Jenneke Klein-Nulend

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)

Adipose tissue-derived mesenchymal stem cell yield and growth characteristics are affected by the tissue-harvesting procedure

BACKGROUND Adipose tissue contains a stromal vascular fraction that can be easily isolated and provides a rich source of adipose tissue-derived mesenchymal stem cells (ASC). These ASC are a potential source of cells for tissue engineering. We studied whether the yield and growth characteristics of ASC were affected by the type of surgical procedure used for adipose tissue harvesting, i.e. resection, tumescent liposuction and ultrasound-assisted liposuction. METHODS Frequencies of ASC in the stromal vascular fraction were assessed in limiting dilution assays. The phenotypical marker profile of ASC was determined, using flow cytometry, and growth kinetics were investigated in culture. ASC were cultured under chondrogenic and osteogenic conditions to confirm their differentiation potential. RESULTS The number of viable cells in the stromal vascular fraction was affected by neither the type of surgical procedure nor the anatomical site of the body from where the adipose tissue was harvested. After all three surgical procedures, cultured ASC did express a CD34+ CD31- CD105+ CD166+ CD45- CD90+ ASC phenotype. However, ultrasound-assisted liposuction resulted in a lower frequency of proliferating ASC, as well as a longer population doubling time of ASC, compared with resection. ASC demonstrated chondrogenic and osteogenic differentiation potential. DISCUSSION We conclude that yield and growth characteristics of ASC are affected by the type of surgical procedure used for adipose tissue harvesting. Resection and tumescent liposuction seem to be preferable above ultrasound-assisted liposuction for tissue-engineering purposes.

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.

Mechanosensation and transduction in osteocytes

The human skeleton is a miracle of engineering, combining both toughness and light weight. It does so because bones possess cellular mechanisms wherein external mechanical loads are sensed. These mechanical loads are transformed into biological signals, which ultimately direct bone formation and/or bone resorption. Osteocytes, since they are ubiquitous in the mineralized matrix, are the cells that sense mechanical loads and transduce the mechanical signals into a chemical response. The osteocytes then release signaling molecules, which orchestrate the recruitment and activity of osteoblasts or osteoclasts, resulting in the adaptation of bone mass and structure. In this review, we highlight current insights in bone adaptation to external mechanical loading, with an emphasis on how a mechanical load placed on whole bones is translated and amplified into a mechanical signal that is subsequently sensed by the osteocytes.

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.

Mechanical stimulation by intermittent hydrostatic compression promotes bone-specific gene expression in vitro

In a previous study of the cellular mechanism underlaying Wolffs law we showed that mechanical stimulation by intermittent hydrostatic compression (IHC) increases bone formation in cultured fetal mouse calvariae compared to non-stimulated cultures. To test whether mechanical stimuli may modulate bone-specific gene expression, we studied the effect of IHC on alkaline phosphatase (AP) expression and enzyme activity as well as collagen and actin mRNA levels in neonatal mouse calvariae and calvarial bone cells. Two cell populations, one resembling osteoprogenitor (OPR) cells and another resembling osteoblasts (OB) were obtained from calvariae by sequential digestion. IHC was applied by intermittently (0.3 Hz) compressing the gas- phase of a closed culture chamber (peak stress 13kPa, peak stress rate 32.5 kPas-1). In control cultures of calvariae as well as OB and OPR cells, AP activity and AP-, collagen-, and actin-mRNA levels all decreased after one or more days, with the exception of OPR cell collagen expression which increased during culture. IHC treatment upregulated AP, collagen and actin expression and AP activity in calvariae and OB cells, but decreased collagen expression in OPR cells. These results suggest that treatment with IHC promotes the osteoblastic phenotype in bone organ cultures and in osteoblasts. Osteoprogenitor cells seem to react somewhat differently to mechanical stress than osteoblasts. The loss of bone-specific gene expression under control culture conditions, in the absence of mechanical stimuli, suggests that the mechanical environment is important in maintaining the differentiated phenotype of bone cells, and that IHC treatment partially restores this environment in bone cell- and organ cultures.