Stepwise mechanical stretching inhibits chondrogenesis through cell-matrix adhesion mediated by integrins in embryonic rat limb-bud mesenchymal cells.

Biomechanical forces are major epigenetic factors that determine the form and differentiation of skeletal tissues, and may be transduced through cell adhesion to the intracellular biochemical signaling pathway. To test the hypothesis that stepwise stretching is translated to molecular signals during early chondrogenesis, we developed a culture system to study the proliferation and differentiation of chondrocytes. Rat embryonic day-12 limb buds were microdissected and dissociated into cells, which were then micromass cultured on a silicone membrane and maintained for up to 7 days. Stepwise-increased stretching was applied to the silicone membrane, which exerted shearing stress on the cultures on day 4 after the initiation of chondrogenesis. Under stretched conditions, type II collagen expression was significantly inhibited by 44% on day 1 and by 67% on day 2, and this difference in type II collagen reached 80% after 3 days of culture. Accumulation of type II collagen protein and the size of the chondrogenic nodules had decreased by 50% on day 3. On the other hand, expression of the non-chondrogenic marker fibronectin was significantly upregulated by 1.8-fold on day 3, while the up-regulation of type I collagen was minimal, even by day 3. The downregulation in the expression of chondrogenic markers was completely recovered when cell-extracellular matrix attachment was inhibited by Gly-Arg-Gly-Asp-Ser-Pro-Lys peptide or by the application of blocking antibodies for alpha2, alpha5 or beta1 integrins. We conclude that shearing stress generated by stepwise stretching inhibits chondrogenesis through integrins, and propose that signal transduction from biomechanical stimuli may be mediated by cell-extracellular matrix adhesion.

Osteoblasts and osteocytes express MMP2 and -8 and TIMP1, -2, and -3 along with extracellular matrix molecules during appositional bone formation.

Our previous studies suggested that a part of bone extracellular matrix (ECM) molecules are degraded and remodeled during embryonic bone formation. In contrast, little is known about ECM remodeling in postnatal appositional bone formation. The present study was designed to investigate expression of matrix metalloproteinases (MMPs) and tissue inhibitors of metalloproteinases (TIMPs) during experimentally initiated appositional bone formation in rats. Expressions of ECM molecules, MMPs, and TIMPs were examined using in situ hybridization. Osteoblasts and osteocytes expressed MMP2 and -8, TIMP1, -2, and -3, as well as type I collagen, osteopontin, and osteocalcin in the course of the appositional bone formation, while they showed few transcripts of MMP13. The results indicated that while osteoblasts and osteocytes in the apposed bone produce ECM molecules, they degrade ECM molecules with MMPs and regulate the degradation by inhibiting the activity of MMPs using TIMPs. Osteoblasts and osteocytes may reorganize the ECM composition to mature the bone matrix in appositional bone formation.

Age-related changes in gene expression patterns of matrix metalloproteinases and their collagenous substrates in mandibular condylar cartilage in rats.

Matrix metalloproteinases (MMPs) have been implicated in physiological cartilage matrix remodelling as well as in pathological and invasive extracellular matrix remodelling of tissue. Age-related changes in the gene expression patterns of MMPs in mandibular condylar cartilages (MCCs) were analysed. We examined the gene expression patterns of Mmp-8 and -13 and their substrates, Col1a1, Col2a1 and Col10a1, in MCC of growing and ageing rats. Temporomandibular joints of male Wistar rats aged 4, 8, 16 and 32 weeks were subjected to in situ hybridization analysis. Histologically, MMCs showed characteristics of growth plate cartilage at ages 4 and 8 weeks, and more closely resembled articular cartilage thereafter. Mmp-8 was expressed in the cells in all cartilaginous cell layers at ages 4 and 8 weeks, and then was localized only in the mature cells at ages 16 and 32 weeks. Whereas Mmp-13 expression was limited to the lowermost hypertrophic chondrocytes in the growth stage, mature chondrocytes instead of hypertrophic chondrocytes expressed Mmp-13 in adult non-hypertrophic MCC. Because Mmp-8 and -13 expression overlapped with Col2a1 and Col10a1, chondrocytes could play a pivotal role in degradation as well as production of the cartilaginous matrix in MCC.

Effect of stretching on gene expression of beta1 integrin and focal adhesion kinase and on chondrogenesis through cell-extracellular matrix interactions.

Differentiation of skeletal tissues, such as bone, ligament and cartilage, is regulated by complex interaction between genetic and epigenetic factors. In the present study, we attempted to elucidate the possible role of cell-extracellular matrix (ECM) adhesion on the inhibitory regulation in chondrogenesis responding to the tension force. The midpalatal suture cartilages in rats were expanded by orthopedic force. In situ hybridization for type I and II collagens, immunohistochemical analysis for fibronectin, alpha5 and beta1 integrins, paxillin, and vinculin, and cytochemical staining for actin were used to demonstrate the phenotypic change of chondrocytes. Immunohistochemical analysis for phosphorylation and nuclear translocation of extracellular signal-regulated kinase (ERK)-1/2 was performed. The role of the cell-ECM adhesion in the response of the chondroprogenitor cells to mechanical stress and the regulation of gene expression of focal adhesion kinase (FAK) and integrins were analyzed by using an in vitro system. A fibrous suture tissue replaced the midpalatal suture cartilage by the expansive force application for 14 days. The active osteoblasts that line the surface of bone matrix in the newly formed suture tissue strongly expressed the type I collagen gene, whereas they did not express the type II collagen gene. Although the numbers of precartilaginous cells expressing alpha5 and beta1 integrin increased, the immunoreactivity of alpha5 integrin in each cell was maintained at the same level throughout the experimental period. During the early response of midpalatal suture cartilage cells to expansive stimulation, formation of stress fibers, reorganization of focal adhesion contacts immunoreactive to a vinculin-specific antibody, and phosphorylation and nuclear translocation of ERK-1/2 were observed. In vitro experiments were in agreement with the results from the in vivo study, i.e. the inhibited expression of type II collagen and upregulation in integrin expression. The arginine-glycine-aspartic acid-containing peptide completely rescued chondrogenesis from tension-mediated inhibition. Thus, we conclude that stretching activates gene expression of beta1 integrin and FAK and inhibits chondrogenesis through cell-ECM interactions of chondroprogenitor cells.

Expression of extracellular matrix molecules, MMPs and TIMPs in alveolar bone, cementum and periodontal ligaments during rat tooth eruption.

Tooth eruption involves extensive degradation and reorganization of extracellular matrix (ECM) components. It is not known how ECM-degrading enzymes are coordinated with each other or how they are regulated in the event. The present study was designed to investigate mRNA expression of inhibitors of metalloproteinases (TIMPs) in comparison with matrix metalloproteinases (MMPs) as well as ECM molecules during rat first molar eruption using in situ hybridization. We also examined how TIMPs are involved in the process of tooth eruption, root formation, cementogenesis and alveolar bone remodelling. Expressions of type-I collagen, osteocalcin, MMPs 2 and 8, and TIMPs 1, 2 and 3 were shown in osteoblasts, osteocytes, cementoblasts, cementocytes and periodontal ligament fibroblasts, and the concomitant high expressions of the ECM molecules, MMPs and TIMPs in alveolar bone, cementum and periodontal ligaments were identified in the middle of first molar eruption. The remodelling of ECM in these periodontal tissues might be regulated through balance among the production of ECM molecules, the degradation of ECM by MMPs and the inhibition of MMPs by TIMPs during tooth eruption.

The extent of odontoblast processes in the dentin is distinct between cusp and cervical regions during development and aging.

The question of whether odontoblast processes extend to the dentinal surface has been widely debated in previous studies. In this study odontoblast processes were investigated in the developing and aging dentin of rats and monkeys (Japanese macaques). For this purpose, F-actin of microfilaments and cellular membranes were stained with phalloidin and DiI, respectively. This dual staining demonstrated that positive signals for odontoblast processes were present in the dentinal surface in both the cusp and cervical regions of the dentin at 2 weeks of age. The tips of doubly positive processes were detectable in the dentinal surface in the cusp region even at 100 weeks of age, whereas in the cervical region they were retracted from the dentinal surface towards the pulp during the period of 3-6 weeks of age. During these stages, phalloidin-positive signals showing retracted odontoblast processes in the cervical region were closely associated with the interglobular dentin that was stained with sWGA-lectin. After 6 weeks of age, no association was observed between the processes and the interglobular dentin, since they were retracted approximately to the inner third portion of the dentinal tubules. This staining pattern can be detected until 100 weeks of age. Moreover, different distribution patterns of odontoblast processes between the two dentinal regions were also confirmed in dentin of monkey teeth. These results suggest that the existence of the regional differences in the extent of the odontoblast processes in the dentin, i.e., the persistence of the processes in the dentinal surface in the cusp region and their retraction from the dentinal surface in the cervical region.

Gene expression of MMP8 and MMP13 during embryonic development of bone and cartilage in the rat mandible and hind limb.

Matrix metalloproteinases (MMPs) 8 and 13 comprise the collagenase subfamily in rats and mice, and only MMP13 has been implicated in degradation of the collagenous matrices during development of bone and cartilage. On the hypothesis that MMP8 is also involved in bone and cartilage development, the present study was designed to investigate gene expression of MMP8 in rat embryonic mandibles and hind limbs. Expression of MMP8 was examined with in situ hybridization and RT-PCR and was compared with that of MMP13. Osteoblastic and chondrocytic cells expressing collagenous matrix molecules were identified using in situ hybridization for collagen Types I and II. The results demonstrated that MMP8 is expressed by osteoblastic progenitors, differentiated osteoblasts, osteocytes, and chondrocytes in the growth plate for the first time. Furthermore, the expression of MMP8 is much broader than that of MMP13, for which expression is confined to differentiated phenotypes of osteoblastic and chondrocytic lineage.