Bone tissue engineering is an interdisciplinary field involving both engineers and cell biologists, whose main purpose is to repair bone anatomical defects and maintain its functions. A novel system that integrates pulsed electromagnetic fields (PEMFs) and bioreactors was applied to bone tissue engineering for regulating osteoblast proliferation and differentiation in'vitro. Osteoblasts were acquired from the calvaria of newborn Wistar rats and isolated after sequential digestion. Poly(DL-lactic-co-glycolic acid) (PLGA) scaffolds were made by the solvent merging/particulate leaching method. Osteoblasts were seeded into porous PLGA scaffolds with 85% porosity and cultured in bioreactors for the 18-day culture period. Cells were exposed to PEMF pulsed stimulation with average (rms) amplitudes of either 0.13, 0.24, or 0.32 mT amplitude. The resulting induced electric field waveform consisted of single, narrow 300 micros quasi-rectangular pulses with a repetition rate of 7.5'Hz. The results showed that PEMF stimulation for 2 and 8 h at .13 mT increased the cell number on days 6 and 12, followed by a decrease on day 18 using 8 h stimulation. However, ALP activity was decreased and then increased on days 12 and 18, respectively. On the other hand, PEMF-treated groups (irrespective of the stimulation time) at 0.32 mT inhibited cell proliferation but enhanced ALP activity during the culture period. These findings suggested that PEMF stimulation with specific parameters had an effect on regulating the osteoblast proliferation and differentiation. This novel integrated system may have potential in bone tissue engineering.

With the use of Helmholtz coils and pulsed electromagnetic field (PEMF) stimulators to generate uniform time varying electromagnetic fields, the effects of extremely low frequency electromagnetic fields on osteoporosis and serum prostaglandin E(2) (PGE(2)) concentration were investigated in bilaterally ovariectomized rats. Thirty-five 3 month old female Sprague-Dawley rats were randomly divided into five different groups: intact (INT), ovariectomy (OVX), aspirin treated (ASP), PEMF stimulation (PEMF + OVX), and PEMF stimulation with aspirin (PEMF + ASP) groups. All rats were subjected to bilateral ovariectomy except those in INT group. Histomorphometric analyses showed that PEMF stimulation augmented and restored proximal tibial metaphyseal trabecular bone mass (increased hard tissue percentage, bone volume percentage, and trabecular number) and architecture (increased trabecular perimeter, trabecular thickness, and decreased trabecular separation) in both PEMF + OVX and PEMF + ASP. Trabecular bone mass of PEMF + OVX rats after PEMF stimulation for 30 days was restored to levels of age matched INT rats. PEMF exposure also attenuated the higher serum PGE(2) concentrations of OVX rats and restored it to levels of INT rats. These experiments demonstrated that extremely low intensity, low frequency, single pulse electromagnetic fields significantly suppressed the trabecular bone loss and restored the trabecular bone structure in bilateral ovariectomized rats. We, therefore, conclude that PEMF may be useful in the prevention of osteoporosis resulting from ovariectomy and that PGE(2) might relate to these preventive effects.

Mechanical perturbations serve as extracellular signals to a variety of cells, including bone cells. Low-intensity pulsed ultrasound produces significant multifunctional effects that are directly relevant to bone formation and resorption. Ultrasound stimulation has been shown to accelerate bone-defect healing and trabecular bone regeneration. In this study, we use an in vitro bone cell culture model to investigate the effect of low-intensity pulsed ultrasound. The rat alveolar mononuclear cell-calvaria osteoblast coculture system was used in this study. Before treatment, the bone cells were cultured for 3 days to facilitate their attachment and differentiation. Then, ultrasound exposure (frequency = 1 MHz, intensity = 0.068 W/cm(2)) or sham exposure for 20 min per day was applied until the end of the experiment. Half of the culture media were obtained on the 4th, 5th, 6th, 7th, 8th, 9th, and 10th days for the analysis of cytokines and biochemical parameters. At the end of the experiment, cells were fixed and stained for identification and quantification of the osteoblast and osteoclast cells. After low-intensity pulse ultrasound stimulation, the osteoblast cell counts were significantly increased, whereas the osteoclast cell counts were significantly decreased. The total alkaline phosphatase amount in the culture medium was increased after 7 days of ultrasound stimulation, and tumor necrosis factor-alpha in ultrasound-stimulated bone cells was significantly increased after the 7th day of culture and reached 474.77% of the control medium on the 10th day of culture. The results of this study suggest that low-intensity ultrasound treatment may have a stimulatory effect on bone-healing processes.

Over the past decade, a large number of biomaterials have been proposed as artificial bone fillers for repairing bone defects. The material most widely used in clinical medicine is hydroxyapatite. The aim of our investigation was to study the effect of hydroxyapatite size mechanism on osteoblasts. The osteoblasts were cultured in vitro with 0.1% (1 mg/mL) of various sized hydroxyapatite particles (0.5-3.0, 37-63, 177-250, and 420-841 microm) for 1 h, 3 h, 1 day, 3 days, and 7 days. The results showed that adding hydroxyapatite particles to osteoblast cultures can significantly affect osteoblast cell count. Osteoblast populations decreased significantly. Osteoblast mean surface areas also changed significantly. Transforming growth factor-beta1 (TGF-beta1) concentrations in culture medium decreased significantly with the addition of hydroxyapatite particles. Prostaglandin E2 (PGE2) concentrations in medium increased significantly. The changes in TGF-beta1 and PGE2 concentration were more significant and persisted longer in smaller-particle groups. The inhibitory effects of hydroxyapatite particles on osteoblast cell cultures were mediated by the increased synthesis of PGE2. Caution should be exercised before using a hydroxyapatite product which could easily break down into fine particles.

Low intensity ultrasound stimulation has been used as a strategy to promote fracture healing. This study investigated the mechanism of ultrasound stimulation in enhancing fracture healing. Forty-five adult New Zealand White rabbits were divided into control, microwave treated, and ultrasound stimulation groups. After anesthesia, transverse osteotomy was created at midportion of the fibula bone. Intravital staining followed by fluorescence microscopic examination of new bone formation in the osteotomy site and biomechanical tests on torsional stiffness of the osteotomy site were performed. The difference between each examination was evaluated and analyzed. After ultrasound stimulation, new bone formation in the osteotomy site of the stimulated limb was 23.1-35.8% faster than that of the sham treated limb; the torsional stiffness of the stimulated limb was 44.4-80.0% higher than that of the sham treated limb. In the group of microwave hyperthermia treatment, the new bone formation was higher than that of the sham treated limb, but the difference was not statistically significant. The difference in torsional stiffness between the microwave hyperthermia treated limbs and the sham treated limb was not quite statistically significant. We demonstrated that low intensity ultrasound stimulation could increase the new bone formation and torsional stiffness. These effects probably are not mediated via hyperthermia.