Growth plate mechanics and mechanobiology. A survey of present understanding

The longitudinal growth of long bones occurs in growth plates where chondrocytes synthesize cartilage that is subsequently ossified. Altered growth and subsequent deformity resulting from abnormal mechanical loading is often referred to as mechanical modulation of bone growth. This phenomenon has key implications in the progression of infant and juvenile musculoskeletal deformities, such as adolescent idiopathic scoliosis, hyperkyphosis, genu varus/valgus and tibia vara/valga, as well as neuromuscular diseases. Clinical management of these deformities is often directed at modifying the mechanical environment of affected bones. However, there is limited quantitative and physiological understanding of how bone growth is regulated in response to mechanical loading. This review of published work addresses the state of knowledge concerning key questions about mechanisms underlying biomechanical modulation of bone growth. The longitudinal growth of bones is apparently controlled by modifying the numbers of growth plate chondrocytes in the proliferative zone, their rate of proliferation, the amount of chondrocytic hypertrophy and the controlled synthesis and degradation of matrix throughout the growth plate. These variables may be modulated to produce a change in growth rate in the presence of sustained or cyclic mechanical load. Tissue and cellular deformations involved in the transduction of mechanical stimuli depend on the growth plate tissue material properties that are highly anisotropic, time-dependent, and that differ in different zones of the growth plate and with developmental stages. There is little information about the effects of time-varying changes in volume, water content, osmolarity of matrix, etc. on differentiation, maturation and metabolic activity of chondrocytes. Also, the effects of shear forces and torsion on the growth plate are incompletely characterized. Future work on growth plate mechanobiology should distinguish between changes in the regulation of bone growth resulting from different processes, such as direct stimulation of the cell nuclei, physico-chemical stimuli, mechanical degradation of matrix or cellular components and possible alterations of local blood supply.     

The role of osteopontin in tendon tissue remodeling after denervation-induced mechanical stress deprivation.

It has been shown that musculoskeletal tissues undergo dynamic tissue remodeling by a process that is quite sensitive to the mechanical environment. However, the detailed molecular mechanism underlying this process remains unclear. We demonstrate here that after denervation-induced mechanical stress deprivation, tendons undergo dynamic tissue remodeling as evidenced by a significant reduction of the collagen fibril diameter. Importantly, the transient up-regulation of osteopontin (OPN) expression was characteristic during the early phase of tendon tissue remodeling. Following this dynamic change of OPN expression, matrix metalloproteinase (MMP)-13 expression was induced, which presumably accounts for the morphological changes of tendon by degrading tendon collagen fibrils. The modulation of MMP-13 expression by OPN was specific, since the expression of MMP-2, which is also known to be involved in tissue remodeling, did not alter in the tendons under the absence or presence of OPN. We also demonstrate that the modulation of MMP-13 expression by OPN is due to the signaling through cell surface receptors for OPN. Thus, we conclude that OPN plays a crucial role in conveying the effect of denervation-induced mechanical stress deprivation to the tendon fibroblasts to degrade the extracellular matrices by regulating MMP-13 expression in tendon fibroblasts.     

FGF upregulates osteopontin in epiphyseal growth plate chondrocytes: Implications for endochondral ossification

Fibroblast growth factor receptor 3 (FGFR3) signaling pathways are essential for normal longitudinal bone growth. Mutations in this receptor lead to various human growth disorders, including Achondroplasia, disproportionately short-limbed dwarfism, characterized by narrowing of the hypertrophic region of the epiphyseal growth plates. Here we find that FGF9, a preferred ligand for FGFR3 rapidly induces the upregulation and secretion of the matrix resident phosphoprotein, osteopontin (OPN) in cultured chicken chondrocytes. This effect was observed as early as two hours post stimulation and at FGF9 concentrations as low as 1.25 ng/ml at both mRNA and protein levels. OPN expression is known to be associated with chondrocyte and osteoblast differentiation and osteoclast activation. Unexpectedly, FGF9 induced OPN was accompanied by inhibition of differentiation and increased proliferation of the treated chondrocytes. Moreover, FGF9 stimulated OPN expression irrespective of the differentiation stage of the cells or culture conditions. In situ hybridization analysis of epiphyseal growth plates from chicken or mice homozygous for the Achondroplasia, G369C/mFGFR3 mutation demonstrated co-localization of OPN expression and osteoclast activity, as evidenced by tartarate resistant acid phosphatase positive cells in the osteochondral junction. We propose that FGF signaling directly activates OPN expression independent of chondrocytes differentiation. This may enhance the recruitment and activation of osteoclasts, and increase in cartilage resorption and remodeling in the chondro-osseus border.     

Osteopontin stimulates tumor growth and activation of promatrix metalloproteinase-2 through nuclear factor-kappa B-mediated induction of membrane type 1 matrix metalloproteinase in murine melanoma cells

Matrix metalloproteinases (MMPs) degrade the extracellular matrix (ECM) and play critical roles in tissue repair, tumor invasion, and metastasis. MMPs are regulated by different cytokines, ECM proteins, and other factors. However, the molecular mechanisms by which osteopontin (OPN), an ECM protein, regulates ECM invasion and tumor growth and modulates MMP activation in B16F10 cells are not well defined. We have purified OPN from human milk and shown that OPN induces pro-MMP-2 production and activation in these cells. Moreover, our data revealed that OPN-induced membrane type 1 (MT1) MMP expression correlates with translocation of p65 (nuclear factor-kappaB (NF-kappaB)) into the nucleus. However, when the super-repressor form of IkappaBalpha (inhibitor of NF-kappaB) was transfected into cells followed by treatment with OPN, no induction of MT1-MMP expression was observed, indicating that OPN activates pro-MMP-2 via an NF-kappaB-mediated pathway. OPN also enhanced cell migration and ECM invasion by interacting with alpha(v)beta(3) integrin, but these effects were reduced drastically when the MMP-2-specific antisense S-oligonucleotide was used to suppress MMP-2 expression. Interestingly, when the OPN-treated cells were injected into nude mice, the mice developed larger tumors, and the MMP-2 levels in the tumors were significantly higher than in controls. The proliferation data indicate that OPN increases the growth rate in these cells. Both tumor size and MMP-2 expression were reduced dramatically when anti-MMP-2 antibody or antisense S-oligonucleotide-transfected cells were injected into the nude mice. To our knowledge, this is the first report that MMP-2 plays a direct role in OPN-induced cell migration, invasion, and tumor growth and that demonstrates that OPN-stimulated MMP-2 activation occurs through NF-kappaB-mediated induction of MT1-MMP.     

Modulation of neonatal growth plate development by ex vivo intermittent mechanical stress

Although growth plate response to mechanical stress has been increasingly studied, our understanding of mechanical modulation of neonatal growth plate is incomplete, especially concerning biochemical changes. This study was designed to explore the cellular and biochemical responses of the cranial base growth plate (CBGP) explant upon cyclic loading. The growth plate with subchondral bone was aseptically isolated from each of 24 neonatal rabbits and fixated in an organ culture system. Cyclic loading was applied to growth plate explants at 200 mN and 1 Hz for 60 min (N=12), whereas control explants were immersed in organ culture for 60 min without mechanical loading (N=12). Computerized image analysis revealed that cyclic loading induced significantly more proliferating chondrocytes than unloaded controls (p<0.001), as well as significantly higher growth plate height at 856+/-30 microm than the unloaded controls at 830+/-36 microm (p<0.05). Immunoblotting with monoclonal antibodies (mAb) disclosed that the average mAb binding area for chondroitin sulfate was significantly higher in the loaded specimens than the unloaded controls at (p<0.001). The average mAb binding area for keratan sulfate was also significantly higher in the loaded specimens than the unloaded controls (p<0.01). Biochemical analysis showed that the average total hyaluronan content of loaded specimens at 0.25+/-0.06 microg/microg DNA was significantly higher than the unloaded controls at 0.09+/-0.05 microg/microg DNA (p<0.01). Taken together, these data suggest that brief doses of cyclic, intermittent forces activate cellular and molecular responses in the CBGP ex vivo. Whether hyaluronan-mediated pathway is involved in the biological responses of growth plate to mechanical loading warrants additional investigations.     

Mechanical effects on skeletal growth

The growth (i.e. increase of external dimensions) of long bones and vertebrae occurs longitudinally by endochondral ossification at the growth plates, and radially by apposition of bone at the periosteum. It is thought that mechanical loading influences the rate of longitudinal growth. The 'Hueter-Volkmann Law' proposes that growth is retarded by increased mechanical compression, and accelerated by reduced loading in comparison with normal values. The present understanding of this mechanism of bone growth modulation comes from a combination of clinical observation (where altered loading and growth is implicated in some skeletal deformities) and animal experiments in which growth plates of growing animals have been loaded. The gross effect of growth modulation has been demonstrated qualitatively and semi-quantitatively. Sustained compression of physiological magnitude inhibits growth by 40% or more. Distraction increases growth rate by a much smaller amount. Experimental studies are underway to determine how data from animal studies can be scaled to other growth plates. Variables include: differing sizes of growth plate, different anatomical locations, different species and variable growth rate at different stages of skeletal maturity. The two major determinants of longitudinal growth are the rate of chondrocytic proliferation and the amount of chondrocytic enlargement (hypertrophy) in the growth direction. It is largely unknown what are the relative changes in these key variables in mechanically modulated growth, and what are the signaling pathways that produce these changes.     

Altered endochondral bone development in matrix metalloproteinase 13-deficient mice

The assembly and degradation of extracellular matrix (ECM) molecules are crucial processes during bone development. In this study, we show that ECM remodeling is a critical rate-limiting step in endochondral bone formation. Matrix metalloproteinase (MMP) 13 (collagenase 3) is poised to play a crucial role in bone formation and remodeling because of its expression both in terminal hypertrophic chondrocytes in the growth plate and in osteoblasts. Moreover, a mutation in the human MMP13 gene causes the Missouri variant of spondyloepimetaphyseal dysplasia. Inactivation of Mmp13 in mice through homologous recombination led to abnormal skeletal growth plate development. Chondrocytes differentiated normally but their exit from the growth plate was delayed. The severity of the Mmp13- null growth plate phenotype increased until about 5 weeks and completely resolved by 12 weeks of age. Mmp13-null mice had increased trabecular bone, which persisted for months. Conditional inactivation of Mmp13 in chondrocytes and osteoblasts showed that increases in trabecular bone occur independently of the improper cartilage ECM degradation caused by Mmp13 deficiency in late hypertrophic chondrocytes. Our studies identified the two major components of the cartilage ECM, collagen type II and aggrecan, as in vivo substrates for MMP13. We found that degradation of cartilage collagen and aggrecan is a coordinated process in which MMP13 works synergistically with MMP9. Mice lacking both MMP13 and MMP9 had severely impaired endochondral bone, characterized by diminished ECM remodeling, prolonged chondrocyte survival, delayed vascular recruitment and defective trabecular bone formation (resulting in drastically shortened bones). These data support the hypothesis that proper ECM remodeling is the dominant rate-limiting process for programmed cell death, angiogenesis and osteoblast recruitment during normal skeletal morphogenesis.     

Mechanical stimulation of osteopontin mRNA expression and synthesis in bone cell cultures

We have shown earlier that mechanical stimulation by intermittent hydrostatic compression (IHC) promotes alkaline phosphatase and procollagen type I gene expression in calvarial bone cells. The bone matrix glycoprotein osteopontin (OPN) is considered to be important in bone matrix metabolism and cell-matrix interactions, but its role is unknown. Here we examined the effects of IHC (13 kPa) on OPN mRNA expression and synthesis in primary calvarial cell cultures and the osteoblast-like cell line MC3T3-E1. OPN mRNA expression declined during control culture of primary calvarial cells, but not MC3T3-E1 cells. IHC upregulated OPN mRNA expression in late released osteoblastic cell cultures, but not in early released osteoprogenitor-like cells. Also, in both proliferating and differentiating MC3T3-E1 cells, OPN mRNA expression and synthesis were enhanced by IHC, differentiating cells being more responsive than proliferating cells. These results suggest a role for OPN in the reaction of bone cells to mechanical stimuli. The severe loss of OPN expression in primary bone cells cultured without mechanical stimulation suggests that disuse conditions down-regulate the differentiated osteoblastic phenotype. J. Cell. Physiol. 170:174–181, 1997. © 1997 Wiley-Liss, Inc.     

Rapid regulation of collagen but not metalloproteinase 1, 3, 13, 14 and tissue inhibitor of metalloproteinase 1, 2, 3 expression in response to mechanical loading of cartilage explants in vitro

This study analyzes the molecular response of articular chondrocytes to short-term mechanical loading with a special focus on gene expression of molecules relevant for matrix turnover. Porcine cartilage explants were exposed to static and dynamic unconfined compression and viability of chondrocytes was assessed to define physiologic loading conditions. Cell death in the superficial layer correlated with mechanical loading and occurred at peak stresses >or=6 MPa and a cartilage compression above 45%. Chondrocytes in native cartilage matrix responded to dynamic loading by rapid and highly specific suppression of collagen expression. mRNA levels dropped 11-fold (collagen 2; 6 MPa, P=0.009) or 14-fold (collagen 1; 3 and 6 MPa, P=0.009) while levels of aggrecan, tenascin-c, matrix metalloproteinases (MMP1, 3, 13, 14), and their inhibitors (TIMP1-3) did not change significantly. Thus, dynamic mechanical loading rapidly shifted the balance between collagen and aggrecan/tenascin/MMP/TIMP expression. A better knowledge of the chondrocyte response to mechanical stress may improve our understanding of mechanically induced osteoarthrits.     

The turnover of mineralized growth plate cartilage into bone may be regulated by osteocytes

During endochondral ossification, growth plate cartilage is replaced with bone. Mineralized cartilage matrix is resorbed by osteoclasts, and new bone tissue is formed by osteoblasts. As mineralized cartilage does not contain any cells, it is unclear how this process is regulated. We hypothesize that, in analogy with bone remodeling, osteoclast and osteoblast activity are regulated by osteocytes, in response to mechanical loading. Since the cartilage does not contain osteocytes, this means that cartilage turnover during endochondral ossification would be regulated by the adjacent bone tissue. We investigated this hypothesis with an established computational bone adaptation model. In this model, osteocytes stimulate osteoblastic bone formation in response to the mechanical bone tissue loading. Osteoclasts resorb bone near randomly occurring microcracks that are assumed to block osteocyte signals. We used finite element modeling to evaluate our hypothesis in a 2D-domain representing part of the growth plate and adjacent bone. Cartilage was added at a constant physiological rate to simulate growth. Simulations showed that osteocyte signals from neighboring bone were sufficient for successful cartilage turnover, since equilibrium between cartilage remodeling and growth was obtained. Furthermore, there was good agreement between simulated bone structures and rat tibia histology, and the development of the trabecular architecture resembled that of infant long bones. Additionally, prohibiting osteoclast invasion resulted in thickened mineralized cartilage, similar to observations in a knock-out mouse model. We therefore conclude that it is well possible that osteocytes regulate the turnover of mineralized growth plate cartilage.     

Protective effects of cilengitide on inflammation in chondrocytes under excessive mechanical stress

Chondrocytes constantly receive external stimuli, which regulates remodeling. An optimal level of mechanical stress is essential for maintaining chondrocyte homeostasis, however, excessive mechanical stress induces inflammatory cytokines and protease, such as matrix metalloproteinases (MMPs). Therefore, excessive mechanical stress is considered to be one of the main causes to cartilage destruction leading to osteoarthritis (OA). Integrins are well‐known as cell adhesion molecules and act as receptors for extracellular matrix (ECM), and are believed to control intracellular signaling pathways both physically and chemically as a mechanoreceptor. However, few studies have focused on the roles and functions of integrins in inflammation caused by excessive mechanical stress. In this study, we examined the relationship between integrins (αVβ3 and αVβ5) and the expression of inflammatory factors under mechanical loading in chondrocytes by using an integrin receptor antagonist (cilengitide). Cilengitide suppressed the gene expression of interleukin‐1β (IL‐1β), tumor necrosis factor‐α (TNF‐α), matrix metalloproteinase‐3 (MMP‐3), and MMP‐13 induced by excessive mechanical stress. In addition, the protein expression of IL1‐β and MMP‐13 was also inhibited by the addition of cilengitide. Next, we investigated the involvement of intracellular signaling pathways in stress‐induced integrin signaling in chondrocytes by using western blotting. The levels of p‐FAK, p‐ERK, p‐JNK, and p‐p38 were enhanced by excessive mechanical stress and the enhancement was suppressed by treatment with cilengitide. In conclusion, this study revealed that excessive mechanical stress may activate integrins αVβ3 and αVβ5 on the surface of chondrocytes and thereby induce an inflammatory reaction by upregulating the expression of IL‐1β, TNF‐α, MMP‐3, and MMP‐13 through phosphorylation of FAK and MAPKs.     

In vitro and clinical data analysis of Osteopontin as a prognostic indicator in colorectal cancer

Osteopontin (OPN) has been shown to promote colorectal cancer (CRC) progression; however, the mechanism of OPN‐induced CRC progression is largely unknown. In this study, we found that OPN overexpression led to enhanced anchorage‐independent growth, cell migration and invasion in KRAS gene mutant cells but to a lesser extent in KRAS wild‐type cells. OPN overexpression also induced PI3K signalling, expression of Snail and Matrix metallopeptidase 9 (MMP9), and suppressed the expression of E‐cadherin in KRAS mutant cells. In human CRC specimens, a high‐level expression of OPN significantly predicted poorer survival in CRC patients and OPN expression was positively correlated with MMP9 expression, and negatively correlated with E‐cadherin expression. Furthermore, we have found that 15 genes were co‐upregulated in OPN highly expression CRC and a list of candidate drugs that may have potential to reverse the secreted phosphoprotein 1 (SPP1) gene signature by connectivity mapping. In summary, OPN is a potential prognostic indicator and therapeutic target for colon cancer.     

Mechanical regulation of HB-GAM expression in bone cells

Bone adaption upon mechanical stimulation is accompanied by changes in gene expression. In this context we investigated the influence of mechanical loading on heparin binding growth associated molecule (HB-GAM) expression, an extracellular matrix molecule which in cell culture has been shown to stimulate the differentiation of osteoblasts. We obtained information on the participating signal transduction pathways using a mitogenic loading regimen. Specific inhibitors of various signal transduction pathways were added to loaded cells and to unloaded controls. By semi-quantitative PCR studies we demonstrated a rapid decrease of HB-GAM expression in primary osteoblasts and SaOs-2 cells by 20-30% upon mechanical loading within 30min. We showed that the RGD-integrin interaction is involved in the regulation of HB-GAM expression. Furthermore, integrity of the cytoskeleton, stretch-activated, and voltage-sensitive Ca(2+) channels as well as gap junctional communication are necessary for the downregulation of HB-GAM expression by mechanical loading.     

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.     

The effect of cyclical compressive loading on gene expression in articular cartilage

Osteoarthritis (OA) develops as a consequence of articular cartilage degeneration possibly initiated by excessive or abnormal loading of the joint, and potentially mediated through a proteinase/proteinase inhibitor imbalance. We have shown previously that physiological loads (0.5 MPa, 1 Hz, 3 hour) elicit increased expression and activation of the matrix metalloproteinases (MMPs) in articular cartilage explants in vitro. The objective of this study was to identify mechanically-regulated genes involved in the observed induction of MMP expression and enhanced activation. Differential RNA Display (DRD) was used to identify mechanically-regulated genes by comparing DRD products derived from loaded and unloaded cartilage. One gene up-regulated in cartilage after 10, 30 and 60 minute loading revealed 83% homology with Mus musculus thymosin beta_4 which is known to induce MMP gene expression. The identification of mechanically regulated genes will greatly enhance our understanding of matrix turnover providing an exciting future in elucidating the role of mechanically-regulated genes in the development of OA.     

Expression of muscle anabolic and metabolic factors in mechanically loaded MLO-Y4 osteocytes

Lack of physical activity results in muscle atrophy and bone loss, which can be counteracted by mechanical loading. Similar molecular signaling pathways are involved in the adaptation of muscle and bone mass to mechanical loading. Whether anabolic and metabolic factors regulating muscle mass, i.e., insulin-like growth factor-I isoforms (IGF-I Ea), mechano growth factor (MGF), myostatin, vascular endothelial growth factor (VEGF), or hepatocyte growth factor (HGF), are also produced by osteocytes in bone in response to mechanical loading is largely unknown. Therefore, we investigated whether mechanical loading by pulsating fluid flow (PFF) modulates the mRNA and/or protein levels of muscle anabolic and metabolic factors in MLO-Y4 osteocytes. Unloaded MLO-Y4 osteocytes expressed mRNA of VEGF, HGF, IGF-I Ea, and MGF, but not myostatin. PFF increased mRNA levels of IGF-I Ea (2.1-fold) and MGF (2.0-fold) at a peak shear stress rate of 44Pa/s, but not at 22Pa/s. PFF at 22 Pa/s increased VEGF mRNA levels (1.8- to 2.5-fold) and VEGF protein release (2.0- to 2.9-fold). Inhibition of nitric oxide production decreased (2.0-fold) PFF-induced VEGF protein release. PFF at 22 Pa/s decreased HGF mRNA levels (1.5-fold) but increased HGF protein release (2.3-fold). PFF-induced HGF protein release was nitric oxide dependent. Our data show that mechanically loaded MLO-Y4 osteocytes differentially express anabolic and metabolic factors involved in the adaptive response of muscle to mechanical loading (i.e., IGF-I Ea, MGF, VEGF, and HGF). Similarly to muscle fibers, mechanical loading enhanced expression levels of these growth factors in MLO-Y4 osteocytes. Although in MLO-Y4 osteocytes expression levels of IGF-I Ea and MGF of myostatin were very low or absent, it is known that the activity of osteoblasts and osteoclasts is strongly affected by them. The abundant expression levels of these factors in muscle cells, in combination with low expression in MLO-Y4 osteocytes, provide a possibility that growth factors expressed in muscle could affect signaling in bone cells.