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Jenneke Klein-Nulend

Publications and source records attributed to Jenneke Klein-Nulend.

18 recordsLinked to original sources
PubMed2006

Osteocytes subjected to pulsating fluid flow regulate osteoblast proliferation and differentiation.

Osteocytes are thought to orchestrate bone remodeling, but it is unclear exactly how osteocytes influence neighboring bone cells. Here, we tested whether osteocytes, osteoblasts, and periosteal fibroblasts subjected to pulsating fluid flow (PFF) produce soluble factors that modulate the proliferation and differentiation of cultured osteoblasts and periosteal fibroblasts. We found that osteocyte PFF conditioned medium (CM) inhibited bone cell proliferation, and osteocytes produced the strongest inhibition of proliferation compared to osteoblasts and periosteal fibroblasts. The nitric oxide (NO) synthase inhibitor N(G)-nitro-L-arginine methyl ester (L-NAME) attenuated the inhibitory effects of osteocyte PFF CM, suggesting that a change in NO release is at least partially responsible for the inhibitory effects of osteocyte PFF CM. Furthermore, osteocyte PFF CM stimulated osteoblast differentiation measured as increased alkaline phosphatase activity, and l-NAME decreased the stimulatory effects of osteocyte PFF CM on osteoblast differentiation. We conclude that osteocytes subjected to PFF inhibit proliferation but stimulate differentiation of osteoblasts in vitro via soluble factors and that the release of these soluble factors was at least partially dependent on the activation of a NO pathway in osteocytes in response to PFF. Thus, the osteocyte appears to be more responsive to PFF than the osteoblast or periosteal fibroblast with respect to the production of soluble signaling molecules affecting osteoblast proliferation and differentiation.

Animals↗
PubMed2006

Polyamines modulate nitric oxide production and COX-2 gene expression in response to mechanical loading in human adipose tissue-derived mesenchymal stem cells.

For bone tissue engineering, it is important that mesenchymal stem cells (MSCs) display a bone cell-like response to mechanical loading. We have shown earlier that this response includes increased nitric oxide (NO) production and cyclooxygenase-2 (COX-2) gene expression, both of which are intimately involved in mechanical adaptation of bone. COX-2 gene expression is likely regulated by polyamines, which are organic cations implicated in cell proliferation and differentiation. This has led to the hypothesis that polyamines may play a role in the response of adipose tissue-derived MSCs (AT-MSCs) to mechanical loading. The aim of this study was to investigate whether genes involved in polyamine metabolism are regulated by mechanical loading and to study whether polyamines modulate mechanical loading-induced NO production and COX-2 gene expression in human AT-MSCs. Human AT-MSCs displayed a bone cell-like response to mechanical loading applied by pulsating fluid flow (PFF), as demonstrated by increased NO production and increased gene expression of COX-2. Furthermore, PFF increased gene expression of spermidine/spermine N (1)-acetyltransferase, which is involved in polyamine catabolism, suggesting that mechanical loading modulates polyamine levels. Finally, the polyamine spermine was shown to inhibit both PFF-induced NO production and COX-2 gene expression, suggesting that polyamines modulate the response of human AT-MSCs to mechanical loading. In conclusion, this is the first study implicating polyamines in the response of human AT-MSCs to mechanical loading, creating opportunities for the use of polyamines in tissue engineering approaches targeting skeletal defects.

Acetyltransferases↗
PubMed2006

Release of nitric oxide, but not prostaglandin E2, by bone cells depends on fluid flow frequency.

Loading frequency is an important parameter for the stimulation of bone formation in vivo. It is still unclear how the information of external loading characteristics is conveyed to osteoblasts and osteoclasts. Osteocytes are thought to detect mechanical loads by sensing fluid flow through the lacuno-canalicular network within bone and to translate this information into chemical signals. The signaling molecules nitric oxide (NO) and prostaglandin E2 (PGE2) are known to play important roles in the adaptive response of bone to mechanical loads. We have investigated the effects of fluid flow frequency on the production of PGE2 and NO in bone cells in vitro. Pulsatile fluid flow with different frequencies stimulated the release of NO by MC3T3-E1 osteoblasts in a dose-dependent manner. In contrast, PGE2 production was enhanced consistently by all fluid flow regimes, independent of flow frequency. This implies that the NO response may play a role in mediating the differential effects of the various loading patterns on bone.

3T3 Cells↗
PubMed2006

Bone cell responses to high-frequency vibration stress: does the nucleus oscillate within the cytoplasm?

Mechanosensing by cells directs changes in bone mass and structure in response to the challenges of mechanical loading. Low-amplitude, high-frequency loading stimulates bone growth by enhancing bone formation and inhibiting disuse osteoporosis. However, how bone cells sense vibration stress is unknown. Hence, we investigated bone cell responses to vibration stress at a wide frequency range (5-100 Hz). We used NO and prostaglandin E2 (PGE2) release, and COX-2 mRNA expression as parameters for bone cell response since these molecules regulate bone adaptation to mechanical loading. NO release positively correlated whereas PGE2 release negatively correlated to the maximum acceleration rate of the vibration stress. COX-2 mRNA expression increased in a frequency-dependent manner, which relates to increased NO release at high frequencies, confirming our previous results. The negatively correlated release of NO and PGE2 suggests that these signaling molecules play different roles in bone adaptation to high-frequency loading. The maximum acceleration rate is proportional to omega3 (frequency=omega/2pi), which is commensurate with the Stokes-Einstein relation for modeling cell nucleus motion within the cytoplasm due to vibration stress. Correlations of NO and PGE2 with the maximum acceleration rate then relate to nucleus oscillations, providing a physical basis for cellular mechanosensing of high-frequency loading.

3T3 Cells↗
PubMed2006

Bio imaging of intracellular NO production in single bone cells after mechanical stimulation.

UNLABELLED: We show the intracellular upregulation of NO production after mechanical stimulation, an essential chemical signal in bone remodeling. This is done in real time using the fluorescent chromophore DAR-4M AM. Differences in cellular response to mechanical stimulation of different regions of a single cell were observed. INTRODUCTION: Osteocytes are the most abundant bone cells that are believed to be the mechanosensors of bone, responding to mechanical stresses in interstitial fluid flow through the canaliculi. Under mechanical load, chemical signals such as NO play a key role in the activity of osteoblasts/osteoclasts that regulate bone remodeling. Despite the importance of NO in signaling, its real-time detection has proved challenging. This is largely because of the short NO half-life (typically approximately 0.1-5 s). Here, we show the upregulation of intracellular NO production in single osteocytes under localized mechanical stimulation. MATERIALS AND METHODS: We used the chromophore DAR-4M AM for NO detection. This is loaded into surface-attached MLO-Y4 osteocyte-like and MC3T3-E1 osteoblast-like cells that are subjected to a localized mechanical stimulation using optical tweezers or a microneedle tip. DAR-4M AM is membrane-permeable and chelates NO, forming a stable, fluorescent compound, which is visible with a rhodamine filter. RESULTS: Nonstimulated MLO-Y4 and MC3T3-E1 cells showed basal NO production levels, as indicated by a gradual increase in their fluorescence intensity. Localized mechanical stimulation of single MC3T3-E1 cells and MLO-Y4 cells by optical tweezers (150-550 pN, 0.5-3 Hz, 1 minute) showed a nearly 15-30% increase, whereas MLO-Y4 cells stimulated by a microneedle (10-20 nN, 1 minute) showed nearly 15-16% increase relative to their nonstimulated state. Furthermore, stimulation of a single cell process by a microneedle resulted in a 2-10% increase in the fluorescence intensity. CONCLUSIONS: NO is essential for mechanically induced bone remodeling and is a meaningful parameter for measuring bone cell activation after mechanical loading. Here we show NO upregulation in individual bone cells after a localized mechanical stimulation. We also show that both the cell body and the cell processes might be involved in mechanosensing. This technique allows characterization of the mechanosensitivity of different parts of a single osteocyte. This opens up the possibility to uncover the complexities and function of single osteocytes in the dynamic process of bone remodeling.

Animals↗
PubMed2005

The effect of cytoskeletal disruption on pulsatile fluid flow-induced nitric oxide and prostaglandin E2 release in osteocytes and osteoblasts.

Fluid flowing through the bone porosity might be a primary stimulus for functional adaptation of bone. Osteoblasts, and osteocytes in particular, respond to fluid flow in vitro with enhanced nitric oxide (NO) and prostaglandin E(2) (PGE(2)) release; both of these signaling molecules mediate mechanically-induced bone formation. Because the cell cytoskeleton is involved in signal transduction, we hypothesized that the pulsatile fluid flow-induced release of NO and PGE(2) in both osteoblastic and osteocytic cells involves the actin and microtubule cytoskeleton. In testing this hypothesis we found that fluid flow-induced NO response in osteoblasts was accompanied by parallel alignment of stress fibers, whereas PGE(2) response was related to fluid flow stimulation of focal adhesions formed after cytoskeletal disruption. Fluid flow-induced PGE(2) response in osteocytes was inhibited by cytoskeletal disruption, whereas in osteoblasts it was enhanced. These opposite PGE(2) responses are likely related to differences in cytoskeletal composition (osteocyte structure was more dependent on actin), but may occur via cytoskeletal modulation of shear/stretch-sensitive ion channels that are known to be dominant in osteocyte (and not osteoblast) response to mechanical loading.

3T3 Cells↗
PubMed2005

Initial stress-kick is required for fluid shear stress-induced rate dependent activation of bone cells.

The shear stress induced by the loading-mediated flow of interstitial fluid through the lacuno-canalicular network is a likely stimulus for bone cell adaptive responses. Furthermore, the magnitude of the cellular response is related to the rate of mechanical loading rather than its magnitude. Thus, bone cells might be very sensitive to sudden stress-kicks, as occuring e.g., during impact loading. There is evidence that cells change stiffness under stress, which might make them more sensitive to subsequent loading. We studied the influence of a stress-kick on the mechanosensitivity of MC3T3-E1 osteoblast-like cells under different peak shear rate conditions, as measured by nitric oxide production. MC3T3-E1 bone cells were treated with steady or pulsating fluid shear stress (PFSS) for 5 min with different peak rates (9.70, 17.5, and 22.0 Pa Hz) using varying frequencies (5 and 9 Hz), and amplitudes (0.70 and 0.31 Pa). PFSS treatment was done with or without fluid flow pretreatment phase, which removed the initial stress-kick by first applying a slow fluid flow increase. Nitric oxide production in response to fluid shear stress was rate dependent, but necessitated an initial stress-kick to occur. This suggests that high-rate stimuli condition bone cells to be more sensitive for high-frequency, low-amplitude loads.

Bone Marrow Cells↗
PubMed2005

Detection of living cells in non-processed but deep-frozen bone allografts.

Impacted morselized donor bone is successfully used to treat bone loss in revision total hip arthroplasties. It is generally thought, but not proven, that the processing and storage at -80 degrees C of the donor bone kills all cells. Because of the risk of contamination and to increase our understanding about the process of new bone formation after revision total hip arthroplasty, the aim of this study was to investigate whether the donor bone does contain vital cells. Samples from 11 femoral heads were obtained according to the American and European standards of bone banking, and tested for their capacity to give rise to proliferating cells, using tissue culture methods. All bone samples were stored at - 80 degrees C for a minimum of 6 months. Bone sample cores were morselized and cultured for 6 weeks. Inverted phase contrast microscopy was used to evaluate cell growth. DNA marker analysis was used to confirm cellular identity. All bank bone samples gave rise to cell growth. The cell cultures showed osteoblastic characteristics in that they expressed high levels of alkaline phosphatase activity. DNA marker analysis showed identical alleles for cultured cells from frozen bone and freshly obtained buccal cells from the same donor, indicating that the cells growing from the banked bone were indeed originating from the donor tissue. It was therefore concluded that -80 degrees C freezing of bone tissue does not routinely kill cells within the tissue.

Alkaline Phosphatase↗
PubMed2005

Dynamic shear stress in parallel-plate flow chambers.

An in vitro model using a parallel-plate fluid flow chamber is supposed to simulate in vivo fluid shear stresses on various cell types exposed to dynamic fluid flow in their physiological environment. The metabolic response of cells in vitro is associated with the wall shear stress. However, parallel-plate flow chambers have not been characterized for dynamic fluid flow experiments. We use a dimensionless ratio h / lambda(v), in determining the exact magnitude of the dynamic wall shear stress, with its oscillating components scaled by a shear factor T. It is shown that, in order to expose cells to predictable levels of dynamic fluid shear stress, two conditions have to be met: (1) h / lambda(v) < 2, where h is the distance between the plates and lambda(v) is the viscous penetration depth; and (2) f(0) < f(c) / m, where the critical frequency f(c) is the upper threshold for this flow regime, m is the highest harmonic mode of the flow, and f(0) is the fundamental frequency of fluid flow.

Animals↗
PubMed2004

A comparison of strain and fluid shear stress in stimulating bone cell responses--a computational and experimental study.

Bone undergoes continuous remodeling in response to mechanical loading. However, the underlying mechanisms by which bone cells respond to their changing mechanical environment, that is, strain in the load-bearing matrix or fluid flow through the canalicular network, are not well understood. It has been established in vitro that bone cells respond differently to substrate strain and fluid shear stress treatments. Uncovering the mechanical basis of these differences represents a significant challenge to our understanding of cellular mechanotransduction and bone remodeling. To investigate this problem, we developed a biomechanical model of an adherent cell, to test the hypothesis that bone cells respond differently to 0.6 Pa fluid shear stress and 1,000 mu(epsilon) substrate strain stimulation because of qualitative and quantitative differences in the cellular deformation caused. Fluid shear stress loading conditions resulted in maximum displacements at the apical surface of the cell approximately 8 times higher than those due to strain at the cell-substrate interface and also caused higher stressing of all parts of the cell. Significantly, this shows that the deforming effects of fluid shear stress and strain on a cellular level are qualitatively different, which may provide a basis for explaining differences in bone cell responses to both stimuli as reported in several studies. Although our approach to modeling the morphology and complex physical environment of an adherent cell is certainly simplified, our results do show independent roles for fluid flow and strain as mechanical stimuli and highlight the importance of deformation on a cellular level in bone physiology.

Adolescent↗
PubMed2004

Shear stress inhibits while disuse promotes osteocyte apoptosis.

Cell apoptosis operates as an organizing mechanism in biology in addition to removing effete cells. We have recently proposed that during bone remodeling, osteocyte apoptosis steers osteonal alignment in relation to mechanical loading of the whole bone [J. Biomech. 36 (2003) 1453]. Here we present evidence that osteocyte apoptosis in cell culture is modulated by shear stress. Under static culture conditions, serum starved osteocytes exposed phosphatidylserine (PS) on their cell membrane 6x more often than periosteal fibroblasts and 3x more often than osteoblasts. Treatment with shear stress reduced the number of osteocytes that exposed PS by 90%, but did not affect the other cell types. Fluid shear stress of increasing magnitude, dose-dependently stimulated Bcl-2 mRNA expression in human bone cells, while shear stress did not change Bax expression. These data suggest that disuse promotes osteocyte apoptosis, while mechanical stimulation by fluid shear stress promotes osteocyte survival, by modulating the Bcl-2/Bax expression ratio.

Adaptation, Physiological↗
PubMed2004

Nitric oxide production by bone cells is fluid shear stress rate dependent.

Shear stress due to mechanical loading-induced flow of interstitial fluid through the lacuno-canalicular network is a likely signal for bone cell adaptive responses. Moreover, the rate (determined by frequency and magnitude) of mechanical loading determines the amount of bone formation. Whether the bone cells' response to fluid shear stress is rate dependent is unknown. Here we investigated whether bone cell activation by fluid shear stress is rate dependent. MC3T3-E1 osteoblastic cells were subjected for 15 min to fluid shear stress of varying frequencies and amplitudes, resulting in peak fluid shear stress rates ranging from 0 to 39.6 Pa-Hz. Nitric oxide production, a parameter for bone cell activation, was found to be linearly dependent on the fluid shear stress rate; the slope was steepest at 5 min (0.11 Pa-Hz(-1)) and decreased to 0.03 Pa-Hz(-1) at 15 min. We conclude that the fluid shear stress rate is an important parameter for bone cell activation.

Animals↗
PubMed2003

Interactive effects of PTH and mechanical stress on nitric oxide and PGE2 production by primary mouse osteoblastic cells.

Parathyroid hormone (PTH) and mechanical stress both stimulate bone formation but have opposite effects on bone resorption. PTH increased loading-induced bone formation in a rat model, suggesting that there is an interaction of these stimuli, possibly at the cellular level. To investigate whether PTH can modulate mechanotransduction by bone cells, we examined the effect of 10-9 M human PTH-(1-34) on fluid flow-induced prostaglandin E2 (PGE2) and nitric oxide (NO) production by primary mouse osteoblastic cells in vitro. Mechanical stress applied by means of a pulsating fluid flow (PFF; 0.6 +/- 0.3 Pa at 5 Hz) stimulated both NO and PGE2 production twofold. In the absence of stress, PTH also caused a twofold increase in PGE2 production, but NO release was not affected and remained low. Simultaneous application of PFF and PTH nullified the stimulating effect of PFF on NO production, whereas PGE2 production was again stimulated only twofold. Treatment with PTH alone reduced NO synthase (NOS) enzyme activity to undetectable levels. We speculate that PTH prevents stress-induced NO production via the inhibition of NOS, which will also inhibit the NO-mediated upregulation of PGE2 by stress, leaving only the NO-independent PGE2 upregulation by PTH. These results suggest that mechanical loading and PTH interact at the level of mechanotransduction.

Animals↗
PubMed2003

Osteocyte and bone structure.

The osteocyte is the most abundant cell type of bone. There are approximately 10 times as many osteocytes as osteoblasts in adult human bone, and the number of osteoclasts is only a fraction of the number of osteoblasts. Our current knowledge of the role of osteocytes in bone metabolism is far behind our insight into the properties and functions of the osteoblasts and osteoclasts. However, the striking structural design of bone predicts an important role for osteocytes in determining bone structure. Over the past several years, the role of osteocytes as the professional mechanosensory cells of bone, and the lacunocanalicular porosity as the structure that mediates mechanosensing have become clear. Strain-derived flow of interstitial fluid through this porosity seems to mechanically activate the osteocytes, as well as ensure transport of cell signaling molecules, nutrients, and waste products. This concept explains local bone gain and loss--as well as remodeling in response to fatigue damage--as processes supervised by mechanosensitive osteocytes. Alignment during remodeling seems to occur as a result of the osteocyte's sensing different canalicular flow patterns around the cutting cone and reversal zone during loading, therefore determining the bone's structure.

Bone Matrix↗
PubMed2003

Strain-derived canalicular fluid flow regulates osteoclast activity in a remodelling osteon--a proposal.

The concept of bone remodelling by basic multicellular units is well established, but how the resorbing osteoclasts find their way through the pre-existing bone matrix remains unexplained. The alignment of secondary osteons along the dominant loading direction suggests that remodelling is guided by mechanical strain. This means that adaptation (Wolff's Law) takes place throughout life at each remodelling cycle. We propose that alignment during remodelling occurs as a result of different canalicular flow patterns around cutting cone and reversal zone during loading. Low canalicular flow around the tip of the cutting cone is proposed to reduce NO production by local osteocytes thereby causing their apoptosis. In turn, osteocyte apoptosis could be the mechanism that attracts osteoclasts, leading to further excavation of bone in the direction of loading. At the transition between cutting cone and reversal zone, however, enhanced canalicular flow will stimulate osteocytes to increase NO production, which induces osteoclast retraction and detachment from the bone surface. Together, this leads to a treadmill of attaching and detaching osteoclasts in the tip and the periphery of the cutting cone, respectively, and the digging of a tunnel in the direction of loading.

Biomechanical Phenomena↗
PubMedDate not supplied

Adipose tissue-derived mesenchymal stem cells acquire bone cell-like responsiveness to fluid shear stress on osteogenic stimulation.

To engineer bone tissue, mechanosensitive cells are needed that are able to perform bone cell-specific functions, such as (re)modeling of bone tissue. In vivo, local bone mass and architecture are affected by mechanical loading, which is thought to provoke a cellular response via loading-induced flow of interstitial fluid. Adipose tissue is an easily accessible source of mesenchymal stem cells for bone tissue engineering, and is available in abundant amounts compared with bone marrow. We studied whether adipose tissue-derived mesenchymal stem cells (AT-MSCs) are responsive to mechanical loading by pulsating fluid flow (PFF) on osteogenic stimulation in vitro. We found that ATMSCs show a bone cell-like response to fluid shear stress as a result of PFF after the stimulation of osteogenic differentiation by 1,25-dihydroxyvitamin D3. PFF increased nitric oxide production, as well as upregulated cyclooxygenase-2, but not cyclooxygenase-1, gene expression in osteogenically stimulated AT-MSCs. These data suggest that AT-MSCs acquire bone cell-like responsiveness to pulsating fluid shear stress on 1,25-dihydroxyvitamin D3-induced osteogenic differentiation. ATMSCs might be able to perform bone cell-specific functions during bone (re)modeling in vivo and, therefore, provide a promising new tool for bone tissue engineering.

Adipose Tissue↗