Journal of Cell 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 Arthritis 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 gene-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 chondrocytes. 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 cartilage specific extracellular matrix 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, Chondrocyte, 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 hyaluronic acid 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-cell adhesion 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), tenascin (Pacifici, 1995), fibronectin (Silbermann et al., progressive joint diseases. 1987), cellular receptors for these matrix proteins 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 glycosaminoglycans 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