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RESEARCH REPORT |
-induced Osteocyte Apoptosis
1 Department of Oral Cell Biology, Academic Centre for Dentistry Amsterdam (ACTA)-Universiteit van Amsterdam and Vrije Universiteit, Van der Boechorststraat 7, NL-1081 BT Amsterdam, The Netherlands; and
2 Department of Orthodontics and Oral Biology, Radboud University Nijmegen Medical Centre, Nijmegen, The Netherlands
* corresponding author, j.kleinnulend{at}vumc.nl
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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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--induced apoptosis in chicken osteocytes, osteoblasts, and periosteal fibroblasts. During fluid stasis, TNF- 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--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.
KEY WORDS: osteocyte • TNF- • mechanical loading • apoptosis • orthodontic tooth movement
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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Osteocytes are derived from osteoblasts that have stopped producing bone matrix, and are literally buried in bone matrix. They are in contact with neighboring osteocytes via long cell processes, located in canaliculi, which are filled with interstitial fluid (van der Plas et al., 1994). This three-dimensional network of interconnected cells is present throughout bone, and it is via this network that the osteocytes are positioned to regulate bone remodeling (Klein-Nulend et al., 1995b; Burger and Klein-Nulend, 1999). When bone is loaded, interstitial fluid is squeezed through the three-dimensional network, resulting in fluid flow. This flow results in a strain-driven movement of interstitial fluid, through the canaliculi and along the osteocyte processes, which is sensed and transduced by osteocytes (Weinbaum et al., 1994; Burger and Klein-Nulend, 1999).
Pulsating fluid flow provokes an immediate nitric oxide (NO) response in osteocytes in vitro (Klein-Nulend et al., 1995a). NO inhibits osteoclast activity (MacIntyre et al., 1991), and mediates adaptive bone formation in vivo (Fox et al., 1996). If bone is unloaded, no flow through the canaliculi will occur, resulting in reduced osteocyte fluid shear stress stimulation, as well as NO production (Klein-Nulend et al., 1995a). In endothelial cells, NO production in response to fluid flow prevents apoptosis (Haendeler et al., 1997). Since osteocytes and endothelial cells respond similarly to mechanical stimulation by fluid shear stress with an up-regulation of endothelial cell nitric oxide synthase (ecNOS) and increased NO production (Klein-Nulend et al., 1995b), we suggest that insufficient NO production, due to insufficient fluid flow, might cause apoptosis in osteocytes (Bakker et al., 2004).
TNF- is a pro-inflammatory cytokine, and induces apoptosis in, among others, fibroblasts and osteoblasts (Pavalko et al., 2003; Alikhani et al., 2004). In bone, it stimulates osteoclastogenesis and inhibits osteoblast function (Bertolini et al., 1986), and elevated TNF- levels are found in osteoporosis, periodontitis (Ralston et al., 1990; Graves and Cochran, 2003), and in the gingival sulcus during orthodontic tooth movement (Uematsu et al., 1996). On the pressure side, lowering of normal strain from the functioning periodontal ligament occurs (Melsen, 1999), which might result in local stasis of extracellular fluid in the canalicular network (Smit and Burger, 2000; Smit et al., 2002), a lack of fluid shear stress on the osteocytes, and reduced NO production. Fluid stasis in combination with TNF- then induces osteocyte apoptosis on the pressure side.
Osteocyte apoptosis could be the signal for osteoclast recruitment to resorb bone on the pressure side, enabling the teeth to move (Burger et al., 2003). Osteoclastic attack is directed toward apoptotic osteocytes (Bronckers et al., 1996; Noble et al., 1997), suggesting a key regulatory role for osteocyte apoptosis in bone remodeling, such as occurs after orthodontic load application.
Here, we studied osteocyte apoptosis induced by TNF- after pulsating fluid flow (PFF) application. PFF, resulting in fluid shear stress on the cells, mimicked the manner in which loading of whole bones is thought to be conveyed to osteocytes in vivo (Weinbaum et al., 1994; Burger and Klein-Nulend, 1999). Static cultures represented disuse conditions. Apoptosis was assessed as caspase-3/7 activity, since this enzyme mediates TNF--induced apoptosis (Jaeschke et al., 1998). The responses of TNF--treated bone cells to mechanical loading were studied by measuring NO production. To validate that NO may modulate apoptosis, we inhibited the release of NO by NG-Nitro-L-Arginine Methyl Ester (L-NAME), and apoptosis was assessed. We hypothesized that mechanical stimulation by fluid flow inhibits TNF--induced apoptosis in osteocytes, but not in osteoblasts or periosteal fibroblasts.
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MATERIALS & METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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-MEM; Gibco, Paisley, Scotland) supplemented with 2% chicken serum (Gibco), 200 µg/mL glutamine (Sigma), 50 µg/mL gentamycin sulfate (Sigma), 50 µg/mL L-ascorbic acid (Merck, Darmstadt, Germany), and 1 mg/mL D-glucose (Merck) at 37°C in a humidified atmosphere of 5% CO2 in air. After induction of apoptosis, cells were subjected to PFF.
Apoptosis
TNF- (Imgen Technologies, Alexandria, VA, USA) at 5, 10, 25, 50, and 100 ng/mL, was added to osteoblasts, seeded at 3000 cells/well in a 96-well plate. Cells were incubated for 6, 16, and 24 hrs with TNF--containing serum-free -MEM with 1% bovine serum albumin.
Pulsating Fluid Flow (PFF)
After the induction of apoptosis with 10 ng/mL TNF- for 16 hrs, osteocytes, osteoblasts, and periosteal fibroblasts were either or not subjected to 1 hr of PFF through a parallel-plate flow chamber containing the cells as described previously (Klein-Nulend et al., 1995b). The cells were subjected to a 5-Hz pulse with a mean shear stress of 0.7 Pa, a pulse amplitude of 0.3 Pa, and a peak shear stress rate of 8.4 Pa/sec. Static cultures were kept in a Petri dish under conditions similar to those for the experimental cultures. After 10 and 30 min of PFF treatment or static culture, medium was collected and assayed for NO production. After 1 hr of PFF treatment or static culture, fresh medium was added, and cells were post-incubated under static conditions for 24 hrs.
Caspase-3/7 Activity and DNA Content
We created a culture well (diameter, 1.5 cm) around the cell spot by securing a silicone rubber incubation ring onto the glass slide. A cell-lysis-based reagent of the Caspase-Glo 3/7 Assay (Promega, Madison, WI, USA) was added for the assessment of caspase-3/7 activity with a luminometer (Berthold Technologies, Bad Wildbad, Germany), according to the manufacturer’s instructions. DNA in the cell lysate was determined by a CyQUANT Cell Proliferation Assay (Molecular Probes Inc., Eugene, OR, USA). Cell apoptosis was expressed in relative light units per ng DNA.
Nitric Oxide
NO was measured as nitrite accumulation in the medium, with Griess reagent (Green et al., 1982) consisting of 1% sulfanilamide, 0.1% naphthylethylene-diamine-dihydrochloride, and 2.5 M H3PO4. Serial dilutions of NaNO2 in medium were used for a standard curve. Absorbance was measured at 540 nm.
Inhibition of Nitric Oxide Production
NO release was inhibited by the addition of 1 mM L-NAME (Sigma). The NO inhibitor was added to the culture medium during exposure to PFF.
Statistics
Treatment-over-control ratios and NO production data were analyzed by a paired t test. Differences were considered significant when p < 0.05.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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, we added various concentrations (from 5 to 100 ng/mL) to osteoblasts for 6, 16, and 24 hrs. These dose-response and time-course studies showed that maximal induction of apoptosis was achieved by 10 ng/mL TNF- after 16 hrs of treatment (Figs. 1a, 1b
). Lower and higher concentrations of TNF- for 16 hrs resulted in less induction of apoptosis (Fig. 1b), and 16 hrs was the earliest time-point at which apoptosis could be detected (Fig. 1a). Similar results were obtained for osteocytes (data not shown). Therefore, 10 ng/mL TNF- for 16 hrs was used to induce apoptosis for fluid flow experiments.
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at 10 ng/mL for 16 hrs increased caspase-3/7 activity in osteocytes and osteoblasts by more than two-fold, compared with untreated cells (Fig. 2). However, TNF- did not affect the caspase-3/7 activity of periosteal fibroblasts (Fig. 2).
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, one-hour PFF was applied to osteocytes, osteoblasts, and periosteal fibroblasts. No cells were removed by the application of PFF, as assessed by total DNA quantification (periosteal fibroblasts, static 21.4 ± 2.9 ng/mL, PFF 19.3 ± 2.0 ng/mL; osteoblasts, static 11.3 ± 1.2 ng/mL, PFF 10.4 ± 1.5 ng/mL; osteocytes, static 3.4 ± 1.0 ng/mL, PFF 4.2 ± 1.2 ng/mL; mean ± SEM of 6-8 experiments). Furthermore, PFF did not result in visible changes in cell shape or alignment in the direction of the flow.
One-hour PFF reduced caspase-3/7 activity in TNF--treated osteocytes by 25% compared with static cultures, at 24 hrs post-incubation without loading (Fig. 3a). However, PFF did not affect caspase-3/7 activity in osteoblasts or periosteal fibroblasts (Fig. 3a). Inhibition of apoptosis in osteocytes subjected to one-hour PFF was detectable 24 hrs post-incubation without loading, but not immediately after PFF treatment (data not shown). To study a possible interrelationship between enhanced NO production resulting from PFF treatment and apoptosis, we subjected cells to PFF in the presence of L-NAME. The addition of 1 mM L-NAME to the culture medium during exposure to PFF blocked the inhibitory effect of PFF on caspase-3/7 activity in TNF--treated osteocytes, but did not affect apoptosis in osteoblasts and periosteal fibroblasts (Fig. 3b).
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-treated osteocytes (Fig. 4a). PFF significantly increased NO production at 30, but not 10, min in osteocytes (Fig. 4a). When data were expressed as PFF-treated-over-control ratios, the increase (4.8-fold) in TNF--treated osteocytes was even more clear (Fig. 4b). Such a significant increase was not seen in osteoblasts (1.8-fold) or periosteal fibroblasts (2.0-fold) (Fig. 4b). The addition of 1 mM L-NAME during PFF inhibited PFF-mediated NO synthesis (data not shown), which has been described previously (Klein-Nulend et al., 1995a).
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS & METHODSRESULTSDISCUSSIONREFERENCES |
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increased apoptosis in osteocytes and osteoblasts, but not in periosteal fibroblasts, under static conditions representing a situation of no fluid shear stress along the cell membrane. We are the first to show that mechanical loading by pulsating fluid flow of physiological magnitude reduces TNF--induced apoptosis in osteocytes, but not in osteoblasts and periosteal fibroblasts. The reduction of apoptosis was not due to removal of pre-apoptotic osteocytes by shear stress, since there was no reduction of DNA content. Inhibition of NO synthesis by L-NAME prevented the PFF-mediated down-regulation of apoptosis in osteocytes, suggesting that PFF-mediated NO release by osteocytes is, at least in part, responsible for the loading-induced inhibition of osteocyte apoptosis. We conclude that, for terminally differentiated osteocytes, fluid shear stress seems to be an important survival factor, while no such sensitivity is expressed by the less mature osteoblasts and periosteal fibroblasts containing osteoprogenitors.
Others have described the inhibition of MLO-Y4 cell apoptosis by stretching (Plotkin et al., 2005), and of rat osteoblast apoptosis by steady fluid shear stress (Pavalko et al., 2003). However, we have subjected our cells to a pulsating fluid shear stress regime, which mimics the manner by which loading of whole bones is thought to be conveyed to the osteocytes in vivo (Weinbaum et al., 1994; Burger and Klein-Nulend, 1999). Earlier, we showed the inhibition of serum-starvation-induced apoptosis by mechanical stimulation (Bakker et al., 2004). During inflammation and orthodontic tooth movement, however, the cytokine TNF- is produced. We found that the inhibitory effect of PFF on TNF--induced osteocyte apoptosis was less pronounced than after serum starvation, suggesting that mechanical loading is less effective in inhibiting TNF--induced apoptosis.
TNF- stimulates apoptosis of murine osteoblasts and osteocytic MLO-Y4 cells (Ahuja et al., 2003). We observed that periosteal fibroblasts fail to undergo apoptosis in response to TNF- at 10 ng/mL. Higher concentrations of TNF- (20 ng/mL) stimulate apoptosis in human fibroblasts (Alikhani et al., 2004), suggesting that TNF- affects apoptosis of mature bone cells, such as osteocytes and osteoblasts, but that periosteal fibroblasts are less sensitive.
The signal transduction pathway leading from fluid shear stress to the inhibition of osteocyte apoptosis is currently unknown, but it is likely that NO is involved. Osteocytes rapidly (within minutes) produce low amounts of NO in response to shear stress (Klein-Nulend et al., 1995a). NO inhibits apoptosis in endothelial cells via the inhibition of caspase-3 (Haendeler et al., 1997). Here, we show that fluid shear stress also stimulates NO production in TNF--treated osteocytes, and that the inhibition of NO production by L-NAME prevented the PFF-mediated down-regulation of apoptosis in osteocytes. This suggests that NO is a mediator of mechanical effects in bone, leading to the inhibition of apoptosis.
Verification of our in vitro results is needed in human bone cells and, ultimately, in whole human bones, before definitive conclusions can be drawn, but a relationship between stress-related osteocyte survival and bone remodeling seems likely. Local fatigue damage of the extracellular matrix results in loss of osteocyte integrity and activates bone remodeling (Verborgt et al., 2000; Burr, 2002). Our results are compatible with this concept if we consider that fatigue damage will lead to reduced canalicular fluid flow (Prendergast and Huiskes, 1996). Reduced canalicular shear stress will promote osteocyte apoptosis, which will attract osteoclasts, thereby activating remodeling (Bronckers et al., 1996).
Our results might offer, at least in part, an explanation for the complex process of orthodontic tooth movement, in which many cells and cytokines are involved. Periodontal ligament cells are stretched or compressed (Davidovitch, 1991), and cytokines such as TNF- and IL-1 are produced (Uematsu et al., 1996). We suggest that, together with TNF- in the gingival sulcus, osteocyte apoptosis is caused on the pressure side by local stress shielding due to decreased functioning of the periodontal ligament (Uematsu et al., 1996; Melsen, 1999; Hamaya et al., 2002), which causes almost complete fluid stasis in the canaliculi of the osteocytes (Smit et al., 2002). Osteoclasts are then attracted by apoptotic osteocytes (Bronckers et al., 1996), resulting in bone resorption and remodeling. On the tension side, increased strain likely results in increased fluid flow, which stimulates osteocytes to produce NO, thereby maintaining osteocyte viability, and osteoblasts to produce new bone. In vivo observations by Hayashi et al.(2002) showed inhibition of orthodontic tooth movement in rats treated with the NO inhibitor L-NAME. This inhibition might be explained by decreased bone formation on the tension side.
In summary, TNF- induces apoptosis of mature bone cells, i.e., osteocytes and osteoblasts, but not immature bone cells of the periosteal fibroblast population. Fluid shear stress inhibits TNF--induced apoptosis specifically in osteocytes, but not in osteoblasts and periosteal fibroblasts. This inhibitory effect is, at least in part, mediated by NO. This suggests a regulatory role for osteocyte apoptosis in osteoclastic bone resorption during bone remodeling, such as occurs after the application of an orthodontic load.
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ACKNOWLEDGMENTS |
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Received August 2, 2005; Last revision June 15, 2006; Accepted June 21, 2006
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