (MMPs) degrade most components of the extracellular matrix (ECM), as well as many non-ECM molecules. MMPs participate in (1). degradation of ECM to allow cell migration; (2). alteration of the ECM microenvironment resulting in alteration in cellular behavior; (3). modulation of biologically active molecules by direct cleavage or release from ECM stores; (4). regulation of the activity of other proteases; and (5). cell attachment, proliferation, differentiation, and apoptosis. We have sought to understand the role of MMPs during development and tissue repair in transgenic mice. Endochondral bone formation presents a particularly interesting developmental challenge. During this process, an avascular tissue (cartilage) is converted into one of the most highly vascularized tissues (bone) in the vertebrate body. Ossification begins with invasion of the calcified hypertrophic cartilage by capillaries. Apoptosis of the terminal hypertrophic chondrocytes, degradation of the cartilage matrix, and deposition of bone matrix by osteoblasts accompanies neovascularization of the growth plate. Remodeling of ECM results in a cavity filled with vascular channels containing hematopoietic cells. Our results reveal that MMP9, MMP13, and vascular endothelial growth factor are key regulators for the remodeling of the skeletal tissues. They coordinate not only matrix degradation, but also the recruitment and differentiation of endothelial cells, osteoclasts, chondroclasts, and osteoprogenitors.

Endochondral bone formation is characterized by the progressive replacement of a cartilage anlagen by bone at the growth plate with a tight balance between the rates of chondrocyte proliferation, differentiation, and cell death. Deficiency of matrix metalloproteinase-9 (MMP-9) leads to an accumulation of late hypertrophic chondrocytes. We found that galectin-3, an in vitro substrate of MMP-9, accumulates in the late hypertrophic chondrocytes and their surrounding extracellular matrix in the expanded hypertrophic cartilage zone. Treatment of wild-type embryonic metatarsals in culture with full-length galectin-3, but not galectin-3 cleaved by MMP-9, mimicked the embryonic phenotype of Mmp-9 null mice, with an increased hypertrophic zone and decreased osteoclast recruitment. These results indicate that extracellular galectin-3 could be an endogenous substrate of MMP-9 that acts downstream to regulate hypertrophic chondrocyte death and osteoclast recruitment during endochondral bone formation. Thus, the disruption of growth plate homeostasis in Mmp-9 null mice links galectin-3 and MMP-9 in the regulation of the clearance of late chondrocytes through regulation of their terminal differentiation.

The extracellular matrix (ECM), once thought to be a static structural component of tissues, is now known to play a complex and dynamic role in a variety of cellular functions in a number of diverse tissues. A significant body of literature attests to the ability of the ECM to communicate both spatial and temporal information to adherent cells, thereby directing cell behavior via interactions between the ECM and cell-surface receptors. Moreover, volumes of experimental data show that a great deal of communication travels in the opposite direction, from the cell to the ECM, allowing for regulation of the cues transmitted by the ECM. As such, the ECM, with respect to its components and their organization, is not a fixed reflection of the state the local microenvironment in which a cell finds itself at a particular time, but rather is able to respond to and effect changes in its local microenvironment. As an example of the developmental consequences of ECM interactions, this review gives an overview of the 'give and take' relationship between the ECM and the cells of the developing skeletal elements, in particular, the chondrocyte.

SUMMARYThe 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-null 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-null mutations, which affect both cartilage and bone in the callus, the Mmp2-null 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-null mice compared with wild type by in situ hybridization, but we observed decreased expression of Timp2 in the calluses of Mmp2-, Mmp9- and Mmp13-null mice. In keeping with the skeletal phenotype of Mmp2-null 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.

Recessive inactivating mutations in human matrix metalloproteinase 2 (MMP2, gelatinase A) are associated with syndromes that include abnormal facial appearance, short stature, and severe bone loss. Mmp2 ؊/؊ mice have only mild aspects of these abnormalities, suggesting that MMP2 function is redundant during skeletal development in the mouse. Here, we report that Mmp2 ؊/؊ mice with additional mutations that render type I collagen resistant to collagenase-mediated cleavage to TC A and TC B fragments (Col1a1 r/r mice) have severe developmental defects resembling those observed in MMP2-null humans. Composite Mmp2 ؊/؊ ;Col1a1 r/r mice were born in expected Mendelian ratios but were half the size of wild-type, Mmp2 ؊/؊ , and Col1a1 r/r mice and failed to thrive. Furthermore, composite Mmp2 ؊/؊ ;Col1a1 r/r animals had very abnormal craniofacial features with shorter snouts, bulging skulls, incompletely developed calvarial bones and unclosed cranial sutures. In addition, trabecular bone mass was reduced concomitant with increased numbers of bone-resorbing osteoclasts and osteopenia. In vitro, MMP2 had a unique ability among the collagenolytic MMPs to degrade mutant collagen, offering a possible explanation for the genetic interaction between Mmp2 and Col1a1 r . Thus, because mutations in the type I collagen gene alter the phenotype of mice with null mutations in Mmp2, we conclude that type I collagen is an important modifier gene for Mmp2.