Electromechanical properties of human osteoarthritic and asymptomatic articular cartilage are sensitive and early detectors of degeneration.

OBJECTIVE: To evaluate cross-correlations of ex vivo electromechanical properties with cartilage and subchondral bone plate thickness, as well as their sensitivity and specificity regarding early cartilage degeneration in human tibial plateau. METHOD: Six pairs of tibial plateaus were assessed ex vivo using an electromechanical probe (Arthro-BST) which measures a quantitative parameter (QP) reflecting articular cartilage compression-induced streaming potentials. Cartilage thickness was then measured with an automated thickness mapping technique using Mach-1 multiaxial mechanical tester. Subsequently, a visual assessment was performed by an experienced orthopedic surgeon using the International Cartilage Repair Society (ICRS) grading system. Each tibial plateau was finally evaluated with µCT scanner to determine the subchondral-bone plate thickness over the entire surface. RESULTS: Cross-correlations between assessments decreased with increasing degeneration level. Moreover, electromechanical QP and subchondral-bone plate thickness increased strongly with ICRS grade (ρ = 0.86 and ρ = 0.54 respectively), while cartilage thickness slightly increased (ρ = 0.27). Sensitivity and specificity analysis revealed that the electromechanical QP is the most performant to distinguish between different early degeneration stages, followed by subchondral-bone plate thickness and then cartilage thickness. Lastly, effect sizes of cartilage and subchondral-bone properties were established to evaluate whether cartilage or bone showed the most noticeable changes between normal (ICRS 0) and each early degenerative stage. Thus, the effect sizes of cartilage electromechanical QP were almost twice those of the subchondral-bone plate thickness, indicating greater sensitivity of electromechanical measurements to detect early osteoarthritis. CONCLUSION: The potential of electromechanical properties for the diagnosis of early human cartilage degeneration was highlighted and supported by cartilage thickness and µCT assessments.

Atomic force microscopy can be used to mechanically stimulate osteoblasts and evaluate cellular strain distributions.

In this study, atomic force microscopy (AFM) was used to mechanically stimulate primary osteoblasts. In response to mechanical force applied by the AFM, the indented cell increased its intracellular calcium concentration. The material properties of the cell could be estimated and the membrane strains calculated. We proceeded to validate this technique experimentally and a 20% error was found between the predicted and the measured diameter of indentation. We also determined the strain distributions within the cell that result from AFM indentation using a simple finite element model. This enabled us to formulate hypotheses as to the mechanism through which cells may sense the applied mechanical strains. Finally, we report the effect of the Poisson ratio and the cell thickness on the strain distributions. Varying the Poisson ratio did not change the order of magnitude of the strains; whereas the cellular thickness dramatically changed the order of magnitude of the cellular strains. We conclude that AFM can be used for controlled mechanical stimulation of osteoblasts and that cellular strain distributions can be computed with a good accuracy when the cell is indented in its highest part.

Vasoactive intestinal peptide (VIP)/pituitary adenylate cyclase-activating peptide receptor subtypes in mouse calvarial osteoblasts: presence of VIP-2 receptors and differentiation-induced expression of VIP-1 receptors.

Three distinct complementary DNAs for vasoactive intestinal peptide (VIP) and pituitary adenylate cyclase-activating peptide (PACAP) receptors have been cloned and designated VIP-1 receptor (VIP-1R), VIP-2 receptor (VIP-2R), and PACAP receptor (PACAP-R). In the present study, we have characterized the binding sites on primary mouse calvarial osteoblasts for VIP and related peptides. By analyzing the cAMP response, the rank order of response observed was PACAP 38 > PACAP 27 > helodermin > VIP > helospectin > glucagon > PHI >>> secretin. The VIP-2R/PACAP-R antagonist, PACAP 6-38, inhibited both VIP- and PACAP-stimulated cAMP formation. Binding studies using an atomic force microscopy (AFM) technique showed high affinity binding for VIP and PACAP 38, but not for secretin. Radioligand binding studies using (125)I-VIP and (125)I-PACAP 38 demonstrated a more specific and higher affinity binding for PACAP 38 than for VIP. Secretin failed to inhibit both (125)I-VIP and (125)I-PACAP 38 binding. RT-PCR demonstrated that undifferentiated mouse calvarial osteoblasts express messenger RNA for VIP-2R, but not for VIP-1R or PACAP-R. When the osteoblasts were cultured for 20 days to induce bone noduli formation, VIP-1R, in addition to VIP-2R, were expressed when the nodules started to mineralize at 12 days. Taken together, these data demonstrate that mouse calvarial osteoblasts express functional VIP-2R with higher affinity binding for PACAP than for VIP and that the VIP-1R expression is induced during osteoblastic differentiation.

Vasoactive intestinal peptide regulates osteoclast activity via specific binding sites on both osteoclasts and osteoblasts.

Clinical and experimental observations, together with immunohistochemical findings, suggest that neuro-osteogenic interactions may occur in the skeleton. In this study, we have examined the effect of vasoactive intestinal peptide (VIP), one of the neuropeptides present in bone, on the activity of the bone-resorbing osteoclast. Effects on bone resorption were assessed by counting the number of pits formed by rat osteoclasts incubated on devitalized slices of bovine cortical bone. Under conditions with an initially sparse density of stromal cells/osteoblasts, VIP caused a rapid cytoplasmic contraction and decreased motility of osteoclasts. This was coupled with a decrease in the number of resorption lacunae and a decrease in the total area resorbed by the osteoclasts in 48-h cultures. Time-course experiments revealed that the inhibitory effects on contraction and motility were transient and that the cells gradually regained their activity, such that, when culture time was prolonged to 120 h, a stimulatory effect by VIP on bone resorption was observed. When osteoclasts were incubated on bone slices, in the presence of an initially large number of stromal cells/osteoblasts, VIP treatment increased the number of resorption pits and total bone area resorbed in 48-h cultures. Using atomic force microscopy, we provide direct evidence that both osteoclasts and stromal cells/osteoblasts bind VIP. Also, VIP was shown to cause a rapid rise of intracellular calcium in osteoclasts and in a proportion (20%) of stromal cells/osteoblasts. Taken together, these data suggest that differentiated osteoclasts are equipped with receptors for VIP that are linked to a transient inhibition of osteoclast activity and, in addition, that stromal cells/osteoblasts have VIP receptors coupled to a delayed stimulation of osteoclastic resorption.

Adapting atomic force microscopy for cell biology.

We present details of our AFM modifications to produce an adaptable imaging system for the cell biologist. We have designed and validated a new inverted microscope interface and a scan head with increased Z-range, based upon the TopoMetrix Explorer AFM. We have utilised these changes, together with home-made glass ball cantilevers, to obtain topographical information over cells with large Z-dimension (over 15 microm high), and mapped calcitonin-calcitonin receptor binding forces in living bone cells. We conclude that modified AFM can be used to evaluate intermolecular events in living cells and that this approach will ensure general application to the study of receptor-ligand interactions under truly physiological conditions.

New technologies in scanning probe microscopy for studying molecular interactions in cells.

Atomic force microscopy (AFM) is a specialised form of scanning probe microscopy, which was invented by Binnig and colleagues in 1986. Since then, AFM has been increasingly used to study biomedical problems. Because of its high resolution, AFM has been used to examine the topography or shape of surfaces, such as during the molecular imaging of proteins. This, combined with the ability to operate under known force regimes, makes AFM technology particularly useful for measuring intermolecular bond forces and assessing the mechanical properties of biological materials. Many of the constraints (e.g. complex instrumentation, slow acquisition speeds and poor vertical range) that previously limited the use of AFM in cell biology are now beginning to be resolved. Technological advances will enable AFM to challenge both confocal laser scanning microscopy and scanning electron microscopy as a method for carrying out three-dimensional imaging. Its use as both a precise micro-manipulator and a measurement tool will probably result in many novel and exciting applications in the future. In this article, we have reviewed some of the current biological applications of AFM, and illustrated these applications using studies of the cell biology of bone and integrin-mediated adhesion.

Intracellular fragmentation of bone resorption products by reactive oxygen species generated by osteoclastic tartrate-resistant acid phosphatase.

Tartrate-resistant acid phosphatase (TRAP) is highly expressed in bone-resorbing osteoclasts and activated macrophages. It has been suggested that a redox-active iron in the binuclear iron center of TRAP could have the capacity to react with hydrogen peroxide to produce highly destructive reactive oxygen species (ROS). Here we show that TRAP can generate ROS in vitro and that cells over-expressing TRAP produce higher amounts of intracellular ROS than their parent cells. We further demonstrate that these ROS can be targeted to destroy collagen and other proteins. In resorbing osteoclasts, TRAP was found in transcytotic vesicles transporting matrix degradation products through the cell, suggesting that TRAP-facilitated fragmentation of endocytosed material takes place in a specific cellular compartment. These results suggest that bone matrix degradation occurs not only extracellularly in the resorption lacunae but also intracellularly in the transcytotic vesicles. We propose that proteins containing redox-active iron could represent a novel mechanism of physiological fragmentation of organic molecules. This mechanism could be important in tissue remodeling and as a defense mechanism of phagocytosing cells.

Single integrin molecule adhesion forces in intact cells measured by atomic force microscopy.

Cross-talk between cells and the extracellular matrix is critically influenced by the mechanical properties of cell surface receptor-ligand interactions; these interactions are especially well defined and regulated in cells capable of dynamically modifying their matrix environment. In this study, attention was focused on osteoclasts, which are absolutely dependent on integrin extracellular matrix receptors in order to degrade bone; other bone cells, osteoblasts, were used for comparison. Integrin binding forces were measured in intact cells by atomic force microscopy (AFM) for several RGD-containing (Arg-Gly-Asp) ligands and ranged from 32 to 97 picoNewtons (pN); they were found to be cell and amino acid sequence specific, saturatable and sensitive to the pH and divalent cation composition of the cellular culture medium. In contrast to short linear RGD hexapeptides, larger peptides and proteins containing the RGD sequence, such as osteopontin (a major non-collagenous bone protein) and echistatin (a high affinity RGD sequence containing antagonist snake venom protein), showed different binding affinities. This demonstrates that the context of the RGD sequence within a protein has considerable influence upon the final binding force for receptor interaction. These data also demonstrate that AFM, as a methodological approach, can be adapted to cell biology studies wherever cell-matrix interactions play a critical role, and, moreover, may have applicability to the analysis of receptor-ligand interactions in cell membranes in general.

The regulation of pHi in osteoclasts is dependent on the culture substrate and on the stage of the resorption cycle.

The present study was performed to clarify the role of vacuolar H+-ATPase in the regulation of the intracellular pH (pHi) in osteoclasts. Bafilomycin A1 or amiloride were added to rat osteoclast cultures to block the H+-ATPases and Na+/H+-exchangers, respectively. Addition of 10(-8) M bafilomycin A1 to osteoclasts cultured on bone induced a rapid decrease of the pHi, while addition of amiloride had only a minor effect. The response to bafilomycin A1 appeared simultaneously with resorption activity and was abolished when resorption was inhibited by calcitonin. Osteoclasts on bone recovered from acid loading caused by propionate in the presence of amiloride, while bafilomycin A1 inhibited this recovery almost completely. The pHi of osteoclasts cultured on glass responded to the addition of amiloride, but was not effected by even high concentrations of bafilomycin A1. In contrast, as little as 10(-10) M bafilomycin A1 caused accumulation of large vesicles in the cytoplasm.

Different calcium sensitivity in osteoclasts on glass and on bone and maintenance of cytoskeletal structures on bone in the presence of high extracellular calcium.

The sensitivity of rat osteoclasts to increased extracellular calcium concentrations ([Ca2+]e) was investigated by single cell measurements of free cytosolic calcium concentrations ([Ca2+]i), by changes in microfilament organization of resorbing osteoclasts, and by in vitro bone resorption assays. Osteoclasts cultured on glass and on bone showed clear differences in their responses, as in 44% and 52% of osteoclasts on glass but in only 21% and 25% of osteoclasts on bone [Ca2+]i increased when [Ca2+]e was increased from 2 mM to 6 or 10 mM via perfusion, respectively. Bone resorption was inhibited without changes in the osteoclast numbers only by 10 mM [Ca2+]e in 2 day cultures. Furthermore, there were no changes in the organization of microfilament structures in resorbing osteoclasts after increased [Ca2+]e (up to 20 mM [Ca2+]e, 30 min incubation). These results suggest that the sensitivity of osteoclasts to increased [Ca2+]e is dependent on their activation phase (resting/migrating vs. resorbing) and that resorbing osteoclasts are not sensitive to increased [Ca2+]e or that the sensing system cannot be reached in polarized resorbing osteoclasts. In contrast, increasing [Ca2+]i through the use of calcium ionophores dispersed specific microfilament structures at the sealing zone transiently in a few minutes. This shows that [Ca2+]i is used as a signaling mechanism to inactivate osteoclasts, with a similar end result on microfilament structures at the sealing zone as caused by increased concentration of cAMP and activation of protein kinase C.

Effects of recombinant human osteogenic protein-1 on the differentiation of osteoclast-like cells and bone resorption.

Recombinant human OP-1 stimulated the formation of tartrate-resistant acid phosphatase (TRAP)-positive multinucleated cells (MNCs) significantly and in a dose-dependent manner in rat bone marrow cell culture. Newly formed MNCs either induced by hOP-1 alone or with 1,25(OH)2D3 were also positive for vitronectin receptor and carbonic anhydrase II. Moreover, OP-1 markedly increased the capacity of 1,25(OH)2D3 to induce osteoclast-like cell formation and bone resorption in bone marrow cultures. 25 pg/ml of calcitonin significantly inhibited both OP-1- and vitamin D3-induced TRAP-positive MNC-formation in marrow cultures, indicating that in both cases the MNC formation was calcitonin sensitive. OP-1 at 5-100 ng/ml did not have any significant effect on bone resorption as studied by pit formation assay. These studies that OP-1 in concert with 1,25(OH)2D3 could have an important role in bone remodeling by exhibiting its effects not only on osteoblast growth and differentiation but also on the recruitment of osteoclasts.