By continuing to browse this site you agree to us using cookies as described in About Cookies
Notice: Wiley Online Library will be unavailable on Saturday 7th Oct from 03.00 EDT / 08:00 BST / 12:30 IST / 15.00 SGT to 08.00 EDT / 13.00 BST / 17:30 IST / 20.00 SGT and Sunday 8th Oct from 03.00 EDT / 08:00 BST / 12:30 IST / 15.00 SGT to 06.00 EDT / 11.00 BST / 15:30 IST / 18.00 SGT for essential maintenance. Apologies for the inconvenience.
Midkine (MK) is a heparin-binding growth/differentiation factor implicated in the control of development and repair of various tissues. Upon fracture of the murine tibia, MK was found to be transiently expressed during bone repair. MK was immunohistochemically detected in spindle-shaped mesenchymal cells at the fracture site on day 4 after fracture and in chondrocytes in the area of endochondral ossification on day 7. MK expression was decreased on day 14 and scarcely seen on day 28 when bone repair was completed. This mode of MK expression is reminiscent of MK expression during development. MK was expressed in hypertrophic chondrocytes of the prebone cartilage rudiments on embryonic day 14 in mouse embryos. MK was also strongly expressed in the epiphyseal growth plate. MK was localized intracellularly during both bone repair and development, and this localization was confirmed by immunoelectron microscopy for embryonic chondrocytes. When MK cDNA was transfected into ATDC5 chondrogenic cells and overexpressed, the majority of transfected cells with strong MK expression showed enhanced chondrogenesis as revealed by increased synthesis of sulfated glycosaminoglycans, aggrecan, and type II collagen. These results suggest that MK plays important roles in chondrogenesis and contributes to bone formation and repair.
Fracture healing proceeds through three distinct phases, namely inflammation, reparation, and remodeling.(1) The healing process generally follows the same pathway as during chondrogenesis and osteogenesis observed in normal development.(1) Various lines of evidence have strongly suggested that several growth factors, such as bone morphogenetic protein,(2–4) fibroblast growth factors,(5) platelet-derived growth factor,(6) insulin-like growth factor,(7) and transforming growth factor-β(8) participate in the healing process.(1) However, the entire process of fracture healing has not been clarified. For example, other as yet unidentified factors may be important in the healing process. Precise molecular understanding of the repair process will contribute to control of the process and promotion of fracture healing. Midkine (MK) is a heparin-binding growth/differentiation factor with a molecular weight of 13 kDa which is rich in cysteine residues and basic amino acids.(9,10) MK has about 50% sequence identity with pleiotrophin,(11) also called heparin-binding growth associated molecule,(12) but has no sequence homology with other known proteins. Retinoic acid-induced heparin-binding protein (RIHB)(13) is regarded as the chicken homolog of MK.(14) MK is considered to be involved in neurogenesis since it promotes neurite outgrowth,(15,16) survival of embryonic neurons,(17) neuroneal differentiation,(18,19) and synapse formation.(20) MK has also been suggested to participate in tooth development.(21) Furthermore, MK promotes fibrinolysis,(22) is chemotactic to neutrophils,(23) and is involved in tumorigenesis and tumor progression.(24–26) It is necessary to evaluate the role of MK in fracture healing first because this molecule has been implicated in tissue repair because it is expressed in earlier stages of repair following experimental cerebral infarction(27) and heart infarction.(28) Furthermore, intraretinal injection of MK prevents retinal degeneration caused by exposure to constant light.(29) Second, MK stimulates collagen synthesis and glycosaminoglycan synthesis in human skin fibroblasts.(30) Third, MK or RIHB expression was reported during chondrogenesis in the chicken(31) and in the mouse.(32) In the present investigation, we at first examined MK expression during fracture repair. Then, we performed an in-depth study of MK expression during bone development in the mouse and compared the mode of MK expression during the repair process to that during normal development. Finally, we overexpressed MK in a chondrogenic cell line to determine whether the correlation between MK expression and chondrogenesis has functional implications.
MATERIALS AND METHODS
Bone fracture model
A modification of the fracture model of Hiltunen(33) was used with ICR mice (Clear Co., Tokyo, Japan) approximately 10 weeks old. A standardized closed diaphyseal fracture was produced in each tibia. Under anesthesia induced by intraperitoneal injection (0.014 ml/g of body weight) of 10% pentobarbital, a 0.2 mm stainless steel rod was introduced into the medullar cavity of the tibia from the tibial tuberosity. The wound was closed and the procedure was repeated on the contralateral side. The prenailed right tibial shaft was fractured manually, while that on the left side was not fractured. The animals were allowed free movement with unrestricted weight bearing in cages after recovery from anesthesia. The animals were sacrificed by cervical dislocation 2, 4, 7, 14, or 28 days after the operation, then bilateral whole tibias were harvested.
ICR mice were spontaneously mated, and the day of the first appearance of a vaginal plug was taken as day 0 of pregnancy; the gestational age of embryos in embryonic days (E) was also confirmed by morphological criteria.(34) The forelimb buds (E10–E16) were cut off from the trunks in Dulbecco's phosphate-buffered saline (PBS), pH 7.4, under a stereo microscope.
Mouse growth plate tissue
Epiphyseal growth plate tissue was collected from the proximal tibia of 2-day-old newborn ICR mice.
All specimens were fixed overnight in 4% paraformaldehyde in PBS, pH 7.4. Harvested tibias were decalcified with 0.5 mol/l of EDTA. After dehydration, the tissues were embedded in paraffin and cut into sections 6-μm-thick through the long axis. Serial sections were mounted on slides, dried overnight, and stored in an airtight box. Sections were stained with hematoxylin and eosin, Alcian blue (pH 1.0), Safranin O, or immunohistochemically.
After dewaxing and blocking of intrinsic peroxidase by 0.3% peroxide in methanol for 30 minutes, the sections were incubated with 1% normal goat serum for 30 minutes. Sections were incubated overnight at 4°C with anti-MK antibody (30 μg/ml in 0.1% bovine serum albumin [BSA] in PBS, pH 7.4) or anti-aggrecan antibody. Control sections were incubated with preimmune rabbit serum. Sections were then incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit immunoglobulin G (IgG) antibody (Jackson Immunoresearch Laboratories, Inc., Westgrove, PA, U.S.A.) (dilution 1:500 in PBS). Peroxidase was visualized by incubation with 3-amino-9-ethylcarbazole (AEC) (DAKO, Carpinteria, CA, U.S.A.) containing 1% H2O2. Light counterstaining was performed with hematoxylin. Crystal/Mount was used for permanent preservation.
Thin-section electron microscopy
Digits of the E16 mouse embryos were fixed with 2.5% glutaraldehyde in 0.05 M phosphate buffer (pH 7.4) for 2 h, and subsequently with 1% OsO4 in the buffer for 1 h. The specimens were washed with 5% sucrose in distilled water and incubated with 3% uranyl acetate in distilled water for 1 h. Then the specimens were dehydrated through a graded ethanol series and embedded in Epok 812 epoxy resin (Oken Shoji, Tokyo, Japan). Ultrathin sections were cut with a diamond knife on a Porter-Blum MT-1 Ultramicrotome (Sorvall, Newtown, CT, U.S.A.), stained with uranyl acetate and lead citrate, and examined with a H-7100 transmission electron microscope (Hitachi, Tokyo, Japan).
Digits of the E16 mouse embryos were fixed with 4% paraformaldehyde in 0.05 M phosphate buffer (pH 7.4) at 4°C for 2 h. The specimens were washed thoroughly in the buffer and incubated with 50 mM NH4Cl in the buffer at 4°C overnight to block free aldehyde groups. After washing in 5% sucrose solution, the specimens were dehydrated in graded concentrations of ethanol and embedded in Lowicryl K4 M resin (TAAB, Berk, U.K.). Lowicryl ultrathin sections cut with the ultramicrotome were picked up on uncoated nickel grids. The sections were preincubated for 15 minutes with 1% BSA in PBS and incubated with rabbit anti-MK antibody diluted 1:50 at room temperature for 1 h. After washing in PBS, the sections were incubated with 10 nm colloidal gold-conjugated goat anti-rabbit IgG antibody (Amersham Japan, Tokyo, Japan) diluted 1:30 at room temperature for 1 h. Then the sections were stained with uranyl acetate and lead citrate and examined with an electron microscope. Control sections were incubated with normal rabbit serum in place of the anti-MK antibody.
Heparin-Sepharose column chromatography and Western blotting
Seven days after fracture, samples of about 1 g of callus tissue were collected from 27 ICR mice. As a control, contralateral muscle around the bone at the same level as the fracture site was harvested. The samples were subjected to heparin-Sepharose column chromatography and Western blotting as described previously.(16) Briefly, they were homogenized and heparin binding proteins were isolated by heparin-Sepharose CL-6B column chromatography, then analyzed by SDS-PAGE. Proteins in the gels were transferred onto nitrocellulose membranes. After overnight incubation in 5% skimmed milk in PBS at 4°C, the nitrocellulose sheets were incubated with anti-MK antibody for 2 h at room temperature. After washing with PBS containing 0.1% Tween 20, membranes were incubated with anti-rabbit IgG conjugated with horseradish peroxidase for 1 h. Enhanced chemiluminescence (Amersham) was used to expose autoradiographic film.
Cell lines and culture conditions
ATDC5, a chondrogenic clonal cell line established by Atsumi,(35) was provided by Riken Cell Bank (Tsukuba, Japan). ATDC5 cells were cultured in maintenance medium consisting of a 1:1 mixture of DMEM and Ham's F-12 medium (ICN Biomedicals, Ltd., Thame, U.K.) containing 5% fetal bovine serum (Dainippon Pharmaceutical Co. Ltd., Osaka, Japan), 10 μg/ml human transferrin (Sigma Chemical Co., St. Louis, MO, U.S.A.) and 3 × 10–8 sodium selenite (Nacalai Tesque, Kyoto, Japan), as previously described.(36) For induction of chondrogenesis, the cells were cultured in differentiation medium consisting of maintenance medium supplemented with 10 μg/ml bovine insulin (Sigma). Cells were maintained at 37°C in a humidified atmosphere of 5% CO2 in air. The medium was replaced every other day.
DNA transfection was performed as described previously.(24) ATDC5 cells were plated at a density of 2 × 105 cells in 60-mm dishes. On the following day, 2.5 μg of either MIW mMK (a mouse MK expression vector under the control of the Rous sarcoma virus enhancer with the chicken β-actin promoter and enhancer) or MIW (an empty vector without any insert)(37) was transfected together with 0.25 μg of pST neo (a neomycin resistance gene expression vector) into ATDC5 cells using lipofectin (GIBCO BRL, Rockville, MD, U.S.A.) according to the manufacturer's instructions. Two days after plating, cells were treated with 400 μg/ml G418 (Sigma). G418-resistant colonies were isolated from the tissue culture dishes after ∼12–15 days of selection.
When ATDC5 cells or their transfectants reached confluency, medium was replaced by serum-free medium containing 20 μg/ml of heparin. Medium was harvested after 24 h. MK concentration in the harvested medium was measured by enzyme-linked immunoassay as described previously.(38)
Staining of cultured cells with Alcian blue, anti-aggrecan antibody, or anti–type II collagen antibody
Cells were inoculated at a density of 8 × 103 cells per well in 8-well glass chamber slides (Nunc, Naperville, IL, U.S.A.). From the next day, cells were incubated in maintenance medium or differentiation medium. Cells were stained 1, 2, and 3 weeks after inoculation. After medium was removed, cells were washed three times with PBS(+) and fixed with Kahle's solution for more than 10 minutes. Then they were rinsed twice with distilled water and incubated with 0.5% Alcian blue (Sigma) in 0.1 N HCl overnight. They were destained with 0.1 N HCl for 10 minutes, rinsed twice with distilled water, and covered with glycerin and a coverslip. Immunostaining was performed as described by Atsumi et al.(35) Briefly, after medium was removed, cells were washed and fixed with methanol at –20°C for 2 minutes. Then they were incubated with anti-aggrecan antibody or anti–type II collagen antibody, and finally with HRP-conjugated goat anti-rabbit IgG antibody or HRP-conjugated goat anti-mouse IgG antibody (Jackson Immunoresearch Laboratories, Inc.), respectively. Peroxidase was visualized by incubation with AEC containing 1% H2O2.
Determination of sulfated glycosaminoglycan synthesis
Sulfated glycosaminoglycan synthesis by transfectants was measured by35S incorporation.(39) Cells were inoculated at a density of 9 × 104 cells in 35-mm dishes, cultured for 2 weeks in differentiation medium, and then incubated in medium containing 10 μCi/ml of35S sulfate (Amersham) for 24 h. After removal of the medium and washing with PBS five times, the cells were incubated with 0.5 ml of 0.25% trypsin at room temperature for 10 minutes, and the resultant cell suspension was collected. The dish was washed with 0.5 ml of PBS. The cell suspension and the eluate were combined, and the cells were removed by centrifugation. Aliquots of 0.4 ml of supernatant from each dish were digested with 1 mg of pronase (Wako Bioproducts, Richmond, VA, U.S.A.) for 3 h at 37°C. The digests were mixed with 0.1 ml of 0.2 M NaCl containing 2 mg of carrier chondroitin sulfate (Seikagaku Kogyo Co., Tokyo, Japan) and 0.5 ml of 1% cetylpyridinium chloride (Wako). After incubation at 37°C for 1 h, the precipitate was collected by centrifugation at 10,000 rpm for 30 minutes. The cetylpyridinium–glycosaminoglycan complex was dissolved in 0.1 ml of 4 M NaCl and reprecipitated with 1.4 ml of 80% aqueous ethanol to obtain purified glycosaminoglycans. These were dissolved in 0.4 ml of water, and radioactivity was measured by liquid scintillation counting.
Northern blot analysis
To prepare mouse aggrecan probe, total RNA was extracted from limb cartilages of newborn mice and a specific fragment corresponding to the interglobular domain of aggrecan was amplified by polymerase chain reaction as described by Glumoff et al.(40) and was32P-labeled by Megaprime DNA labeling system (RPN 1607; Amersham). Total RNA was prepared from cells cultured in 10-cm dishes for 2 weeks in differentiation medium, subjected to agarose gel electrophoresis, and transferred to nitrocellulose membrane as described previously.(41) After hybridization and washing,(41) the membranes were exposed to an imaging plate and then the radioactivity on the membrane was determined with a radioimage analyzer (BAS 2000, Fuji Photo Film Co., Tokyo, Japan).
Antibodies and other materials and methods
Rabbit anti-MK antibody was purified by affinity chromatography on an MK-column as described previously.(16) Preimmune serum was taken from same rabbit used to produce anti-MK antibody and was similarly affinity-purified on an MK-column(16); the elute obtained was used as the control reagent. Neutralization of anti-MK antibody by MK was performed as described previously.(42) Anti-aggrecan (anticartilage-specific proteoglycan) antibody was kindly provided by Prof. K. Kimata, Aichi Medical School, Japan.(43) Anti–type II collagen monoclonal antibody was obtained from Chemicon International Inc. (Temecula, CA, U.S.A.). Protein content of cultured cells was determined by BCA protein assay (Pierce Chemical Co., Rockford, IL, U.S.A.) using BSA as a standard.
MK expression in bone and cartilage formation
MK expression during fracture repair
We examined MK expression during repair of the fractured mouse tibia by immunohistochemical procedures. On day 2 after fracture, anti-MK immunoreactivity was scarcely observed in blood clots. On day 4 at the early stage of fracture healing, spindle-shaped mesenchymal cells were proliferating in the fractured area (Fig. 1a). MK was expressed in some mesenchymal cells at the fracture site and weakly in thickened periosteum adjacent to the site of the fracture (Fig. 1b). On day 7, chondrocytes were proliferating in the fractured area, and intramembranous bone formation could be observed at the periosteum close to the site of the fracture. At this stage, MK immunoreactivity was strongly detected in cytoplasm of hypertrophic chondrocytes (Fig. 1d), and chondrocytes could be identified by staining with Safranin O (Fig. 1c) or with antiaggrecan antibody (Fig. 1f). No staining was observed in controls, which were stained by the control reagent obtained from preimmune serum (Fig. 1e). Other chondrocytes were occasionally weakly positive, and osteocytes in the area of intramembranous ossification showed weak staining (data not shown). Western blotting analysis confirmed that the MK-immunoreactive material in the callus on day 7 was indeed MK (Fig. 2). On day 14, chondrocytes had partially been replaced by newly formed trabecular bone, and immunostaining was observed at similar sites as observed on day 7 but was less intense. On day 28, chondrocytes were completely replaced by trabecular bone, and fractured bones were united (Fig. 1g). MK was scarcely detected in trabecular bone at this stage (Fig. 1h). Therefore, we concluded that during fracture repair, MK was strongly expressed in chondrocytes.
MK expression during chondrogenesis in limb buds and in epiphyseal growth plates
We then examined whether MK expression during fracture repair is related to that during normal development. As the first sign of bone formation in limb buds, we found condensation of mesenchyme which was weakly stained with Alcian blue on E12. At and before this stage, MK was not detected. On E14, the condensed mesenchymal cells in the center core of limb buds became separated and formed the prebone cartilage rudiments. MK was expressed in hypertrophic chondrocytes in the central part of the prebone cartilage rudiments. On E16, MK expression was increased in the same area as on E14 (Fig. 3). MK was also expressed in myocytes and epithelium. In epiphyseal growth plates, MK immunoreactivity was observed in the same area as where extracellular matrix was intensely stained with Alcian blue (Figs. 4a and 4b). MK expression was most intense in chondrocytes in the resting zone (Fig. 4c) of the mouse epiphyseal growth plate, but MK was also localized in proliferative (Fig. 4d) and hypertrophic zone (Fig. 4e). Extracellular matrix was not stained.
Transfection with MK cDNA enhanced chondrogenesis in ATDC5 cells
We then examined causal relationship between MK expression and chondrogenesis. When MK was added to ATDC5 chondrogenic cells, we observed no enhancement of chondrogenesis. Therefore, we examined the effects of MK by DNA transfection. We first transfected either a mouse MK expression vector (MIW mMK) or control vector (MIW) together with pST neo (a neomycin resistance gene expression vector) into ATDC5 cells. Colonies on tissue culture dishes were then isolated after ∼12–15 days of G418 selection. To examine MK expression, the amount of MK in medium of each clone was measured by enzyme-linked immunoassay. Five clones that expressed MK at the highest level were used for the experiments (Table 1). Five clones randomly selected from colonies grown after transfection with MIW and neo were used as controls. Cells were cultured with or without insulin, and after 1, 2, and 3 weeks they were stained with Alcian blue. Three transfectants (MK25, MK26, and MK29) were stained more intensely than control cells when they were cultured in the medium with insulin (the differentiation medium). After 3 weeks in culture without insulin, the three clones were weakly stained, although the five control clones and parental ATDC5 were not stained (Fig. 5). Promoted proteoglycan synthesis by MK transfectants was revealed by measurement of35S incorporation into proteoglycans. Three clones that showed intense staining incorporated more35S than control clones. BCA protein assay showed little difference between MK transfectants and control cells (Table 1). This indicated that the promoted proteoglycan synthesis was not due to increased cell proliferation. Therefore, we concluded that MK enhanced biosynthesis of glycosaminoglycans, probably by promoting chondrogenesis. We also noted that not all the transfectants producing high levels of MK were promoted for chondrogenesis. Some intrinsic factor of the recipient cells may give influence on MK action.
Table Table 1.. 35S-Labeled Glycosaminoglycan Synthesis by MK Transfectants or Mock Transfected Cells
To verify further the increased chondrogenesis in the three clones transfected with MK cDNA, three clones as well as three control clones cultured in the differentiation medium were stained with anti-aggrecan (Fig. 6A) or antitype II collagen (Fig. 6B) antibodies, since aggrecan and type II collagen are established markers of chondrocytes.(44) Indeed, the three clones (M25, M26, and M29) expressed aggrecan and type II collagen much more strongly than control clones. We also determined the amount of aggrecan mRNA by Northern blot analysis and confirmed that clones M25, M26, and M29 expressed much more aggrecan mRNA than control clones when they were cultured for 2 weeks in differentiation medium (Fig. 7). Thus, we were able to conclude that chondrogenesis was promoted in these clones transfected with MK cDNA.
Immunoelectron microscopic localization of MK in chondrocytes in limb buds
In chondrocytes, MK was present intracellularly both during fracture repair and development. Furthermore, exogenously added MK had no effect on ATDC5 cells, while that delivered endogeneously by DNA transfection was effective. These observations made it necessary to locate precisely MK within cells. Intracellular localization of MK was analyzed by electron microscopy in MK-expressing chondrocytes of the prebone cartilage rudiments on E16. Rough endoplasmic reticulum and Golgi apparatus were well developed in the MK-expressing cells (Figs. 8a and 8b). Gold particles were distributed in rough endoplasmic reticulum–rich regions (Fig. 8c) and other cytoplasmic regions (Fig. 8d). There were only a few gold particles in the lumen of rough endoplasmic reticulum (Fig. 8c). No immunoreactivity was observed upon control staining with normal rabbit serum (Figs. 8e and 8f) or staining with neutralized antibody (data not shown). Very few gold particles were seen in the nuclei.
MK was found to be expressed from the early stages of fracture repair in the mouse tibia. Scarcely any MK was detected in the inflammatory stages. Although MK has also been suggested to participate in inflammation in relation to rheumatoid arthritis,(23) it may not be involved in normal inflammation preceding bone repair. However, the mode of MK expression during the reparative stage, i.e., strong expression in proliferative chondrocytes, suggested a role in chondrogenesis. In normal development, MK was also expressed in chondrocytes in both the limb bud and growth plates. The mode of MK expression in the fractured area was similar to that in the growth plates; in both cases MK was detectable in the area in which extracellular matrix was abundant. The mode of expression in limb buds was somewhat different, with MK present in the central part of the prebone cartilage rudiments where calcification and angiogenesis first occur. Similar findings have already been reported for RIHB.(31) Although cellular mechanisms of cartilage elongenation are considered to be the same in the growth plate and in the embryonic limb, these observation might imply some differences between them. Furthermore, taking into consideration its angiogenic activity,(45) MK may also play a role in vascularization. In this context, it is noteworthy that basic fibroblast growth factor, which is present in growth plates in a manner similar to MK, is considered to act as a chemotactic signal for vessel proliferation.(46)
cDNA transfection and overexpression of MK cDNA in ATDC5 chondrogenic cells indicated that MK enhances chondrogenesis. Increased staining with Alcian blue, increased synthesis of sulfated glycosaminoglycans, aggrecan, and type II collagen were observed in the transfected cells. It should be noted that not all the transfected cells expressing high levels of MK showed up-regulated chondrogenesis. Some factors in the recipient cells determined susceptibility to MK. A similar phenomenon was observed following oncogenic transformation of NIH3T3 cells by transfection with MK cDNA.(24) We also noted that MK exogenously added to the culture of ATDC5 had no effect. RIHB was also reported to be ineffective in promotion of chondrogenesis.(47) The effectiveness of cDNA transfection rather than addition to culture medium to demonstrate MK activity has been reported; NIH3T3 cells oncogenically transformed with MK cDNA introduction were readily detached from substratum, while exogenously added MK showed no such activity.(24) Angiogenic activity of MK was also found by DNA transfection,(45) while we failed to detect any angiogenic activity following addition of MK to the culture medium (unpublished results). These differences in activities can be best explained by differences in delivery of MK. Exogenously added MK may be degraded by proteases and/or taken up by cells, while MK was constantly produced by the transfected cells.
We noted that during both fracture repair and normal development, MK was mainly located intracellularly. Immunoelectron microscopy revealed the intracellular localization of MK in the region rich in rough endoplasmic reticulum and other cytoplasmic regions of embryonic chondrocytes. In the region rich in endoplasmic reticulum, MK was not frequently found in the lumen. There are two ways to interpret the result in terms of the mode of action. First, MK may not be produced and targeted to the secretory pathway through rough endoplasmic reticulum and may be rather synthesized in the cytoplasm. MK immunoreactivity revealed by immunoelectron microscopy might correspond to MK on the way of synthesis, and also to MK, which is internalized and in the process of degradation. It is likely that MK is rapidly synthesized and degrade in these cells and that a small amount of MK, which remains extracellularly, exerts the effect. Promotion of chondrogenesis by MK may require its continuous presence, which may not be achieved by the addition to the medium. Alternatively, MK immunoreactivity detected in the cytoplasm could be internalized MK, and this MK might play a role to exert its function, possibly by interacting with some cytoplasmic structure. Our biochemical data that callus MK, which should principally be intracellular, was the mature form devoid of the signal sequence is consistent with the view. However, further experiments are needed to know whether this mechanism of MK action is present. Our observation that MK enhances chondrogenesis may have clinical implications. With the design of a delivery system to continuously supply MK to the site of injury, fracture repair may be enhanced.
We thank Prof. Koji Kimata for the gift of anti-aggrecan antibody. This work was supported by grants-in-aid for scientific research from the Ministry of Education, Science, Sports and Culture (09557016, 08457035, and 10178102), especially grant-in-aid for COE Research.