Cornelis M. Semeins

Sensitivity of osteocytes to biomechanical stress in vitro.

It has been known for more than a century that bone tissue adapts to functional stress by changes in structure and mass. However, the mechanism by which stress is translated into cellular activities of bone formation and resorption is unknown. We studied the response of isolated osteocytes derived from embryonic chicken calvariae to intermittent hydrostatic compression as well as pulsating fluid flow, and compared their response to osteoblasts and periosteal fibroblasts. Osteocytes, but not osteoblasts or periosteal fibroblasts, reacted to 1 h pulsating fluid flow with a sustained release of prostaglandin E2. Intermittent hydrostatic compression stimulated prostaglandin production to a lesser extent: after 6 and 24 h in osteocytes and after 6 h in osteoblasts. These data provide evidence that osteocytes are the most mechanosensitive cells in bone involved in the transduction of mechanical stress into a biological response. The results support the hypothesis that stress on bone causes fluid flow in the lacunar‐canalicular system, which stimulates the osteocytes to produce factors that regulate bone metabolism.—Klein‐Nulend, J., van der Plas, A., Semeins, C. M., Ajubi, N. E., Frangos, J. A., Nijweide, P. J., Burger, E. H. Sensitivity of osteocytes to biomechanical stress in vitro. FASEB J. 9, 441–445 (1995)

Pulsating Fluid Flow Stimulates Prostaglandin Release and Inducible Prostaglandin G/H Synthase mRNA Expression in Primary Mouse Bone Cells

Bone tissue responds to mechanical stress with adaptive changes in mass and structure. Mechanical stress produces flow of fluid in the osteocyte lacunar‐canalicular network, which is likely the physiological signal for bone cell adaptive responses. We examined the effects of 1 h pulsating fluid flow (PFF; 0.7 ± 0.02 Pa, 5 Hz) on prostaglandin (PG) E2, PGI2, and PGF2α production and on the expression of the constitutive and inducible prostaglandin G/H synthases, PGHS‐1, and PGHS‐2, the major enzymes in the conversion of arachidonic acid to prostaglandins, using mouse calvarial bone cell cultures. PFF treatment stimulated the release of all three prostaglandins under 2% serum conditions, but with a different time course and to a different extent. PGF2α was rapidly increased 5–10 minutes after the onset of PFF. PGE2 release increased somewhat more slowly (significant after 10 minutes), but continued throughout 60 minutes of treatment. The response of PGI2 was the slowest, and only significant after 30 and 60 minutes of treatment. In addition, PFF induced the expression of PGHS‐2 but not PGHS‐1. One hour of PFF treatment increased PGHS‐2 mRNA expression about 2‐fold relative to the induction by 2% fresh serum given at the start of PFF. When the addition of fresh serum was reduced to 0.1%, the induction of PGHS‐2 was 8‐ to 9‐fold in PFF‐treated cells relative to controls. This up‐regulation continued for at least 1 h after PFF removal. PFF also markedly increased PGHS activity, measured as the conversion of arachidonic acid into PGE2. One hour after PFF removal, the production of all three prostaglandins was still enhanced. These results suggest that prostaglandins are important early mediators of the response of bone cells to mechanical stress. Prostaglandin up‐regulation is associated with an induction of PGHS‐2 enzyme mRNA, which may subsequently provide a means for amplifying the cellular response to mechanical stress.

Osteocyte morphology in fibula and calvaria --- is there a role for mechanosensing?

INTRODUCTION External mechanical forces on cells are known to influence cytoskeletal structure and thus cell shape. Mechanical loading in long bones is unidirectional along their long axes, whereas the calvariae are loaded at much lower amplitudes in different directions. We hypothesised that if osteocytes, the putative bone mechanosensors, can indeed sense matrix strains directly via their cytoskeleton, the 3D shape and the long axes of osteocytes in fibulae and calvariae will bear alignment to the different mechanical loading patterns in the two types of bone. MATERIALS AND METHODS We used confocal laser scanning microscopy and nano-computed tomography to quantitatively determine the 3D morphology and alignment of long axes of osteocytes and osteocyte lacunae in situ. RESULTS Fibular osteocytes showed a relatively elongated morphology (ratio lengths 5.9:1.5:1), whereas calvarial osteocytes were relatively spherical (ratio lengths 2.1:1.3:1). Osteocyte lacunae in fibulae had higher unidirectional alignment than the osteocyte lacunae in calvariae as demonstrated by their degree of anisotropy (3.33 and 2.10, respectively). The long axes of osteocyte lacunae in fibulae were aligned parallel to the principle mechanical loading direction, whereas those of calvarial osteocyte lacunae were not aligned in any particular direction. CONCLUSIONS The anisotropy of osteocytes and their alignment to the local mechanical loading condition suggest that these cells are able to directly sense matrix strains due to external loading of bone. This reinforces the widely accepted role of osteocytes as mechanosensors, and suggests an additional mode of mechanosensing besides interstitial fluid flow. The relatively spherical morphology of calvarial osteocytes suggests that these cells are more mechanosensitive than fibular osteocytes, which provides a possible explanation of efficient physiological load bearing for the maintenance of calvarial bone despite its condition of relative mechanical disuse.

Low-intensity ultrasound stimulates endochondral ossification in vitro

Animal and clinical studies have shown an acceleration of bone healing by the application of low‐intensity ultrasound. The objective of this study was to examine in vitro the influence of low‐intensity ultrasound on endochondral ossification of 17‐day‐old fetal mouse metatarsal rudiments.

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.

Osteocyte morphology in human tibiae of different bone pathologies with different bone mineral density — Is there a role for mechanosensing?☆

Matrix strains due to external loading are different in bones of different pathologies with different bone mineral density (BMD), and are likely sensed by the osteocytes, the putative bone mechanosensors. The mechanosensitivity of osteocytes appears to be strongly influenced by their morphology. In this study, we explored the possibility that osteocyte morphology might play a role in various bone pathologies with different BMD. Confocal laser scanning microscopy and nano-CT were used to quantitatively determine 3D morphology and alignment of osteocytes and osteocyte lacunae in human proximal tibial bone with relatively low (osteopenic), medium (osteoarthritic), and high (osteopetrotic) BMD. Osteopenic osteocytes were relatively large and round (lengths 8.9:15.6:13.4 microm), osteopetrotic osteocytes were small and discoid shaped (lengths 5.5:11.1:10.8 microm), and osteoarthritic osteocytes were large and elongated (lengths 8.4:17.3:12.2 microm). Osteopenic osteocyte lacunae showed 3.5 fold larger volume and 2.2 fold larger surface area than osteoarthritic lacunae, whereas osteopetrotic lacunae were 1.9 fold larger and showed 1.5 fold larger surface area than osteoarthritic lacunae. Osteopetrotic osteocyte lacunae had lower alignment than osteopenic and osteoarthritic lacunae as indicated by their lower degree of anisotropy. The differences in 3D morphology of osteocytes and their lacunae in long bones of different pathologies with different BMD might reflect an adaptation to matrix strain due to different external loading conditions. Moreover, since direct mechanosensing of matrix strain likely occurs by the cell bodies, the differences in osteocyte morphology and their lacunae might indicate differences in osteocyte mechanosensitivity. The exact relationship between osteocyte morphology and bone architecture, however, is complex and deserves further study.

Pulsating fluid flow modulates gene expression of proteins involved in Wnt signaling pathways in osteocytes

Strain‐derived flow of interstitial fluid activates signal transduction pathways in osteocytes that regulate bone mechanical adaptation. Wnts are involved in this process, but whether mechanical loading modulates Wnt signaling in osteocytes is unclear. We assessed whether mechanical stimulation by pulsating fluid flow (PFF) leads to functional Wnt production, and whether nitric oxide (NO) is important for activation of the canonical Wnt signaling pathway in MLO‐Y4 osteocytes. MC3T3‐E1 osteoblasts were studied as a positive control for the MLO‐Y4 osteocyte response to mechanical loading. MLO‐Y4 osteocytes and MC3T3‐E1 osteoblasts were submitted to 1‐h PFF (0.7 ± 0.3 Pa, 5 Hz), and postincubated (PI) without PFF for 0.5–3 h. Gene expression of proteins related to the Wnt canonical and noncanonical pathways were studied using real‐time polymerase chain reaction (PCR). In MLO‐Y4 osteocytes, PFF upregulated gene expression of Wnt3a, c‐jun, connexin 43, and CD44 at 1–3‐h PI. In MC3T3‐E1 osteoblasts, PFF downregulated gene expression of Wnt5a and c‐jun at 0.5–3‐h PI. In MLO‐Y4 osteocytes, gene expression of PFF‐induced Wnt target genes was suppressed by the Wnt antagonist sFRP4, suggesting that loading activates the Wnt canonical pathway through functional Wnt production. The NO inhibitor L‐NAME suppressed the effect of PFF on gene expression of Wnt target genes, suggesting that NO might play a role in PFF‐induced Wnt production. The response to PFF differed in MC3T3‐E1 osteoblasts. Because Wnt signaling is important for bone mass regulation, osteocytes might orchestrate loading‐induced bone remodeling through, among others, Wnts.

Inhibition of osteocyte apoptosis by fluid flow is mediated by nitric oxide

Bone unloading results in osteocyte apoptosis, which attracts osteoclasts leading to bone loss. Loading of bone drives fluid flow over osteocytes which respond by releasing signaling molecules, like nitric oxide (NO), that inhibit osteocyte apoptosis and alter osteoblast and osteoclast activity thereby preventing bone loss. However, which apoptosis-related genes are modulated by loading is unknown. We studied apoptosis-related gene expression in response to pulsating fluid flow (PFF) in osteocytes, osteoblasts, and fibroblasts, and whether this is mediated by loading-induced NO production. PFF (0.7+/-0.3Pa, 5Hz, 1h) upregulated Bcl-2 and downregulated caspase-3 expression in osteocytes. l-NAME attenuated this effect. In osteocytes PFF did not affect p53 and c-Jun, but l-NAME upregulated c-Jun expression. In osteoblasts and fibroblasts PFF upregulated c-Jun, but not Bcl-2, caspase-3, and p53 expression. This suggests that PFF inhibits osteocyte apoptosis via alterations in Bcl-2 and caspase-3 gene expression, which is at least partially regulated by NO.

Fluid shear stress inhibits TNFalpha-induced osteocyte apoptosis.

Bone tissue can adapt to orthodontic load. Mechanosensing in bone is primarily a task for the osteocytes, which translate the canalicular flow resulting from bone loading into osteoclast and osteoblast recruiting signals. Apoptotic osteocytes attract osteoclasts, and inhibition of osteocyte apoptosis can therefore affect bone remodeling. Since TNF-α is a pro-inflammatory cytokine with apoptotic potency, and elevated levels are found in the gingival sulcus during orthodontic tooth movement, we investigated if mechanical loading by pulsating fluid flow affects TNF-α-induced apoptosis in chicken osteocytes, osteoblasts, and periosteal fibroblasts. During fluid stasis, TNF-α increased apoptosis by more than two-fold in both osteocytes and osteoblasts, but not in periosteal fibroblasts. One-hour pulsating fluid flow (0.70 ± 0.30 Pa, 5 Hz) inhibited (−25%) TNF-α-induced apoptosis in osteocytes, but not in osteoblasts or periosteal fibroblasts, suggesting a key regulatory role for osteocyte apoptosis in bone remodeling after the application of an orthodontic load.

Donor age and mechanosensitivity of human bone cells

Abstract: With increasing age the human skeleton decreases in density, thereby compromising its load-bearing capacity. Mechanical loading activates bone formation, but an age-dependent decrease in skeletal mechanoresponsiveness has been described in rats. In this paper we examine whether age-related bone loss is reflected by a decrease in the mechanosensitivity of isolated bone cells from human donors. Bone cell cultures were obtained from 39 donors (males and females) between 7 and 85 years of age. Cultures were challenged with 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) or mechanically stressed by treatment with pulsating fluid flow (PFF; 0.7 ± 0.03 Pa at 5 Hz for 1 h). The growth capacity of the bone-derived cell population almost halved between 7 and 85 years of age. Basal alkaline phosphatase activity of the cells increased with donor age, while the response to 1,25(OH)2D3, measured as stimulated osteocalcin production, decreased with age. Together this suggests that the cell cultures from older donors represented a more mature, slower-growing cell population than the cultures from young donors. All cell cultures responded to mechanical stress with enhanced release of prostaglandin E2 (PGE2) and I2 (PGI2). The magnitude of the response was positively correlated with donor age, cell cultures from older donors showing a higher response than cultures from younger donors. There was also a positive correlation between time to reach confluency and mechanosensitivity, i.e., the PGE2 response to PFF treatment was higher in bone cell cultures with a slower growth rate. We conclude that bone cell cultures from older donors have a lower proliferative capacity and a higher degree of osteoblastic maturation than younger donors. The higher degree of osteoblastic maturation explains the higher response of the cultures to mechanical stress, in line with earlier studies on chicken bone cells. This study found no evidence for loss of mechanosensitivity with donor age. The reduced growth capacity might, however, be a factor in age-related bone loss.