Jiro Nagatomi

Frequency- and Duration-Dependent Effects of Cyclic Pressure on Select Bone Cell Functions

The present study demonstrated unique correlations between characteristic parameters of mechanical loading and osteoblast functions. Specifically, osteoblast proliferation was dependent on the frequency and on the duration of the applied cyclic pressure stimulus: decreased cell proliferation was only observed when these cells were exposed to cyclic pressure at 1.0-Hz (but not at 0.25-Hz) frequency for 1 h (but not for 20 min) daily for 5 days. In contrast, endothelial cells were not responsive to cyclic pressure, whereas fibroblast proliferation increased under similar test conditions. Most important, cyclic pressure affected various osteoblast genes differently: exposure of osteoblasts to cyclic pressure (at 1.0-Hz frequency for 1 h daily) resulted in enhanced transcription and translation of alkaline phosphatase after 5 days; the same mechanical stimulus, however, did not affect osteopontin mRNA expression during the same time periods. These findings provide cellular and molecular level information, which is not only important in elucidating the correlation between mechanical loading and bone homeostasis, but can be useful in development of new technology in skeletal tissue engineering.

Cyclic pressure affects osteoblast functions pertinent to osteogenesis.

AbstractIn an attempt to elucidate the cellular/molecular correlations between mechanical stimuli and new bone formation, the present in vitro study used a custom-made laboratory setup and examined the effects of cyclic pressure on select functions of osteoblasts pertinent to osteogenesis. The results demonstrated that, compared to controls (no pressure), mRNA expression for type-I collagen (the main constituent of the organic phase of bone) was enhanced when osteoblasts were exposed to cyclic pressure (10–40 kPa at 1.0 Hz) for 1 h daily for up to 19 consecutive days. In addition, compared to controls, both deposition of collagen and accumulation of calcium (one of the major components of the inorganic phase of bone) increased significantly (p < 0.05) following exposure of osteoblast cultures to cyclic pressure for 19 days. Since the amounts of total DNA in controls and in osteoblast cultures exposed to cyclic pressure were similar at all time points tested, it was concluded that increased collagen and calcium concentrations in cultures resulted from enhanced osteoblast function (and not from increased number of cells); the presence of increased amounts of collagen affected the subsequent increased accumulation of calcium. These results provide evidence that daily exposure to cyclic pressure for various time periods (up to 19 days) affect osteoblast functions pertinent to bone formation.

Effects of Cyclic Pressure on Bone Marrow Cell Cultures

The present in-vitro study used bone marrow cell cultures and investigated the effects of cyclic pressure on osteoclastic bone resorption. Compared to control (cells maintained under static conditions), the number of tartrate resistant acid phosphatase (TRAP)-positive, osteoclastic cells was significantly (p<0.05) lower when, immediately upon harvesting, bone marrow cells were exposed to cyclic pressure (10-40 kPa at 1.0 Hz). In contrast, once precursors in bone marrow cells differentiated into osteoclastic cells under static culture conditions for 7 days, subsequent exposure to the cyclic pressure of interest to the present study did not affect the number of osteoclastic cells. Most important, exposure of bone marrow cells to cyclic pressure for 1 h daily for 7 consecutive days resulted in significantly (p<0.05) lower osteoclastic bone resorption and in lowered mRNA expression for interleukin-1 (IL-1) and tumor necrosisfactor-a (TNF-a), cytokines that are known activators of osteoclast function. In addition to unique contributions to osteoclast physiology, the present study provided new evidence of a correlation between mechanical loading and bone homeostasis as well as insight into the molecular mechanisms of bone adaptation to mechanical loading, namely cytokine-mediated control of osteoclast functions.

Role of cytoskeletal components in stress-relaxation behavior of adherent vascular smooth muscle cells.

A number of recent studies have demonstrated the effectiveness of atomic force microscopy (AFM) for characterization of cellular stress-relaxation behavior. However, this techniques recent development creates considerable need for exploration of appropriate mechanical models for analysis of the resultant data and of the roles of various cytoskeletal components responsible for governing stress-relaxation behavior. The viscoelastic properties of vascular smooth muscle cells (VSMCs) are of particular interest due to their role in the development of vascular diseases, including atherosclerosis and restenosis. Various cytoskeletal agents, including cytochalasin D, jasplakinolide, paclitaxel, and nocodazole, were used to alter the cytoskeletal architecture of the VSMCs. Stress-relaxation experiments were performed on the VSMCs using AFM. The quasilinear viscoelastic (QLV) reduced-relaxation function, as well as a simple power-law model, and the standard linear solid (SLS) model, were fitted to the resultant stress-relaxation data. Actin depolymerization via cytochalasin D resulted in significant increases in both rate of relaxation and percentage of relaxation; actin stabilization via jasplakinolide did not affect stress-relaxation behavior. Microtubule depolymerization via nocodazole resulted in nonsignificant increases in rate and percentage of relaxation, while microtubule stabilization via paclitaxel caused significant decreases in both rate and percentage of relaxation. Both the QLV reduced-relaxation function and the power-law model provided excellent fits to the data (R(2)=0.98), while the SLS model was less adequate (R(2)=0.91). Data from the current study indicate the important role of not only actin, but also microtubules, in governing VSMC viscoelastic behavior. Excellent fits to the data show potential for future use of both the QLV reduced-relaxation function and power-law models in conjunction with AFM stress-relaxation experiments.

Do cement lines restrict osteoclastic bone resorption

Bone is constantly remodeled throughout life, but whether the microstructure of the bone affects resorption is unknown. In this present study, longitudinal and transverse devitalized bone slices were cultured in-vitro with rat bone marrow cells to investigate the influence of bone microstructure in directing and regulating bone resorption by osteoclasts. After the 14-day culture period, the average size of resorption areas produced in the longitudinal slices was found to be three times larger than in the transverse slices (p < 0.05). In addition, bone resorption pro-files were consistent with the microstructure of the bone slices. In the longitudinal bone sections resorption areas proceeded more longitudinally than transversely and resorption areas in transverse bone sections were more circular, either contained inside or outside the cement line surrounding the osteon. These results show that the bone microstructure influences osteoclastic resorption and that the cement lines may be the microstructural element that directs and regulates osteoclastic resorption.

Mechanical regulation of bone homeostasis: effects of cyclic pressure on bone cell function in vitro

The present in vitro study exposed rat osteoblasts and osteoclast-precursors in bone marrow cell populations to controlled regimes of cyclic pressure and examined various cell functions that are pertinent to bone homeostasis. The results provided evidence that osteoblasts are sensitive to the frequency of the applied cyclic pressure stimulus. Specifically, compared to controls (static conditions) and cells exposed to cyclic pressure at 0.25 Hz frequency, osteoblast proliferation was significantly (p < 0.05) lower, but alkaline phosphatase mRNA expression and enzyme activity were enhanced, only when these cells were exposed to cyclic pressure at 1.0 Hz frequency for 1 hour daily for 5 consecutive days. Furthermore, the results of the present study demonstrated that the timing of application of the cyclic pressure was critical for osteoclastic cell formation from precursors in bone marrow. Exposure of bone marrow cells to cyclic pressure immediately upon harvesting led to decreased formation of osteoclastic cells from their precursors; in contrast, the number of osteoclastic cells was not affected when the cyclic pressure was applied after 7 days of culture under static conditions.

Cyclic pressure affects bone cell proliferation

Success of load-bearing dental/orthopedic implants relies on appropriate integration of the surrounding tissues at the interface. Following surgically induced trauma, bone tissue at the implant site undergoes a healing/regeneration process, which involves multiple cell types. Although the general sequence of wound healing is well known, the cellular/molecular-level responses to mechanical loading at the bone-implant interface are still poorly understood. In order to elucidate some of the events taking place during bone healing/remodeling under mechanical loading, the present study used in vitro cellular models and investigated and compared the effects of cyclic pressure on the proliferation of osteoblasts, fibroblasts and endothelial cells.