Henry J. Donahue

Differential effect of steady versus oscillating flow on bone cells

Loading induced fluid flow has recently been proposed as an important biophysical signal in bone mechanotransduction. Fluid flow resulting from activities which load the skeleton such as standing, locomotion, or postural muscle activity are predicted to be dynamic in nature and include a relatively small static component. However, in vitro fluid flow experiments with bone cells to date have been conducted using steady or pulsing flow profiles only. In this study we exposed osteoblast-like hFOB 1.19 cells (immortalized human fetal osteoblasts) to precisely controlled dynamic fluid flow profiles of saline supplemented with 2% fetal bovine serum while monitoring intracellular calcium concentration with the fluorescent dye fura-2. Applied flows included steady flow resulting in a wall shear stress of 2 N m(-2), oscillating flow (+/-2 Nm(-2)), and pulsing flow (0 to 2 N m(-2)). The dynamic flows were applied with sinusoidal profiles of 0.5, 1.0, and 2.0 Hz. We found that oscillating flow was a much less potent stimulator of bone cells than either steady or pulsing flow. Furthermore, a decrease in responsiveness with increasing frequency was observed for the dynamic flows. In both cases a reduction in responsiveness coincides with a reduction in the net fluid transport of the flow profile. Thus. these findings support the hypothesis that the response of bone cells to fluid flow is dependent on chemotransport effects.

Substrate deformation levels associated with routine physical activity are less stimulatory to bone cells relative to loading-induced oscillatory fluid flow

Although it is well accepted that bone tissue metabolism is regulated by external mechanical loads, it remains unclear to what load-induced physical signals bone cells respond. In this study, a novel computer-controlled stretch device and parallel plate flow chamber were employed to investigate cytosolic calcium (Ca2+i) mobilization in response to a range of dynamic substrate strain levels (0.1-10 percent, 1 Hz) and oscillating fluid flow (2 N/m2, 1 Hz). In addition, we quantified the effect of dynamic substrate strain and oscillating fluid flow on the expression of mRNA for the bone matrix protein osteopontin (OPN). Our data demonstrate that continuum strain levels observed for routine physical activities (< 0.5 percent) do not induce Ca2+i responses in osteoblastic cells in vitro. However, there was a significant increase in the number of responding cells at larger strain levels. Moreover, we found no change in osteopontin mRNA level in response to 0.5 percent strain at 1 Hz. In contrast, oscillating fluid flow predicted to occur in the lacunar-canalicular system due to routine physical activities (2 N/m2, 1 Hz) caused significant increases in both Ca2+i and OPN mRNA. These data suggest that, relative to fluid flow, substrate deformation may play less of a role in bone cell mechanotransduction associated with bone adaptation to routine loads.

Functional gap junctions between osteocytic and osteoblastic cells.

Morphological evidence shows that osteocytes, bone cells that exist enclosed within bone matrix, are connected to one another and to surface osteoblasts via gap junctions; however, it is unknown whether these gap junctions are functional. Using a newly established murine osteocytic cell line MLO‐Y4, we have examined functional gap junctional intercellular communication (GJIC) between osteocytic cells and between osteocytic and osteoblastic cells. In our hands, MLO‐Y4 cells express phenotypic characteristics of osteocytic cells including a stellate morphology, low alkaline phosphatase activity, and increased osteocalcin messenger RNA (mRNA) compared with osteoblastic cells. Northern and Western blot analysis revealed that MLO‐Y4 cells express abundant connexin 43 (Cx43) mRNA and protein, respectively. Lucifer yellow dye transferred from injected to adjacent cells suggesting that osteocytic cells were functionally coupled via gap junctions. Functional GJIC between osteocytic and osteoblastic (MC3T3‐E1) cells was determined by monitoring the passage of calcein dye between the two cell types using a double labeling technique. The ability of bone cells to communicate a mechanical signal was assessed by mechanically deforming the cell membrane of single MLO‐Y4 cells, cocultured with MC3T3‐E1 cells. Deformation induced calcium signals in MLO‐Y4 cells and those elicited in neighboring MC3T3‐E1 cells were monitored with the calcium sensitive dye Fura‐2. Our results suggest that osteocytic MLO‐Y4 cells express functional gap junctions most likely composed of Cx43. Furthermore, osteocytic and osteoblastic cells are functionally coupled to one another via gap junctions as shown by the ability of calcein to pass between cells and the ability of cells to communicate a mechanically induced calcium response. (J Bone Miner Res 2000;15:209–217)

Fluid Shear-Induced ATP Secretion Mediates Prostaglandin Release in MC3T3-E1 Osteoblasts

ATP is rapidly released from osteoblasts in response to mechanical load. We examined the mechanisms involved in this release and established that shear‐induced ATP release was mediated through vesicular fusion and was dependent on Ca2+ entry into the cell through L‐type voltage‐sensitive Ca2+ channels. Degradation of secreted ATP by apyrase prevented shear‐induced PGE2 release.

Oscillating fluid flow activation of gap junction hemichannels induces ATP release from MLO-Y4 osteocytes.

Mechanical loads are required for optimal bone mass. One mechanism whereby mechanical loads are transduced into localized cellular signals is strain‐induced fluid flow through lacunae and canaliculi of bone. Gap junctions (GJs) between osteocytes and osteoblasts provides a mechanism whereby flow‐induced signals are detected by osteocytes and transduced to osteoblasts. We have demonstrated the importance of GJ and gap junctional intercellular communication (GJIC) in intracellular calcium and prostaglandin E2 (PGE2) increases in response to flow. Unapposed connexons, or hemichannels, are themselves functional and may constitute a novel mechanotransduction mechanism. Using MC3T3‐E1 osteoblasts and MLO‐Y4 osteocytes, we examined the time course and mechanism of hemichannel activation in response to fluid flow, the composition of the hemichannels, and the role of hemichannels in flow‐induced ATP release. We demonstrate that fluid flow activates hemichannels in MLO‐Y4, but not MC3T3‐E1, through a mechanism involving protein kinase C, which induces ATP and PGE2 release.. J. Cell. Physiol. 212: 207–214, 2007.

Human foetal osteoblastic cell response to polymer-demixed nanotopographic interfaces

Nanoscale cell–substratum interactions are of significant interest in various biomedical applications. We investigated human foetal osteoblastic cell response to randomly distributed nanoisland topography with varying heights (11, 38 and 85 nm) produced by a polystyrene (PS)/polybromostyrene polymer-demixing technique. Cells displayed island-conforming lamellipodia spreading, and filopodia projections appeared to play a role in sensing the nanotopography. Cells cultured on 11 nm high islands displayed significantly enhanced cell spreading and larger cell dimensions than cells on larger nanoislands or flat PS control, on which cells often displayed a stellate shape. Development of signal transmitting structures such as focal adhesive vinculin protein and cytoskeletal actin stress fibres was more pronounced, as was their colocalization, in cells cultured on smaller nanoisland surfaces. Cell adhesion and proliferation were greater with decreasing island height. Alkaline phosphatase (AP) activity, an early stage marker of bone cell differentiation, also exhibited nanotopography dependence, i.e. higher AP activity on 11 nm islands compared with that on larger islands or flat PS. Therefore, randomly distributed island topography with varying nanoscale heights not only affect adhesion-related cell behaviour but also bone cell phenotype. Our results suggest that modulation of nanoscale topography may be exploited to control cell function at cell–biomaterial interfaces.

Surface energy effects on osteoblast spatial growth and mineralization

While short-term surface energy effects on cell adhesion are relatively well known, little is revealed as regards its later stage effects on cell behavior. We examined surface energy effects on osteoblastic cell growth and mineralization by using human fetal osteoblastic (hFOB) cells cultured on plasma-treated quartz (contact angle, theta=0 degrees) and octadecyltrichlorosilane (OTS)-treated quartz (theta=113 degrees). hFOB cells formed a homogeneous cell layer on plasma-treated quartz, while those cultured on OTS-treated quartz produced randomly distributed clump-like structures that were filled with cells (confirmed by confocal microscopy). Mineral deposition by hFOB cells was spatially homogeneous when cultured on hydrophilic surfaces. Furthermore, cells on hydrophilic surfaces exhibited increased mineralized area as well as enhanced mineral-to-matrix ratio (assessed by Fourier transform infrared spectroscopy), relative to cells on hydrophobic surfaces. Experiments using other types of osteoblast-like cells (MC3T3-E1, MG63, and SAOS-2) revealed more or less similar effects in spatial growth morphology. It was concluded that hydrophilic surfaces induce homogeneous spatial osteoblastic cell growth and mineral deposition and enhance the quantity (e.g., area) and quality (e.g., mineral-to-matrix ratio) of mineralization relative to hydrophobic surfaces. Our data suggest that surface energy effects on osteoblastic cell differentiation, especially mineralization, may be correlated with surface energy dependent changes in spatial cell growth.

Effect of COX-2-Specific Inhibition on Fracture-Healing in the Rat Femur

BACKGROUND Nonsteroidal anti-inflammatory medications have been shown to delay fracture-healing. COX-2-specific inhibitors such as celecoxib have recently been approved for human use. Our goal was to determine, mechanically, histologically, morphologically, and radiographically, whether COX-2-specific inhibition affects bone-healing. METHODS A nondisplaced unilateral fracture was created in the right femur of fifty-seven adult male rats. Rats were given no drug, indomethacin (1 mg/kg/day), or celecoxib (3 mg/kg/day) daily, starting on postoperative day 1. Fractures were analyzed at four, eight, and twelve weeks after creation of the fracture. Callus and bridging bone formation was assessed radiographically. The amounts of fibrous tissue, cartilage, woven bone, and mature bone formation were determined histologically. Morphological changes were assessed to determine fibrous healing, callus formation, and bone-remodeling. Callus strength and stiffness were assessed biomechanically with three-point bending tests. RESULTS At four weeks, only the indomethacin group showed biomechanical and radiographic evidence of delayed healing. Although femora from rats treated with celecoxib appeared to have more fibrous tissue than those from untreated rats at four and eight weeks, radiographic signs of callus formation, mechanical strength, and stiffness did not differ significantly between the groups. By twelve weeks, there were no significant differences among the three groups. CONCLUSIONS Postoperative administration of celecoxib, a COX-2-specific inhibitor, did not delay healing as seen at twelve weeks following fracture in adult rat femora. At four and eight weeks, fibrous healing predominated in the celecoxib group as compared with the findings in the untreated group; however, mechanical strength and radiographic signs of healing were not significantly inhibited. CLINICAL RELEVANCE Many orthopaedists rely on narcotic analgesia for postfracture and postoperative pain, despite deleterious side effects and morbidity. Traditional nonsteroidal anti-inflammatory medications have been shown to delay fracture union. This effect may be smaller with COX-2-specific inhibitors.

Gap Junctional Intercellular Communication Contributes to Hormonal Responsiveness in Osteoblastic Networks

To evaluate whether intercellular coupling via connexin43 gap junction channels modulates hormonal responsiveness of cells in contact, we have created osteoblastic cell lines deficient in connexin43. Osteoblastic ROS 17/2.8 cells were transfected with a plasmid containing an antisense cDNA construct to rat connexin43. Control transfection did not alter cell-to-cell coupling nor connexin43 mRNA or protein expression relative to nontransfected ROS 17/2.8 cells. In contrast, stable transfection with an antisense connexin43 cDNA resulted in two clones, RCx4 and RCx16, which displayed significant decreases in connexin43 mRNA and protein expression and were dramatically deficient in cell-to-cell coupling. Phenotypically, all transfectants retained osteoblastic characteristics. However, cells rendered connexin43-deficient through antisense transfection displayed a dramatic attenuation in the cAMP response to parathyroid hormone. Alterations in hormonal responses were not due to changes in parathyroid hormone receptor number or binding kinetics nor to alterations in adenylyl cyclase activity. These results indicate that gap junctions may be required for mediating hormonal signals. Furthermore, these experiments support a regulatory role for connexin43-mediated intercellular communication in the modulation of hormonal responses within elaborately networked bone cells.

From streaming-potentials to shear stress: 25 years of bone cell mechanotransduction.

Mechanical loads are vital regulators of skeletal mass and architecture as evidenced by the increase in bone formation following the addition of exogenous loads and loss of bone mass following their removal. While our understanding of the molecular mechanisms by which bone cells perceive changes in their mechanical environment has increased rapidly in recent years, much remains to be learned. Here, we outline the effects of interstitial fluid flow, a potent biophysical signal induced by the deformation of skeletal tissue in response to applied loads, on bone cell behavior. We focus on the molecular mechanisms by which bone cells are hypothesized to perceive interstitial fluid flow, the cell signaling cascades activated by fluid flow, and the use of this signal in tissue engineering protocols.