Gene Expression and Distribution of Connective Tissue Growth Factor (CCN2/CTGF) During Secondary Ossification Center Formation

Abstract

CCN2/connective tissue growth factor (CCN2/CTGF) is a critical signaling modulator of mesenchymal tissue development. This study investigated the localization and expression of CCN2/CTGF as a factor supporting angiogenesis and chondrogenesis during development of secondary ossification centers in the mouse tibial epiphysis. Formation of the secondary ossification center was initiated by cartilage canal formation and blood vessel invasion at 7 days of age, and onset of ossification was observed at 14 days. In situ hybridization showed that CCN2/CTGF mRNA was distinctively expressed in the region of the cartilage canal and capsule-attached marginal tissues at 7 days of age, and distinct expression was also observed in proliferating chondrocytes around the marrow space at 14 days of age. Immunostaining showed that CCN2/CTGF was distributed broadly around the expressed cells located in the central region of the epiphysis, where the chondrocytes become hypertrophic and the cartilage canal enters into the hypertrophic mass. Furthermore, an overlapping distribution of metalloproteinase (MMP)9 and CCN2/CTGF was found in the secondary ossification center. These findings suggest that the CCN2/CTGF is involved in establishing epiphyseal vascularization and remodeling, which eventually determines the secondary ossification center in the developing epiphysial cartilage.

CCN2/connective tissue growth factor (CTGF) is a cysteine-rich extracellular matrix (ECM)-associated protein of 36-38 kDa. It belongs to the CCN family that consists of six distinct members (CTGF/Fisp12/Hcs24/CCN2, Cyr61/Cef10/CCN1, Nov/CCN3, ELM-1/WISP-1/CCN4, rCOP-1/WISP-2/CTGF-3/CCN5, and WISP-3/CCN6) (Bork 1993; Brigstock 1999; Lau and Lam 1999; Takigawa et al. 2003, 2005; Perbal 2004). The mitogenic and chemotactic effects of CCN2/CTGF on fibroblasts have been studied in relation to fibrotic disorders (Igarashi et al. 1996; Allen et al. 1999; Abou-Shady et al. 2000; Yokoi et al. 2001; Dean et al. 2005). In recent years, CCN2/CTGF mRNA expression and/or protein production has been shown in endothelial cells, fibroblasts, chondrocytes, vascular smooth muscle cells, and osteoblasts during the course of embryonic development and normal body growth (Surveyor and Brigstock 1999; Friedrichsen et al. 2005; Schutze et al. 2005). CCN2/CTGF has also been implicated in tumor angiogenesis, wound healing, and fracture repair (Igarashi et al. 1993; Kondo et al. 2002; Nakata et al. 2002; Moritani et al. 2003).

Previously, a cDNA specific in a human chondrosarcoma-derived cell line, HCS-2/8 (Takigawa et al. 1989), was cloned by differential display PCR. After the identification of the cDNA clone as ccn2/ctgf, CCN2/CTGF was shown to be highly expressed in hypertrophic chondrocytes (Nakanishi et al. 1997). Subsequent studies showed that recombinant CCN2/CTGF promoted the proliferation and differentiation of chondrocytes and osteoblasts in vitro (Nakanishi et al. 2000; Nishida et al. 2000; Safadi et al. 2003). Furthermore, it stimulated the proliferation, migration, and tube formation of vascular endothelial cells in vitro and angiogenesis in vivo (Shimo et al. 1998, 1999; Babic et al. 1999). Interestingly, CCN2/CTGF increased the expression of a number of metalloproteinases (MMPs) that play roles in the vascular invasive processes in human umbilical vein endothelial cells (Kondo et al. 2002). These MMPs function to promote angiogenesis by regulating endothelial cell attachment, proliferation, migration, and growth, either directly or indirectly by the release of growth factors stored within the ECM. Indeed, MMP9 is regarded as a key factor of growth plate angiogenesis (Vu et al. 1998). These findings indicate that CCN2/CTGF plays a critical role in endochondral ossification, possibly through collaboration with these MMPs. Indeed, a knockout mutation in ccn2/ctgf resulted in major skeletal defects caused by the impaired chondrocyte proliferation, vascular invasion, and matrix remodeling in the hypertrophic layers of the growth plate, which was accompanied by reduced MMP9 production (Ivkovic et al. 2003). However, no study has thus far comprehensively described the behavior and function of CCN2/CTGF among different cells involved in the development of a certain mesenchymal tissue in vivo.

The secondary ossification center is a feasible target for the study of the integrated function of CCN2/CTGF in mesenchymal tissues, because its formation involves various types of CCN2/CTGF producing/target cells (i.e., chondrocytes, osteoblasts, vascular endothelial cells) that support epiphyseal vascularization and endochondral ossification process. The formation of the secondary ossification center begins morphologically with cartilage canal formation, which is associated with peripheral vascular proliferation that invaginates from the perichondrium. This process provides a physical and biological background to support and nourish the joints that are exposed to mechanical stress immediately after birth, whereas the primary ossification center is engaged in longitudinal bone growth. Therefore, these two ossification centers play distinct roles in skeletal development through similar, but different, biological processes. Notably, subsequent ossification, which is characterized by disintegration of hypertrophic chondrocyte and formation of bone trabeculae accompanied by vascular invasion, takes place adjacent to preexisting cartilage canals (Karaplis 2002). A previous study presented evidence that CCN2/CTGF was a central growth factor in endochondral ossification, acting in paracrine and matricrine manners in the growth cartilage. However, the functional significance and role of CCN2/CTGF during earlier events, such as the formation of the secondary ossification center, are not known yet.

This study investigated the temporal and spatial gene expression and localization of CCN2/CTGF during the formation of the secondary ossification center and determined that CCN2/CTGF collaborates with other molecules in establishing epiphyseal vascularization. These data suggest that CCN2/CTGF is therefore involved in establishing epiphyseal vascularization, as well as determining and developing the secondary ossification center in the mouse tibia.

Materials and Methods

Tissue Preparation

To prepare samples for immunostaining and in situ hybridization, 20 mice at 1, 7, 14, 21, and 28 days postnatal were killed. Knee joints with the surrounding soft tissues were dissected and fixed with 4% paraformaldehyde in 0.1 M PBS (pH 7.4), decalcified with 10% EDTA, and embedded in paraffin according to an established procedure. Longitudinal sections of 7.0 μm thickness were cut on a microtome and mounted on slide glasses. The sections were stored at 4C until subsequent use. The Animal Committee of Okayama University Dental School approved all of the procedures.

Immunostaining

Two different antibodies were used as primary antibodies: One was a polyclonal anti-CCN2/CTGF serum that was raised in rabbits by immunization with a synthetic peptide of CCN2/CTGF, as described previously (Kubota et al. 2001). The other is a rabbit polyclonal antibody against mouse MMP9, which was commercially available (AB19047; Chemicon International, Temecula, CA). The paraffin sections were soaked in xylene to remove the paraffin and dehydrated in a graded series of ethanol (100% to 70%). After deparaffinization, the sections were rinsed in tap water. Endogenous peroxidase activity was quenched by immersion in 3% hydrogen peroxide in methanol for 30 min. After being washed in 0.1 M Tris-buffered saline (TBS; pH 7.4) for 5 min, the sections were incubated with 10% normal serum of the same species that produced the primary antibody for 1 hr at room temperature to eliminate nonspecific binding. Thereafter, the sections were incubated overnight with a 1:100 dilution of each primary antibody at 4C. After a wash in TBS with 0.1% Tween 20, the sections were incubated with a peroxidase-conjugated anti-goat IgG as a secondary antibody (PI-1000; Vector, Burlingame, CA) for 60 min at room temperature and washed in TBS. Finally, color development was performed using 3,3′-diaminobenzidine tetrachloride (Dojindo; Tokyo, Japan). The sections were also counterstained with hematoxylin and mounted. Controls samples were processed with the omission of the primary antibody or incubation with a normal rabbit serum (Dako; Tokyo, Japan) instead.

Confocal Laser-scanning Microscopy

The sections were also scanned for fluorescence using Radiance 2100K2 model (Bio-Rad Japan; Tokyo, Japan) to detect emission by nuclear staining (Sytox Green) or by antibody staining (Alexa Fluor). Sections were deparaffinized in xylene and rehydrated through graded ethanol to water, blocked in a blocking buffer (5% non-fat milk in TBS), and incubated overnight with a 1:100 dilution of primary anti-CCN2/CTGF antibody at 4C, and subsequently with a 1:800 dilution of goat anti-rabbit secondary antibody (IgG) conjugated to Alexa Fluor 568 (Molecular Probes; Eugene, OR) for 40 min at room temperature, after which samples were counterstained in Sytox Green (Molecular Probes) for 20 min at room temperature and washed in TBS. Confocal laser-scanning microscopy was performed using an argon/krypton laser (excitation wavelength, 488 and 568 nm). The images were enhanced using the Adobe Photoshop software program (San Jose, CA).

Preparation of Probes for In Situ Hybridization

For the generation of sense and antisense mouse CCN2/CTGF probes, a CCN2/CTGF cDNA (10kbp) that included the full length of the open reading frame, which was obtained from restriction enzymatic digestion and the fragment purification of pcDNA3.1(−)-ctgf (Kubota et al. 2000), was subcloned into a pGEM-3Zf plasmid (Promega; Madison, WI). For the generation of sense and antisense mouse collagen type X (α1) probes, a cDNA of mouse collagen type X α1 (640 bp), which was obtained from restriction enzymatic digestion and fragment purification of a PCR product, was subcloned into pGEM-7Zf(+) plasmid (Promega). Using these plasmids as templates, digoxigenin (DIG)-labeled sense and antisense riboprobes were synthesized with T7 or SP6 polymerase and DIG-11-UTP using a DIG-RNA labeling kit (Roche Molecular Systems; Mannheim, Germany). The synthesized probes were processed into pieces measuring ∼150 bp in length by hydrolysis for use in in situ hybridization.

Figure 1

Histological features and the localization of CCN2/connective tissue growth factor (CTGF) mRNA and protein in the tibial epiphysis at 1 day of age. Serial sections were stained with hematoxylin and eosin (A, H), hybridized with mRNA probes for CCN2/CTGF (B, D, E, I), hybridized with mRNA probes for type X collagen (C, J), or reacted with anti-CCN2/CTGF antibody (F, K). Control section was incubated with a normal rabbit serum (G, L). A higher magnification of the boxed regions in A-C is shown in H, D, E, I, and J, respectively. (D, E) CCN2/CTGF mRNA was detected in the perichondrium (arrowheads) on the surface of the epiphysis and in the ligament (asterisk). (F) CCN2/CTGF protein was detected in proliferating chondrocytes facing the perichondrium (arrowheads). (H-L) At a zone of the future epiphyseal plate, CCN2/CTGF protein was broadly detected from proliferation chondrocytes to hypertrophic chondrocytes, whereas CCN2/CTGF mRNA was detected in the chondrocytes facing toward those expressing type X collagen. pc, perichondrium. Bars: A-D = 200 μm; E-L = 100 μm.

In Situ Hybridization

In situ hybridization was performed, using the DIG-labeled sense and antisense CCN2/CTGF riboprobes as previously described (Nakanishi et al. 1997). In brief, deparaffinized and rehydrated sections were incubated with 10 μg/ml proteinase K for 15 min at 37C. The sections were hybridized for 16 hr at 50C. The hybridization solution contained 50% deionized formamide, 1× Denhardt solution, 10% dextran sulfate, 0.25% SDS, 1 mM EDTA, 600 mM NaCl, and 1 μg/ml labeled probe. Hybridization was performed in a humidified chamber. After hybridization, the sections were washed and immunoreacted with a diluted anti-digoxigenin Fab fragment conjugated with alkaline phosphatase (Roche) at room temperature for 1 hr. The sections were stained with nitroblue tetrazolium chloride and 5-bromo-4-chloro-3-indolyphosphate at room temperature. Under the conditions applied, the control hybridization incubated with the sense probe showed no detectable signals.

Figure 2

Histological features and the localization of CCN2/CTGF mRNA and protein in the tibial epiphysis at 7 days of age; sections facing toward the canal blind end. Serial sections were stained with hematoxylin and eosin (A, C), hybridized with mRNA probes for CCN2/CTGF (B, D, G), hybridized with mRNA probes for type X collagen (E, H), or reacted with anti-CCN2/CTGF antibody (F). A higher magnification of the boxed regions in A and B is shown in C and D, respectively. (G, H) Higher magnification of the regions invaded by blood vessels is shown in D and E, respectively. (C, E) The hypertrophic differentiation of chondrocytes in the central portion of epiphysis was observed around the blind end of the cartilage canal. (D) CCN2/CTGF mRNA was detected in the region of cartilage canal and capsule-attached marginal tissues. Note the CCN2/CTGF signals at the blood vessel invasion area (arrowheads). (F) CCN2/CTGF protein was detected in hypertrophic chondrocytes around the cartilage canal. (I-K) The localization of CCN2/CTGF in chondrocytes adjacent to the cartilage canal. (L-N) Confocal laser-scanning microscopic analysis of CCN2/CTGF in chondrocytes at a proliferation zone of the future epiphyseal plate. (J, M) Red labeling with CCN2/CTGF antibody and Alexa fluor-conjugated secondary antibody. (I, L) Green, Sytox green-stained cell nuclei. (K, N) Merged images of I and J, and L and M, respectively. In K, CCN2/CTGF is in both the pericellular matrix and the nuclei, whereas CCN2/CTGF is mainly in the pericellular matrix in N. hc, hypertrophic chondrocytes. Bars: A, B = 200 μm; C-F = 100 μm; G, H = 50 μm; I-N = 10 μm.

Results

Developmental Time Course of the Secondary Ossification Center in Mouse Tibiae

Initially, the secondary ossification center formation process was monitored before the study of stage-specific distribution of CCN2/CTGF, after a postnatal time course. At 1 day, the developing tibial epiphysis was mostly composed of resting cartilage (Figure 1A), and a cartilage canal was not yet present. However, in the superficial layer, chondrocytes adjacent to the capsule attachment tended to be proliferating. At 7 days of age, the chondrocytes in the central region of the epiphysis became hypertrophic, expressing type X collagen (Figure 2E). At the same time, the cartilage canal formed by an invagination from the perichondrium was observed with the growth of the epiphysis in the horizontal direction (Figure 3A). At this stage, fibrovascular proliferation started from the perichondrium close to the capsule attachment. The canal space was continuously lined by endothelial cells and was filled with a number of immature vasculatures (Figure 3D). Hypertrophic chondrocytes and resorption cartilage appeared around the cartilage canal and were particularly prominent near the canal blind end. At 14 days of age, the marrow space increased in size with the resorption of the hypertrophic chondrocytes, and a secondary ossification center was observed with the formation of thin bone trabeculae (Figure 4A). Along the boundary of the articular cartilage and secondary ossification center, an immature but polarized growth plate-like structure with continuously aligned chondrocytes was observed, but no such structure was observed at the metaphysial side of the marrow space (Figure 4B). Consistent with this histological feature, chondrocytes expressing type X collagen were observed only along the articular side of the ossification center (Figure 4J). Finally, at 28 days of age, the ossification center expanded, and the walls of marrow cavity were formed by thick bone trabeculae (Figure 5A). Groups of hypertrophic chondrocytes in the central region of the epiphysis were mostly replaced by bone marrow except at the marginal region of both lateral sides. At this stage, the formation of articular cartilage and the epiphyseal plate can be eventually clearly distinguished.

Expression of ccn2/ctgf and Distribution of CCN2/CTGF During Cartilage Canal Invasion

The spatiotemporal patterns of CCN2/CTGF expression were examined during the formation of the ossification center by in situ hybridization. Initially (1 day of age), ccn2/ctgf was expressed in perichondrium adjacent to the capsule attachment, in the capsule itself, and in a zone of proliferating cartilage of the future epiphyseal plate before the cartilage canal invasion (Figures 1D, 1E, and 1I). No signals were yet observed in the central region of the epiphysis. At 7 days of age, ccn2/ctgf was strongly expressed in the region of cartilage canal and capsule-attached marginal cartilage (Figures 2B and 3B). The signals were detected in the cells adjacent to cartilage canal and were located at the center of the hypertrophic region of the epiphysis (Figure 2G). Most signals in chondrocytes were from those juxtaposing the cartilage canal, and distinct signals were also observed in the endothelial cell on walls of the cartilage canal (Figure 3I). It should be noted that chondrocytes expressing ccn2/ctgf were located in the areas facing toward, but distinct from those chondrocytes expressing type X collagen. Immunostaining of the serial sections showed that CCN2/CTGF molecules were distributed in a different pattern from that of the mRNA. At 1 day of age, positive staining was observed in the proliferating chondrocytes of the superficial layer adjacent to the capsule attachment and perichondrium (Figure 1F). At 7 days of age, CCN2/CTGF protein was detected broadly in both proliferating and hypertrophic chondrocytes around the cartilage canal and the area of blood vessel invasion (Figures 2F and 3G). Positive signals were also present in endothelial cells in the cartilage canal (Figure 3K). A confocal laser-scanning microscopic analysis showed that CCN2/CTGF was localized in both the pericellular matrix and the nuclei of the chondrocytes adjacent to the cartilage canal (Figure 2K). In contrast, at a zone of proliferating cartilage of the future epiphyseal plate, CCN2/CTGF protein was broadly detected from proliferating chondrocytes to hypertrophic chondrocytes over the ones expressing type X collagen. The chondrocytes expressing ccn2/ctgf were detected in the areas facing toward but distinct from those chondrocytes expressing type X collagen (Figures 1I-1K). A confocal laser-scanning microscopic analysis showed that CCN2/CTGF was principally present in the pericellular matrix of proliferating chondrocytes of the future epiphyseal plate with a few signals in the nuclei (Figure 2N). This localization and gene expression pattern of CCN2/CTGF in the epiphyseal plate and its prototype was maintained throughout the observation period.

Figure 3

The histological features and localization of CCN2/CTGF mRNA and protein in the tibial epiphysis at 7 days of age; cross-section of the cartilage canal. Serial sections were stained with hematoxylin and eosin (A, D), hybridized with mRNA probes for CCN2/CTGF (B, E, I), hybridized with mRNA probes for type X collagen (C, F, J), reacted with anti-CCN2/CTGF antibody (G, K), or incubated with a normal rabbit serum (H: control). Higher magnification of the boxed regions in A-C is shown in D-F, respectively. Certain areas in E and F were further magnified and are shown in I and J, respectively. (D) The cartilage canal was formed with peripheral vascular proliferation that invaginates from perichondrium. The hypertrophic differentiation of chondrocytes was observed around the blind end of cartilage canal. (E, I) CCN2/CTGF mRNA was detected form chondrocytes juxtaposing the cartilage canal and endothelial cells on the walls of the cartilage canal (arrowheads). (G, K) CCN2/CTGF protein was detected in the hypertrophic chondrocytes around the cartilage canal and endothelial cells. Bars: A-C = 200 μm; D-K = 100 μm.

Figure 4

The histological features and localization of CCN2/CTGF mRNA and protein in the tibial epiphysis at 14 days of age. Serial sections were stained with hematoxylin and eosin (A-D), hybridized with mRNA probes for CCN2/CTGF (E-H), hybridized with mRNA probes for type X collagen (I-L), reacted with anti-CCN2/CTGF antibody (M-O), or incubated with a normal rabbit serum (P-R: control). A higher magnification of the boxed regions in A, E, and I is shown in B-D, F-H, and J-L, respectively. CCN2/CTGF mRNA was detected in chondrocytes in the region of the articular surface and the distal side of the marrow cavity (arrowheads) and one layer of the cartilage zone in the future epiphyseal plate (arrows). (M-O) CCN2/CTGF protein was detected in a number of chondrocytes including proliferating chondrocytes and hypertrophic chondrocytes around the secondary ossification center. Specific regions are indicated as follows: pz, zone of proliferating cartilage; os, ossification center. Bars: A, E, I = 200 μm; B-D, F-H, J-R = 100 μm.

Figure 5

The histological features and localization of CCN2/CTGF mRNA and protein in the tibial epiphysis at 28 days of age. Serial sections were stained with hematoxylin and eosin (A), hybridized with mRNA probes for CCN2/CTGF (B), hybridized with mRNA probes for type X collagen (C), or reacted with anti-CCN2/CTGF antibody (D). Control sections were incubated with a normal rabbit serum (E). A higher magnification of the lateral sides of marrow cavity of A-E is shown in F-J, respectively. The ossification center expanded with progressive resorption of hypertrophic chondrocytes around the marrow cavity. The epiphyseal growth plate can be clearly distinguished. CCN2/CTGF mRNA was detected in the chondrocytes in the region of the articular surface (B). CCN2/CTGF protein was strongly detected in chondrocytes at the lateral sides of the marrow cavity (D) but was weakly detected in chondrocytes near both the articular surface and the distal side of the marrow cavity (arrowheads). hc, hypertrophic chondrocytes. Bar = 100 μm.

Expression of ccn2/ctgf and Distribution of CCN2/CTGF During Development of the Secondary Ossification Center

After the formation of the cartilage canal, a strong ccn2/ctgf expression was distinctly observed in relatively immature chondrocytes in the region of both the articular surface and a specific layer of the early proliferating cartilage zone in the growth plate at 14 days of age (Figure 4E). A relatively low level of expression was also detected at the border of the epiphysial marrow space (Figures 4F and 4H). This spatial gene expression pattern was observed until 21 days of age (data not shown). However, the CCN2/CTGF signals decreased gradually as the ossification progressed, and eventually, ccn2/ctgf mRNA signals became limited in chondrocytes in the region of articular surface at 28 days of age (Figure 5B). Even though there was a limited distribution of the ccn2/ctgf expression, immunostaining showed strong and diffuse CCN2/CTGF signals among the chondrocytes around the ossification center at 14 days of age (Figure 4M). However, the CCN2/CTGF that was strongly detected in the hypertrophic chondrocytes on day 7 decreased because of the replacement of the corresponding hypertrophic cartilage by bone marrow. Next, at 28 days of age, the number of positively stained cells further decreased, because of the disintegration of hypertrophic chondrocyte and formation of bone trabeculae in its place (Figure 5D). The CCN2/CTGF-positive hypertrophic chondrocytes were localized in a quite limited area adjacent to the ossification center. Throughout the observation period of 28 days, CCN2/CTGF that accumulated in/around the hypertrophic chondrocytes tended to be preserved for a long time period.

Production of MMP9 in the Cartilage Canal and Secondary Ossification Center in Early Stages of Secondary Ossification Center Formation

Because MMP9 is known to be critically involved in endochondral ossification (Vu et al. 1998), the distribution of MMP9 molecules during the formation of the ossification center was also examined. Positive immunostaining of MMP9 was clearly observed during the formation of the cartilage canal (Figure 6A) and an enlargement of the marrow space (Figure 6D). The MMP9 protein was strongly detected at the blind end of the canal and the wall of the forming marrow space. Furthermore, the MMP9 protein was also detected in the hypertrophic chondrocytes adjacent to the cartilage canal, where CCN2/CTGF protein was detected as well.

Discussion

This study clarified that CCN2/CTGF exhibits highly dynamic patterns of expression that are temporally and spatially restricted during postnatal formation of the secondary ossification center in mouse long bones. These expression patterns suggest that the factor is engaged in establishing epiphyseal vascularization and remodeling of the secondary ossification center. These experiments showed that the CCN2/CTGF protein was present at three sites: the surface of the epiphysis, proliferating and hypertrophic layers in the growth plate and secondary ossification center, and articular cartilage. At these sites, relatively immature chondrocytes expressing ccn2/ctgf were localized in the areas facing to, but distinct from, those of chondrocytes accumulating CCN2/CTGF and expressing type X collagen. This suggests CCN2/CTGF to be associated with the terminal differentiation and ossification of these chondrocytes, which is consistent with the findings on CCN2/CTGF during endochondral ossification at the growth plate.

Figure 6

Immunohistochemical staining of MMP9 in the tibial epiphysis at 7 days of age (A-C) and 14 days of age (D-F). (B) Higher magnification of the area of the canal blind end in A. MMP9 protein was detected abundantly in the canal blind ends and also in chondrocytes (arrowheads) adjacent to the canal. The canal blind end was accompanied by resorption of cartilage matrix. (E) Higher magnification of the lateral side of the marrow cavity in D. The MMP9 protein was detected at the wall of forming marrow space and chondrocytes (arrowheads) near marrow space. (C, F) Control sections incubated with a normal rabbit serum. Bars: A, C, D, F = 100 μm; B, E = 50 μm.

The expression pattern of ccn2/ctgf in the secondary ossification center, however, is different from that at growth plate in the diaphysis. First, the strongest ccn2/ctgf expression occurs in relatively immature chondrocytes, in contrast to the prehypertrophic and hypertrophic chondrocytes in the growth plate. Second, ccn2/ctgf is expressed in the cells adjacent to cartilage canal formation, which is uniquely observed in secondary ossification formation. Because CCN2/CTGF is a matrix-associated secreted protein, it either stays at the site of production for a long time or can be transported away to execute functions elsewhere. Interestingly, CCN2/CTGF accumulated in the central region of the mouse tibial epiphysis, where chondrocytes become hypertrophic, and the cartilage canals pass into the hypertrophic cell mass to supply the mesenchymal cells for bone formation. This spatiotemporal expression and distribution pattern suggests that CCN2/CTGF is therefore related, not only to chondrocyte proliferation, but also to epiphyseal vascularization remodeling.

There is significant evidence indicating that CCN2/CTGF plays a role in the regulation of the endothelial cell function and angiogenesis. CCN2/CTGF regulates the production and/or activity or other angiogenic molecules, such as vascular endothelial growth factor (Inoki et al. 2002; Kondo et al. 2006), or possibly, basic fibroblast growth factor (Brigstock 2002). Moreover, CCN2/CTGF is intrinsically active in in vivo assays for angiogenic activity (Babic et al. 1999; Shimo et al. 1999). In fact, CCN2/CTGF knockout mice exhibited vascular defects during endochondral bone formation (Ivkovic et al. 2003).

It should be noted that CCN2/CTGF may also affect ECM stability or integrity. CCN2/CTGF increases the expression of an MMP in vascular endothelial cells (Kondo et al. 2002). The MMPs are extracellular endopeptidases that regulate cell growth, migration, and ECM remodeling. In addition, the MMPs also have been implicated in a number of physiological and pathological processes including normal and tumor angiogenesis (Bergers et al. 2000; Engsig et al. 2000; Fang et al. 2000). These data together suggest that CCN2/CTGF can drive the balance of proteinases and their respective endogenous inhibitors toward increased proteolysis of the ECM, which eventually promotes vascular endothelial cell migration. This study showed the overlapping distribution of MMP9 and CCN2/CTGF during the formation of the ossification center, which suggested that CCN2/CTGF was produced in the perichondrium and cartilage around the canal and reached their target by diffusion to provoke MMP9 production. In addition, Davoli et al. (2001) previously reported the histozymographic reactions and immunolocalization of active MMP9 during the development of microcirculation of the secondary ossification center in the rat humeral head. Three immunoreactive sites were identified: the walls on the blind end of cartilage canal, segments of the wall around forming marrow space, and along the edges of the wall containing groups of hypertrophic chondrocytes. In this study, MMP9 was observed not only these sites but also in hypertrophic chondrocytes around cartilage canal and marrow space in the central portion of the epiphysis. This broader distribution of reactiveness might be because of the characteristic of the primary antibody that recognizes both the pro-form and active form of mouse MMP9 (the 95-to 105-kDa gelatinase). Supported by the in vivo and in vitro findings above, it is reasonable to hypothesize that MMPs, including MMP9, are playing major roles as cartilage excavators during the formation of the ossification center.

After serial changes during the development of the ossification center, ccn2/ctgf expression decreased along the diffuse ossification of the epiphysis, whereas immunostaining for CCN2/CTGF remained strong in proliferating and hypertrophic chondrocytes for a long time. It has been widely recognized that CCN2/CTGF is an ECM-associated growth factor, which is best represented by the fact that heparan sulfate proteoglycans were critically required for CCN2/CTGF to exert its function (Gao and Brigstock 2004). After being secreted and thereafter diffusing from the source, CCN2/CTGF is stably trapped in the ECM, even after the gene expression ceases. Therefore, the expression pattern of CCN2/CTGF mRNA and the distribution of CCN2/CTGF proteins do not always coincide, as observed in this study. Furthermore, CCN2/CTGF was also detected in the nuclei of certain chondrocytes. Although CCN2/CTGF has been reported to be taken up into the nucleus of chondrocytic cells (Kawata et al. 2006), its functional significance is unknown. The role of the nuclear CCN2/CTGF is currently being studied.

In conclusion, CCN2/CTGF first accumulated at the center of mouse tibial epiphysis on formation of the cartilage canal. Thereafter, it was distributed at the locations toward the secondary ossification center development. There was also an overlapping distribution of MMP-9 and CCN2/CTGF in the secondary ossification center. These findings suggest that CCN2/CTGF is an important factor for establishing epiphyseal vascularization in determining the secondary ossification center formation, together with MMP9.