Two new, near-infrared, fluorescent probes as potential tools for imaging bone repair.

A precise imaging technique to evaluate osteogenesis, osteodifferentiation, and osseointegration following peri-implant surgery is in high clinical demand. Herein, we report the generation of two new, near-infrared (NIR) fluorescent probes for use in the molecular imaging of bone repair. The first probe aims to monitor the in vitro differentiation of human mesenchymal stem cells (MSCs) into osteoblasts. A NIR fluorochrome was conjugated to a cyclic peptide that binds to integrin α5ß1, a factor that promotes osteogenesis in MSCs and therefore functioned as an osteoblast-specific marker. The second probe aims to monitor osteogenesis, and was generated by conjugating the drug pamidronate to a NIR fluorescent gold nanocluster. Pamidronate specifically binds to hydroxyapatite (HA), a mineral present in bone that is produced by osteoblasts, and therefore provides a functional marker for new bone formation. Our results show that both probes bind to their specific targets in vitro-differentiated osteoblasts, and not to undifferentiated MSCs, and emit NIR fluorescence for functional detection. This in vitro work demonstrates the ability of these probes to bind to active osteoblasts and their mineral deposits and highlight their potential utility as clinical tools for the imaging of the osseointegration process at the molecular level.

Studies of photokilling of bacteria using titanium dioxide nanoparticles.

Metal pins used to apply skeletal traction or external fixation devices protruding through skin are susceptible to the increased incidence of pin site infection. In this work, we tried to establish the photokilling effects of titanium dioxide (TiO2) nanoparticles on an orthopedic implant with an in vitro study. In these photocatalytic experiments, aqueous TiO2 was added to the tested microorganism. The time effect of TiO2 photoactivation was evaluated, and the loss of viability of five different bacteria suspensions (Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, Enterococcus hirae, and Bacteroides fragilis) was examined by the viable count procedure. The bactericidal effect of TiO2 nanoparticle-coated metal plates was also tested. The ultraviolet (UV) dosage used in this experiment did not affect the viability of bacteria, and all bacteria survived well in the absence of TiO2 nanoparticles. The survival curve of microorganisms in the presence of TiO2 nanoparticles showed that nearly complete killing was achieved after 50 min of UV illumination. The formation of bacterial colonies above the TiO2 nanoparticle-coated metal plates also decreased significantly. In this study, we clearly demonstrated the bactericidal effects of titanium dioxide nanoparticles. In the presence of UV light, the titanium dioxide nanoparticles can be applicable to medical facilities where the potential for infection should be controlled.

Comparison of ultrasound and electromagnetic field effects on osteoblast growth.

This study compares the mechanisms of ultrasound (US) on osteoblast proliferation with those of pulsed electromagnetic field (PEMF), by different signal transduction pathway inhibitors. The cells were stimulated for 15 min under US or for 2 h under PEMF exposure. Twenty-four h after the beginning of stimulation, the cells were harvested and used for mitochondrial activity test (MTT) analysis. The results showed that there are different transduction pathways for US and PEMF stimulation that lead to an upgrade of osteoblast proliferation, although their pathways all lead to an increase in cytocolic Ca2+ and activation of calmodulin. These findings offer a biochemical mechanism to support the process of ultrasound and PEMF-induced enhanced healing of bone fractures.

Pulsed electromagnetic fields stimulation affects osteoclast formation by modulation of osteoprotegerin, RANK ligand and macrophage colony-stimulating factor.

Electromagnetic stimulation has been documented to treat recalcitrant problems of musculoskeletal system. Yet, the underlying mechanisms are not completely understood. In this study, we investigated effect of pulsed electromagnetic fields (PEMF) with parameters modified from clinical bone growth stimulator on osteoclast formation, bone resorption, and cytokines associated with osteoclastogenesis. Marrow cells were harvested from both femora and tibiae of 6 week-old mice and cultured in 8-well chamber slides or 16-well calcium phosphate apatite-coated multitest slides. After 1-day incubation, marrow cells were exposed to PEMF at different electric field intensities for 2h/day and continued for 9 days. Osteoprotegerin (OPG), receptor activator of NFkappaB-ligand (RANKL) and macrophage colony-stimulating factor (M-CSF) concentrations of each group were determined after PEMF stimulation. Osteoclast identity was confirmed by both tartrate resistant acid phosphatase (TRAP) stain and bone resorption assay. A statistically significant increase and decrease of osteoclastogenesis and bone resorption areas were found when exposed to PEMF with different intensities. Besides, consistent correlations among OPG, RANKL, M-CSF, osteoclast numbers, and bone resorption after exposure to different intensities of PEMF were observed. These data demonstrated that PEMF with different intensities could regulate osteoclastogenesis, bone resorption, OPG, RANKL, and M-CSF concentrations in marrow culture system.

The effect of Gu-Sui-Bu (Drynaria fortunei J. Sm) immobilized modified calcium hydrogenphosphate on bone cell activities.

In our previous study, we have validated the efficacy and the safety of Gu-Sui-Bu [Drynaria fortunei (Kunze) J. Sm.] by the bone cells culture. However, a satisfactory delivery system for Gu-Sui-Bu must be developed before it can be used in clinical medicine. In this study, we try to use modified calcium hydrogenphosphate (MCHP) bioceramic as a carrier to transport Gu-Sui-Bu into the bone cell culture system. Toward this goal, we evaluated the effect of a Gu-Sui-Bu-immobilized modified calcium hydrogenphosphate (GI-MCHP) on the bone cells activities. THE CHINESE MEDICINE: Gu-Sui-Bu [Drynaria fortunei (kunze) J. Sm] was extracted and then immobilized on the surface of MCHP. The rat osteoblasts-osteoclasts co-culture system was used as the experimental model. After the cells grew to 80% confluence, different sizes of GI-MCHP particles were tested. The mitochondria activity of the bone cells after exposure was determined by colorimetric assay. Biochemical markers such as lactate dehydrogenase (LDH), alkaline phosphatase (ALP), acid phosphatase (ACP) and prostaglandin E(2) titer were analyzed to evaluate the bone cells activities. Histomorphometric study of osteoclasts activities and the phenotype expression of osteoblasts were also evaluated. There is no detectable titer of LDH secretion into the medium and no significant change in the intracellular ALP content. The ALP titer in the culture medium did increase significantly at 3 days' culture, while there is a significant decrease in the intracellular ACP content and significant increase in the ACP titer in the medium. The concentrations of PGE(2) in tested medium are always significantly higher than that of control medium during the 7 days' culture. At the end of 7 days' culture, the PGE(2) concentrations in the tested medium were still 4.74 times that of the control medium. After GI-MCHP treatment on bone cells, the size of the osteoclasts seems decreased and their cell integrity seems lost, while the osteoblasts phenotype expression was relatively preserved. From this study, we demonstrated that Gu-Sui-Bu [Drynaria fortunei (Kunze) J. Sm.] immobilized MCHP has well preserved the potential beneficial effects of Gu-Sui-Bu on the bone cells culture.

The influence on gene-expression profiling of osteoblasts behavior following treatment with the ionic products of sintered beta-dicalcium pyrophosphate dissolution.

Sintered dicalcium pyrophosphate (SDCP) is biocompatible to bone tissue both in the in vivo and in vitro model. However, the molecular mechanisms that mediated these processes have yet to be identified. In this study, we investigated the influence of SDCP ions on in vitro osteoblasts behavior. The powder of sintered beta-dicalcium pyrophosphate (SDCP) was dissolved by HCl and then diluted into different concentration of solutions by culture medium used in the osteoblast cell culture. The effects of various concentration of SDCP on bone cell activities were evaluated by using MTT assay. For the differentiation of osteoblasts, alkaline phosphatase (AP) staining, von Kossa stain for mineralized nodules and bone markers messenger ribonucleic acid (mRNA) isolation and identification were performed at 3h, days 1, 3, 7 and 14. In the presence of 10(-8)M SDCP for 14 days, the osteoblasts population was still significantly higher than that of control. In the qualitative analysis for the formation of AP staining colonies and mineralization nodules formation were not affected by SDCP ions. When osteoblasts cultured in the presence of 10(-8)M SDCP ions, the osteocalcin mRNA expression was up-regulated; while the collagen, osteonectin and osteopontin mRNA expression were down-regulated. In this study, we demonstrated that the elevated concentration of calcium and pyrophosphate ions can activate genes of the bone cells. This study will contribute to a better understanding of cell/biomaterial interactions and mechanisms that SDCP affect the bone cells.

Optimum intensities of ultrasound for PGE(2) secretion and growth of osteoblasts.

This study compared the effects of different intensity ultrasound (US) on osteoblasts in the far-field model with effects of the near-field model from the literature, to understand the relations between prostaglandin E(2) (PGE(2)) and osteoblast growth. We used an in vitro model to investigate the effects of 1-MHz, pulsed 1:4, and five different spatial-average temporal-peak intensity (150, 300, 600, 1200 and 2400 mW/cm(2)) US stimulations in far-field exposure (240 mm) on osteoblasts for 15 min. Optimum intensity in this study was 600 mW/cm(2), and cell density and PGE(2) secretion could be significantly stimulated at this intensity. This research may indicate that the growth of osteoblasts by US stimulation was, at least partly, due to increases in the synthesis and secretion of PGE(2). This well-controlled model can lead to further research on the biologic mechanisms for US.

Effects of ultrasound on osteotomy healing in a rabbit fracture model.

This study investigated the effects of ultrasound (US) at different frequencies on fracture healing over a three-week period in a rabbit fibular fracture model. Forty-five adult New Zealand White rabbits were divided into five groups: a control group and four groups treated with US frequencies of 0.5, 1.0, 1.5 and 2.0 MHz (0.5 W/cm(2), 200-µs burst, pulsed 1:4). After anesthesia, transverse osteotomy was performed on the fibula bone. This was followed by intravital staining and fluorescence microscopic examination of new bone formation and biomechanical tests of torsional stiffness at the osteotomy site. Results showed that total new bone formation and torsional stiffness of the fibula were greater in all US-treated groups than in the control group. No significant difference was found between any of the four US-treated groups. The US treatment also enhanced bone growth of the sham-treated contralateral fracture site. These results suggest that US treatment at 0.5, 1.0, 1.5 or 2.0 MHz can enhance fracture healing in a rabbit model. Furthermore, the effects of US on fracture healing at present parameters might not be confined locally.

Effects of low-intensity pulsed ultrasound, dexamethasone/TGF-beta1 and/or BMP-2 on the transcriptional expression of genes in human mesenchymal stem cells: chondrogenic vs. osteogenic differentiation.

The effects of low-intensity pulsed ultrasound (LIPUS) on the differentiation of human mesenchymal stem cells (hMSCs) were investigated in this study. hMSCs were subjected to LIPUS with or without dexamethasone/transforming growth factor-beta1 (TD) or bone morphogenetic protein-2 (BMP-2) and the effects of this treatment were assessed. TD-treated hMSCs exhibited characteristic chondrogenic morphology and increased messenger RNA (mRNA) expression of chondrogenic markers and LIPUS enhanced the chondrogenic differentiation of hMSCs treated with TD. The expression of Runx2, an osteogenic transcription factor was not altered in either TD treatment group; however, a significant increase was detected in the LIPUS only group. The osteogenic appearance exhibited 3 days after LIPUS and/or BMP-2 treatment. Increases in the mRNA expression levels of osteogenic markers, Runx2 and ALP were also detected. There was no additive or altered effect with combined LIPUS and BMP-2 treatment. LIPUS alone can increase osteogenic differentiation of hMSCs and LIPUS enhances TD-mediated chondrogenic differentiation of hMSCs. Clinically, LIPUS may differentially influence bone vs. cartilage repair.

Effects of functional electrical stimulation cycling exercise on bone mineral density loss in the early stages of spinal cord injury.

OBJECTIVE: To determine whether bone mineral density loss after spinal cord injury can be attenuated by an early intervention with functional electrical stimulation cycling exercises (FESCE) and to ascertain whether the effect persists after FESCE is discontinued. DESIGN: A prospective study. SUBJECTS: Twenty-four individuals with spinal cord injury, 26-52 days after spinal cord injury, were divided into FESCE or control groups. METHODS: FESCE was applied in the initial 3 months and then suspended in the subsequent 3 months. Bone mineral density in the femoral neck and distal femur was measured using dual energy X-ray absorptiometry before training, immediately after the initial 3 months of training, and at the end of the subsequent 3 months. RESULTS: The bone mineral density decrease rate in the distal femur in the FESCE group was significantly less than that in the control group during the initial 3 months. However, there was no significant difference in the subsequent 3 months. CONCLUSION: FESCE in the early stages of spinal cord injury can partly attenuate bone mineral density loss in the distal femur. However, bone mineral density loss in the distal femur cannot be ameliorated completely by FESCE. In addition, the effect on the attenuation of bone loss in the distal femur faded once FESCE was discontinued.

Pulsed electromagnetic fields affect osteoblast proliferation and differentiation in bone tissue engineering.

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.

Cytokine release from osteoblasts in response to different intensities of pulsed electromagnetic field stimulation.

We use an in-vitro osteoblast cell culture model to investigate the effects of low-frequency (7.5 Hz) pulsed electromagnetic field (PEMF) stimulation on osteoblast population, cytokines (prostaglandin E(2) (PGE(2)), transforming growth factor beta1(TGFbeta1), and alkaline phosphatase (ALP) activity to find the optimal intensity of PEMF for osteoblast growth. The results demonstrate that PEMF can stimulate osteoblast growth, release of TGFbeta1, and, in addition, an increase of ALP activity. The synthesis and release of PGE(2) in the culture medium are reduced with increasing numbers of cells. Higher intensity does not necessarily mean increased osteoblast growth, and the most efficient intensity is about 2 mV/cm in this case. Although the lower intensities of the PEMF are yet to be determined, the results of this study can shed light on the mechanisms of PEMF stimulation on non union fracture therapy and osteoporosis prevention in the future.

Pulsed electromagnetic fields accelerate apoptotic rate in osteoclasts.

Selective control of cell function by applying specifically configured, low-energy, time-varying electromagnetic fields (EMF) has added a new, exciting dimension to biology and medicine. However, the mechanism involved is less clear. In our study, we investigated the effect of pulsed electromagnetic fields (PEMF) on induction of osteoclasts apoptosis. A statistically significant increase of apoptotic rate in osteoclasts (48 hr after isolation) was found when exposed to 7.5 Hz PEMF with induced electric fields intensity of 3.0 muv/cm for 8 (105%, p < 0.001) and 16 hr (30%, p < 0.05). However, exposure of osteoclasts to PEMF for only 1 hr showed no statistically significant differences. These findings suggest that PEMF have the ability to speed up apoptosis of osteoclasts derived from primary osteoblasts and bone marrow cells cocultures. This in vitro study, therefore, could be considered as groundwork for in vivo PEMF applications on some osteoclasts-associated bone diseases such as osteoporosis.

Pulsed electromagnetic fields prevent osteoporosis in an ovariectomized female rat model: a prostaglandin E2-associated process.

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.

Effect of pulse-burst electromagnetic field stimulation on osteoblast cell activities.

Electric stimulation has been used successfully to treat a wide range of bone disorders. However, the mechanism by which the electric fields can influence the bone cells behavior remains poorly understood. The purpose of this research was to assess the possible mechanism of the stimulatory effect of pulsed electromagnetic field (PEMF) on bone cells. A PEMF with a frequency of 15 Hz (1 G [0.1 mT]; electric field strength 2 mV/cm) were applied to neonatal mouse calvarial bone cell cultures for 14 days. The temporal effects of PEMF on the osteoblasts were evaluated by the status of proliferation, differentiation, mineralization, and gene expression on the 3rd, 5th, 7th, and 14th days of culture. Our results demonstrated that PEMF stimulation significantly increased the osteoblasts' proliferation by 34.0, 11.5, and 13.3% over the control group after 3, 5, and 7 days' culture. Although the alkaline phosphatase (ALP) staining and the mineralization nodules formation did not change, the ALP activity of the bone cells decreased significantly after PEMF stimulation. Under the PEMF stimulation, there was no effect on the extracellular matrix synthesis, while the osteoprotegerin (OPG) mRNA expression was up regulated and the receptor activator of NF-kappaB ligand (RANKL) mRNA expression were down regulated, compared to the control. In conclusion, the treatment by PEMF of osteoblasts may accelerate cellular proliferation, but did not affect the cellular differentiation. The effect of PEMF stimulation on the bone tissue formation was most likely associated with the increase in the number of cells, but not with the enhancement of the osteoblasts' differentiation.

Effects of different intensities of extremely low frequency pulsed electromagnetic fields on formation of osteoclast-like cells.

Over the past 30 years, the beneficial therapeutic effects of selected low energy, time varying electromagnetic fields (EMF) have been documented with increasing frequency to treat therapeutically resistant problems of the musculoskeletal system. However, the underlying mechanisms at a cellular level are still not completely understood. In this study, the effects of extremely low frequency pulsed electromagnetic fields (ELF-PEMF) on osteoclastogenesis, cultured from murine bone marrow cells and stimulated by 1,25(OH)(2)D(3), were examined. Primary bone marrow cells were cultured from mature Wistar rats and exposed to ELF-PEMF stimulation daily for 7 days with different intensities of induced electric field (4.8, 8.7, and 12.2 micro V/cm rms) and stimulation times (0.5, 2, and 8 h/day). Recruitment and authentication of osteoclast-like cells were evaluated, respectively, by determining multinuclear, tartrate resistant acid phosphatase (TRAP) positive cells on day 8 of culture and by the pit formation assay. During the experiments, cytokines such as tumor necrosis factor-alpha (TNF-alpha), interleukin 1-beta (IL-1beta), and prostaglandin-E(2) (PGE(2)) were assayed using the enzyme linked immunosorbent assay (ELISA). These findings suggest that ELF-PEMF can both enhance (approximately 50%) and suppress (approximately 27%) the formation of osteoclast-like cells in bone marrow culture, depending on the induced electric field intensity. In addition, consistent correlations were observed between TNF-alpha, IL-1beta, and osteoclast-like cell number after exposure to different induced electric field intensities of ELF-PEMF. This in vitro study could be considered as groundwork for in vivo ELF-PEMF clinical applications on some osteoclast-associated bone diseases.

Study of thermal effects of ultrasound stimulation on fracture healing.

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.