sign in sign up
<<
Home , Aggrecan

Journal of Science 111, 2067-2076 (1998) 2067 Printed in Great Britain © The Company of Biologists Limited 1998 JCS3791

Compressive force promotes Sox9, type II collagen and aggrecan and inhibits IL-1β expression resulting in chondrogenesis in mouse embryonic limb bud mesenchymal cells

Ichiro Takahashi, Glen H. Nuckolls, Katsu Takahashi, Osamu Tanaka, Ichiro Semba, Ralph Dashner, Lillian Shum and Harold C. Slavkin* Craniofacial Development Section, National Institute of and Musculoskeletal and Skin Diseases, National Institutes of Health, Bethesda, MD, USA *Author for correspondence (e-mail: [email protected])

Accepted 19 May; published on WWW 30 June 1998

SUMMARY

The initial modeling and subsequent development of the apparent acceleration in the rate and extent of skeleton is controlled by complex -environment chondrogenesis. Quantitatively, there was a significant 2- to interactions. Biomechanical forces may be one of the major 3-fold increase in type II collagen and aggrecan expression epigenetic factors that determine the form and beginning at day 5 of culture and the difference was differentiation of skeletal tissues. In order to test the maintained through 10 days of cultures. Compressive force hypothesis that static compressive forces are transduced also causes an elevated level of Sox9, a transcriptional into molecular signals during early chondrogenesis, we activator of type II collagen. In contrast, the expression and have developed a unique three-dimensional collagen gel cell accumulation of IL-1β, a transcriptional repressor of type culture system which is permissive for the proliferation and II collagen was down-regulated. We conclude that static differentiation of . Mouse embryonic day 10 compressive forces promote chondrogenesis in embryonic (E10) limb buds were microdissected and dissociated into limb bud mesenchyme, and propose that the signal cells which were then cultured within a collagen gel matrix transduction from a biomechanical stimuli can be mediated and maintained for up to 10 days. Static compressive forces by a combination of positive and negative effectors of were exerted onto these cultures. The time course for specific macromolecules. expression pattern and level for cartilage specific markers, type II collagen and aggrecan, and regulators of chondrogenesis, Sox9 and IL-1β, were analyzed and Key words: Biomechanical force, , Type II collagen, compared with non-compressed control cultures. Under Aggrecan, Sox9, IL-1β, Competitive PCR, Mouse embryo, 3- compressive conditions, histological evaluation showed an Dimensional collagen gel culture

INTRODUCTION Indeed, mathematical modeling predicts that biomechanical forces generated by cell migration and cell division create cell- The musculoskeletal system including bone, cartilage, skeletal cell and/or cell-extracellular matrix (ECM) interactions which muscles and ligaments responds to biomechanical stimuli by contribute to early patterning and morphogenesis in many altering their metabolism, cellular and cytoskeletal organization, tissues and organs, including neural tube (Jacobson and rate of proliferation and state of differentiation during Gordon, 1976) and precartilaginous mesenchymal development. For example, exercise induces physiological condensation (Edelstein-Keshet and Ermentrout, 1990; Murray changes in muscles, bone and articular cartilage, and static force and Oster, 1984; Ngwa and Maini, 1995; Totafurno and applied during orthodontic treatments induces bone remodeling, Bjerknes, 1995). During chondrogenesis, it was proposed that differentiation of chondrocytes and of other connective tissue the osmotic changes during synthesis and cells (Asano, 1986; McNamara and Carlson, 1979; Takahashi et degradation could produce sufficient swelling and deswelling al., 1995). In addition, excessive or inappropriate force loading to regulate the balance between cell-cell versus cell-ECM is a contributing factor in common skeletal degenerative contacts (Oster et al., 1985). diseases. The development of these tissues during embryonic Experimental models, though limited, have generally development may also be regulated by biomechanical stimuli, supported these theories. Paralyzed chicken embryos failed to contributing to the initial modeling of the three-dimensional form the clavicles and the quadratojugal, which is associated structure of the skeleton (van Limborgh, 1982). with the failure to activate chondrogenic markers, illustrating 2068 I. Takahashi and others the prerequisite of physical movements in the initial stages of hypothesis that compression promoted chondrogenesis is a chondrogenesis (Fang and Hall, 1995; Hall, 1979; Hall and response of molecular determinants to biomechanical forces. Herring, 1990). In vitro models using intermittent compressive Due to the complexity of differential mechanical properties force accelerated the maturation of chondrocytes (van Kampen among different cell populations and local matrix components, et al., 1985; van’t Veen et al., 1995; Veldhuijzen et al., 1987). and normal movements, an in vitro three-dimensional collagen However, the function of static compressive force in the early gel cell culture system was developed. We report that static molecular differentiation of chondroprogenitor cells frequently compressive force promotes the expression of two cartilage encountered in orthodontic and orthopedic treatments is as yet specific markers, type II collagen and aggrecan. This unknown. accelerated chondrogenesis was associated with an Morphogenesis of the skeletal system begins with the upregulation of the positive regulator Sox9, and condensation of undifferentiated embryonic mesenchymal downregulation of the negative regulator IL-1β. An cells (Hall, 1988). Condensation is promoted by a regulated understanding of the biomechanical stimuli that regulate balance between cell-ECM and cell- involving cartilage development and maintenance can contribute to the type I and type III collagen (von der Mark and von der Mark, improved management of skeletal malformations and 1977), (Pacifici, 1995), fibronectin (Silbermann et al., progressive joint diseases. 1987), cellular receptors for these matrix such as integrins (Camper et al., 1997), and other non-integrin adhesion molecules (Noonan et al., 1996), such as cell-cell MATERIALS AND METHODS adhesion molecules N-CAM and N-cadherin (Oberlender and Tuan, 1994; Tavella et al., 1994; Tsonis et al., 1994). After Cell culture and force loading system condensation, the differentiating chondrocytes change their Timed-pregnant Swiss Webster mice (with day of detection of vaginal cell shape and alter their cell adhesion properties. sperm plug designated as day 0 of gestation) were purchased (Harlan Chondrocytes express type II collagen (Mizoguchi et al., 1990; Sprague Dawley, Inc., Indianapolis, Indiana). At gestation day 10, von der Mark and von der Mark, 1977), aggrecan (Vornehm et pregnant mice were sacrificed and the E10 stage embryos were al., 1996), and associated tissue-specific isolated. Fore- and hindlimb buds were microdissected in ice-cold (Takahashi et al., 1996). As these matrix molecules are phosphate buffered saline (PBS), placed on ice, and subsequently they were washed three times in cold PBS, dissociated in 0.25 mg/ml deposited between cells, cell-cell contacts are lost (Tavella et trypsin EDTA (Life Technologies Gibco BRL, Inc., Gaithersburg, al., 1994). New proliferating chondrocytes establish the pattern MD) and 0.25 mg/ml collagenase type 2 (Washington Biochemical of subsequent bone formation. At the epiphyseal growth plate, Corporation, Freehold, NJ) in 0.1 M PBS for 15 minutes at 37°C, and the chondrocytes become hypertrophic, the cartilage matrix cell numbers were determined using a hemocytometer. Subsequently, becomes calcified, and the cartilage is replaced by bone in the cells were collected by centrifugation at 200 g for 15 minutes at 4°C process of endochondral ossification. At other sites, such as the and resuspended in Dulbecco’s modified Eagle’s medium (DMEM; formation of synovial joints, chondrocytes maintain the Life Technologies Gibco BRL, Inc., Gaithersburg, MD) at 4.5×107 or cartilage matrix through adulthood. Thus, skeletal development 5.0×107 cells/ml for compressed and control groups. Cells were provides mechanical stability and mobility, and is likely to cultured in three-dimensional collagen gel system (described below) respond reciprocally to biomechanical stimuli in order to adapt in DMEM supplemented with 10% heat inactivated fetal bovine serum (HyClone Laboratories Inc, Logan, UT), 2.4 mg/ml of Hepes (ICN to the range of force exerted and motion. The ECM component Biomedicals Inc., Aurora, OH), 0.2% bicarbonate, 2 mM glutamine is conceivably the transducer of biomechanical stimuli into (Sigma, St Louis, MO), 100 units of penicillin, 100 µg of transcriptional controls. streptomycin and 0.25 µg of amphotericin (Life Technologies Gibco Signal transduction initiated by growth factors and BRL, Inc., Gaithersburg, MD). cytokines, and subsequent transcriptional controls contribute In order to determine the effect of static compressive force on the broadly to the regulation of cartilage and bone development. differentiation of limb bud mesenchymal cells, cells were embedded However, one such cytokine, interleukin 1-beta (IL-1β), has and cultured in a three-dimensional collagen gel system to mimic in been demonstrated to inhibit chondrocyte differentiation by vivo conditions (Yasui et al., 1982) as illustrated in Fig. 1. A collagen directly suppressing transcription of the cartilage specific gel stock was prepared by dissolving type I collagen from calf skin marker type II collagen gene, and also inhibits (Sigma, St Louis, MO) in 1 mM acetic acid at a concentration of 3.33 mg/ml and dialyzed against deionized water for 6 hours. The collagen production leading to decreased synthesis gel cultures were assembled by mixing 10 volumes of 2× medium, 9 of another cartilage specific marker, aggrecan (Chandrasekhar volumes of collagen stock solution and 1 volume of cell suspension et al., 1994; Gibson et al., 1984; Goldring et al., 1994a,b). IL- to give a final cell density of 2.25×106 cells/ml for compressed gels 1β also induces the expression of matrix metalloproteases, and 2.5×106 cells/ml for control gel at a collagen gel concentration of molecules involved in the degradation of cartilage matrix 1.5 mg/ml. These gels were plated in 96-well plates and allowed to during hypertrophy (Yang and Gerstenfeld, 1997). In contrast polymerized for 1 hour, after which the gels were transferred to 24- to IL-1β being a negative regulator of chondrogenesis, the well plates so that an agarose supporting ring could be cast around transcription factor Sox9 is a positive regulator in this process. the cultures. Static compressive forces were applied by the use of a Sox9 is expressed in precartilaginous condensing mesenchyme weight placed on the lid of the culture plate exerted through a well and maturing cartilage (Wright et al., 1995; Zhao et al., 1997). insert which fitted on top of the collagen gel as illustrated in Fig. 1. The weight per unit surface area of the collagen gel was converted to Sox9 binds directly to an enhancer element in the first intron compressive pressure as kiloPascals (kPa). Since our collagen gel of the collagen II gene (Lefebvre et al., 1996; Ng et al., 1997), culture system is unique, the amount of force used in our experiments; and upregulates the type II collagen expression (Bell et al., 1, 1.5, and 2 kPa was empirically determined to yield a 20-30% 1997). deformation of the gel as used in previous studies (Buschmann et al., In this investigation, studies were designed to test the 1995; Kim et al., 1994, 1996). Cultures were incubated at 37°C, 5% Compressive force promotes chondrogenesis 2069

CO2 for up to 10 days with daily medium changes. Control non- and 5′-CCTGGGGA-AGGCATTAGAAACAGT-3′ for secondary compressed cultures were assembled without force loading. PCR based on the IL-1β cDNA sequence (Gray et al., 1986). Forty cycles of primary amplification followed by 10 cycles of secondary Isolation of total RNA and DNA, and reverse transcription amplification were also empirically determined to optimize for Total RNAs were isolated from both compressed and control gels at amplification linearity. 0, 1, 3, 5, 7 and 10 days of culture, and from E9, 10, 11, 12 and 14 After PCR amplification, products were electrophoresed on 2.0% mouse limb buds using a guanidine isothiocyanate, phenol- agarose gel in Tris acetate buffer, stained with ethidium bromide, chloroform extraction based total RNA isolation kit (5 prime 3 prime imaged with Charged Coupled Device camera, then pixel depth was Inc., Boulder, CO) according to specifications from the manufacturer. converted to optical density and quantitated with the aid of NIH image Total RNA was transcribed into cDNA using SuperScript II reverse software package (NIH, Bethesda, MD). The amount of type II transcriptase (Life Technologies Gibco BRL, Inc., Gaithersburg, MD). collagen and aggrecan mRNAs were calculated from the standard Genomic DNA was also isolated from samples at day 0, 3, 5 and 7 of curve generated from competitive PCR and the amount of Sox9 and culture using routine phenol-chloroform extraction and ethanol IL-1β mRNA was quantitated relative to β-actin expression. precipitation methods. A series of predetermined cell numbers were used for DNA content to generate a standard curve of cell number Histology, immunohistochemistry and ELISA versus total DNA content. Total cell numbers in the experimental limb Collagen gel supported cell cultures at day 5 and 7 were fixed with bud cell cultures were calculated using this standard curve. 4% paraformaldehyde in PBS for 12 hours. Subsequently, specimens were thoroughly rinsed in 0.01 M PBS, infiltrated through a graded Quantitative and semi-quantitative reverse transcription- series of sucrose and acrylamide, embedded first in 10% acrylamide polymerase chain reaction (RT-PCR) gel and then in Tissue Freezing Medium (Triangle Biomedical In order to accurately quantitate the expression of chondrogenic Sciences, Durham, NC). Ten µm-thick sections were collected and markers, type II collagen and aggrecan, competitive RT-PCR was subjected to either Toluidine Blue staining or indirect fluorescence performed (Siebert and Larrick, 1992). Amplimers designed for type immunohistochemistry for type II and X collagens (gift from Dr II collagen were 5′-TGAAGACATCCGCAGCCCC-3′ and 5′- Sasano), IL-1β and IL-1RI (Santa Cruz Biotechnology, Inc., Santa ATAATGGGAAGGCGGGAGG-3′, based on the mouse type II Cruz, CA). Immunohistochemical staining was performed as collagen genomic DNA sequence (Metsaranta et al., 1991) and for previously described (Mizoguchi et al., 1990). Total cell number and aggrecan 5′-GTCCCTGGTCAGCCCCGCTTG-3′ and 5′-CACTGA- the number of IL-1β positive cells were counted in a 270 µm by 310 CACACCTCGGAAGCA-3′ based on the mouse aggrecan cDNA µm area for every third or fourth serial section for 10 sections of each sequences (Watanabe et al., 1995). Competitors bearing identical 5′ compressed and control sample at day 7 of culture. and 3′ amplification arms of type II collagen and aggrecan sequences, IL-1β in conditioned medium from day 3, 5, 7 and 10 of culture but with an altered internal sequence, were constructed using the PCR was assayed using Quantikine M mouse IL-1β immunoassay kit MIMIC construction kit (Clontech, Palo Alto, CA) according to (R&D System, Minneapolis, MN) according to specifications from the specifications from the manufacturer. Total RNA from E10 mouse manufacturer. limb buds was extracted, reverse transcribed and subjected to PCR amplification for both type II collagen and aggrecan. PCR products Statistical analysis of target templates and competitors for type II collagen and aggrecan Transcript expression levels for type II collagen, aggrecan, IL-1β and were subcloned into pCRII vector (Invitrogen, Carlsbad, CA), Sox9 as well as levels of IL-1β between compressed and amplified and transcribed into cRNA using an RNA transcription kit control groups over the time course of study were subjected to (Stratagene, La Jolla, CA). Standard curves of the logarithm of statistical analyses using Student’s t-test. Statistical significance was template versus the ratio of template to competitors were generated deduced at P<0.05. for type II collagen and aggrecan. Correlation formulae were deduced (Zhao et al., 1996) from the standard curves and regressions of 0.9885 and 0.9606 were calculated for type II collagen and aggrecan, respectively. PCR amplifications were carried out by using ampliTaq RESULTS (Life Technologies Gibco BRL, Inc., Gaithersburg, MD) and DNA Thermal cycler 480 (Perkin Elmer, Branchburg, NJ). Thermal cycling Compressive force accelerates the rate and extent programs for quantitative PCR were 5 minutes at 95°C for of chondrogenic nodule formation in vitro denaturation, 45 seconds at 55°C for annealing, and 2 minutes at 72°C We developed an in vitro three-dimensional model system for for extension for the first cycle, followed by similar cycles changing culturing dissociated mouse embryonic limb bud mesenchyme only the denaturation step to 45 seconds and a final extension cycle in collagen gels which mimicks early in vivo chondrogenesis of 10 minutes. Cycle numbers were empirically determined to be 27 and 32 for type II collagen and aggrecan, respectively, to optimize for (Fig. 1). A static compressive force of 1 kPa was applied and signal and amplification linearity. evidence of chondrogenesis was assayed using histological and Semi-quantitative RT-PCR method was performed to evaluate the molecular markers. The rate and extent of chondrogenesis were expression levels of Sox9 and IL-1β relative to that of β-actin. compared with that of control non-compressed cultures. Amplimers designed for Sox9 were 5′-AGAAAGACCACCCC- Control and compressed cultures exhibited the formation of GATTA-3′ and 5′-ACTGGGACG-ACATGGAGAAG-3′ based on the cartilaginous nodules accompanied by cartilage specific matrix Sox9 cDNA sequence (Wright et al., 1995), and that for β-actin were deposition as reflected by metachromatic Toluidine Blue 5′-TGCTGAT-GCCGTAACTGCC-3′ and 5′-TGAGGTAGTCCGT- staining (Fig. 2A to D). By day 5, compressed cultures ′ CAGGTCC-3 based on the actin cDNA sequence (Alonso et al., contained more chondrogenic aggregates and deposited more 1986). Twenty-eight cycles of amplification for Sox9 and 22 cycles cartilaginous matrix than controls. By day 7, compressed for β-actin were empirically determined to optimize for signal and amplification linearity. Nested PCR was performed to quantitate the cultures contained cartilage lacunae indicative of maturing expression level of IL-1β, since its expression level was relatively low. chondrocytes that were not seen in control cultures. In addition, Amplimers designed for IL-1β were 5′-GGGATGATGATG- cartilaginous nodule chondrocytes in compressed cultures ATAACCTGCTG-3′ and 5′-TTCTTGTGACCCTGAGCGACCTG-3′ showed aggregated linear alignment oriented perpendicular to for primary PCR and 5′-GGGATGATGATGATAACCTGCTGG-3′ the direction of force applied, whereas control cultures showed 2070 I. Takahashi and others

Load

Compressive force

Well insert

Medium 0.8 m Millipore filter

Agarose gel Cells in collagen gel

Fig. 1. Diagrammatic representation of the three-dimensional collagen gel cell culture model. Static compressive force was loaded by the use of a well insert bearing the weight of the plate cover and calculated load. Dissociated mouse embryonic limb bud mesenchymal cells were embedded in collagen gel matrix supported by a cast agarose gel ring. dispersed and more randomly distributed cells in the matrix. Cartilage ECM synthesis was characterized by positive immunocytochemical reaction to both type II and X collagen in the chondrocytes in both control and compressed cultures Fig. 2. Biomechanical stimuli accelerated chondrogenesis. Toluidine on days 5 and 7. Consistent with Toluidine Blue staining, there Blue staining of 10 µm sections from collagen gel cell culture of was more cartilage specific matrix accumulation in compressed day 5 control (A), day 5 compressed (B), day 7 control (C) and day cultures (Fig. 2E and F). Furthermore, type X collagen 7 compressed samples (D). Metachromasia indicates cartilage accumulation in the ECM surrounding matured chondrocytes matrix deposition. Arrowheads indicate mature chondrocytes in were observed in compressed cultures at day 7 (Fig. 2G and cartilage lacunae in D that were not observed in control cultures (C). H). Immunohistochemical staining for type II collagen at day 5 in control (E), and compressed culture (F), and type X collagen at day Compressive force promotes the expression of type 7 in control (G), and compressed culture (H). White arrowheads II collagen and aggrecan indicates accumulation of type X collagen in chondrocytes. Bars, 100 µm. In order to quantitate the accelerated process of chondrogenesis under static compressive force, a quantitative RT-PCR method was used to assess type II collagen or aggrecan mRNA. The sensitivity and effective range of our quantitative RT-PCR mRNA levels in compressed cultures was 1.9-fold higher at 5 methods was 0.002 to 5.95 pg/µg of total RNA. The linearity days than controls (which increased to a 3.4-fold elevation at was reflected by r=0.994 and r=0.980 for type II collagen and day 10). aggrecan, respectively. The acceleration of type II collagen expression by Differentiating chondrocytes went through an initial phase compressive force was found to be directly proportional to the of cell condensation and subsequently begin to deposit magnitude of compressive force applied (Fig. 3C). When 1.5 cartilage specific matrix. Consistent with histological and 2 kPa of force was loaded, type II collagen mRNA observations, matrix molecule transcripts were apparent by RT- increased 1.5- and 3.8-fold, respectively, over transcript levels PCR by day 3 of culture, with a major increase in transcript detected in samples subjected to 1 kPa of force magnitude. In levels between day 3 and 5. Quantitation of type II collagen comparison to the control (non-compressed) samples, a transcripts in compressed cultures by competitive RT-PCR maximum force used in our experiments of 2kPa of force revealed a 5-fold increase on day 3 when compared with magnitude produced an 8.3-fold increase in type II collagen control cultures. This upregulation was maintained and mRNA. significant between control and compressed cultures through A collagen gel matrix under compressive force is likely to 10 days of culture; the difference at day 10 being 2-fold (Fig. become more compact when compared with non-compressed 3A). Consistent with the profile of type II collagen expression, matrix. Compaction may lead to changes in gel density and cell aggrecan mRNA, was also upregulated (Fig. 3B). Aggrecan number per unit volume. It has been previously reported that Compressive force promotes chondrogenesis 2071

0.6 14 A A ** )

6 12 0.5 * 10 * 0.4 8 0.3 6 g of total RNA 0.2 4 pg of type II collagen Numbers of cells (x10 2 mRNA/ 0.1 ** 0 0 0357 35710 Days in cultutre Days in culture 0.5 B B ** 250 1 0.4 0.9 * 200 0.8 0.7 0.3 150 0.6 0.5 g of total RNA 0.2 100 0.4 0.3 pg of aggrecan 50 0.2 0.1 mRNA/ 0.1 Relative thickness of gels

0 0 compressed/control groups

0 01357 Ratio of gel thickness between 35710 Days in cultutre Days in culture 0.7 1.8 ** C 1.6 C 0.6 1.4 0.5 1.2 1 0.4 g of total RNA

0.8 g total RNA ** 0.3 0.6 ** 0.2 0.4 pg of type II collagen mRNA/

0.2 pg of type II collagen mRNA/ 0.1 0 0 1.0 1.5 2.0 0 kPa 357 Days in cultutre Fig. 3. Compressive forces increased type II collagen and aggrecan Fig. 4. Changes in cell number and gel density had no effects on expression assayed by competitive RT-PCR. Time course of type II chondrogenesis. Time course of changes in cell numbers under collagen mRNA expression level (A) and aggrecan mRNA control and compressed cultures (A), and changes in thickness of expression level (B), and force magnitude dependency of type II gels expressed as relative thickness of gels, and as the ratio of gel collagen mRNA expression (C). Open and solid bars represent thickness between compressed and control groups (B). Open and control and compressed cultures, respectively. Standard deviation solid bars represent control and compressed cultures, respectively. bars are placed on top of each respective data bar. Statistically Expression level of type II collagen mRNA under various significant differences were indicated by *P<0.05, **P<0.01, n=4-5 combinations of cell number and gel concentration (C). Dotted, (A,B), n=3 (C). open, hatched and solid bars represent 2.25×106 cells/ml and 1.5 mg of gel/ml, 2.5×106 cells/ml and 1.5 mg of gel/ml, 2.25×106 cells/ml and 1.95 mg of gel/ml, and 2.5×106 cells/ml and 1.95 mg of gel/ml, respectively. Standard deviation bars are placed on top of each high cell density per se promotes chondrogenesis (Solursh and respective data bar. No statistical differences (P=0.05) were detected Meier, 1974). Therefore, in order to examine the primary effect among groups. All experiments were done in triplicate. of compression on chondrogenesis, we should control for changes in gel density and cell number in our three- dimensional collagen gel cell culture system. Two strategies compressed cultures beginning on day 3 and through day 7 were used: (1) we monitored and compared the changes in gel (Fig. 4A). The volume of both compressed and non- density and cell number in non-compressed versus compressed compressed culture gels exhibited a phase increase from day 0 collagen gels over the time course of our studies; (2) we used through day 3, and subsequently a gradual phase of decrease the observed differences to make compensatory adjustments through day 7 (Fig. 4B). The volume stabilized and remained and assayed for effects on the rate and extent of relatively constant after day 10 (data not shown). We suggest chondrogenesis. that these gel volume changes are due to the increase in cell The total cell number increased linearly in all cultures. Non- number and/or changes in the hydration state of the gels for compressed cultures contained 20% more cells than the first (expansion) phase, followed by a decrease in gel 2072 I. Takahashi and others thickness in the second phase. Compressed gels that did not compression is a direct effect attributable to the force applied contain any cells exhibited expansion to a lesser extent (data and not secondarily mediated by associated changes in cell not shown). The maximum difference in volume between number or gel density found in compressed cultures. compressed and non-compressed gels was 30%. Based upon these observations, we then assayed type II Sox9 increase in chondrocytes is subjected to force collagen mRNA levels under various combinations of loading increased cell number and collagen gel concentration (based We observed that compressive forces promote chondrogenesis on the differences detected in the earlier experiments). Since in embryonic limb bud mesenchyme cells through experiments the volume of non-compressed gels was approximately 30% designed to test the increase of type II collagen as a regulated more than compressed, we varied the collagen concentration event mediated through positive regulators of type II collagen by this amount, increasing from 1.5 to 1.95 mg collagen gel gene transcription. We analyzed the pattern of Sox9 expression matrix/ml in control cultures. The total cell number was 20% during embryonic limb development in vivo. In vivo expression more in non-compressed than compressed cultures. From these of Sox9 in the mouse embryo was detectable at low levels at results, we varied the cell density by 10%; increasing from E9. Sox9 expression peaked at E11 and then decreased through 2.25×106 to 2.5×106 cells/ml in control cultures. Fig. 4C shows E14. At E11, Sox9 expression preceded that of the onset of an a time course of type II collagen transcript level in non- exponential accumulation of type II collagen transcripts which compressed cultures with combinations of cell number and occurred between E12-14 (Fig. 5A). Sox9 was expressed in collagen gel density adjustments. No significant difference in control and compressed cultures (Fig. 5B). Similar to in vivo type II collagen expression was detected among groups at each profiles, Sox9 transcript levels peaked at day 3 of culture in time point. Therefore, chondrogenesis advanced by both groups and then decreased. However, in compressed cultures, despite an overall decrease in , Sox9 transcript level was maintained at an elevated state as compared with control cultures through day 10. The ratio between 0.3 5 compressed and control levels was 2.9 (day 5) and then reached A 6.9 at day 10. Statistically significant differences in Sox9 4 expression in control and compressed cultures were detectable at days 7 and 10. 0.2 3 IL-1β decreases in chondrocytes under compression β 2 g of total RNA IL-1 is a direct suppresser of type II collagen and aggrecan 0.1 gene expression in chondrocytes (Goldring et al., 1994a,b). IL- β 1 1 mRNA expression and the accumulation of the activated pg of type II collagen mRNA/ protein in conditioned culture media of control and compressed cultures were assayed by semi-quantitative RT-PCR and Relative expression level of Sox9 0 0 ELISA, respectively. Compressed cultures showed 9 1011121314 β Embryonic days significantly less IL-1 transcript expression when compared with controls at days 3, 5 and 7 (Fig. 6A). The difference was 2.5 B largest at day 5; compressed cultures only had 1/4 of the expression level of the control value. The overall protein 2 accumulation increased from day 3 to 7, and then stabilized after day 7 in both control and compressed groups (Fig. 6B). 1.5 Peak RNA expression on day 5 (Fig. 6A) was followed by peak protein accumulation 48 hours later (Fig. 6B). However, compressed cultures showed significantly less IL-1β 1 ** * accumulation than that of contol cultures; compressed culture had 20% less IL-1β than control cultures at day 5 and 7. This 0.5 is consistent with a lower RNA expression level beginning at day 3 prior to a detectable decrease in protein accumulation Relative expression level of Sox9 0 which was first observed at day 5. Since the total cell numbers 135710 in compressed cultures were slightly lower than those of Days in culture controls over the course of the culture period as shown in Fig. 4, it is possible that the difference observed in IL1-β protein Fig. 5. Biomechanical compressive stimuli increased Sox9 mRNA accumulation only reflected the difference in total cell number expression. In vivo expression of Sox9 transcripts (solid line) was rather than expression level in each cell. Therefore, we counted assayed by semi-quantitative RT-PCR in mouse limb bud. Expression the number of IL-1β expressing cells on the immunostained peaked at E11, in advance of an exponential increase in type II sections (Fig. 6D and F) in both control and compressed collagen mRNA expression (dashed line) between E12 to E14 (A). samples at day 7 of culture to standardize for the accumulation Time course of in vitro Sox9 mRNA expression (B). Open and solid β bars represent control and compressed cultures, respectively. of protein per cell. The number of cells expressing IL-1 in Standard deviation bars are for respective data bar. Statistically compressed and control cultures were (2.8±0.9)×106 and significant differences were indicated by *P<0.05, **P<0.01. (2.7±0.4)×106 per gel (n=3), respectively, the two values not Numbers of the samples: n=3 (A), n=4 (B). being statistically different (P=0.74). This suggests that indeed Compressive force promotes chondrogenesis 2073 β 6 the difference in the accumulation of IL-1 in the culture media A was indicative of the expression level itself. Both IL-1β and its receptor, IL-1RI, were detectable by immunocytochemistry, 5 demonstrating that they were coexpressed in chondrocytes at day 7 in both control and compressed groups (Fig. 6D and F). 4 Therefore, IL-1β may exert its action by an autocrine and/or paracrine mode. 3 DISCUSSION 2 ** ** Using a unique three-dimensional collagen gel cell culture * 1 system that mimicks much of the cell adhesion interactions of embryonic tissues, we have determined that static compressive Relative expression level of IL-1 0 force promotes chondrogenesis during embryonic limb bud 35710 mesenchymal cell differentiation. The rate and extent of type II collagen and aggrecan expression in differentiating Days in culture chondrocytes were significantly increased under compressive 45 B forces. Further, we also determined that this accelerated pattern 40 of chondrogenesis was accompanied by up-regulation of Sox9 35 ** expression (a transcriptional activator of type II collagen), and down-regulation of IL-1β (a repressor of both type II collagen 30 and aggrecan). The balance of signaling between a / ml of 25 combination of intermediate activator and repressor suggests that the biomechanical stimuli and subsequent transduction 20 * resulting in differential gene activation is a tightly regulated 15 process as summarized in Fig. 7. pg of IL-1

conditioned medium 10 Since the mesenchymal cells in our experiments are derived from embryonic mouse limb buds just prior to chondrogenesis 5 in vivo, we assume that our in vitro model system reflects a 0 dynamic range of signaling circuits required for normal 35710 differentiation of chondrocytes from mesenchymal cells. This Days in culture culture system contains a heterogeneous population of cells

Fig. 6. Biomechanical compressive stimuli inhibited expression and accumulation of IL-1β. Time course of amount of IL-1β transcripts assayed by semi-quantitative RT-PCR (A), and of active IL-1β accumulation in conditioned culture media assayed by ELISA (B). Open and solid bars represent control and compressed cultures, respectively. Standard deviation bars are for each respective data bar. Statistically significant differences were indicated by *P<0.05, and **P<0.01. Numbers of the samples in each time point were 5. Immunocytochemistry for IL-1β and IL-1RI of day 7 control (D) and compressed (F) cultures. Respective phase contrast images are shown in C and E. IL-1β and IL-1RI were identified by rhodamine-conjugated and fluorescein-conjugated secondary antibodies, respectively. Arrowheads indicate double positive round- shaped chondrocytes within cartilaginous nodules. Arrows indicate non-chondrocytic cells positive only for IL-1RI. Bar, 50 µm. 2074 I. Takahashi and others

Differentiating Mature Hypertrophic Chondrogenesis chondrocytes chondrocytes chondrocytes

Sox9 Type II collagen Type X collagen Aggrecan Fig. 7. Proposed mechanism for static compressive Molecular force regulation of initial chondrogenesis. Static events compressive force supported the expression of two IL-1 GAGs cartilage-specific markers, type II collagen and aggrecan. This differential gene expression was MMPs associated with an up-regulation of the positive Compressive regulator Sox9 and down-regulation of the negative Supports the progression of chondrogenesis regulator IL-1β. IL-1β has been found to inhibit force GAGs biosynthesis and induce MMPs. including epithelial and precursor cells for bone, cartilage, higher Sox9 expression could account for the increase in type muscle and other connective tissues. The acceleration of II collagen transcript accumulation that was observed at later chondrogenesis observed in compressed cultures is likely due time points. However, our data also suggest that other to both an increase in the number of cells differentiating into transcription factors play an important role in regulating type chondrocytes as well as an increase in the expression of II collagen transcription, since we observed a statistically cartilage-specific ECM proteins associated with each significant stimulation in type II collagen expression at the day chondrocyte. Immunohistochemical analyses revealed that 3 time point while no difference in Sox9 expression was chondrocytes were more numerous and that these cells detected until day 5 (Figs 3A, 4C and 5). exhibited a more advanced state of differentiation as IL-1β expression and accumulation were suppressed in characterized by type II and X collagen deposition in the ECM compressed cultures as a direct consequence of the under compression as compared to non-compressed controls. biomechanical stimuli (Fig. 6). This is consistent with the Increased chondrocytes may reflect increased proliferation of promotion of chondrogenesis, since IL-1β has been shown to the prechondrocyte sub-population, or a recruitment of cells inhibit type II collagen transcription and glycosaminoglycan from other cell lineages to become chondrocytes in response production leading to decreased aggrecan synthesis (Frisbie to compressive force stimuli. and Nixon, 1997). Further, IL-1β induces the cartilage These experiments demonstrate responsiveness of the degrading enzymes matrix metalloproteases (MMPs) (Arner developmental process of chondrogenesis to external and Tortorella, 1995; Yang and Gerstenfeld, 1997). Decreased biomechanical stimuli. During skeletogenesis, the initial IL-1β expression could lead to a greater accumulation of pattern of most of the skeleton is first established as cartilage matrix, consistent with our histological and cartilaginous tissue which is subsequently replaced with bone immunohistochemical findings. Immunostaining revealed by the process of endochondral ossification. Epigenetic factors coexpression of IL-1β and IL-1RI in chondrocytes, whereas such as compressive force that stimulate chondrogenesis can neighboring non-chondrocytic cells expressed only the have a significant impact on long term three-dimensional receptor (Fig. 6). This suggests that IL-1β may regulate modeling of the structural elements in bones. Indeed, from our chondrogenesis through either a paracrine or autocrine histological observations, the aggregated linear alignment of feedback loop, and the response of chondrocytes to chondrocytes appeared to orient perpendicular to the direction compressive force could be conveyed to neighboring non- of compressive force delivery and suggests that the chondrocytic cells via a diffusible cytokine. construction of bony pillars in the epiphyseal region of long Studies of the effects of compressive force on mature articular bones directed to the plane of weight bearing could also be cartilage (Buschmann et al., 1995; Lee and Bader, 1997) reveal controlled by similar biomechanical stimuli prior to and/or significant differences with our results with embryonic during bone deposition. This interpretation is consistent with mesenchymal cells. Dynamic, periodic loading of articular Wolff’s law which states that directional modeling of bony cartilage up-regulates the incorporation of 3H-proline and 35S- pillars during ossification is a result of resistance to sulfate into cartilage ECM, while static compressive force down- biomechanical stress. regulates cartilage matrix deposition (Kim et al., 1994) and Two morphoregulatory factors were identified to be involved aggrecan expression (Kim et al., 1996). In vitro studies with in controlling chondrogenesis in response to compressive mandibular condyle cartilage, non-terminally differentiated biomechanical stimuli. Sox9 expression patterns showed a growth plate cartilage, showed similar results. In contrast, similar peak level for expression in both compressed and collagen and biosynthesis was up-regulated in control cultures (Fig. 5). However, a more gradual decrease in postnatal growth plate cartilage of mandibular condyle cultured expression level was observed only in compressed cultures under compressive forces (Copray et al., 1985). Further, subsequent to the peak (Fig. 5B). Thus, a higher Sox9 mRNA compaction and low oxygen tension lead to chondrogenesis in level in compressed cultures was effectively sustained over the periosteal membrane cultures (Bassette and Herrmann, 1961). same time course when compared with controls. If Sox9 is a Compressive stimulation on midpalatal suture cartilage in rats limiting factor in transcriptional regulation of type II collagen, promoted maturation and hypertrophy of precartilaginous Compressive force promotes chondrogenesis 2075 undifferentiated mesenchymal cells in vivo (Saitoh et al., 1997). B. (1995). Mechanical compression modulates matrix biosynthesis in In vivo studies in experimentally paralyzed chicken embryos chondrocyte/agarose culture. J. Cell Sci. 108, 1497-1508. (Hall and Herring, 1990) and muscular dysgenic mice (Herring Camper, L., Heinegard, D. and Lundgren-Akerlund, E. (1997). Integrin α2β1 is a receptor for the cartilage matrix protein chondroadherin. J. Cell and Lakars, 1981) demonstrate that decreased compression of Biol. 138, 1159-1168. developing bones by the associated skeletal muscles leads to Chandrasekhar, S., Harvey, A. K., Johnson, M. G. and Becker, G. W. decreased chondrogenesis. The effects of compressive forces on (1994). /SPARC is a product of articular chondrocytes/cartilage chondrogenesis are, therefore, dependent on the specific type of and is regulated by cytokines and growth factors. Biochim. Biophys. Acta 1221, 7-14. , the stage of cartilage maturation, the site of force Chrzanowska-Wodnicka, M. and Burridge, K. (1996). Rho-stimulated loading, and the types of forces exerted upon the cartilage. The contractility drives the formation of stress fibers and focal adhesions. J. Cell effect of static compressive force on chondrocytes differentiation Biol. 133, 1403-1415. from mesenchymal cells in this culture model reveals that Copray, J. C., Jansen, H. W. and Duterloo, H. S. (1985). Effects of important changes occurs during differentiation pathways compressive forces on proliferation and matrix synthesis in mandibular condylar cartilage of the rat in vitro. Arch. Oral Biol. 30, 299-304. between the detection of force signals and cellular responses to Edelstein-Keshet, L. and Ermentrout, G. B. (1990). Contact response of those epigenetic signals. cells can mediate morphogenetic pattern formation. Differentiation 45, 147- In summary, we demonstrate that biomechanical 159. compressive stimuli accelerated the progression of Fang, J. and Hall, B. K. (1995). Differential expression of neural cell chondrogenesis in mouse limb mesenchymal cells through adhesion molecule (NCAM) during osteogenesis and secondary β chondrogenesis in the embryonic chick. Int. J. Dev. Biol. 39, 519-528. regulating the expression of Sox9 and IL-1 as positive and Frisbie, D. D. and Nixon, A. J. (1997). Insulin-like growth factor 1 and negative regulators, respectively, for the expression of cartilage corticosteroid modulation of chondrocyte metabolic and mitogenic activities specific macromolecules. Possible mechanisms of how in interleukin 1-conditioned equine cartilage. Am. J. Vet. Res. 58, 524-530. compressive stimuli are translated into biochemical signals Gibson, G. J., Beaumont, B. W. and Flint, M. H. (1984). Synthesis of a low molecular weight collagen by chondrocytes from the presumptive might involve integrin or non-integrin ECM receptors, cell-cell calcification region of the embryonic chick sterna: the influence of culture adhesion molecules or alteration of cytoskeletal architecture with collagen gels. J. Cell Biol. 99, 208-216. mediated by MAP kinase (Banes et al., 1995; Kimura et al., Goldring, M. B., Birkhead, J. R., Suen, L. F., Yamin, R., Mizuno, S., 1996), rho kinase (Chrzanowska-Wodnicka and Burridge, Glowacki, J., Arbiser, J. L. and Apperley, J. F. (1994a). Interleukin-1 1996) or focal adhesion associated tyrosine kinase signal beta-modulated gene expression in immortalized chondrocytes. J. Clin. Invest. 94, 2307-2316. transduction pathways (Yamada and Miyamoto, 1995; Yamada Goldring, M. B., Fukuo, K., Birkhead, J. R., Dudek, E. and Sandell, L. J. and Geiger, 1997). However, the molecular mechanisms (1994b). Transcriptional suppression by interleukin-1 and interferon-gamma remain as yet unkown. Further investigations are necessary to of type II collagen gene expression in human chondrocytes. J. Cell. clarify the molecular mechanisms by which biomechanical Biochem. 54, 85-99. Gray, P. W., Glaister, D., Chen, E., Goeddel, D. V. and Pennica, D. (1986). stimuli affect skeletogenesis through mechano-biochemical Two interleukin 1 in the mouse: Cloning and expression of the cDNA translation and transcriptional regulation. Our model system of for murine interleukin 1-beta. J. Immunol. 137, 3644-3648. chondrogenesis under compressive load provides the Hall, B. K. (1979). Selective proliferation and accumulation of opportunity for testing the interplay of ECM or cell-cell chondroprogenitor cells as the mode of action of biomechanical factors adhesion receptor and growth factor receptors in controlling during secondary chondrogenesis. Teratology 20, 81-91. Hall, B. K. (1988). Patterning of connective tissues in the head: discussion chondrocyte differentiation. report. In Craniofacial Development. Development 103, 171-174. Hall, B. K. and Herring, S. W. (1990). Paralysis and growth of the We are grateful to Dr Yasuyuki Sasano (Second Department of Oral musculoskeletal system in the embryonic chick. J. Morphol. 206, 45-56. Anatomy, School of Dentistry, Tohoku University) for the generous Herring, S. W. and Lakars, T. C. (1981). Craniofacial development in the gift of antibodies against type II and X collagens. This work was absence of muscle contraction. J. Craniofac. Genet. Dev. Biol. 1, 341-357. supported by NIH grant Z01-AR41114-02-BCTB. Jacobson, A. G. and Gordon, R. (1976). Changes in the shape of the developing vertebrate nervous system analyzed experimentally, mathematically and by computer simulation. J. Exp. Zool. 197, 191-246. Kim, Y. J., Sah, R. L., Grodzinsky, A. J., Plaas, A. H. and Sandy, J. D. REFERENCES (1994). Mechanical regulation of cartilage biosynthetic behavior: physical stimuli. Arch. Biochem. Biophys. 311, 1-12. Alonso, S., Minty, A., Bourlet, Y. and Buckingham, M. (1986). Comparison Kim, Y. J., Grodzinsky, A. J. and Plaas, A. H. (1996). Compression of of three actin-coding sequences in the mouse; evolutionary relationships cartilage results in differential effects on biosynthetic pathways for between the actin genes of warm-blooded vertebrates. J. Mol. Evol. 23, 11- aggrecan, link protein, and hyaluronan. Arch. Biochem. Biophys. 328, 331- 22. 340. Arner, E. C. and Tortorella, M. D. (1995). Signal transduction through Kimura, K., Ito, M., Amano, M., Chihara, K., Fukata, Y., Nakafuku, M., chondrocyte integrin receptors induces matrix synthesis Yamamori, B., Feng, J., Nakano, T., Okawa, K. et. al. (1996). Regulation and synergizes with interleukin-1. Arthritis Rheum. 38, 1304-1314. of myosin phosphatase by Rho and Rho-associated kinase (Rho-kinase). Asano, T. (1986). The effects of mandibular retractive force on the growing Science 273, 245-248. rat mandible. Am. J. Orthod. Dentofacial Orthop. 90, 464-474. Lee, D. A. and Bader, D. L. (1997). Compressive strains at physiological Banes, A. J., Tsuzaki, M., Yamamoto, J., Fischer, T., Brigman, B., Brown, frequencies influence the metabolism of chondrocytes seeded in agarose. J. T. and Miller, L. (1995). Mechanoreception at the cellular level: the Orthop. Res. 15, 181-188. detection, interpretation, and diversity of responses to mechanical signals. Lefebvre, V., Zhou, G., Mukhopadhyay, K., Smith, C. N., Zhang, Z., Biochem. Cell Biol. 73, 349-365. Eberspaecher, H., Zhou, X., Sinha, S., Maity, S. N. and de Bassette, C. A. L. and Herrmann, I. (1961). Influence of oxygen Crombrugghe, B. (1996). An 18-base-pair sequence in the mouse concentration and mechanical factors on differentiation of connective tissue proalpha1(II) collagen gene is sufficient for expression in cartilage and binds in vitro. Nature 190, 460-461. nuclear proteins that are selectively expressed in chondrocytes. Mol. Cell Bell, D. M., Leung, K. K., Wheatley, S. C., Ng, L. J., Zhou, S., Ling, K. Biol. 16, 4512-4523. W., Sham, M. H., Koopman, P., Tam, P. P. and Cheah, K. S. (1997). McNamara, J. Jr and Carlson, D. S. (1979). Quantitative analysis of SOX9 directly regulates the type-II collagen gene. Nat. Genet. 16, 174-178. temporomandibular joint adaptations to protrusive function. Am. J. Orthod. Buschmann, M. D., Gluzband, Y. A., Grodzinsky, A. J. and Hunziker, E. 76, 593-611. 2076 I. Takahashi and others

Metsaranta, M., Toman, D., de Crombrugghe, B. and Vuorio, E. (1991). Totafurno, J. and Bjerknes, M. (1995). Morphogenesis and mechanical Mouse type II collagen gene. Complete nucleotide sequence, exon structure, instability of a prestressed tissue. Biochem. Cell Biol. 73, 565-574. and . J. Biol. Chem. 266, 16862-16869. Tsonis, P. A., Del Rio-Tsonis, K., Millan, J. L. and Wheelock, M. J. (1994). Mizoguchi, I., Nakamura, M., Takahashi, I., Kagayama, M. and Mitani, Expression of N-cadherin and alkaline phosphatase in chick limb bud H. (1990). An immunohistochemical study of localization of type I and type mesenchymal cells: regulation by 1, 25-dihydroxyvitamin D3 or TGF-beta II collagens in mandibular condylar cartilage compared with tibial growth 1. Exp. Cell Res. 213, 433-437. plate. Histochemistry 93, 593-599. van Kampen, G. P., Veldhuijzen, J. P., Kuijer, R., van de Stadt, R. J. and Murray, J. D. and Oster, G. F. (1984). Cell traction models for generating Schipper, C. A. (1985). Cartilage response to mechanical force in high- pattern and form in morphogenesis. J. Math. Biol. 19, 265-279. density chondrocyte cultures. Arthritis Rheum. 28, 419-424. Ng, L. J., Wheatley, S., Muscat, G. E., Conway-Campbell, J., Bowles, J., van Limborgh, J. (1982). Factors controlling skeletal morphogenesis. In Wright, E., Bell, D. M., Tam, P. P., Cheah, K. S. and Koopman, P. (1997). Factors and Mechanisms Influencing Bone Growth (ed. A. D. Diyan and B. SOX9 binds DNA, activates transcription, and coexpresses with type II G. Saruct), pp. 1-17. New York: Alde R. Liss. collagen during chondrogenesis in the mouse. Dev. Biol. 183, 108-121. van’t Veen, S. J., Hagen, J. W., van Ginkel, F. C., Prahl-Andersen, B. and Ngwa, G. A. and Maini, P. K. (1995). Spatio-temporal patterns in a Burger, E. H. (1995). Intermittent compression stimulates cartilage mechanical model for mesenchymal morphogenesis. J. Math. Biol. 33, 489- mineralization. Bone 17, 461-465. 520. Veldhuijzen, J. P., Huisman, A. H., Vermeiden, J. P. and Prahl-Andersen, Noonan, K. J., Stevens, J. W., Tammi, R., Tammi, M., Hernandez, J. A. B. (1987). The growth of cartilage cells in vitro and the effect of intermittent and Midura, R. J. (1996). Spatial distribution of CD44 and hyaluronan in compressive force. A histological evaluation. Connect. Tissue Res. 16, 187- the proximal tibia of the growing rat. J. Orthop. Res. 14, 573-581. 196. Oberlender, S. A. and Tuan, R. S. (1994). Spatiotemporal profile of N- von der Mark, K. and von der Mark, H. (1977). The role of three genetically cadherin expression in the developing limb mesenchyme. Cell Adhes. distinct collagen types in endochondral ossification and calcification of Commun. 2, 521-537. cartilage. J. Bone Joint Surg. [Br], 458-464. Oster, G. F., Murray, J. D. and Maini, P. K. (1985). A model for Vornehm, S. I., Dudhia, J., Von der Mark, K. and Aigner, T. (1996). chondrogenic condensations in the developing limb: the role of extracellular Expression of collagen types IX and XI and other major cartilage matrix matrix and cell tractions. J. Embryol. Exp. Morphol. 89, 93-112. components by human fetal chondrocytes in vivo. Matrix Biol. 15, 91-98. Pacifici, M. (1995). Tenascin-C and the development of articular cartilage. Watanabe, H., Gao, L., Sugiyama, S., Doege, K., Kimata, K. and Yamada, Matrix Biol. 14, 689-698. Y. (1995). Mouse aggrecan, a large cartilage proteoglycan: protein sequence, Saitoh, S., Takahashi, I., Mizoguchi, I., Kagayama, M. and Mitani, H. gene structure and promoter sequence. Biochem. J. 308, 433-440. (1997). Effects od compressive force on the growth and differentaition of Wright, E., Hargrave, M. R., Christiansen, J., Cooper, L., Kun, J., Evans, midpalatal suture cartilage in rats. J. Jpn. Orthod. Soc. 56, 372-382. T., Gangadharan, U., Greenfield, A. and Koopman, P. (1995). The Sry- Siebert, P. D. and Larrick, J. W. (1992). Competitive PCR. Nature 359, 557- related gene Sox9 is expressed during chondrogenesis in mouse embryos. 558. Nat. Genet. 9, 15-20. Silbermann, M., Reddi, A. H., Hand, A. R., Leapman, R. D., Von der Yamada, K. M. and Geiger, B. (1997). Molecular interactions in cell adhesion Mark, K. and Franzen, A. (1987). Further characterisation of the complexes. Curr. Opin. Cell Biol. 9, 76-85. extracellular matrix in the mandibular condyle in neonatal mice. J. Anat. Yamada, K. M. and Miyamoto, S. (1995). Integrin transmembrane signaling 151, 169-188. and cytoskeletal control. Curr. Opin. Cell Biol. 7, 681-689. Solursh, M. and Meier, S. (1974). Effects of cell density on the expression Yang, R. and Gerstenfeld, L. C. (1997). Structural analysis and of differentiation by chick embryo chondrocytes. J. Exp. Zool. 187, 311- characterization of tissue and hormonal responsive expression of the avian 322. bone sialoprotein (BSP) gene. J. Cell. Biochem. 64, 77-93. Takahashi, I., Mizoguchi, I., Nakamura, M., Kagayama, M. and Mitani, Yasui, N., Osawa, S., Ochi, T., Nakashima, H. and Ono, K. (1982). Primary H. (1995). Effects of lateral pterygoid muscle hyperactivity on culture of chondrocytes embedded in collagen gels. Exp. Cell Biol. 50, 92- differentiation of mandibular condyles in rats. Anat. Rec. 241, 328-336. 100. Takahashi, I., Mizoguchi, I., Sasano, Y., Saitoh, S., Ishida, M., Kagayama, Zhao, J., Bu, D., Lee, M., Slavkin, H. C., Hall, F. L. and Warburton, D. M. and Mitani, H. (1996). Age-related changes in the localization of (1996). Abrogation of transforming growth factor-beta type II receptor glycosaminoglycans in condylar cartilage of the mandible in rats. Anat. stimulates embryonic mouse lung branching morphogenesis in culture. Dev. Embryol. 194, 489-500. Biol. 180, 242-257. Tavella, S., Raffo, P., Tacchetti, C., Cancedda, R. and Castagnola, P. Zhao, Q., Eberspaecher, H. and De Crombrugghe, B. (1997). Parallel (1994). N-CAM and N-cadherin expression during in vitro chondrogenesis. expression of Sox9 and Col2a1 in cells undergoing chondrogenesis. Dev. Exp. Cell Res. 215, 354-362. Dynam. 209, 377-386.