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Effects of Near-Field Ultrasound Stimulation on New Bone Formation and Osseointegration of Dental Titanium Implants In Vitro and In Vivo
Ultrasound in Med. & Biol., Vol. 37, No. 3, pp. 403–416, 2011 Copyright Ó 2011 World Federation for Ultrasound in Medicine & Biology Printed in the USA. All rights reserved 0301-5629/$ - see front matter
doi:10.1016/j.ultrasmedbio.2010.12.004
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Original Contribution EFFECTS OF NEAR-FIELD ULTRASOUND STIMULATION ON NEW BONE FORMATION AND OSSEOINTEGRATION OF DENTAL TITANIUM IMPLANTS IN VITRO AND IN VIVO SHIH-KUANG HSU,* WEN-TAO HUANG,y BAI-SHUAN LIU,z SHIH-MIAO LI,zx HSIEN-TE CHEN,{k and CHEN-JUNG CHANGz * Department of Dental Technology and Material Sciences, Central Taiwan University of Science and Technology, Taichung, Taiwan; y Department of Radiological Technology, Yuanpei University, Hsinchu, Taiwan; z Laboratory of Tissue-Engineering, Department of Medical Imaging and Radiological Technology, Central Taiwan University of Science and Technology, Taichung, Taiwan; x Institute of Biotechnology, National Tsing Hua University, Hsinchu, Taiwan; { Department of Orthopaedic Surgery, China Medical University Hospital; School of Chinese Medicine, China Medical University, Taichung, Taiwan; and k Department of Materials Science and Engineering, Feng Chia University, Taichung, Taiwan (Received 16 June 2010; revised 1 November 2010; in final form 3 December 2010)
Abstract—A near-field ultrasound stimulation system was designed for use in in vitro and in vivo trials. The intensity of ultrasound was studied to optimize the osseointegration of the dental titanium implant into the adjacent bone. MG63 osteoblast-like cells were seeded on commercial purity titanium (CP-Ti) plate, and then sonicated for 3 min/day at a frequency of 1 MHz and intensities of 0.05, 0.15 and 0.30 W/cm2, using either pulsed or continuous ultrasound. Cells were analyzed to determine viability (inhibition of (3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) reduction) and alkaline phosphatase (ALP). Tissue culture was performed in vitro by placing a CP-Ti plate in a cultured rat neonatal calvarial defect in response to ultrasound stimulation. In the in vivo trial, screw-shaped CP-Ti implants were inserted into the metaphysis of rabbit tibia, and then stimulated by ultrasound for 10 min daily for 30 d. All samples were processed for histomorphometric evaluation and analyzed by image system. Color Doppler ultrasonography was inspected to evaluate the supply of blood flow. Pulsed ultrasound groups had higher MTT and ALP than control. Tissue culture indicated that pulsed ultrasound groups promoted cell migration and new bone regeneration more effectively than in the control. In animal study, blood flow and mature type I collagen fibers were more prevalent around titanium implants, and bone formation was accelerated in pulsed ultrasound groups. In conclusion, low-intensity pulsed ultrasound at 0.0520.3 W/cm2 may accelerate cell proliferation and promote the maturation of collagen fibers and support osteointegration. (E-mail: [email protected]) Ó 2011 World Federation for Ultrasound in Medicine & Biology. Key Words: Near-field ultrasound, Osseointegration, Titanium, Dental implant.
Titanium is the ideal metal for intraosseous dental implants, whose surfaces allow gradual bone formation that is osteoconductive, rather than osteoinductive (Buser et al. 1999). The formation of bone on implants that are partially located in the bone marrow cavity under the compact bone starts at the endosteum (Trisi et al. 2002). Low-intensity ultrasound (0.03 to 1 W/cm2) accelerated tissue repair (Dyson and Brookes 1983; Harris 1992; Cook et al. 2001). At the cellular level, ultrasound promoted collagen synthesis in fibroblasts, bone matrix formation in osteoblast cells and aggrecan mRNA expression in chondrocytes (Webster et al. 1980; Doan et al. 1999; Reher et al. 1997; Yang et al. 1996; Parvizi et al. 1999; Zhang et al. 2002). At the cytokine level,
INTRODUCTION Effective and rapid bone implant osseointegration provides early fixation with long-term implant stability, which is a prerequisite to the clinical success of dental and maxillofacial surgery (LeGeros and Craig 1993). Effective implant osseointegration may minimize the risk of aseptic loosening, a serious complication in reconstructive surgery and joint replacement, reducing patient morbidity (Morra et al. 2003).
Address correspondence to: Chen-Jung Chang, Ph.D., Laboratory of Tissue-Engineering, Department of Medical Imaging and Radiological Technology, Central Taiwan University of Science and Technology, Taichung, Taiwan. E-mail: [email protected] 403
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ultrasound increased the production of interleukin-1 beta (IL-1b), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), nitric oxide (NO) and prostaglandin E2 (PGE2) from osteoblast cells possibly via a calcium-signaling pathway (Reher et al. 2002; Li et al. 2002; Parvizi et al. 2002). The local stimulation of hard tissues has also been evaluated. Wang et al. (2003) used a rat femoral fracture model and observed accelerated fracture repair at 21 d. The treated limbs received 200-ms bursts of 1.5 or 0.5 MHz sine waves repeated at 1.0 KHz, at an intensity of 30 mW/cm2 (spatial average temporal average intensity, ISATA). The stiffness of the treated fractures exceeded that of the control fractures, but the difference was significant only when the 1.5-MHz signal was applied. Yang et al. (1996) also evaluated several mechanical, biochemical and genetic parameters after treating the fractures with 0.5-MHz ultrasound at 50 mW/cm2 (ISATA). After three weeks, the mean maximum torque and torsion stiffness were statistically significantly greater than those in the control. Biochemical analysis failed to demonstrate significant differences in cell number, collagen or calcium content. Dyson and Brookes (1983) established that the best treatment period for effectively accelerating the repair of fibula fractures using 1.5- or 3-MHz, pulsed, 0.5 W/cm2 (ISATA) levels of ultrasound, is the first two weeks of repair (inflammatory phase). Pilla et el. (1990) indicated that low-intensity ultrasound (1.5 MHz, pulsed 0.03 W/cm2 (ISATA)) stimulates fracture repair in rabbits to such an extent that the treated limbs reached their maximum strength 17 days after injury, which compares with 28 d for the controls. However, they also identified a deleterious effect when ultrasound was applied at 1.0 W/cm2. In this study, MG63 cells were seeded on a titanium plate and sonicated at a frequency of 1 MHz and an intensity of 0.05, 0.15 or 0.30 W/cm2 (ISATA) using either pulsed or continuous ultrasound. Cells were exposed to ultrasound for 3 min per day and were then analyzed to determine cell viability. Cellular behavior was analyzed by evaluating the biochemical markers of the osteoblastic phenotype, including alkaline phosphatase activity (ALP), and by performing a cell cycle analysis. Tissue culture was also performed in vitro by placing a commercial purity titanium (CP-Ti) plate in a cultured rat neonatal calvarial defect; it was exposed to lowintensity ultrasound and observed by optical microscopy and scanning electric microscopy (SEM). In an animal study, screw-shaped smooth titanium implants were inserted into the metaphysis of rabbit tibia, and then ultrasonically stimulated for 10 min daily for 30 d. Color Doppler ultrasonography was used at various times after implantation to evaluate the blood flow supply. Finally, all samples were processed for histology and analyzed under a microscope.
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MATERIALS AND METHODS Cell cultures The MG63 osteoblast-like cells, originally isolated from a human osteosarcoma, were purchased from the Food Industry Research and Development Institute (60279, FIRDI, Hsinchu, Taiwan). The cells were incubated as adherent monolayers in Dulbecco’s minimum essential medium (DMEM) (Gibco Life Technologies Ltd; Paisley, UK), 10% heat-inactivated fetal calf serum (FCS) (PAA Laboratories; Linz, Austria), 2 mM L-glutamine (Gibco), 100 U/mL penicillin (Gibco) and 100 mg/mL streptomycin (Gibco) at 37 C in a humidified atmosphere of 5% CO2 in air. The medium was replaced twice weekly until the cells were present at a high density. They were detached from the monolayer by incubating them with trypsin-EDTA (0.25% trypsin, 1 mM EDTA) (Gibco) for 5 min at 37 C, centrifuged and recultured in DMEM until required. Ultrasound treatment A clinical ultrasound exposure system (Enraf Nonius Sonoplus 492, Rotterdam, the Netherlands) with a transducer (geometric surface 1.15cm2, effective radiating area 0.8 cm2) was calibrated, and a needle hydrophone (TNU001A, NTR Systems, Seattle, WA, USA) was used to measure the width (20 mm) and the length (20 mm) of the intensity field at the site of 18 mm away from the surface of the transducer, with a pitch of scanning 0.1 mm (Fig. 1). The 3-D and color 2-D scan acoustic intensity field map were obtained from Broadsound Corporation, Hsinchu, Taiwan. MG63 osteoblastlike cells (3 3 104 cells/well), seeded on sterilized CP-Ti plate (TP270, ASTM Grade 1/, Sumitomo Metals, Osaka, Japan), were cultured in 24-well plates and given one day to attach before they were exposed to ultrasound. The sterilized ultrasound transducer, operated at a frequency of 1.0 MHz, was secured over the culture well, which was filled with 3 mL of DMEM and 2.5% fetal bovine serum (FBS). The transducer head was lowered vertically in the culture plate, just touching the surface of the medium. Accordingly, the distance between the transducer head and the bottom of the dish was approximately 18 mm (Fig. 2). A pulsed mode of 2 ms ‘‘on’’ and 8 ms ‘‘off’ and a continuous mode were applied. To evaluate the effects of ultrasound on osteoblast cells, six separate treatment groups at exposure doses of 0 (control), 0.05 W/cm2, 0.15 W/cm2 and 0.30 W/cm2 (spatial-average, temporal-peak intensity, ISATP), in either continuous or pulsed mode for 3 min each day for 5 d, were studied. Cells were also cultured on a polystyrene culture plate without Cp-Ti plate, for comparison. The TiCon and Con groups underwent the same experimental treatments without stimulation
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Fig. 1. (a) The acoustic intensity field maps of ultrasound in near- and far-field. (b) The 3-D acoustic intensity field map of near-field ultrasound in continuous mode, 0.5 W/cm2 (Broadsound Corporation). (c) Color 2-D scan of an acoustic intensity field map of near-field ultrasound. The color bar represents linear scale of acoustic intensity (dB). And the maximum value of the acoustic intensity field map is defined 0 dB. Measure site: 18 mm from the surface of the transducer. Measure field: width (20 mm) and length (20 mm) of the intensity field.
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specimens were then studied using a SEM (FE-SEM, JSM 1025, JEOL).
Fig. 2. A picture of the ultrasound stimulation system used during cell culture trial. The whole system was placed in a sterile 37 C CO2 incubator.
by ultrasound. The ultrasound stimulation system was placed in a sterile 37 C CO2 incubator. The temperature rise of the culture media in all plates is ,0.3 C after 3 min ultrasound exposure. Every experiment was repeated four times (Table 1).
Morphological evaluation of seeded osteoblast cells After 5 d of treatment with ultrasound, the osteoblast cells that adhere to the CP-Ti plates were fixed with 10% formaldehyde in phosphate buffered saline (PBS) for 24 h for morphological evaluation. After they were washed several times with PBS, the cells were dehydrated using an ascending alcohol series over 30 min, and then dried in a critical point drier (CPD). They were then coated with gold to a thickness of around 500 3 1028 cm using a Hitachi IB-2 coater, in a high vacuum at 0.1 Torr, a high voltage of 1.0 kV and an applied current of 50 mA. All Table 1. The parameters of ultrasound treatments (frequency 1 MHz) Stimulate mode
Spatial average intensity (W/cm2)
Cp-Ti alloy
Con P5 P15 P30 C5 C30
Sham stimulate Pulse 1:4 (20%) Pulse 1:4 (20%) Pulse 1:4 (20%) Continuous (100%) Continuous (100%)
0 0.05 0.15 0.30 0.05 0.30
2 2 2 2 2 2
TiCon TiP5 TiP15 TiP30 TiC5 TiC30
Sham stimulate Pulse 1:4 (20%) Pulse 1:4 (20%) Pulse 1:4 (20%) Continuous (100%) Continuous (100%)
0 0.05 0.15 0.30 0.05 0.30
1 1 1 1 1 1
Group
Stimulation time: 3 min/d, n 5 4 cultures under each condition.
MTT assay MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl2H-tetrazolium bromide) assay quantifies mitochondrial activity by measuring the amount of a dark blue formazan product that is formed by the reduction of the tetrazolium ring of MTT. The reduction of MTT is thought to occur primarily in the mitochondria by the action of succinate dehydrogenase and constitutes an effective index of mitochondrial function (Mosmann 1983). In this investigation, osteoblast cells were treated with ultrasound for 5 d and the cell viability was evaluated using the MTT assays at 1, 3 and 5 d. Briefly, 4 h after the incubation of MTT (Sigma, St. Louis, MO, USA) at 37 C, MTT solution was removed and the insoluble dark blue formazan product was dissolved in dimethylsulfoxide (Sigma). Then, 100 mL was aspirated and poured into another 96-well cell culture plate to measure the change in absorbance at 570 nm using a microplate reader (Molecular Devices, Emax, Sunnyvale, CA, USA). ALP activity After harvesting, culture media were decanted; the cell layers were washed twice with PBS and then removed using a cell scraper. The cells were centrifuged, the pellets were washed and the cell lysates were prepared by vortexing in 500 mL deionized water plus 25 mL 1% Trito X-100, before they were homogenized by sonification. After the cells had been lysed, their total protein content was determined using a commercially available kit (Micro/Macro BCA; Pierce Chemical Co., Rockford, IL, USA). Their ALP activity was assayed by converting a colorless r-nitrophenyl phosphate to a colored r-nitrophenol, following the manufacturer’s protocol (Sigma). The color change was measured spectrophotometrically at 405 nm (Molecular Devices, MWG Biotech, Ebersberg, Germany), and the amount of enzyme that was released by the cells was quantified by comparison with a standard curve. ALP levels were normalized to the total protein content of the cells at the end of the experiment. Cell cycle analysis by flow cytometry After treatment with ultrasound, 53105 cells were collected and incubated with ice-cold PBS. The cells were then fixed with ethanol, collected and washed with PBS by centrifugation (1500 rpm, 10 min). One milliliter PBS and 200 mL RNAase A (100 mg/mL) (Sigma) were added and incubated for 30 min at 37 C to suspend the cells. The cells were dyed with 5 mL of propidium iodide (10 mg/mL) (Sigma) in the dark. The number of cells at different phases of the cell cycle was determined using
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a flow cytometer (BD FACScan, San Jose, CA, USA) after they were passed through a cell filter. In vitro neonatal calvarial tissue culture Calvarias were harvested from neonatal 3-d-old Sprague-Dawley rats. The rats were sacrificed by administering an overdose of pentothal (0.5 to 1.0 mL); the calvaria were then dissected, and the endocranial and extracranial periostea were completely removed to minimize fibroblastic impurity. The calvaria were immediately placed in PBS. A round defect with a diameter of 7 mm was then created in the central area of each parietal bone using a sterilized steel stick. A 6 3 6 3 1-mm square area CP-Ti plates were then placed in of the defect. The tissue culture unit was transferred to Fitton-Jackson’s modified Bigger’s culture medium in a 12-well cell culture plate, supplemented with 10% FBS, 20 mL/mL 200 mM glutamine, 10 mL/mL penicillin/streptomycin (500 U/mL), 25 mL/mL HEPES 1 M solution, 10 mL/mL b-glycerol-2-phosphate 1 M solution and 50 mg/ml L-ascorbic acid. Four groups—TiCon, TiP15, TiP30 and TiC5—were chosen based on the results of cell studies. The ultrasound treatments were 3 min/d and lasted for 35 d (5 wk). The medium was replaced every 3 d. Each tissue culture unit was observed under an optical microscope (Axioskop HAL 100, Carl Zeiss, Jena, Germany) weekly. After five weeks of culturing, the CP-Ti plates were removed before von Kossa’s stain. The von Kossa’s stain was used to elucidate the mineralized matrix formation. Using a histomorphometric method, we performed quantitative evaluation of the regenerated mineralized bone. The histomorphometric evaluation was performed under a light microscope (ECLIPSE E200, Nikon, Tokyo, Japan) with an image analysis system (Image-Pro Lite, Media Cybernetics, Silver Spring, MD, USA). The images were captured using a digital camera (COOLPIX 8400, Nikon, Tokyo, Japan) that was attached to the microscope and displayed on a computer monitor. Then the newly mineralized bone tissue was calculated and expressed as the percentage of the ingrown bone tissue that filled the created calvarial bone defect. In addition, the morphology of the osteoblast cells that were attached to the CP-Ti samples was studied using a SEM. Every experiment was repeated three times. Surgical procedure and ultrasound stimulation Thirty-six adult male New Zealand White rabbits, weighing 3.5–4.0 kg, were used in this study. Either the left or right tibia of the rabbits was implanted with a titanium screw-type implant with an external diameter of 3.6 mm and a length of 8 mm. All implants were sterilized using gamma radiation. General anesthesia was induced by an intramuscular injection of a combination of ketamine (Imalgene 1000, Merial Laboratoire de Toulouse,
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France) and xylazine (Rompun, Bayer, Taipei, Taiwan). After an incision had been made in the skin, fascia and periosteum, the proximal tibia was exposed by making a muscle-splitting incision. The osteotomies at the implant site were prepared in the usual manner. All drilling procedures were performed until the implants were in their final positions, in which the top surface of each implant was 2 mm above the cortical bone surface and under sterile saline irrigation. The wound was closed in layers using 4-0 Dexon sutures. Twenty-four hours after surgery, an ultrasonic stimulator was applied around the incision site on the rabbit, using a probe with an area of 1.77 cm2 (diameter 5 1.5 cm). The six treatment groups (TiCon, TiP5, TiP15, TiP30, TiC5 and TiC30) were performed 10 min daily for 20 and 30 d (6 implants/time period/group). The probe was gently applied near the incision site to prevent inflammation. Aquasonic gel was used as a coupling medium before treatment. The control underwent sham stimulation and identical treatment, using a dummy transducer without ultrasonic output. The animals were housed in temperature (22.1 C) and humidity (45%)-controlled rooms with 12-h light cycles; they had access to food and water ad libitum. All procedures were consistent with the guidelines of the Animal Care and Use Committee of the authors’ university. Color Doppler ultrasonography and histomorphometric evaluation After 5, 10, 15 and 25 d, blood flows around the CP-Ti implants were evaluated and calculated using a Color Doppler ultrasound system (Logiq Book, GE, Waukesha, WI, USA) and a 10-MHz transducer. The animals were sacrificed by intravenous injections of air under general anesthesia at 20 and 30 d after surgery. The proximal tibias that contained the implants were removed and the screw implants were removed before histological exam. The tibias were then fixed in 4% formaldehyde, dehydrated using an ascending series of alcohols and embedded for decalcified sectioning. Decalcified sections were prepared on a plane parallel to the long axis of each implant and contained the central part of the implants. The sections were stained with hematoxylin-eosin (H&E) and Masson’s trichrome stain, and histomorphometric evaluation was performed according to the method of Park et al. (2007). The percentage of periosteal bone to implant contact (PBIC%) and connective tissue to implant contact (CTIC%) over the total length of the implant was measured at days 20 and 30 (6 implants/time period/ group). PBIC% and CTIC% were measured as the percentage of the length of periosteal bone and connective tissue in direct contact with the implant surface, respectively.
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Statistical analysis Results were expressed as mean 6 standard deviation for each group of samples. All data were analyzed by analysis of variance, followed by the post hoc analytic method of Scheffe.
RESULTS Morphological evaluation of seeded osteoblast cells Figure 3 (a–f) presents SEM photographs of MG63 cells attached to the CP-Ti after 5 d of ultrasound treatment. In TiP5, TiP15 and the TiCon, the MG63 cells on the CP-Ti surface were flat and some of them had a few filopodia. In TiC5 and TiC30, the MG63 cells were more spherical than were those of the TiCon, TiP5,
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TiP15 and TiP30 groups. The cells in TiP30 group were spindle-shaped, totally flat and widely spread. MTT assay Figure 4 presents the MTT assay for MG63 cell viability. On the first day, TiP5 (0.279 6 0.017) had statistically higher cell viability than the TiCon (0.182 6 0.031). On day 3, the P5 (0.423 6 0.016) and P15 (0.490 6 0.013) had a statistically higher viability than Control (0.357 6 0.017). The TiP5 (0.433 6 0.020) and TiP15 (0.449 6 0.032) cells had a higher viability than TiCon (0.273 6 0.025). On day 5, P5 (0.795 6 0.023), P15 (0.876 6 0.018) and P30 (0.778 6 0.019) had a statistically higher viability than Control (0.648 6 0.020), and TiP5 (0.749 6 0.026), TiP15 (0.930 6 0.018), TiP30
Fig. 3. SEM photographs of osteoblast cells attached to the CP-Ti after 5 d of ultrasound treatment. (a) TiCon, (b) TiP5, (c) TiP15, (d) TiP30, (e) TiC5 and (f) TiC30 (5003).
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Fig. 4. MTT assay for MG63 cell viability on days 1, 3 and 5 in Con, P5, P15, P30, C5, C30, TiCon, TiP5, TiP15, TiP30, TiC5 and TiC30. Error bars represent mean 6 SE, n 5 4 cultures under each condition. *Groups significantly differ from control group (p , 0.05). #Groups with significantly differ polystyrene and Ti-beded group (p , 0.05).
(0.912 6 0.024) and TiC5 (0.859 6 0.031) had a higher viability than TiCon (0.581 6 0.017). In addition, TiP30 and TiC5 had a statistically higher value than P30 and C5 (0.561 6 0.040), respectively. These indicated that when the cells were cultured on the Cp-Ti plates, the ultrasound stimulation increased cell viability. ALP activity In Fig. 5, no statistical difference was found between any of the stimulated groups and the control in ALP specific activity on the first or third day. On the fifth day, TiP5 (0.46 6 0.03), TiP15 (0.42 6 0.04) and TiP30 (0.50 6 0.02) had higher ALP activities than TiCon (0.34 6 0.03). TiC30 (0.22 6 0.03) had a statistically lower ALP activity than TiCon. Interestingly, on days 3 and 5, all of the groups with polystyrene had markedly higher ALP values than all the groups with CP-Ti implants. Proliferation index of MG63 cells To study the effect of ultrasonic stimulation on the proliferation index ([G2M1S]/[G0G11G2M1S],%) of the MG63 cells on the Ti-beded culture plates, the various phases of the cell cycle were determined by flow cytometry analyses. In Fig. 6, no statistically significant difference was observed between the TiCon and any of the stimulated groups on the first or fifth days. The TiP5 (61.15 6 1.74%) and TiP15 (65.21 6 1.62%) had higher proliferation index than the TiCon (53.12 6 1.22%) on the third day.
Tissue culture Neonatal rat calvarial tissue culture. Figure 7 (a, b) presents optical photomicrographs and SEM photographs of the osteoblast cells that were attached to, and migrated on, the later side and upper surfaces of the CP-Ti plates in TiCon, TiP15, TiP30 and TiC5. Under optical microscopic observations, the number of cells and the rate of cell migration in TiP15, TiP30 and TiC5 exceeded that in TiCon in each period. In addition, the cells in the stimulated groups were aligned in one direction after three weeks. In the SEM photographs, the CP-Ti material exhibited effective osteoconduction, as demonstrated by the morphology of the osteoblast cells that grew toward the CP-Ti surfaces. The layers of the osteoblast cells in the TiCon that formed on the lateral side of the CP-Ti plates were thinner and sparser than those in the TiP15, TiP30 and TiC5 groups. These results reveal that ultrasonic stimulation may accelerate the migration and regeneration of osteoblast cells. Figure 8 shows photographs of formed mineralized bone matrix (red) and dense mineralized nodule (black), as revealed by von Kossa’s stain after five weeks. The mineralized bone matrix grew from the margins of the calvarial bone defect toward the surface of the CP-Ti plates. Table 2 statistically analyzes the quantities of regenerated mineralized bone tissues associated with test groups. The percentages of regenerated mineralized bone tissues in TiP15 (1.22 6 0.43%), TiP30 (1.82 6 0.36%) and TiC5 (1.08 6 0.26%) were statistically higher (p , 0.05) than in TiCon (0.68 6 0.21%). In
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Fig. 5. ALP activity for MG63 cell on days 1, 3 and 5 in Con, P5, P15, P30, C5, C30, TiCon, TiP5, TiP15, TiP30, TiC5 and TiC30. Error bars represent mean 6 SE, n 5 4 cultures under each condition. *Groups significantly differ from control group (p , 0.05). #Groups significantly differ from polystyrene and Ti-beded group (p , 0.05).
addition, TiP30 displayed higher mineralized bone matrix (p , 0.05) than other groups. TiP15 (0.046 6 0.019%) and TiP30 (0.042 6 0.016%) exhibited more dense mineralized nodule (p , 0.05) than other groups. Animal study Blood flow in color Doppler sonography. The blood flows in the injured area were evaluated using a color Doppler (LOGIQ Book Ultrasound, GE Healthcare)
Fig. 6. Proliferation index ([G2M1S]/[G0G11G2M1S], %) of osteoblast cells attached to the CP-Ti after 1, 3 and 5 days of ultrasound treatment in TiCon, TiP5, TiP15, TiP30, TiC5 and TiC30. Error bars represent mean 6 SE, n 5 4 cultures under each condition. *Groups significantly differ from control group (p , 0.05).
ultrasonograph from 5–25 d (Fig. 9). The number of blood flows were calculated at days 5, 10, 15 and 25 (Table 3). On the fifth day, small blood flows were observed at the interface between the Cp-Ti implant and the bone tissue in all groups. On the tenth day, TiP5 (4.3 6 1.2), TiP15 (4.8 6 1.6), TiC5 (4.1 6 1.8) and TiC30 (4.5 6 1.7) had a higher number of blood flows (p , 0.05) than the control (2.2 6 0.9). On the fifteenth day, the blood flows around the implants were clearly reduced. Finally, a few blood flows were observed around the implant and few blood flow was observed in TiP30 (0.5 6 0.8) or TiC5 (0.5 6 0.8). Histological evaluation, periosteal bone- and connective tissue-to-implant contact percentage (PBIC% and CTIC%) Figure 10 shows the radiographic appearance of implant placed in the tibia. Figure 11 presents the sections of all groups stained with H&E and Masson’s trichrome stain. The trichrome method of staining undecalcified tissues was modified to stain decalcified bone sections. Table 4 presented the PBIC% and CTIC% at days 20 and 30. In all groups, new bone formed in the root of the screw thread. On day 20, TiP5 (77.2 6 19.7, 57.8 6 9.8), TiP15 (67.5 6 9.1, 74.2 6 12.2), TiC5 (77.5 6 14.7, 62.3 6 15.9) and TiC30 (72.2 6 7.3, 60.3 6 16.1) exhibited higher PBIC% and CTIC% (p , 0.05) than TiCon (48.2 6 12.2, 39.5 6 6.4). After 30 d, TiP5 (66.1 6 11.7), TiP15 (73.9 6 14.1) and TiP30 (63.4 6 13.6) exhibited more new periosteal bone (red area) in the
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Fig. 7. The optical photomicrographs on the third week and SEM photographs on the fifth week of the osteoblast cells that were attached to, and migrated on, the lateral side and upper surfaces of the CP-Ti plates in (a) TiCon, (b) TiP15, (c) TiP30 and (d) TiC5. Arrowhead 5 cavities in the lateral side of the CP-Ti plates (dashed line).
image of H&E stain along the implant to the bone marrow cavity (p , 0.05) than did TiCon (50.2 6 8.2). Trichrome staining revealed the growth of mass connective tissue
(blue area) over the surface of the implant, and its extension to the bone marrow cavity. TiP5 (63.1 6 12.4), TiP15 (57.3 6 19.1), TiP30 (43.4 6 13.6), TiC5
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Fig. 8. Photographs of new bone stained with von Kossa in (a) TiCon, (b) TiP15, (c) TiP30 and (d) TiC5, that formed close to the CP-Ti plates that were co-cultured with rat calvaria and treated by ultrasound for five weeks (403).
(50.3 6 11.6) and TiC30 (49.4 6 16.2) displayed higher CTIC% (p , 0.05) than TiCon (25.3 6 14.4). In general, the TiC5, TiC30 and TiCon exhibited less new periosteal bone formation around the implants than did the other groups. DISCUSSION In vitro study In this study, the morphology of MG63 cells was evaluated by SEM micrographic observation. In TiP3, TiP15 and TiCon, the cells showed an obvious spreading appearance and good cell activity (distinguishable from the morphology of the cells). It has been reported that the filopodia is the evidence of cell adhesion to material surface (Nolte et al. 2001). The spreading of filopodia then grows to be thin membranes as the cell adhesion continues, and the cells become flattened on the Ti plate. This is a good index to the quality of cell growth. In
addition, the presence of filopodia on the flattened cells also indicated the good cell activity. On the contrary, the MG63 cells in TiC5 and TiC30 have not yet begun spreading at the stage of growth, and the morphology of the adhered cells was of spherical shape. These results indicate that the pulsed ultrasound groups greatly outperformed the continuous ultrasound groups in the intensity range 0.0520.3 W/cm2. MTT assay was used to evaluate the disruption of a critical biochemical function. The MTT assay results suggest that pulsed ultrasound (0.05 to 0.30 W/cm2) and low-intensity continuous ultrasound (0.05 W/cm2) positively affected the viability of MG63 cells on day 5. In addition, Con displayed similar viability with TiCon. The results showed to have no toxic effects of Cp-Ti plates on viability of primary rat osteoblast cells. The cytotoxicity tests performed with MG63 also suggested the absence of cellular damage, as reported elsewhere (Ramires et al. 2001). TiP30 and TiC5 had statistically
Table 2. Area of mineralized bone ingrowth as percentage of total (defective area) TiCon (%) Mineralized bone (red) Dense mineralized nodule (black)
0.68 6 0.21 0.022 6 0.015
TiP15 (%) 1.22 6 0.43* 0.046 6 0.019*y
Error bars represent mean 6 SE, n 5 3 cultures under each condition. * Groups significantly differ from control group (p , 0.05). y Groups with significantly differ between ultrasound stimulation groups (p , 0.05).
TiP30 (%)
TiC5 (%) y
1.82 6 0.36* 0.042 6 0.016*y
1.08 6 0.26* 0.011 6 0.016
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Fig. 9. The blood flows in the injured area on days 5, 15 and 25 were evaluated using a color Doppler ultrasonograph in (a) TiCon and (b) TiP15.
higher values than P30 and C5, respectively. These results indicated that the low-intensity ultrasonic stimulation at certain intensity may increase the viability of the MG63 cells that attached to the Cp-Ti plates. Many studies suggest that in MG63 cells ALP activity can be considered an osteoblastic phenotypic
marker and, therefore, as an index of osteoblastic differentiation (Jaiswal et al. 1997; Bruder et al. 1998; Chumakova et al. 2006). In addition, Ramires et al. (2002) declared that the ALP activity of the seeded cells was strongly correlated with the substrates that the cells attach to. In this study, cells with polystyrene (from
Table 3. Number of blood flows Day
TiCon
TiP5
TiP15
TiP30
TiC5
TiC30
5 10 15 25
2.3 6 1.4 2.2 6 0.9 1.2 6 1.0 1.0 6 0.9
2.5 6 0.8 4.3 6 1.2* 1.0 6 1.1 1.0 6 1.0
1.8 6 0.8 4.8 6 1.6* 0.7 6 0.5 1.0 6 0.9
1.6 6 0.5 2.3 6 1.4 1.3 6 0.8 0.5 6 0.8
2.6 6 1.5 4.1 6 1.8* 1.8 6 1.2 0.5 6 0.8
2.3 6 1.7 4.5 6 1.7* 1.3 6 1.0 1.7 6 1.0
Error bars represent mean 6 SE, n 5 6 implants/group. * Groups significantly differ from control group (p , 0.05).
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Because the ratio of (G2M1S) to (G2M1G0G11S) is used as a proliferation index to evaluate the proliferative status of cells, flow cytometric analysis of the cell cycle was performed in this study to compare the proliferation index of MG63 cells that were exposed to low-intensity ultrasound with the TiCon after various periods of culture. The results reveal a significant difference between the pulsed ultrasound groups (0.05 to 0.15 W/cm2) and the TiCon on the third day, confirming that ultrasonic stimulation positively affected the in vitro proliferation of MG63 cells.
Fig. 10. Radiographic appearance of implant placed in the tibia.
Con to C30) exhibited significantly higher ALP activity than cells with CP-Ti plates (from TiCon to TiC30), on days 3 and 5. These results were consistent with the report of Ramires et al. However, as for the cells cultured on CP-Ti plates, the pulsed ultrasound groups (TiP5, TiP15 and TiP30) exhibited higher ALP activity than TiCon on the fifth day. These results revealed that the pulsed ultrasound may increase the ALP activity when the cells were cultured on the CP-Ti plates.
Fig. 11. Histological evaluation of TiP15 implant 20 d after implantation, then stained with H&E (left) and Masson’s trichrome stain (right). Red area 5 periosteal bone; blue area 5 connective tissue (403).
Tissue culture In a tissue culture trial, optical photomicrographs indicated that the cell number and the rate of cell migration were higher in TiP15, TiP30 and TiC5 than in the TiCon after three weeks. Interestingly, the cells in all ultrasound-stimulated groups were aligned in a single direction, consistent with the observations of Ikegame et al. (2001). In addition, after von Kossa’s stain on the fifth week, the regenerated bone tissues grew faster from the margins of the calvarial bone defect toward the surface of the CP-Ti plates in TiP15, TiP30 and TiC5, than in the TiCon. The amount of formed mineralized bone matrix in TiP15 and TiP30 significantly exceeded the amounts formed in TiCon, which is consistent with the results of ALP. In the SEM photographs, the osteoblast cells were layered on the lateral side and the upper surface of the CP-Ti plates. The layers of the lateral side in TiCon were thinner and sparser than those in TiP15, TiP30 and TiC5. Many cavities were observed on the lateral side of the plates in TiCon, but the plates in TiP15, TiP30 and TiC5 were all covered with layers of cells. These results demonstrated that ultrasonic stimulation may accelerate the migration of osteoblast cells. In vivo study Rawool et al. (2003) suggested that low-intensity pulsed ultrasound may influence the angiogenesis phase of fracture healing. Performing a color Doppler assessment, they found that 10 days of treatment with ultrasound increased the vascularity at the osteotomy site of dog ulnae by 33%. They also suggested exposure to ultrasound produced mechanical vibrations that may have increased the permeability of the cell membrane to calcium ions, increasing the micromechanical fluid pressure of blood flow. In this study, several blood flows were observed at the interface between the Cp-Ti implant and bone tissue after 10 d. TiP5, TiP15, TiC5 and TiC30 exhibited more blood flows than the control. These findings were consistent with those of Rawool et al. Many in vivo studies suggest that low-intensity pulsed ultrasound does not influence the remodeling phase of fracture healing, but does affect the earlier inflammatory or
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Table 4. Percentage of PBIC% and CTIC% at days 20 and 30 PBIC%
CTIC%
Day
20
30
20
30
TiCon (%) TiP5 (%) TiP15 (%) TiP30 (%) TiC5 (%) TiC30 (%)
48.2 6 12.2 77.2 6 19.7* 67.5 6 9.1* 58.4 6 12.6 77.5 6 14.7* 72.2 6 7.3*
50.2 6 8.2 66.1 6 11.7* 73.9 6 14.1* 63.4 6 13.6* 55 6 12.8 60.0 6 18.3
39.5 6 6.4 57.8 6 9.8 74.2 6 12.2 43.8 6 7.4 62.3 6 15.9 60.3 6 16.1
25.3 6 14.4 63.1 6 12.4* 57.3 6 19.1* 43.4 6 13.6* 50.3 6 11.6* 49.4 6 16.2*
Error bars represent mean 6 SE, n 5 6 tibias/time period/group. * Groups significantly differ from control group (p , 0.05).
callus-formation phases of healing and, most strongly, endochondral ossification, as well as the angiogenesis phase (Pilla et al. 1990; Wang et al. 1994; Hantes et al. 2004; Yang et al. 1996; Rawool et al. 2003). Positive effects of ultrasonic treatment on the maturation of bone regenerated during distraction osteogenesis have also been observed in animal models, with considerably increased callus formation, mineral content and regenerated bond stiffness (Eberson et al. 2003; Claes et al. 2005). In an animal study, low-intensity pulsed ultrasound promoted bone regeneration under rapid distraction, and this effect was dosedependent (Chan et al. 2006). In the in vivo trial, the histological evaluation reveals that of the cells treated with pulsed ultrasound at intensity 0.15 W/cm2 was higher than that of the control on the thirtieth day. In this study, histological evaluation on day 30 yielded similar results. Upon exposure to 0.05–0.30 W/cm2 pulsed ultrasound, mass periosteal bone and connective tissue grew over the surface of the implant extending to the bone marrow cavity. Lowintensity ultrasound may accelerate the maturation of collagen fibers and support osteointegration. With respect to the effects of these two substrates (polystyrene and Cp-Ti plate), to which cells were attached on the viability of cells and the ALP activity under exposure to ultrasound, the ALP value differed remarkably between cells on the polystyrene and Cp-Ti substrates. However, the applied ultrasound intensities in these two groups were equal and the roughness of the substrate surfaces was similar. Therefore, the acoustic impedances of the substrates were identified as the main factors that affected the results. However, further studies are required. In addition, ultrasonic stimulation promoted ALP production. CONCLUSIONS In the in vivo trial, the histological evaluation reveals that the cells treated with pulsed ultrasound at intensity 0.15 W/cm2 was higher than that of the control on the thirtieth day. In this study, histological evaluation on
day 30 yielded similar results. Upon exposure to 0.05–0.30 W/cm2 pulsed ultrasound, mass periosteal bone and connective tissue grew over the surface of the implant, extending to the bone marrow cavity. Lowintensity ultrasound may accelerate the maturation of collagen fibers and support osteointegration. With respect to the effects of these two substrates (polystyrene and Cp-Ti plate), to which cells were attached on the viability of cells and the ALP activity under exposure to ultrasound, the ALP value differed remarkably between cells on the polystyrene and Cp-Ti substrates. However, the applied ultrasound intensities in these two groups were equal and the roughness of the substrate surfaces was similar. Therefore, the acoustic impedances of the substrates were identified as the main factors that affected the results. However, further studies are required. In addition, ultrasonic stimulation promoted ALP production. A direct and near-field ultrasound stimulation system was used in in vitro and in vivo trials. The intensity of ultrasound stimulation was studied to optimize the osseointegration of the dental implant into the adjacent bone. We demonstrate that low-intensity pulsed ultrasound may increase cell viability and ALP activity, regardless of whether the cells are seeded on CP-Ti plates. The results of tissue culture show that lowintensity ultrasound promotes cell migration and new bone mineralization. In the animal study, more mature type I collagen fibers and mineralized bone formation are observed at the interface between Cp-Ti implant and bone tissue when low-intensity pulsed ultrasound stimulation was applied. Acknowledgments—The authors would like to thank the National Science Council of the Republic of China, Taiwan, for financially supporting this research under Contract No. NSC-99-2632-13-166001-MY3.
REFERENCES Bruder SP, Jaiswal N, Ricalton NS, Mosca JD, Kraus KH, Kadiyala S. Mesenchymal stem cells in osteobiology and applied bone regeneration. Clin Orthop Relat Res 1998;S247–S256.
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Buser D, Nydegger T, Oxland T, Cochran DL, Schenk RK, Hirt HP, Snetivy D, Nolte LP. Interface shear strength of titanium implants with a sandblasted and acid-etched surface: A biomechanical study in the maxilla of miniature pigs. J Biomed Mater Res 1999;45:75–83. Chan CW, Qin L, Lee KM, Cheung WH, Cheng JC, Leung KS. Dosedependent effect of low-intensity pulsed ultrasound on callus formation during rapid distraction osteogenesis. J Orthop Res 2006;24: 2072–2079. Chumakova OV, Liopo AV, Evers BM, Esenaliev RO. Effect of 5-fluorouracil, optison and ultrasound on MCF-7 cell viability. Ultrasound Med Biol 2006;32:751–758. Claes L, R€ uter A, Mayr E. Low-intensity ultrasound enhances maturation of callus after segmental transport. Clin Orthop Relat Res 2005;430:189–194. Cook SD, Salkeld SL, Popich-Patron LS, Ryaby JP, Jones DG, Barrack RL. Improved cartilage repair after treatment with low intensity pulsed ultrasound. Clin Orthop 2001;391:S231–S243. Doan N, Reher P, Meghji S, Harris M. In vitro effects of therapeutic ultrasound on cell proliferation, protein synthesis, and cytokine production by human fibroblasts, osteoblasts, and monocytes. J Oral Maxillofac Surg 1999;57:409–419. Dyson M, Brookes M. Stimulation of bone repair by ultrasound. Ultrasound Med Biol 1983;(Suppl. 2):61–66. Eberson CP, Hogan KA, Moore DC, Ehrlich MG. Effect of low-intensity ultrasound stimulation on consolidation of the regenerate zone in a rat model of distraction osteogenesis. J Pediatr Orthop 2003;23:46–51. Hantes ME, Mavrodontidis AN, Zalavras CG, Karantanas AH, Karachalios T, Malizos KN. Low-intensity transosseous ultrasound accelerates osteotomy healing in a sheep fracture model. J Bone Joint Surg Am 2004;86-A(10):2275–2282. Harris M. The conservative management of osteoradionecrosis of the mandible with ultrasound therapy. Br J Oral Maxillofac Surg 1992;30:313–318. Ikegame M, Ishibashi O, Yoshizawa T, Shimomura J, Komori T, Ozawa H, Kawashima H. Tensile stress induces bone morphogenetic protein 4 in preosteoblastic and fibroblastic cells, which later differentiate into osteoblasts leading to osteogenesis in the mouse calvariae in organ culture. J Bone Miner Res 2001;16:24–32. Jaiswal N, Haynesworth SE, Caplan AI, Bruder SP. Osteogenic differentiation of purified, culture-expanded human mesenchymal stem cells in vitro. J Cell Biochem 1997;64:295–312. LeGeros RZ, Craig RG. Strategies to affect bone remodeling: Osteointegration. J Bone Miner Res 1993;8(S2):S583–S596. Li JG, Chang WH, Lin JC, Sun JS. Optimum intensities of ultrasound for PGE2 secretion and growth of osteoblasts. Ultrasound Med Biol 2002;28:683–690. Morra M, Cassinelli C, Cascardo G, Cahalan P, Cahalan L, Fini M, Giardino R. Surface engineering of titanium by collagen immobilization. Surface characterizationand in vitro and in vivo studies. Biomaterials 2003;24:4639–4654. Mosmann T. Rapid colorimetric assay for growth and survival: Application to proliferation and cytotoxicity assays. J Immunol Methods 1983;65:55–63.
Volume 37, Number 3, 2011 Nolte PA, Klein-Nulend J, Albers GH, Marti RK, Semeins CM, Goei SW, Burger EH. Low-intensity ultrasound stimulates endochondral ossification in vitro. J Orthop Res 2001;19:301–307. Park JE, Park KB, Suh JY. Effects of calcium ion incorporation on bone healing of Ti6Al4V alloy implants in rabbit tibiae. Biomaterials 2007;28:3306–3313. Parvizi J, Wu CC, Lewallen DG, Greenleaf JF, Bolander ME. Lowintensity ultrasound stimulates proteoglycan synthesis in rat chondrocytes by increasing aggrecan gene expression. J Orthop Res 1999;17:488–494. Parvizi J, Parpura V, Greenleaf JF, Bolander ME. Calcium signaling is required for ultrasound-stimulated aggrecan synthesis by rat chondrocytes. J Orthop Res 2002;20:51–57. Pilla AA, Mont MA, Nasser PR, Kahn SA, Figueiredo M, Kaufman JJ, Siffert RS. Non-invasive low-intensity pulsed ultrasound accelerates bone healing in the rabbit. J Orthop Trauma 1990;4:246–253. Ramires PA, Romito A, Cosentino F, Milella E. The influence of titania/ hydroxyapatite composite coatings on in vitro osteoblasts behaviour. Biomaterials 2001;22:1467–1474. Ramires PA, Cosentino F, Milella E, Torricelli P, Giavaresi G, Giardino R. In vitro response of primary rat osteoblasts to titania/ hydroxyapatite coatings compared with transformed human osteoblast-like cells. J Mater Sci Mater Med 2002;13:797–801. Rawool NM, Goldberg BB, Forsberg F, Winder AA, Hume E. Power Doppler assessment of vascular changes during fracture treatment with low-intensity ultrasound. J Ultrasound Med 2003;22: 145–153. Reher P, Elbeshir el-NI, Harvey W, Meghji S, Harris M. The stimulation of bone formation in vitro by therapeutic ultrasound. Ultrasound Med Biol 1997;23:1251–1258. Reher P, Harris M, Whiteman M, Hai HK, Meghji S. Ultrasound stimulates nitric oxide and prostaglandin E2 production by human osteoblasts. Bone 2002;31:236–241. Trisi P, Lazzara R, Rao W, Rebaudi A. Bone-implant contact and bone quality: Evaluation of expected and actual bone contact and machined and osseotite implants surfaces. Int J Periodontics Restorative Dent 2002;22:535–545. Wang SJ, Lewallen DG, Bolander ME, Chao EY, Ilstrup DM, Greenleaf JF. Low intensity ultrasound treatment increases strength in a rat femoral fracture model. J Orthop Res 1994;12:40–47. Wang CJ. An overview of shock wave therapy in musculoskeletal disorders. Chang Gung Med J 2003;26:220–232. Webster DF, Havery W, Dyson M, Pond JB. The role of cavitation in the ‘in vitro’ stimulation of collagen synthesis in fibroblasts. Ultrasonics 1980;18:33–37. Yang KH, Parvizi J, Wang SJ, Lewallen DG, Kinnick RR, Greenleaf JF, Bolander ME. Exposure to low-intensity ultrasound increases aggrecan gene expression in a rat femur fracture model. J Orthop Res 1996;14:802–809. Zhang ZJ, Huckle J, Francomano CA, Spencer RG. The influence of pulsed low-intensity ultrasound on matrix production of chondrocytes at different stages of differentiation: An explant study. Ultrasound Med Biol 2002;28:1547–1553.
doi:10.1016/j.ultrasmedbio.2010.12.004
d
Original Contribution EFFECTS OF NEAR-FIELD ULTRASOUND STIMULATION ON NEW BONE FORMATION AND OSSEOINTEGRATION OF DENTAL TITANIUM IMPLANTS IN VITRO AND IN VIVO SHIH-KUANG HSU,* WEN-TAO HUANG,y BAI-SHUAN LIU,z SHIH-MIAO LI,zx HSIEN-TE CHEN,{k and CHEN-JUNG CHANGz * Department of Dental Technology and Material Sciences, Central Taiwan University of Science and Technology, Taichung, Taiwan; y Department of Radiological Technology, Yuanpei University, Hsinchu, Taiwan; z Laboratory of Tissue-Engineering, Department of Medical Imaging and Radiological Technology, Central Taiwan University of Science and Technology, Taichung, Taiwan; x Institute of Biotechnology, National Tsing Hua University, Hsinchu, Taiwan; { Department of Orthopaedic Surgery, China Medical University Hospital; School of Chinese Medicine, China Medical University, Taichung, Taiwan; and k Department of Materials Science and Engineering, Feng Chia University, Taichung, Taiwan (Received 16 June 2010; revised 1 November 2010; in final form 3 December 2010)
Abstract—A near-field ultrasound stimulation system was designed for use in in vitro and in vivo trials. The intensity of ultrasound was studied to optimize the osseointegration of the dental titanium implant into the adjacent bone. MG63 osteoblast-like cells were seeded on commercial purity titanium (CP-Ti) plate, and then sonicated for 3 min/day at a frequency of 1 MHz and intensities of 0.05, 0.15 and 0.30 W/cm2, using either pulsed or continuous ultrasound. Cells were analyzed to determine viability (inhibition of (3-(4,5-Dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) reduction) and alkaline phosphatase (ALP). Tissue culture was performed in vitro by placing a CP-Ti plate in a cultured rat neonatal calvarial defect in response to ultrasound stimulation. In the in vivo trial, screw-shaped CP-Ti implants were inserted into the metaphysis of rabbit tibia, and then stimulated by ultrasound for 10 min daily for 30 d. All samples were processed for histomorphometric evaluation and analyzed by image system. Color Doppler ultrasonography was inspected to evaluate the supply of blood flow. Pulsed ultrasound groups had higher MTT and ALP than control. Tissue culture indicated that pulsed ultrasound groups promoted cell migration and new bone regeneration more effectively than in the control. In animal study, blood flow and mature type I collagen fibers were more prevalent around titanium implants, and bone formation was accelerated in pulsed ultrasound groups. In conclusion, low-intensity pulsed ultrasound at 0.0520.3 W/cm2 may accelerate cell proliferation and promote the maturation of collagen fibers and support osteointegration. (E-mail: [email protected]) Ó 2011 World Federation for Ultrasound in Medicine & Biology. Key Words: Near-field ultrasound, Osseointegration, Titanium, Dental implant.
Titanium is the ideal metal for intraosseous dental implants, whose surfaces allow gradual bone formation that is osteoconductive, rather than osteoinductive (Buser et al. 1999). The formation of bone on implants that are partially located in the bone marrow cavity under the compact bone starts at the endosteum (Trisi et al. 2002). Low-intensity ultrasound (0.03 to 1 W/cm2) accelerated tissue repair (Dyson and Brookes 1983; Harris 1992; Cook et al. 2001). At the cellular level, ultrasound promoted collagen synthesis in fibroblasts, bone matrix formation in osteoblast cells and aggrecan mRNA expression in chondrocytes (Webster et al. 1980; Doan et al. 1999; Reher et al. 1997; Yang et al. 1996; Parvizi et al. 1999; Zhang et al. 2002). At the cytokine level,
INTRODUCTION Effective and rapid bone implant osseointegration provides early fixation with long-term implant stability, which is a prerequisite to the clinical success of dental and maxillofacial surgery (LeGeros and Craig 1993). Effective implant osseointegration may minimize the risk of aseptic loosening, a serious complication in reconstructive surgery and joint replacement, reducing patient morbidity (Morra et al. 2003).
Address correspondence to: Chen-Jung Chang, Ph.D., Laboratory of Tissue-Engineering, Department of Medical Imaging and Radiological Technology, Central Taiwan University of Science and Technology, Taichung, Taiwan. E-mail: [email protected] 403
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ultrasound increased the production of interleukin-1 beta (IL-1b), basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), nitric oxide (NO) and prostaglandin E2 (PGE2) from osteoblast cells possibly via a calcium-signaling pathway (Reher et al. 2002; Li et al. 2002; Parvizi et al. 2002). The local stimulation of hard tissues has also been evaluated. Wang et al. (2003) used a rat femoral fracture model and observed accelerated fracture repair at 21 d. The treated limbs received 200-ms bursts of 1.5 or 0.5 MHz sine waves repeated at 1.0 KHz, at an intensity of 30 mW/cm2 (spatial average temporal average intensity, ISATA). The stiffness of the treated fractures exceeded that of the control fractures, but the difference was significant only when the 1.5-MHz signal was applied. Yang et al. (1996) also evaluated several mechanical, biochemical and genetic parameters after treating the fractures with 0.5-MHz ultrasound at 50 mW/cm2 (ISATA). After three weeks, the mean maximum torque and torsion stiffness were statistically significantly greater than those in the control. Biochemical analysis failed to demonstrate significant differences in cell number, collagen or calcium content. Dyson and Brookes (1983) established that the best treatment period for effectively accelerating the repair of fibula fractures using 1.5- or 3-MHz, pulsed, 0.5 W/cm2 (ISATA) levels of ultrasound, is the first two weeks of repair (inflammatory phase). Pilla et el. (1990) indicated that low-intensity ultrasound (1.5 MHz, pulsed 0.03 W/cm2 (ISATA)) stimulates fracture repair in rabbits to such an extent that the treated limbs reached their maximum strength 17 days after injury, which compares with 28 d for the controls. However, they also identified a deleterious effect when ultrasound was applied at 1.0 W/cm2. In this study, MG63 cells were seeded on a titanium plate and sonicated at a frequency of 1 MHz and an intensity of 0.05, 0.15 or 0.30 W/cm2 (ISATA) using either pulsed or continuous ultrasound. Cells were exposed to ultrasound for 3 min per day and were then analyzed to determine cell viability. Cellular behavior was analyzed by evaluating the biochemical markers of the osteoblastic phenotype, including alkaline phosphatase activity (ALP), and by performing a cell cycle analysis. Tissue culture was also performed in vitro by placing a commercial purity titanium (CP-Ti) plate in a cultured rat neonatal calvarial defect; it was exposed to lowintensity ultrasound and observed by optical microscopy and scanning electric microscopy (SEM). In an animal study, screw-shaped smooth titanium implants were inserted into the metaphysis of rabbit tibia, and then ultrasonically stimulated for 10 min daily for 30 d. Color Doppler ultrasonography was used at various times after implantation to evaluate the blood flow supply. Finally, all samples were processed for histology and analyzed under a microscope.
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MATERIALS AND METHODS Cell cultures The MG63 osteoblast-like cells, originally isolated from a human osteosarcoma, were purchased from the Food Industry Research and Development Institute (60279, FIRDI, Hsinchu, Taiwan). The cells were incubated as adherent monolayers in Dulbecco’s minimum essential medium (DMEM) (Gibco Life Technologies Ltd; Paisley, UK), 10% heat-inactivated fetal calf serum (FCS) (PAA Laboratories; Linz, Austria), 2 mM L-glutamine (Gibco), 100 U/mL penicillin (Gibco) and 100 mg/mL streptomycin (Gibco) at 37 C in a humidified atmosphere of 5% CO2 in air. The medium was replaced twice weekly until the cells were present at a high density. They were detached from the monolayer by incubating them with trypsin-EDTA (0.25% trypsin, 1 mM EDTA) (Gibco) for 5 min at 37 C, centrifuged and recultured in DMEM until required. Ultrasound treatment A clinical ultrasound exposure system (Enraf Nonius Sonoplus 492, Rotterdam, the Netherlands) with a transducer (geometric surface 1.15cm2, effective radiating area 0.8 cm2) was calibrated, and a needle hydrophone (TNU001A, NTR Systems, Seattle, WA, USA) was used to measure the width (20 mm) and the length (20 mm) of the intensity field at the site of 18 mm away from the surface of the transducer, with a pitch of scanning 0.1 mm (Fig. 1). The 3-D and color 2-D scan acoustic intensity field map were obtained from Broadsound Corporation, Hsinchu, Taiwan. MG63 osteoblastlike cells (3 3 104 cells/well), seeded on sterilized CP-Ti plate (TP270, ASTM Grade 1/, Sumitomo Metals, Osaka, Japan), were cultured in 24-well plates and given one day to attach before they were exposed to ultrasound. The sterilized ultrasound transducer, operated at a frequency of 1.0 MHz, was secured over the culture well, which was filled with 3 mL of DMEM and 2.5% fetal bovine serum (FBS). The transducer head was lowered vertically in the culture plate, just touching the surface of the medium. Accordingly, the distance between the transducer head and the bottom of the dish was approximately 18 mm (Fig. 2). A pulsed mode of 2 ms ‘‘on’’ and 8 ms ‘‘off’ and a continuous mode were applied. To evaluate the effects of ultrasound on osteoblast cells, six separate treatment groups at exposure doses of 0 (control), 0.05 W/cm2, 0.15 W/cm2 and 0.30 W/cm2 (spatial-average, temporal-peak intensity, ISATP), in either continuous or pulsed mode for 3 min each day for 5 d, were studied. Cells were also cultured on a polystyrene culture plate without Cp-Ti plate, for comparison. The TiCon and Con groups underwent the same experimental treatments without stimulation
Effects of near-field US stimulation on new bone formation d S.-K. HSU et al.
Fig. 1. (a) The acoustic intensity field maps of ultrasound in near- and far-field. (b) The 3-D acoustic intensity field map of near-field ultrasound in continuous mode, 0.5 W/cm2 (Broadsound Corporation). (c) Color 2-D scan of an acoustic intensity field map of near-field ultrasound. The color bar represents linear scale of acoustic intensity (dB). And the maximum value of the acoustic intensity field map is defined 0 dB. Measure site: 18 mm from the surface of the transducer. Measure field: width (20 mm) and length (20 mm) of the intensity field.
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specimens were then studied using a SEM (FE-SEM, JSM 1025, JEOL).
Fig. 2. A picture of the ultrasound stimulation system used during cell culture trial. The whole system was placed in a sterile 37 C CO2 incubator.
by ultrasound. The ultrasound stimulation system was placed in a sterile 37 C CO2 incubator. The temperature rise of the culture media in all plates is ,0.3 C after 3 min ultrasound exposure. Every experiment was repeated four times (Table 1).
Morphological evaluation of seeded osteoblast cells After 5 d of treatment with ultrasound, the osteoblast cells that adhere to the CP-Ti plates were fixed with 10% formaldehyde in phosphate buffered saline (PBS) for 24 h for morphological evaluation. After they were washed several times with PBS, the cells were dehydrated using an ascending alcohol series over 30 min, and then dried in a critical point drier (CPD). They were then coated with gold to a thickness of around 500 3 1028 cm using a Hitachi IB-2 coater, in a high vacuum at 0.1 Torr, a high voltage of 1.0 kV and an applied current of 50 mA. All Table 1. The parameters of ultrasound treatments (frequency 1 MHz) Stimulate mode
Spatial average intensity (W/cm2)
Cp-Ti alloy
Con P5 P15 P30 C5 C30
Sham stimulate Pulse 1:4 (20%) Pulse 1:4 (20%) Pulse 1:4 (20%) Continuous (100%) Continuous (100%)
0 0.05 0.15 0.30 0.05 0.30
2 2 2 2 2 2
TiCon TiP5 TiP15 TiP30 TiC5 TiC30
Sham stimulate Pulse 1:4 (20%) Pulse 1:4 (20%) Pulse 1:4 (20%) Continuous (100%) Continuous (100%)
0 0.05 0.15 0.30 0.05 0.30
1 1 1 1 1 1
Group
Stimulation time: 3 min/d, n 5 4 cultures under each condition.
MTT assay MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl2H-tetrazolium bromide) assay quantifies mitochondrial activity by measuring the amount of a dark blue formazan product that is formed by the reduction of the tetrazolium ring of MTT. The reduction of MTT is thought to occur primarily in the mitochondria by the action of succinate dehydrogenase and constitutes an effective index of mitochondrial function (Mosmann 1983). In this investigation, osteoblast cells were treated with ultrasound for 5 d and the cell viability was evaluated using the MTT assays at 1, 3 and 5 d. Briefly, 4 h after the incubation of MTT (Sigma, St. Louis, MO, USA) at 37 C, MTT solution was removed and the insoluble dark blue formazan product was dissolved in dimethylsulfoxide (Sigma). Then, 100 mL was aspirated and poured into another 96-well cell culture plate to measure the change in absorbance at 570 nm using a microplate reader (Molecular Devices, Emax, Sunnyvale, CA, USA). ALP activity After harvesting, culture media were decanted; the cell layers were washed twice with PBS and then removed using a cell scraper. The cells were centrifuged, the pellets were washed and the cell lysates were prepared by vortexing in 500 mL deionized water plus 25 mL 1% Trito X-100, before they were homogenized by sonification. After the cells had been lysed, their total protein content was determined using a commercially available kit (Micro/Macro BCA; Pierce Chemical Co., Rockford, IL, USA). Their ALP activity was assayed by converting a colorless r-nitrophenyl phosphate to a colored r-nitrophenol, following the manufacturer’s protocol (Sigma). The color change was measured spectrophotometrically at 405 nm (Molecular Devices, MWG Biotech, Ebersberg, Germany), and the amount of enzyme that was released by the cells was quantified by comparison with a standard curve. ALP levels were normalized to the total protein content of the cells at the end of the experiment. Cell cycle analysis by flow cytometry After treatment with ultrasound, 53105 cells were collected and incubated with ice-cold PBS. The cells were then fixed with ethanol, collected and washed with PBS by centrifugation (1500 rpm, 10 min). One milliliter PBS and 200 mL RNAase A (100 mg/mL) (Sigma) were added and incubated for 30 min at 37 C to suspend the cells. The cells were dyed with 5 mL of propidium iodide (10 mg/mL) (Sigma) in the dark. The number of cells at different phases of the cell cycle was determined using
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a flow cytometer (BD FACScan, San Jose, CA, USA) after they were passed through a cell filter. In vitro neonatal calvarial tissue culture Calvarias were harvested from neonatal 3-d-old Sprague-Dawley rats. The rats were sacrificed by administering an overdose of pentothal (0.5 to 1.0 mL); the calvaria were then dissected, and the endocranial and extracranial periostea were completely removed to minimize fibroblastic impurity. The calvaria were immediately placed in PBS. A round defect with a diameter of 7 mm was then created in the central area of each parietal bone using a sterilized steel stick. A 6 3 6 3 1-mm square area CP-Ti plates were then placed in of the defect. The tissue culture unit was transferred to Fitton-Jackson’s modified Bigger’s culture medium in a 12-well cell culture plate, supplemented with 10% FBS, 20 mL/mL 200 mM glutamine, 10 mL/mL penicillin/streptomycin (500 U/mL), 25 mL/mL HEPES 1 M solution, 10 mL/mL b-glycerol-2-phosphate 1 M solution and 50 mg/ml L-ascorbic acid. Four groups—TiCon, TiP15, TiP30 and TiC5—were chosen based on the results of cell studies. The ultrasound treatments were 3 min/d and lasted for 35 d (5 wk). The medium was replaced every 3 d. Each tissue culture unit was observed under an optical microscope (Axioskop HAL 100, Carl Zeiss, Jena, Germany) weekly. After five weeks of culturing, the CP-Ti plates were removed before von Kossa’s stain. The von Kossa’s stain was used to elucidate the mineralized matrix formation. Using a histomorphometric method, we performed quantitative evaluation of the regenerated mineralized bone. The histomorphometric evaluation was performed under a light microscope (ECLIPSE E200, Nikon, Tokyo, Japan) with an image analysis system (Image-Pro Lite, Media Cybernetics, Silver Spring, MD, USA). The images were captured using a digital camera (COOLPIX 8400, Nikon, Tokyo, Japan) that was attached to the microscope and displayed on a computer monitor. Then the newly mineralized bone tissue was calculated and expressed as the percentage of the ingrown bone tissue that filled the created calvarial bone defect. In addition, the morphology of the osteoblast cells that were attached to the CP-Ti samples was studied using a SEM. Every experiment was repeated three times. Surgical procedure and ultrasound stimulation Thirty-six adult male New Zealand White rabbits, weighing 3.5–4.0 kg, were used in this study. Either the left or right tibia of the rabbits was implanted with a titanium screw-type implant with an external diameter of 3.6 mm and a length of 8 mm. All implants were sterilized using gamma radiation. General anesthesia was induced by an intramuscular injection of a combination of ketamine (Imalgene 1000, Merial Laboratoire de Toulouse,
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France) and xylazine (Rompun, Bayer, Taipei, Taiwan). After an incision had been made in the skin, fascia and periosteum, the proximal tibia was exposed by making a muscle-splitting incision. The osteotomies at the implant site were prepared in the usual manner. All drilling procedures were performed until the implants were in their final positions, in which the top surface of each implant was 2 mm above the cortical bone surface and under sterile saline irrigation. The wound was closed in layers using 4-0 Dexon sutures. Twenty-four hours after surgery, an ultrasonic stimulator was applied around the incision site on the rabbit, using a probe with an area of 1.77 cm2 (diameter 5 1.5 cm). The six treatment groups (TiCon, TiP5, TiP15, TiP30, TiC5 and TiC30) were performed 10 min daily for 20 and 30 d (6 implants/time period/group). The probe was gently applied near the incision site to prevent inflammation. Aquasonic gel was used as a coupling medium before treatment. The control underwent sham stimulation and identical treatment, using a dummy transducer without ultrasonic output. The animals were housed in temperature (22.1 C) and humidity (45%)-controlled rooms with 12-h light cycles; they had access to food and water ad libitum. All procedures were consistent with the guidelines of the Animal Care and Use Committee of the authors’ university. Color Doppler ultrasonography and histomorphometric evaluation After 5, 10, 15 and 25 d, blood flows around the CP-Ti implants were evaluated and calculated using a Color Doppler ultrasound system (Logiq Book, GE, Waukesha, WI, USA) and a 10-MHz transducer. The animals were sacrificed by intravenous injections of air under general anesthesia at 20 and 30 d after surgery. The proximal tibias that contained the implants were removed and the screw implants were removed before histological exam. The tibias were then fixed in 4% formaldehyde, dehydrated using an ascending series of alcohols and embedded for decalcified sectioning. Decalcified sections were prepared on a plane parallel to the long axis of each implant and contained the central part of the implants. The sections were stained with hematoxylin-eosin (H&E) and Masson’s trichrome stain, and histomorphometric evaluation was performed according to the method of Park et al. (2007). The percentage of periosteal bone to implant contact (PBIC%) and connective tissue to implant contact (CTIC%) over the total length of the implant was measured at days 20 and 30 (6 implants/time period/ group). PBIC% and CTIC% were measured as the percentage of the length of periosteal bone and connective tissue in direct contact with the implant surface, respectively.
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Statistical analysis Results were expressed as mean 6 standard deviation for each group of samples. All data were analyzed by analysis of variance, followed by the post hoc analytic method of Scheffe.
RESULTS Morphological evaluation of seeded osteoblast cells Figure 3 (a–f) presents SEM photographs of MG63 cells attached to the CP-Ti after 5 d of ultrasound treatment. In TiP5, TiP15 and the TiCon, the MG63 cells on the CP-Ti surface were flat and some of them had a few filopodia. In TiC5 and TiC30, the MG63 cells were more spherical than were those of the TiCon, TiP5,
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TiP15 and TiP30 groups. The cells in TiP30 group were spindle-shaped, totally flat and widely spread. MTT assay Figure 4 presents the MTT assay for MG63 cell viability. On the first day, TiP5 (0.279 6 0.017) had statistically higher cell viability than the TiCon (0.182 6 0.031). On day 3, the P5 (0.423 6 0.016) and P15 (0.490 6 0.013) had a statistically higher viability than Control (0.357 6 0.017). The TiP5 (0.433 6 0.020) and TiP15 (0.449 6 0.032) cells had a higher viability than TiCon (0.273 6 0.025). On day 5, P5 (0.795 6 0.023), P15 (0.876 6 0.018) and P30 (0.778 6 0.019) had a statistically higher viability than Control (0.648 6 0.020), and TiP5 (0.749 6 0.026), TiP15 (0.930 6 0.018), TiP30
Fig. 3. SEM photographs of osteoblast cells attached to the CP-Ti after 5 d of ultrasound treatment. (a) TiCon, (b) TiP5, (c) TiP15, (d) TiP30, (e) TiC5 and (f) TiC30 (5003).
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Fig. 4. MTT assay for MG63 cell viability on days 1, 3 and 5 in Con, P5, P15, P30, C5, C30, TiCon, TiP5, TiP15, TiP30, TiC5 and TiC30. Error bars represent mean 6 SE, n 5 4 cultures under each condition. *Groups significantly differ from control group (p , 0.05). #Groups with significantly differ polystyrene and Ti-beded group (p , 0.05).
(0.912 6 0.024) and TiC5 (0.859 6 0.031) had a higher viability than TiCon (0.581 6 0.017). In addition, TiP30 and TiC5 had a statistically higher value than P30 and C5 (0.561 6 0.040), respectively. These indicated that when the cells were cultured on the Cp-Ti plates, the ultrasound stimulation increased cell viability. ALP activity In Fig. 5, no statistical difference was found between any of the stimulated groups and the control in ALP specific activity on the first or third day. On the fifth day, TiP5 (0.46 6 0.03), TiP15 (0.42 6 0.04) and TiP30 (0.50 6 0.02) had higher ALP activities than TiCon (0.34 6 0.03). TiC30 (0.22 6 0.03) had a statistically lower ALP activity than TiCon. Interestingly, on days 3 and 5, all of the groups with polystyrene had markedly higher ALP values than all the groups with CP-Ti implants. Proliferation index of MG63 cells To study the effect of ultrasonic stimulation on the proliferation index ([G2M1S]/[G0G11G2M1S],%) of the MG63 cells on the Ti-beded culture plates, the various phases of the cell cycle were determined by flow cytometry analyses. In Fig. 6, no statistically significant difference was observed between the TiCon and any of the stimulated groups on the first or fifth days. The TiP5 (61.15 6 1.74%) and TiP15 (65.21 6 1.62%) had higher proliferation index than the TiCon (53.12 6 1.22%) on the third day.
Tissue culture Neonatal rat calvarial tissue culture. Figure 7 (a, b) presents optical photomicrographs and SEM photographs of the osteoblast cells that were attached to, and migrated on, the later side and upper surfaces of the CP-Ti plates in TiCon, TiP15, TiP30 and TiC5. Under optical microscopic observations, the number of cells and the rate of cell migration in TiP15, TiP30 and TiC5 exceeded that in TiCon in each period. In addition, the cells in the stimulated groups were aligned in one direction after three weeks. In the SEM photographs, the CP-Ti material exhibited effective osteoconduction, as demonstrated by the morphology of the osteoblast cells that grew toward the CP-Ti surfaces. The layers of the osteoblast cells in the TiCon that formed on the lateral side of the CP-Ti plates were thinner and sparser than those in the TiP15, TiP30 and TiC5 groups. These results reveal that ultrasonic stimulation may accelerate the migration and regeneration of osteoblast cells. Figure 8 shows photographs of formed mineralized bone matrix (red) and dense mineralized nodule (black), as revealed by von Kossa’s stain after five weeks. The mineralized bone matrix grew from the margins of the calvarial bone defect toward the surface of the CP-Ti plates. Table 2 statistically analyzes the quantities of regenerated mineralized bone tissues associated with test groups. The percentages of regenerated mineralized bone tissues in TiP15 (1.22 6 0.43%), TiP30 (1.82 6 0.36%) and TiC5 (1.08 6 0.26%) were statistically higher (p , 0.05) than in TiCon (0.68 6 0.21%). In
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Fig. 5. ALP activity for MG63 cell on days 1, 3 and 5 in Con, P5, P15, P30, C5, C30, TiCon, TiP5, TiP15, TiP30, TiC5 and TiC30. Error bars represent mean 6 SE, n 5 4 cultures under each condition. *Groups significantly differ from control group (p , 0.05). #Groups significantly differ from polystyrene and Ti-beded group (p , 0.05).
addition, TiP30 displayed higher mineralized bone matrix (p , 0.05) than other groups. TiP15 (0.046 6 0.019%) and TiP30 (0.042 6 0.016%) exhibited more dense mineralized nodule (p , 0.05) than other groups. Animal study Blood flow in color Doppler sonography. The blood flows in the injured area were evaluated using a color Doppler (LOGIQ Book Ultrasound, GE Healthcare)
Fig. 6. Proliferation index ([G2M1S]/[G0G11G2M1S], %) of osteoblast cells attached to the CP-Ti after 1, 3 and 5 days of ultrasound treatment in TiCon, TiP5, TiP15, TiP30, TiC5 and TiC30. Error bars represent mean 6 SE, n 5 4 cultures under each condition. *Groups significantly differ from control group (p , 0.05).
ultrasonograph from 5–25 d (Fig. 9). The number of blood flows were calculated at days 5, 10, 15 and 25 (Table 3). On the fifth day, small blood flows were observed at the interface between the Cp-Ti implant and the bone tissue in all groups. On the tenth day, TiP5 (4.3 6 1.2), TiP15 (4.8 6 1.6), TiC5 (4.1 6 1.8) and TiC30 (4.5 6 1.7) had a higher number of blood flows (p , 0.05) than the control (2.2 6 0.9). On the fifteenth day, the blood flows around the implants were clearly reduced. Finally, a few blood flows were observed around the implant and few blood flow was observed in TiP30 (0.5 6 0.8) or TiC5 (0.5 6 0.8). Histological evaluation, periosteal bone- and connective tissue-to-implant contact percentage (PBIC% and CTIC%) Figure 10 shows the radiographic appearance of implant placed in the tibia. Figure 11 presents the sections of all groups stained with H&E and Masson’s trichrome stain. The trichrome method of staining undecalcified tissues was modified to stain decalcified bone sections. Table 4 presented the PBIC% and CTIC% at days 20 and 30. In all groups, new bone formed in the root of the screw thread. On day 20, TiP5 (77.2 6 19.7, 57.8 6 9.8), TiP15 (67.5 6 9.1, 74.2 6 12.2), TiC5 (77.5 6 14.7, 62.3 6 15.9) and TiC30 (72.2 6 7.3, 60.3 6 16.1) exhibited higher PBIC% and CTIC% (p , 0.05) than TiCon (48.2 6 12.2, 39.5 6 6.4). After 30 d, TiP5 (66.1 6 11.7), TiP15 (73.9 6 14.1) and TiP30 (63.4 6 13.6) exhibited more new periosteal bone (red area) in the
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Fig. 7. The optical photomicrographs on the third week and SEM photographs on the fifth week of the osteoblast cells that were attached to, and migrated on, the lateral side and upper surfaces of the CP-Ti plates in (a) TiCon, (b) TiP15, (c) TiP30 and (d) TiC5. Arrowhead 5 cavities in the lateral side of the CP-Ti plates (dashed line).
image of H&E stain along the implant to the bone marrow cavity (p , 0.05) than did TiCon (50.2 6 8.2). Trichrome staining revealed the growth of mass connective tissue
(blue area) over the surface of the implant, and its extension to the bone marrow cavity. TiP5 (63.1 6 12.4), TiP15 (57.3 6 19.1), TiP30 (43.4 6 13.6), TiC5
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Fig. 8. Photographs of new bone stained with von Kossa in (a) TiCon, (b) TiP15, (c) TiP30 and (d) TiC5, that formed close to the CP-Ti plates that were co-cultured with rat calvaria and treated by ultrasound for five weeks (403).
(50.3 6 11.6) and TiC30 (49.4 6 16.2) displayed higher CTIC% (p , 0.05) than TiCon (25.3 6 14.4). In general, the TiC5, TiC30 and TiCon exhibited less new periosteal bone formation around the implants than did the other groups. DISCUSSION In vitro study In this study, the morphology of MG63 cells was evaluated by SEM micrographic observation. In TiP3, TiP15 and TiCon, the cells showed an obvious spreading appearance and good cell activity (distinguishable from the morphology of the cells). It has been reported that the filopodia is the evidence of cell adhesion to material surface (Nolte et al. 2001). The spreading of filopodia then grows to be thin membranes as the cell adhesion continues, and the cells become flattened on the Ti plate. This is a good index to the quality of cell growth. In
addition, the presence of filopodia on the flattened cells also indicated the good cell activity. On the contrary, the MG63 cells in TiC5 and TiC30 have not yet begun spreading at the stage of growth, and the morphology of the adhered cells was of spherical shape. These results indicate that the pulsed ultrasound groups greatly outperformed the continuous ultrasound groups in the intensity range 0.0520.3 W/cm2. MTT assay was used to evaluate the disruption of a critical biochemical function. The MTT assay results suggest that pulsed ultrasound (0.05 to 0.30 W/cm2) and low-intensity continuous ultrasound (0.05 W/cm2) positively affected the viability of MG63 cells on day 5. In addition, Con displayed similar viability with TiCon. The results showed to have no toxic effects of Cp-Ti plates on viability of primary rat osteoblast cells. The cytotoxicity tests performed with MG63 also suggested the absence of cellular damage, as reported elsewhere (Ramires et al. 2001). TiP30 and TiC5 had statistically
Table 2. Area of mineralized bone ingrowth as percentage of total (defective area) TiCon (%) Mineralized bone (red) Dense mineralized nodule (black)
0.68 6 0.21 0.022 6 0.015
TiP15 (%) 1.22 6 0.43* 0.046 6 0.019*y
Error bars represent mean 6 SE, n 5 3 cultures under each condition. * Groups significantly differ from control group (p , 0.05). y Groups with significantly differ between ultrasound stimulation groups (p , 0.05).
TiP30 (%)
TiC5 (%) y
1.82 6 0.36* 0.042 6 0.016*y
1.08 6 0.26* 0.011 6 0.016
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Fig. 9. The blood flows in the injured area on days 5, 15 and 25 were evaluated using a color Doppler ultrasonograph in (a) TiCon and (b) TiP15.
higher values than P30 and C5, respectively. These results indicated that the low-intensity ultrasonic stimulation at certain intensity may increase the viability of the MG63 cells that attached to the Cp-Ti plates. Many studies suggest that in MG63 cells ALP activity can be considered an osteoblastic phenotypic
marker and, therefore, as an index of osteoblastic differentiation (Jaiswal et al. 1997; Bruder et al. 1998; Chumakova et al. 2006). In addition, Ramires et al. (2002) declared that the ALP activity of the seeded cells was strongly correlated with the substrates that the cells attach to. In this study, cells with polystyrene (from
Table 3. Number of blood flows Day
TiCon
TiP5
TiP15
TiP30
TiC5
TiC30
5 10 15 25
2.3 6 1.4 2.2 6 0.9 1.2 6 1.0 1.0 6 0.9
2.5 6 0.8 4.3 6 1.2* 1.0 6 1.1 1.0 6 1.0
1.8 6 0.8 4.8 6 1.6* 0.7 6 0.5 1.0 6 0.9
1.6 6 0.5 2.3 6 1.4 1.3 6 0.8 0.5 6 0.8
2.6 6 1.5 4.1 6 1.8* 1.8 6 1.2 0.5 6 0.8
2.3 6 1.7 4.5 6 1.7* 1.3 6 1.0 1.7 6 1.0
Error bars represent mean 6 SE, n 5 6 implants/group. * Groups significantly differ from control group (p , 0.05).
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Because the ratio of (G2M1S) to (G2M1G0G11S) is used as a proliferation index to evaluate the proliferative status of cells, flow cytometric analysis of the cell cycle was performed in this study to compare the proliferation index of MG63 cells that were exposed to low-intensity ultrasound with the TiCon after various periods of culture. The results reveal a significant difference between the pulsed ultrasound groups (0.05 to 0.15 W/cm2) and the TiCon on the third day, confirming that ultrasonic stimulation positively affected the in vitro proliferation of MG63 cells.
Fig. 10. Radiographic appearance of implant placed in the tibia.
Con to C30) exhibited significantly higher ALP activity than cells with CP-Ti plates (from TiCon to TiC30), on days 3 and 5. These results were consistent with the report of Ramires et al. However, as for the cells cultured on CP-Ti plates, the pulsed ultrasound groups (TiP5, TiP15 and TiP30) exhibited higher ALP activity than TiCon on the fifth day. These results revealed that the pulsed ultrasound may increase the ALP activity when the cells were cultured on the CP-Ti plates.
Fig. 11. Histological evaluation of TiP15 implant 20 d after implantation, then stained with H&E (left) and Masson’s trichrome stain (right). Red area 5 periosteal bone; blue area 5 connective tissue (403).
Tissue culture In a tissue culture trial, optical photomicrographs indicated that the cell number and the rate of cell migration were higher in TiP15, TiP30 and TiC5 than in the TiCon after three weeks. Interestingly, the cells in all ultrasound-stimulated groups were aligned in a single direction, consistent with the observations of Ikegame et al. (2001). In addition, after von Kossa’s stain on the fifth week, the regenerated bone tissues grew faster from the margins of the calvarial bone defect toward the surface of the CP-Ti plates in TiP15, TiP30 and TiC5, than in the TiCon. The amount of formed mineralized bone matrix in TiP15 and TiP30 significantly exceeded the amounts formed in TiCon, which is consistent with the results of ALP. In the SEM photographs, the osteoblast cells were layered on the lateral side and the upper surface of the CP-Ti plates. The layers of the lateral side in TiCon were thinner and sparser than those in TiP15, TiP30 and TiC5. Many cavities were observed on the lateral side of the plates in TiCon, but the plates in TiP15, TiP30 and TiC5 were all covered with layers of cells. These results demonstrated that ultrasonic stimulation may accelerate the migration of osteoblast cells. In vivo study Rawool et al. (2003) suggested that low-intensity pulsed ultrasound may influence the angiogenesis phase of fracture healing. Performing a color Doppler assessment, they found that 10 days of treatment with ultrasound increased the vascularity at the osteotomy site of dog ulnae by 33%. They also suggested exposure to ultrasound produced mechanical vibrations that may have increased the permeability of the cell membrane to calcium ions, increasing the micromechanical fluid pressure of blood flow. In this study, several blood flows were observed at the interface between the Cp-Ti implant and bone tissue after 10 d. TiP5, TiP15, TiC5 and TiC30 exhibited more blood flows than the control. These findings were consistent with those of Rawool et al. Many in vivo studies suggest that low-intensity pulsed ultrasound does not influence the remodeling phase of fracture healing, but does affect the earlier inflammatory or
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Table 4. Percentage of PBIC% and CTIC% at days 20 and 30 PBIC%
CTIC%
Day
20
30
20
30
TiCon (%) TiP5 (%) TiP15 (%) TiP30 (%) TiC5 (%) TiC30 (%)
48.2 6 12.2 77.2 6 19.7* 67.5 6 9.1* 58.4 6 12.6 77.5 6 14.7* 72.2 6 7.3*
50.2 6 8.2 66.1 6 11.7* 73.9 6 14.1* 63.4 6 13.6* 55 6 12.8 60.0 6 18.3
39.5 6 6.4 57.8 6 9.8 74.2 6 12.2 43.8 6 7.4 62.3 6 15.9 60.3 6 16.1
25.3 6 14.4 63.1 6 12.4* 57.3 6 19.1* 43.4 6 13.6* 50.3 6 11.6* 49.4 6 16.2*
Error bars represent mean 6 SE, n 5 6 tibias/time period/group. * Groups significantly differ from control group (p , 0.05).
callus-formation phases of healing and, most strongly, endochondral ossification, as well as the angiogenesis phase (Pilla et al. 1990; Wang et al. 1994; Hantes et al. 2004; Yang et al. 1996; Rawool et al. 2003). Positive effects of ultrasonic treatment on the maturation of bone regenerated during distraction osteogenesis have also been observed in animal models, with considerably increased callus formation, mineral content and regenerated bond stiffness (Eberson et al. 2003; Claes et al. 2005). In an animal study, low-intensity pulsed ultrasound promoted bone regeneration under rapid distraction, and this effect was dosedependent (Chan et al. 2006). In the in vivo trial, the histological evaluation reveals that of the cells treated with pulsed ultrasound at intensity 0.15 W/cm2 was higher than that of the control on the thirtieth day. In this study, histological evaluation on day 30 yielded similar results. Upon exposure to 0.05–0.30 W/cm2 pulsed ultrasound, mass periosteal bone and connective tissue grew over the surface of the implant extending to the bone marrow cavity. Lowintensity ultrasound may accelerate the maturation of collagen fibers and support osteointegration. With respect to the effects of these two substrates (polystyrene and Cp-Ti plate), to which cells were attached on the viability of cells and the ALP activity under exposure to ultrasound, the ALP value differed remarkably between cells on the polystyrene and Cp-Ti substrates. However, the applied ultrasound intensities in these two groups were equal and the roughness of the substrate surfaces was similar. Therefore, the acoustic impedances of the substrates were identified as the main factors that affected the results. However, further studies are required. In addition, ultrasonic stimulation promoted ALP production. CONCLUSIONS In the in vivo trial, the histological evaluation reveals that the cells treated with pulsed ultrasound at intensity 0.15 W/cm2 was higher than that of the control on the thirtieth day. In this study, histological evaluation on
day 30 yielded similar results. Upon exposure to 0.05–0.30 W/cm2 pulsed ultrasound, mass periosteal bone and connective tissue grew over the surface of the implant, extending to the bone marrow cavity. Lowintensity ultrasound may accelerate the maturation of collagen fibers and support osteointegration. With respect to the effects of these two substrates (polystyrene and Cp-Ti plate), to which cells were attached on the viability of cells and the ALP activity under exposure to ultrasound, the ALP value differed remarkably between cells on the polystyrene and Cp-Ti substrates. However, the applied ultrasound intensities in these two groups were equal and the roughness of the substrate surfaces was similar. Therefore, the acoustic impedances of the substrates were identified as the main factors that affected the results. However, further studies are required. In addition, ultrasonic stimulation promoted ALP production. A direct and near-field ultrasound stimulation system was used in in vitro and in vivo trials. The intensity of ultrasound stimulation was studied to optimize the osseointegration of the dental implant into the adjacent bone. We demonstrate that low-intensity pulsed ultrasound may increase cell viability and ALP activity, regardless of whether the cells are seeded on CP-Ti plates. The results of tissue culture show that lowintensity ultrasound promotes cell migration and new bone mineralization. In the animal study, more mature type I collagen fibers and mineralized bone formation are observed at the interface between Cp-Ti implant and bone tissue when low-intensity pulsed ultrasound stimulation was applied. Acknowledgments—The authors would like to thank the National Science Council of the Republic of China, Taiwan, for financially supporting this research under Contract No. NSC-99-2632-13-166001-MY3.
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