Elisabeth H. Burger

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)

Mechanotransduction in bone—role of the lacuno-canalicular network

The capacity of bone tissue to alter its mass and structure in response to mechanical demands has long been recognized but the cellular mechanisms involved remained poorly understood. Over the last several years significant progress has been made in this field, which we will try to summarize. These studies emphasize the role of osteocytes as the professional mechanosensory cells of bone, and the lacuno‐canalicular porosity as the structure that mediates mechanosensing. Strain‐derived flow of interstitial fluid through this porosity seems to mechanically activate the osteocytes, as well as ensuring transport of cell signaling molecules and nutrients and waste products. This concept allows an explanation of local bone gain and loss, as well as remodeling in response to fatigue damage, as processes supervised by mechanosensitive osteocytes.—Burger, E. H., Klein‐Nulend, J. Mechanotransduction in bone—role of the lacuno‐canalicular network. FASEB J. 13 (Suppl.), S101–S112 (1999)

The production of nitric oxide and prostaglandin E2 by primary bone cells is shear stress dependent

Loading-induced flow of interstitial fluid through the lacuno-canalicular network is a likely signal for bone cell adaptive responses. However, the nature of the stimulus that activates the cell is debated. Candidate stimuli include wall shear stress, streaming potentials, and chemotransport. We have addressed the nature of the flow-derived cell stimulus by comparing variations in fluid transport with variations in wall shear stress, using nitric oxide (NO) and prostaglandin E(2) (PGE(2)) production as a parameter of bone cell activation. Adult mouse long bone cell cultures were treated for 15min with or without pulsating fluid flow using the following regimes: Low PFF, mean flow rate 0.20 cm(3)/s, 3 Hz, shear stress 0.4+/-0.12 Pa; Medium PFF, 0.33 cm(3)/s, 5 Hz, 0.6+/-0.27 Pa; and High PFF, 0.63 cm(3)/s, 9Hz, 1.2+/-0.37 Pa. In some Low PFF experiments, 2.8% neutral dextran (mol. wt. 4.98x10(4)) was added to the flow medium to increase the viscosity, thereby increasing the wall shear stress 3-fold to a level similar of the High PFF stimulus, but without affecting streaming potentials or chemotransport. NO and PGE(2) production were stimulated by Low, Medium, and High PFF in a dose-dependent manner. Application of Low PFF using dextran-supplemented medium, enhanced both the NO and PGE(2) response by 3-fold, to a level mimicking the response to High PFF at normal viscosity. These results show that the production of NO and PGE(2) by bone cells can be enhanced in a dose-dependent manner by fluid flow of increasing wall shear stress. Therefore, the stimulus leading to NO and PGE(2) production is the flow-derived shear stress, and not streaming potentials or chemotransport.

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.

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 (Wolffs 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.

Mechanotransduction of bone cells in vitro: mechanobiology of bone tissue.

Mechanical force plays an important role in the regulation of bone remodelling in intact bone and bone repair. In vitro, bone cells demonstrate a high responsiveness to mechanical stimuli. Much debate exists regarding the critical components in the load profile and whether different components, such as fluid shear, tension or compression, can influence cells in differing ways. During dynamic loading of intact bone, fluid is pressed through the osteocyte canaliculi, and it has been demonstrated that fluid shear stress stimulates osteocytes to produce signalling molecules. It is less clear how mechanical loads act on mature osteoblasts present on the surface of cancellous or trabecular bone. Although tissue strain and fluid shear stress both cause cell deformation, these stimuli could excite different signalling pathways. This is confirmed by our experimental findings, in human bone cells, that strain applied through the substrate and fluid flow stimulate the release of signalling molecules to varying extents. Nitric oxide and prostaglandin E2 values increased by between two- and nine-fold after treatment with pulsating fluid flow (0.6±0.3 Pa). Cyclic strain (1000 μstrain) stimulated the release of nitric oxide two-fold, but had no effect on prostaglandin E2. Furthermore, substrate strains enhanced the bone matrix protein collagen I two-fold, whereas fluid shear caused a 50% reduction in collagen I. The relevance of these variations is discussed in relation to bone growth and remodelling. In applications such as tissue engineering, both stimuli offer possibilities for enhancing bone cell growth in vitro.

Demonstration of tartrate-resistant acid phosphatase in un-decalcified, glycolmethacrylate-embedded mouse bone: a possible marker for (pre)osteoclast identification.

Fixed, undecalcified mouse long bones were embedded in glycol methacrylate (GMA), sectioned, and incubated for acid phosphatase in the presence or absence of tartrate, to investigate the feasibility of tartrate-resistant acid phosphatase as a histochemical marker for osteoclast identification. Naphthol AS-BI phosphate was used as the substrate and hexazonium pararosanaline as coupler. Cytocentrifuge preparations of mouse, rat, and quail bone marrow or frozen and GMA sections of mouse splenic tissue were used as controls to specify acid phosphatase activity. After adequate fixation, acid phosphatase activity sensitive to tartrate inhibition (TS-AP) was demonstrated in macrophages from spleen, bone marrow, and loose connective tissue surrounding bone rudiments. Acid phosphatase activity resistant to tartrate inhibition (TR-AP), was detected in multi-nuclear osteoclasts and in some mononuclear cells from bone marrow and periosteum. In cytocentrifuge preparations and frozen sections of mouse spleen, TR-AP was demonstrated after simultaneous incubation with substrate and tartrate. In GMA sections, however, TR-AP could only be demonstrated after pre-incubation with tartrate before application of substrate. We suggest that histochemical demonstration of TR-AP versus TS-AP on GMA-embedded bone sections by means of a pre-incubation method can be used as an identification marker of (pre)osteoclasts. Plastic embedding is recommended for its excellent preservation of morphology and enzyme activity.

Signal transduction pathways involved in fluid flow-induced PGE2 production by cultured osteocytes

To maintain its structural competence, the skeleton adapts to changes in its mechanical environment. Osteocytes are generally considered the bone mechanosensory cells that translate mechanical signals into biochemical, bone metabolism-regulating stimuli necessary for the adaptive process. Prostaglandins are an important part of this mechanobiochemical signaling. We investigated the signal transduction pathways in osteocytes through which mechanical stress generates an acute release of prostaglandin E2 (PGE2). Isolated chicken osteocytes were subjected to 10 min of pulsating fluid flow (PFF; 0.7 +/- 0.03 Pa at 5 Hz), and PGE2 release was measured. Blockers of Ca2+ entry into the cell or Ca2+ release from internal stores markedly inhibited the PFF-induced PGE2 release, as did disruption of the actin cytoskeleton by cytochalasin B. Specific inhibitors of Ca2+-activated phospholipase C, protein kinase C, and phospholipase A2 also decreased PFF-induced PGE2 release. These results are consistent with the hypothesis that PFF raises intracellular Ca2+ by an enhanced entry through mechanosensitive ion channels in combination with Ca2+- and inositol trisphosphate (the product of phospholipase C)-induced Ca2+ release from intracellular stores. Ca2+ and protein kinase C then stimulate phospholipase A2 activity, arachidonic acid production, and ultimately PGE2 release.To maintain its structural competence, the skeleton adapts to changes in its mechanical environment. Osteocytes are generally considered the bone mechanosensory cells that translate mechanical signals into biochemical, bone metabolism-regulating stimuli necessary for the adaptive process. Prostaglandins are an important part of this mechanobiochemical signaling. We investigated the signal transduction pathways in osteocytes through which mechanical stress generates an acute release of prostaglandin E2(PGE2). Isolated chicken osteocytes were subjected to 10 min of pulsating fluid flow (PFF; 0.7 ± 0.03 Pa at 5 Hz), and PGE2release was measured. Blockers of Ca2+ entry into the cell or Ca2+ release from internal stores markedly inhibited the PFF-induced PGE2 release, as did disruption of the actin cytoskeleton by cytochalasin B. Specific inhibitors of Ca2+-activated phospholipase C, protein kinase C, and phospholipase A2 also decreased PFF-induced PGE2 release. These results are consistent with the hypothesis that PFF raises intracellular Ca2+ by an enhanced entry through mechanosensitive ion channels in combination with Ca2+- and inositol trisphosphate (the product of phospholipase C)-induced Ca2+ release from intracellular stores. Ca2+ and protein kinase C then stimulate phospholipase A2activity, arachidonic acid production, and ultimately PGE2 release.

Is BMU‐Coupling a Strain‐Regulated Phenomenon? A Finite Element Analysis

Histologically, two types of bone reconstruction are distinguished: modeling and remodeling. Modeling changes the amount of bone and determines its geometrical form in relation to the prevailing mechanical loads and their resulting deformation (strain). Remodeling renews existing bone in a sequence of resorption and formation. However, in both processes the cells responsible for resorption and formation are the same: osteoclasts and osteoblasts. We studied if there is a relation between the activity of these cells and the deformation of the local bone tissue during remodeling. Two finite element models were built on a microscopic, supracellular level: (1) a secondary osteon in cortical bone and (2) a Howships lacuna in a trabecula. Both models were loaded in the “natural,” that is, longitudinal direction. Equivalent strains were determined as a measure for the deformation of the bone tissue. In the first model, the strain field around the osteon showed a region of decreased deformation in front of the tunnel, just where osteoclasts excavate cortical bone tissue. Behind the cutting cone, elevated strain levels appear in the tunnel wall at locations where osteoblasts are active. The second model showed that a local excavation of a loaded trabecula leads to higher strains at the bottom of the lacuna, where resorption is stopped and osteoblasts are recruited to refill the gap. However, in the direction of loading reduced strain levels appear, just where resorption continues to proceed along the trabecular surface. We conclude that at the tissue level, strain distributions occur during the remodeling process that show a relationship to the activity of osteoblasts and osteoclasts. This suggests that BMU coupling, that is, the subsequent activation of osteoclasts and osteoblasts during remodeling, is a strain‐regulated phenomenon. (J Bone Miner Res 2000;15: 301–307)

The effect of cage stiffness on the rate of lumbar interbody fusion: an in vivo model using poly(l-lactic Acid) and titanium cages.

Study Design. A goat interbody fusion model using poly-(L-lactic acid) and titanium cages was designed to evaluate the effect of cage stiffness on lumbar interbody fusion. Objective. To investigate the effect of cage stiffness on the rate of interbody fusion. Summary of Background Data. Various types of cages considerably exceed the stiffness of vertebral bone, which ultimately may lead to postoperative complications. To avoid these complications, poly-(L-lactic acid) cages with limited stiffness have been designed. The mechanical integrity of the cages remains intact for at least 6 months. Methods. Interbody fusions were performed at L3–L4 of 15 Dutch milk goats, and one of three cages was randomly implanted: 1) a titanium cage (n = 3), 2) a stiff poly-(L-lactic acid) cage (n = 6), or 3) a flexible poly-(L-lactic acid) cage (n = 6). Interbody fusion was assessed radiographically by three independent observers 3 and 6 months after surgery. Results. At 3 months, all the poly-(L-lactic acid) specimens showed ingrowth of new bone, but with radiolucency in the fusion mass. At 6 months, solid arthrodesis was observed in four of six poly-(L-lactic acid) specimens, advanced ingrowth in one specimen, and infection in one specimen. Titanium cages showed ingrowth of bone, but with radiolucency in the fusion mass. Interbody fusion using poly-(L-lactic acid) cages showed a significantly higher rate statistically (P = 0.016) and more complete fusion than titanium cages of the same design. Conclusions. The reduced stiffness of poly-(L-lactic acid) cages showed enhanced interbody fusion, as compared with titanium cages after 6 months. Bioabsorbable poly-(L-lactic acid) cages thus may be a viable alternative to current interbody cage devices, thereby avoiding the concomitant problems related to their excessive stiffness. However, the bioabsorbability of the poly-(L-lactic acid) cages awaits investigation in a long-term study currently underway.