Biomedical subjects

Christopher R Jacobs

Publications and source records attributed to Christopher R Jacobs.

15 recordsLinked to original sources

PubMed2006

Mechanically induced intracellular calcium waves in osteoblasts demonstrate calcium fingerprints in bone cell mechanotransduction.

An early response to mechanical stimulation of bone cells in vitro is an increase in intracellular calcium concentration ([Ca (2+)](i)). This study analyzed the [Ca (2+)](i) wave area, magnitude, duration, rise time, fall time, and time to onset in individual osteoblasts for two identical bouts of mechanical stimulation separated by a 30-min rest period. The area under the [Ca (2+)](i) wave increased in the second loading bout compared to the first. This suggests that rest periods may potentiate mechanically induced intracellular calcium signals. Furthermore, many of the [Ca (2+)](i) wave parameters were strongly, positively correlated between the two bouts of mechanical stimulation. For example, in individual primary osteoblasts, if a cell had a large [Ca (2+)](i) wave area in the first bout it was likely to have a large [Ca (2+)](i) wave area in the second bout (r (2) = 0.933). These findings support the idea that individual bone cells have "calcium fingerprints" (i.e., a unique [Ca (2+)](i) wave profile that is reproducible for repeated exposure to a given stimulus).

Animals↗

PubMed2006

Oscillatory fluid flow-induced shear stress decreases osteoclastogenesis through RANKL and OPG signaling.

Physical activity creates deformation in bone that leads to localized pressure gradients that drive interstitial fluid flow. Due to the cyclic nature of the applied load, this flow is oscillatory by nature. Oscillatory fluid flow (OFF) may lead to positive bone remodeling through effects on both osteoblasts and osteoclasts but its effect on osteoclastogenesis is poorly understood. In this study, the effects of OFF on expression of receptor activator of NF-kappaB ligand (RANKL) and osteoprotegerin (OPG), two important regulators of osteoclast differentiation, were investigated. In addition, its effect on osteoclast formation was quantified. ST-2 murine bone marrow stromal cells were plated on glass slides and cultured with 1,25-dihydroxyvitamin D(3) to express RANKL. Cells were exposed to various durations of OFF resulting in a peak shear stress of 1 Pa. Time course and dose-response studies were performed and real-time RT-PCR was used to quantify levels of RANKL, OPG mRNA. ST-2 cells exposed to OFF were also co-cultured with RAW 264.7 monocytes and osteoclast number quantified. Decrease in RANKL/OPG was maximal immediately after end of flow and there existed a significant increase in OPG and decrease in RANKL with increasing load duration of up to 2 h. OFF resulted in a decrease in osteoclast formation by ST-2 cells co-cultured with RAW 264.7 cells compared to co-culture of control (non-loaded) ST-2 cells with RAW 264.7 cells. These results suggest that indeed OFF is a potent regulator of bone remodeling, and that shift towards positive bone remodeling mediated by loading-induced fluid flow may occur via suppression of the formation of osteoclasts.

Animals↗

PubMed2005

Effects of short-term recovery periods on fluid-induced signaling in osteoblastic cells.

It is well known that cyclic mechanical loading can produce an anabolic response in bone. In vivo studies have shown that the insertion of short-term recovery periods (10-15 s) into mechanical loading profiles led to an increased osteogenic response compared to continuous cyclic loading of bone. Although this is suggestive of temporal processing at the bone cell level, there is little evidence to support such a hypothesis. Therefore, the current study investigated the cellular mechanism of bone's response to rest inserted vs. continuous mechanical loading. Cell responses to rest inserted mechanical loading were quantified by applying oscillatory fluid flow (OFF) to osteoblastic cells and quantifying real-time intracellular calcium [Ca2+]i, prostaglandin E2 (PGE2) release, and osteopontin (OPN) mRNA levels. Cells were exposed to OFF (1 Hz) at shear stresses of 1 and 2 Pa with rest periods of 5, 10, and 15s inserted every 10 loading cycles. The insertion of 10 and 15s rest periods into the flow profile resulted in multiple [Ca2+]i responses by individual cells, increased [Ca2+]i response magnitudes, and increased overall percent of cells responding compared to continuously loaded control groups. We determined the source of the multiple calcium responses to be from intracellular stores. In addition, rest inserted OFF led to similar levels of PGE2 release and increased levels of relative OPN mRNA compared to cells exposed to continuous OFF. Our study suggests that the cellular mechanism of bone adaptation to rest inserted mechanical loading may involve modulation of intracellular levels of calcium (frequency, magnitude, percent of cells responding).

Adaptation, Physiological↗

PubMed2004

Capsaicin-sensitive sensory neurons contribute to the maintenance of trabecular bone integrity.

UNLABELLED: This investigation used capsaicin to selectively lesion unmyelinated sensory neurons in rats. Neuronal lesioning induced a loss of trabecular integrity, reduced bone mass and strength, and depleted neuropeptides in nerve and bone. These data suggest that capsaicin-sensitive sensory nerves contribute to trabecular bone integrity. INTRODUCTION: Familial dysautomia is an autosomal recessive disease in which patients suffer from unmyelinated sensory neuron loss, reduced BMD, and frequent fractures. It has been proposed that the loss of neurotransmitters synthesized by unmyelinated neurons adversely affects bone integrity in this hereditary syndrome. The purpose of this study was to determine whether small sensory neurons are required for the maintenance of bone integrity in rats. MATERIALS AND METHODS: Ten-month-old male Sprague-Dawley rats were treated with either capsaicin or vehicle. In vivo DXA scanning and micro CT scanning, and histomorphometry were used to evaluate BMD, structure, and cellular activity. Bone strength was measured in distal femoral sections. Body weight and gastrocnemius/soleus weights were measured and spontaneous locomotor activity was monitored. Peroneal nerve morphometry was evaluated using light and electron microscopy. Substance P and calcitonin gene-related peptide (CGRP) content in the sciatic nerve and proximal tibia were determined by enzyme immunoassay (EIA). Substance P signaling was measured using a sciatic nerve stimulation extravasation assay. RESULTS: Four weeks after capsaicin treatment, there was a loss of BMD in the metaphyses of the tibia and femur. In the proximal tibia, the osteoclast number and surface increased, osteoblast activity and bone formation were impaired, and trabecular bone volume and connectivity were diminished. There was also a loss of bone strength in the distal femur. No changes occurred in body weight, 24-h grid-crossing activity, weight bearing, or muscle mass after capsaicin treatment, indicating that skeletal unloading did not contribute to the loss of bone integrity. Capsaicin treatment destroyed 57% of the unmyelinated sensory axons, reduced the substance P and CGRP content in the sciatic nerve and proximal tibia, and inhibited neurogenic extravasation. CONCLUSION: These results support the hypothesis that capsaicin-sensitive sensory neurons contribute to the maintenance of trabecular bone integrity. Capsaicin-sensitive neurons have efferent functions in the tissues they innervate, effects mediated by transmitters released from the peripheral nerve terminals. We postulate that the deleterious effects of capsaicin treatment on trabecular bone are mediated by reductions in local neurotransmitter content and release.

Absorptiometry, Photon↗

PubMed2004

The sensitivity of tibiofemoral contact pressure to the size and shape of the lateral and medial menisci.

In an effort to prevent degeneration of articular cartilage associated with meniscectomies, both meniscal allografts and synthetic replacements have been studied. A number of biomechanical criteria may be important for a meniscal replacement to restore normal tibiofemoral contact pressure in the knee joint and hence be clinically successful. One of these criteria is geometric similarity. The objectives of the current study were to: determine the sensitivity of the contact variables of the tibial plateau to the transverse depth and width of both the lateral and medial menisci; determine the sensitivity of the contact variables of the tibial plateau to the cross-sectional width and height of the lateral and medial menisci; and determine the tolerances on each of the four parameters for both menisci. To satisfy these objectives, a previously developed finite element model of the tibiofemoral joint was used to compute the contact pressure distribution on the tibial plateau. The effect of the above-mentioned geometric parameters on the contact behavior was studied by perturbing the finite element model. Results showed that the contact variables are similarly sensitive to both the transverse and cross-sectional parameters of the menisci. Additionally the medial meniscal parameters have a greater effect on the contact variables than do the lateral meniscal parameters. Finally, less than a 0.5 mm change in the medial meniscal height and greater than a 1 mm change in the lateral meniscal height could be tolerated before the relative difference in the contact variables from those for the original geometry exceeded 10%. Thus in the design or selection of meniscal replacements, each of the four parameters should be measured when sizing a replacement tissue. Also tighter tolerances should be placed on the medial meniscal parameters compared to the lateral meniscal parameters.

Adult↗

PubMed2004

Oscillatory fluid flow affects human marrow stromal cell proliferation and differentiation.

Mechanical loading is an important regulator of bone formation and bone loss. Decreased osteoblast number and function are important cellular mechanisms by which mechanical disuse leads to decreased bone formation. Decreased osteoblast number may be a result of decreased osteoprogenitor proliferation, differentiation, or both. However, the effects of cellular level physical signals on osteoprogenitors are not well understood. In this study, we examined the effects of loading induced oscillatory fluid flow (OFF), a potent regulator of osteoblastic cell function, on marrow stromal cells (MSCs). MSCs subjected to OFF exhibited increased intracellular Ca2+ mobilization. In addition, MSCs exhibited increased proliferation and increased mRNA levels for osteopontin and osteocalcin genes. Collagen I and core binding factor 1 mRNA levels did not change. MSCs subjected to OFF also exhibited decreased alkaline phosphatase activity. These results suggest that MSCs are mechanosensitive and that Ca2+ may play a role in the signaling pathway.

Alkaline Phosphatase↗

PubMed2004

Substance P signaling contributes to the vascular and nociceptive abnormalities observed in a tibial fracture rat model of complex regional pain syndrome type I.

Wrist and ankle fractures are the most frequent causes of complex regional pain syndrome (CRPS type I). The current study examined the temporal development of vascular, nociceptive and bony changes after distal tibial fracture in rats and compared these changes to those observed after cast immobilization in intact normal rats. After baseline testing the right distal tibial was fractured and the hindlimb casted. A control group was simply casted without fracturing the tibia. After 4 weeks the casts were removed and the rats retested. Subsequent testing was performed at 6, 8, 10, 16, and 20 weeks after onset of treatment. Distal tibial fracture or cast immobilization alone generated chronic hindlimb warmth, edema, spontaneous protein extravasation, allodynia, and periarticular osteoporosis, changes resembling those observed in CRPS. Hindlimb warmth and allodynia resolved much more quickly after cast immobilization than after fracture. Previously we observed that the substance P receptor (NK(1)) antagonist LY303870 reversed vascular and nociceptive changes in a sciatic section rat model of CRPS type II. Postulating that facilitated substance P signaling may also contribute to the vascular and nociceptive abnormalities observed after tibial fracture or cast immobilization, we attempted to reverse these changes with LY303870. Hindpaw warmth, spontaneous extravasation, edema, and allodynia were inhibited by LY303870. Collectively, these data support the hypotheses that the distal tibial fracture model simulates CRPS, immobilization alone can generate a syndrome resembling CRPS, and substance P signaling contributes to the vascular and nociceptive changes observed in these models.

Animals↗

PubMed2004

Cycle number and waveform of fluid flow affect bovine articular chondrocytes.

Many studies have shown that a loading-induced (bio)physical signal regulates chondrocyte behavior. In a recent study our group has demonstrated the shear stress level- and frequency-dependent effect of sinusoidal oscillatory fluid flow on bovine articular chondrocyte (BAC) cytosolic calcium concentration ([Ca(2+)](i)), neglecting the fact that chondrocytes are not likely to see these ideal waveform in vivo or in vitro. Furthermore, possible overload of articular cartilage or excessive shear stress in chondrocyte cultures are more likely to be of a short nature. Therefore, in this study we choose to investigate a saw-tooth waveform oscillating fluid flow at varying exposure times in comparison to the established sinusoidal oscillatory waveform. [Ca(2+)](i), as an early signaling molecule, was quantified using the fluorescent dye fura-2. BAC were exposed to 1 Hz sinusoidal or saw-tooth waveform oscillating fluid flow at 2.2 Pa flow rates in a parallel plate flow chamber for 8 different loading times. As little as 5 cycles of oscillatory fluid flow were sufficient to increase [Ca(2+)](i) significantly over baseline. The number of responding cells could not be increased any further after a sufficient number of cycles (11), regardless of the waveform. Furthermore, a saw-tooth waveform appeared to be more stimulatory than regular sinusoidal oscillating flow at higher cycle numbers. BAC appear to be able to respond to these biophysical stimuli in a differentiated manner. This ability might give every single chondrocyte the capability to maintain its territory autonomously, since chondrocytes distributed in articular cartilage without the possibility to interact, e.g., via cell processes.

Animals↗

PubMed2003

A substance P receptor (NK1) antagonist enhances the widespread osteoporotic effects of sciatic nerve section.

The long-term effects of sciatic nerve section on bone mineral density (BMD) were studied using dual-energy X-ray absorptiometry (DEXA) in skeletally mature rats. Unilateral sciatic neurectomy caused the rapid loss of cancellous bone in the proximal and distal femur and tibia in the ipsilateral hindlimb and, to a lesser extent, in the contralateral intact hindlimb. The reduction in BMD rapidly progressed for 4 weeks after sciatic section and then gradually stabilized with no evidence of recovery at 12 weeks. The development of osteoporosis in the contralateral intact hindlimb was a novel finding. There was no evidence of disuse in the normal contralateral hindlimb after unilateral sciatic section; grid-crossing activity over a 24-h interval was unchanged and there was no reduction in weight bearing on the contralateral normal hindpaw during the stance phase of ambulation. Unilateral peripheral nerve lesions have well-documented effects on substance P content and function in the corresponding contralateral intact nerve. We hypothesized that after sciatic section a reduction in substance P signaling might contribute to bone loss in the contralateral hindlimb. Daily administration of the substance P receptor (NK1) antagonist LY303870 for 2 weeks caused significant loss of cancellous bone in the denervated and the contralateral hindlimb, evidence that substance P signaling sustained bone density after nerve section. After sciatic neurectomy there was a 33% reduction in sciatic nerve stimulation-evoked extravasation in the contralateral intact hindlimb, indicating transmedian inhibition of substance P signaling after nerve injury. Furthermore, there was a 50% reduction in the substance P content in both tibias after unilateral sciatic section. Collectively, these data support the hypothesis that a widespread reduction in substance P content in bone contributes to the osteoporotic effects of sciatic neurectomy and that residual substance P signaling maintains bone integrity after nerve section in both the denervated and contralateral intact hindlimb.

Absorptiometry, Photon↗

PubMed2003

How the stiffness of meniscal attachments and meniscal material properties affect tibio-femoral contact pressure computed using a validated finite element model of the human knee joint.

In an effort to prevent degeneration of articular cartilage associated with meniscectomies, both meniscal allografts and synthetic replacements are subjects of current interest and investigation. The objectives of the current study were to (1) determine whether a transversely isotropic, linearly elastic, homogeneous material model of the meniscal tissue is necessary to achieve a normal contact pressure distribution on the tibial plateau, (2) determine which material and boundary condition (attachments) parameters affect the contact pressure distribution most strongly, and (3) set tolerances on these parameters to restore the contact pressure distribution to within a specified error. To satisfy these objectives, a finite element model of the tibio-femoral joint of a human cadaveric knee (including both menisci) was used to study the contact pressure distribution on the tibial plateau. To validate the model, the contact pressure distribution on the tibial plateau was measured experimentally in the same knee used to create the model. Within physiologically reasonable bounds on five material parameters and four attachment parameters associated with a meniscal replacement, an optimization was performed under 1200 N of compressive load on the set of nine parameters to minimize the difference between the experimental and model results. The error between the experimental and model contact variables was minimized to 5.4%. The contact pressure distribution of the tibial plateau was sensitive to the circumferential modulus, axial/radial modulus, and horn stiffness, but relatively insensitive to the remaining six parameters. Consequently, both the circumferential and axial/radial moduli are important determinants of the contact pressure distribution, and hence should be matched in the design and/or selection of meniscal replacements. In addition, during surgical implantation of a meniscal replacement, the horns should be attached with high stiffness bone plugs, and the attachments of the transverse ligament and deep medial collateral ligament should be restored to minimize changes in the contact pressure distribution, and thereby possibly prevent the degradation of articular cartilage.

Adult↗

PubMed2003

Osteoblastic cells have refractory periods for fluid-flow-induced intracellular calcium oscillations for short bouts of flow and display multiple low-magnitude oscillations during long-term flow.

Partitioning a daily mechanical stimulus into discrete loading bouts enhances bone formation in rat tibiae (J. Bone Mineral Res. 15(8) (2000) 1596). We hypothesized that a refractory period exists in primary rat osteoblastic cells, during which fluid-flow-induced [Ca(2+)](i) oscillations are insensitive to additional short bouts (2 min) of fluid flow. Because the frequency of [Ca(2+)](i) oscillations is believed to be important for regulating cellular activity and long-term fluid flow alters gene expression in bone cells, we also hypothesized that long-term (15 min) oscillating fluid flow produces multiple [Ca(2+)](i) oscillations in osteoblastic cells. Primary osteoblastic cells from rat long bones were exposed to 2 min of oscillating fluid flow that produced shear stresses of 2 Pa at 2 Hz. After a rest period of 5, 30, 60, 300, 600, 900, 1800, or 2700 s, the cells were exposed to a second 2-min bout of flow. A 600 s rest period was required to recover the percentage of cells responding to fluid flow and a 900 s rest period was required to recover the [Ca(2+)](i) oscillation magnitude. The magnitude and shape of the two [Ca(2+)](i) oscillations were strikingly similar for individual cells after a 900 s rest period. During 15 min of continuous oscillating flow, some individual cells displayed between 1 and 9 oscillations subsequent to the initial [Ca(2+)](i) oscillation. However, only 54% of the cells that responded initially displayed subsequent [Ca(2+)](i) oscillations during long-term flow and the magnitude of the subsequent oscillations was only 28% of the initial response.

Adaptation, Physiological↗

PubMed2003

Finite element analysis as a tool for parametric prosthetic foot design and evaluation. Technique development in the solid ankle cushioned heel (SACH) foot.

In this study, we developed an approach for prosthetic foot design incorporating motion analysis, mechanical testing and computer analysis. Using computer modeling and finite element analysis, a three-dimensional (3D), numerical foot model of the solid ankle cushioned heel (SACH) foot was constructed and analyzed based upon loading conditions obtained from the gait analysis of an amputee and validated experimentally using mechanical testing. The model was then used to address effects of viscoelastic heel performance numerically. This is just one example of the type of parametric analysis and design enabled by this approach. More importantly, by incorporating the unique gait characteristics of the amputee, these parametric analyses may lead to prosthetic feet more appropriately representing a particular user's needs, comfort and activity level.

Amputation, Surgical↗

PubMed2003

Fluid flow induced PGE2 release by bone cells is reduced by glycocalyx degradation whereas calcium signals are not.

It has been hypothesized that bone cells have a hyaluronic acid (HA) rich glycocalyx (cell coat or pericellular matrix) and that this contributes to bone cell mechanotransduction via fluid flow. The glycocalyx of bone cells of the MC3T3-E1 osteoblastic cell line and the MLO-Y4 osteocytic cell line were characterized. Alcian blue staining and lectin binding experiments suggested that these cells have a glycocalyx rich in HA. Sulphated proteoglycans were not detected. Staining with hyaluronic acid binding protein and degradation by hyaluronidase confirmed that HA was a major component of the glycocalyx. We subjected cells, with and without hyaluronidase treatment, to oscillating fluid flow under standardized in vitro conditions. There was no effect of glycocalyx degradation on the intracellular calcium signal, in either cell type, in terms of the percentage of cells responding (40-80%) or the magnitude of the response (2-5 times baseline). However, a 4-fold fluid flow induced increase in PGE2 was eliminated by hyaluronidase pre-treatment in MLO-Y4 cells. We conclude that under these conditions the calcium and PGE2 responses occur via different pathways. An intact glycocalyx is not necessary in order to initiate a calcium signal in response to oscillating fluid flow. However, in osteocyte-like cells the PGE2 pathway is more dependent on mechanical signals transmitted through the glycocalyx.

Animals↗

PubMed2002

P2Y purinoceptors are responsible for oscillatory fluid flow-induced intracellular calcium mobilization in osteoblastic cells.

We previously found that oscillatory fluid flow activated MC3T3-E1 osteoblastic cell Ca(2+)(i) mobilization via the inositol 1,4,5-trisphosphate pathway in the presence of 2% fetal bovine serum (FBS). However, the molecular mechanism of fluid flow-induced Ca(2+)(i) mobilization is unknown. In this study, we first demonstrated that oscillatory fluid flow in the absence of FBS failed to increase [Ca(2+)](i) in MC3T3-E1 cells. Apyrase (10 units/ml), which rapidly hydrolyzes 5' nucleotide triphosphates to monosphophates, prevented the fluid flow induced increases in [Ca(2+)](i) in the presence of FBS. Adding ATP or UTP to flow medium without FBS restored the ability of fluid flow to increase [Ca(2+)](i), suggesting that ATP or UTP may mediate the effect of fluid flow on [Ca(2+)](i). Furthermore, adenosine, ADP, UDP, or adenosine 5'-O-(3-thiotriphosphate) did not induce Ca(2+)(i) mobilization under oscillatory fluid flow without FBS. Pyridoxal phosphate 6-azophenyl-2,4'-disulfonic acid, an antagonist of P2X purinoceptors, did not alter the effect of fluid flow on the Ca(2+)(i) response, whereas pertussis toxin, a G(i/o)-protein inhibitor, inhibited fluid flow-induced increases in [Ca(2+)](i) in the presence of 2% FBS. Thus, by the process of elimination, our data suggest that P2Y purinoceptors (P2Y2 or P2Y4) are involved in the Ca(2+)(i) response to fluid flow. Finally, a decreased percentage of MC3T3-E1 osteoblastic cells treated with P2Y2 antisense oligodeoxynucleotides responded to fluid flow with an increase in [Ca(2+)](i), and an increased percentage of ROS 17/2.8 cells, which do not normally express P2Y2 purinoceptors, transfected with P2Y2 purinoceptors responded to fluid flow in the presence of 2% FBS, confirming that P2Y2 purinoceptors are responsible for oscillatory fluid flow-induced Ca(2+)(i) mobilization. Our findings shed new light of the molecular mechanisms responsible for oscillatory fluid flow-induced Ca(2+)(i) mobilization in osteoblastic cells.

3T3 Cells↗

PubMed2002

A finite element model of the human knee joint for the study of tibio-femoral contact.

As a step towards developing a finite element model of the knee that can be used to study how the variables associated with a meniscal replacement affect tibio-femoral contact, the goals of this study were 1) to develop a geometrically accurate three-dimensional solid model of the knee joint with special attention given to the menisci and articular cartilage, 2) to determine to what extent bony deformations affect contact behavior, and 3) to determine whether constraining rotations other than flexion/extension affects the contact behavior of the joint during compressive loading. The model included both the cortical and trabecular bone of the femur and tibia, articular cartilage of the femoral condyles and tibial plateau, both the medial and lateral menisci with their horn attachments, the transverse ligament, the anterior cruciate ligament, and the medial collateral ligament. The solid models for the menisci and articular cartilage were created from surface scans provided by a noncontacting, laser-based, three-dimensional coordinate digitizing system with an root mean squared error (RMSE) of less than 8 microns. Solid models of both the tibia and femur were created from CT images, except for the most proximal surface of the tibia and most distal surface of the femur which were created with the three-dimensional coordinate digitizing system. The constitutive relation of the menisci treated the tissue as transversely isotropic and linearly elastic. Under the application of an 800 N compressive load at 0 degrees of flexion, six contact variables in each compartment (ie., medial and lateral) were computed including maximum pressure, mean pressure, contact area, total contact force, and coordinates of the center of pressure. Convergence of the finite element solution was studied using three mesh sizes ranging from an average element size of 5 mm by 5 mm to 1 mm by 1 mm. The solution was considered converged for an average element size of 2 mm by 2 mm. Using this mesh size, finite element solutions for rigid versus deformable bones indicated that none of the contact variables changed by more than 2% when the femur and tibia were treated as rigid. However, differences in contact variables as large as 19% occurred when rotations other than flexion/extension were constrained. The largest difference was in the maximum pressure. Among the principal conclusions of the study are that accurate finite element solutions of tibio-femoral contact behavior can be obtained by treating the bones as rigid. However, unrealistic constraints on rotations other than flexion/extension can result in relatively large errors in contact variables.

Adult↗