J Oral Maxillofac Surg 57:409419, 1999

In Vitro Effects of Therapeutic Ultrasound on Cell Proliferation, Protein Synthesis,

and Cytokine Production by Human Fibroblasts, Osteoblasts, and Monocytes

Nghiem Doan, MPH, * Peter Rehem, DDS, MSc, f

Sajeda Meghji, BSc, Mphil, PbD,$

and Malcolm Harris, BDS, MD, FDSRCS, FFDRCSIJ

Purpose: The aim of this study was to evaluate several in vitro effects of ultrasound that could revert or prevent the hypoxia, hypovascularity, and hypocellularity observed in osteoradionecrosis.

Materials and Methods: Two different ultrasound machines were evaluated, a “traditional” (1 MHz, pulsed 1:4) and a “long wave” (45 kHz, continuous) machine, tested at various intensities. Ultrasound was applied to human gingival fibroblasts, mandibular osteoblasts, and monocytes. The assays performed were cell proliferation (DNA synthesis), collagen and noncollagenous protein (NCP) synthesis, and cytokine production (ELISA) involving interleukin (IL) lp, IL-6, and IL-S, tumor necrosis factor 01 (TNFa), basic fibroblast growth factor (bFGF), and vascular endothelial growth factor (VEGF).

Results: Both ultrasound machines induced increased cell proliferation in fibroblasts and osteoblasts, between 35% and 52%. The collagen and NCP synthesis were also significantly enhanced to levels up to 112%, the best results being with the 45-kHz machine. The ELISA results showed a slight stimulation of IL-l p by all cell types; there was no difference in IL-6 and TNFol levels. The angiogenesis-related cytokines evaluated were significantly stimulated: IL-8 and bFGF production was enhanced in osteoblasts, and VEGF production was stimulated in all three cell types. Both ultrasound machines produced the same results, with the recommended intensities being 15 and 30 mW/cm 2(sA) for the 45-kHz ultrasound, and 0.1 and 0.4 W/cm2(sApA) for the 1 MHz ultrasound.

Conclusions: Therapeutic ultrasound induces in vitro cell proliferation, collagen/NCP production, bone formation, and angiogenesis. These findings support its use in prospective clinical trials for the prevention and treatment of osteoradionecrosis.

Osteoradionecrosis (ORN) is a serious long-term com- plication of radiation therapy involving a triad of hypovascularity, hypocellularity, and hypoxia. l The incidence of ORN of the jaws varies, with reported

*MSc Student, Oral and Maxillofacial Surgery, Department of Oral

and Maxillofacial Surgery, Eastman Dental Institute/UCL, London,

UK.

tAssistant Professor, Federal University of Minas Gerais, Belo

Horizonte, Brazil; and PhD Student, Department of Oral

and Maxillofacial Surgery, Eastman Dental Institute/UCL, London,

UK.

*Senior Lecturer, Department of Oral and Maxillofacial Surgery,

Eastman Dental Institute/lJCL, London, UK.

SHead of Department, Department of Oral and Maxillofacial

Surgery, Eastman Dental Institute/UCL, London, UK.

ranges from 0.7* to 44.2%.3 With adequate prevention, the incidence is still approximately 2% to 5%.4 Treat- ment options for ORN include 1) antibiotics and curettage5Jj; 2) hyperbaric oxygen (HBO) therapy,

Supported by a scholarship (to P.R.) from CAPES (no. 0469-95/5),

Ministry of Education, Brazil.

Address correspondence and reprint requests to Dr Meghji: Depart-

ment of Oral and Maxillofacial Surgery, Eastman Dental Institute for Oral

Health Care Science, 256 Gray’s Inn Rd, London WClX SLD, United

Kingdom; e-mail: s.meghji@eastman.ucl.ac.uk

o 1999Amer1can Association of Oral and Max~llofacial Surgeons

02786386/57040010$3.00/0

409

410 IN VITRO EFFECTS OF THERAPEUTIC ULTRASOUND

with or without surgery’? 3) debridment and local flaps; 4) resection and reconstruction9J0; and re- cently, 5) therapeutic ultrasound.‘l

Harrisll has suggested the use of ultrasound as an important means of revascularization of mandibular osteoradionecrosis. The patients were treated with ultrasound (3 MHz, pulsed 1:4, 1 W/cm*@~*)) for 40 sessions of 15 min/d. Ten of 21 (48%) cases showed healing when treated with debridement and ultra- sound alone, 11 cases remained unhealed after ultra- sound therapy and after debridement were covered with a local flap, and only one case needed mandibu- lar resection and reconstruction. In contrast, using conventional HBO therapy and surgery for treatment of ORN, Marx8 reported that only 15% of the cases achieved complete healing, and 70% required resec- tion and major reconstruction.

It is clear that HBO does not cure all cases of osteoradionecrosis. The use of HBO has also been severely limited by the following factors: 1) very few centers have access to HBO; 2) the cost of treatment is high; 3) the treatment may be hazardous for the patient; and 4) it is very time consuming.*J1J2 There- fore, the search has continued for a therapy that is practical and accessable. The use of ultrasound ap- pears to be a preferable option. l 1

Ultrasound (US) can be defined as sound wave or pressure wave with a frequency above the limit of the human hearing range (16 to 20 kHz).13 The unit of ultrasound is the Hertz or cycle per second. Being a propagating pressure wave, US is capable of transfer- ring mechanical energy into the tissues.‘* The energy of the US signal is absorbed, propagated, or reflected, depending on its frequency.15 US can be divided into three types: 1) Diagnostic US uses a frequency be- tween 3 and 5 MHz and low intensity (1 to 50 mW/cm2) to avoid tissue heating; 2) Disruptive ultra- sound, such as those used in ultrasonic cleaning devices, uses a very low frequency (20 to 60 kHz), and high intensity above 8 W/cm2; and 3) Therapeutic US, as used in medicine and physiotherapy, usually uses frequencies between 1 and 3 MHz and intensities of 0.1 to 2 .O W/cm2 csApA). Recently, a new US device has been developed that, instead of using the traditional frequencies of 1 to 3 MHz, uses “long wave” ultra- sound at 45 kHz.16 This lower frequency/long wave- length combination gives a widely divergent field shape, with the treated volume effectively in the far field region. This wave penetrates much deeper into the tissues, reaching areas as deep as several centime- ters, instead of millimeters as with the megahertz machines. To minimize heating effects, it uses low intensities (5 to 50 mW/cm2@*)). For clarity, the intensity measurements used here for continuous ultrasound, are spatially averaged intensity (SA), and for pulsed ultrasound are spatial average pulsed aver- aged (SAPA).

Therapeutic US can exert its physical effects on the cells and tissues by thermal and nonthermal mecha- nisms. Thermal effects are used in physiotherapy for the treatment of acute injuries, strains, and pain relief. Nonthermal effects are used in the stimulation of tissue regeneration, *‘,18 healing of varicose ulcers19 and pressure sores,2o blood flow in chronically isch- emit muscles,21 protein synthesis in fibroblasts,22-24 and tendon repair. 25 Ultrasound affects bone by induc- tion of bone formation in vitro26 and acceleration of bone repair in animals14J7-30 and humans.31J2

It has been shown that the nonthermal effects can result in healing of mandibular osteoradionecrosis. l 1 The principal value is the induction of angiogenesis, as shown by Young and Dyson.33 The new capillary formation involves activation, degradation of base- ment membrane, migration and proliferation of endo- thelial cells from preexisting venules, capillary tube formation, and maturation of new capillaries.3* Thera- peutic angiogenesis is used to reduce unfavorable tissue effects caused by local hypoxia, including osteoradionecrosis, and to enhance tissue repair.s5

The purpose of this study was to clarify the role of therapeutic US in osteoradionecrosis. Cell prolifera- tion assay (DNA synthesis) was used as a model to address the hypocellularity; collagen and noncollag- enous protein synthesis assays were used as a model for connective tissue formation, including bone forma- tion; and to address the hypovascularity and hypoxia questions, the production of inflammatory cytokines and known angiogenic factors was evaluated. To determine the most effective ultrasound regimen, two US machines (1 MHz and 45 kHz) were tested at several intensities.

Material and Methods

CELL CULTURES

The three cell types used were human mandibular osteo- blasts, gingival fibroblasts, and peripheral blood mono- nuclear cells (monocytes).

Fibroblasts and Osteoblasts The osteoblasts were cultured from bone specimens

obtained from patients undergoing surgical removal of third molars using the split bone technique. All patients had no known disease and were 20 to 30 years old. Gingival fibroblasts were obtained in a similar manner. The bone and gingival specimens were rinsed several times with phosphate- buffered saline (PBS-GIBCO, Grand Island, NY), minced, and cultured in 75-cm2 cultured flasks using Dulbecco’s modified Eagle medium (DMEM) supplemented with: 1) heat-inactivated foetal bovine serum (HIFBS), lO%(v/v) (Sigma, St Louis, MO); 2) freshly prepared L-ascorbic acid, 50 pg/mL (Sigma); 3) L-glutamine, 2 mmol/L (Sigma); and 4) penicillin/streptomycin, 100 U/mL each (Gibco BRL). The flasks were transferred into a humidified 5% C02/95% air incubator at 37°C. The media were changed twice a week. After approximately 10 days, the cells started to grow out of the explants. When the cells were confluent, they were trypsinized (0.25% w/v trypsin in PBS) and divided 1 into 3.

DOAN ET AL 411

The fibroblasts were used between the 6th and IOth passages and the osteoblasts between the 4th and 8th passages. The osteoblastic feature was confirmed with alkaline phosphatase staining. The cells were plated in six-well plates (Corning, Acton, MA), at 3 X lo5 cells/well (1.5 X lo5 for the cell proliferation assay) and returned to the incubator. The next day, the media were changed using 5 mL of 2%(v/v) HIFBS, and the cells were left for at least 1 hour before the ultrasound treatment was applied.

Peripheral Blood Mononuclear Cells (Monocytes) Monocytes (PMN) were prepared by Ficoll density gradi-

ent centrifugation. Monocytes were extracted from thor- oughly screened donated human blood obtained from the North London Transfusion Centre. The blood was diluted in equal parts with RPM1 1640 medium (Sigma), and 35 mL of this blood/RPM1 suspension was carefully layered over 15 mL Ficoll-Histopaque 1077 (Sigma). After centrifugation (1,500g for 30 minutes), the mononuclear cell layer was collected in Falcon tubes, washed with RPM1 1640 medium, and centrifuged at 1,500g for 15 minutes. This wash step was repeated and the pellet resuspended in RPM1 1640 medium containing 2% (v/v) HIFBS, L-glutamine, 2 mmol/L (Sigma), and penicillin/streptomycin, 100 U/mL each (Gibco BRL). The PMNs were counted at 5 X lo6 cells/l.5 mL/well in six-well plates. Plates were incubated for 1 to 2 hours to allow the monocytes to adhere, washed once with the prepared medium, and finally each well was filled with 5 mL medium with 2% HIFBS.

THE ULTRASOUND MACHINES EVALUATED

The two US machines evaluated were a “traditional” ultrasound machine that uses a frequency of 1 or 3 MHz, and a “long wave” machine that uses a frequency of 45 kHz. The ‘traditional’ US machine was a Therasonic 1032 unit pro- duced by Electra-Medical Supplies (EMS, Oxfordshire, United Kingdom). This apparatus can be set to work with 1 or 3 MHz and can deliver an intensity ranging from 0.1 to 2.0 W/cm2. It also has a pulsing facility and can be set to continuous or pulsed mode, pulsing 1:2, 1:4, or 1:9. The machine has an electronic control panel, a facility to do an electronic check each time it is switched on, and an alert signal if there is no coupling gel or liquid. The handset head has a flat surface and an effective radiating area of approxi- mately 2.0 cm2. The apparatus was set to 1 MHz, pulsed 1:4 (2 ms ‘on’ and 8 ms ‘o@), and the intensities evaluated were 0.1, 0.4, 0.7, and 1.0 Watts/cm2 (sApA). Several calibrations were performed during the experiments, but at least one was done before and after a set of assays. The calibration was performed at the Department of Medical Physics, University College London. At each calibration, a full elec- tronic checkup was performed, according to the manufactur- er’s manual. The acoustic output power was measured/ calibrated using a precision ultrasound balance (EMS model 67). After setup of the balance and warming up of the US machine, the measurements were taken at 0.2, 0.4,0.6, 0.8, 1.0, and 1.2 Watts/cm*. The calibration was considered in the admissible range if the error in accuracy of the output readings was 10% or less.

The “long wave” US machine (45 kHz) used in this study was a Phys-Assist unit, produced by Orthosonics Ltd (Ashbur- ton, Devon). This apparatus has a fixed frequency of 45 kHz and can deliver an intensity ranging from 5 to 50 mW/cmz GA). It does not need a pulsing facility and works only in “continuous” mode, which at low intensities does not produce harmful tissue damage. The machine also has an electronic control panel with a liquid crystal display, and a facility to calibrate itself each time it is switched on. The

handset head type is conic and has an effective radiating area of approximately 12.8 cm2. The intensities evaluated were 5, 15, 30, and 50 mW/cm2 @*). Calibration was performed several times during the experiments, but at least one was done before and after a set of assays. The calibration was performed at Orthosonics Ltd., Ashburton, Devon, and was considered satisfactory if the acoustic intensity ranged from 45 to 55 mW/cmz at power 4, with corresponding values at the other settings.

THE ULTRASOUND APPLICATION MODEL

A thermostatically controlled water bath (Electrothermal, London, England), was used to maintain a constant tempera- ture of 37°C during the assays. The tank has an internal diameter of 20 cm and a depth of 5 cm and was covered on the inferior and side walls with ultrasound-absorbing rub- ber. It was filled with distilled, deionized, demineralized water, changed before each experiment. The cells used in the experiments were prepared in six-well culture plates, which had a diameter of 35 mm, and the plate thickness was 1 mm. The plates were placed floating directly over the water surface, taking care not to let any air bubbles form between the plate and the water surface (Fig 1). The transducer was held by a microscope stand, which was placed over a rotating platform/shaker (Edmund Biihler, Germany, model KL2) set to 30 rotations/min. In this way, the transducer was constantly moved while the US was applied, avoiding the production of standing waves. The whole apparatus (water bath, transducer head, and rotating platform) was set up in a sterile air flow cabinet (Microflow Pathfinder; Intermed, Hamshire, United Kingdom). The transducer head was swabbed with 70% isopropyl alcohol BB (Azowipe; Vernon Carus, Preston, United Kingdom), left to dry, and immersed vertically into the culture well, just touching the surface of the medium. Each well of the culture plate had 5 mL medium, and in this way the distance between the transducer head and the cells/bones was approximately 5 to 6 mm. The transducer head from the 45-kHz US machine has a conical shape; therefore, only about 2 to 3 cm2 of the total area of 12.8 cm* was immersed. However, because most of the energy comes through the center of the head, we believe that there was not much energy loss. Insonation was applied to five wells (n = 5) for each intensity. Each well was insonated for 5 minutes, and the control group was treated in the same way, but with the US generator switched off. To concentrate the medium, 2

Ultrasound

transducer

Ultrasound absorb,ng Thermostatlcalb waer tank ($20 cm)

rubber controlled heater Electrothermal. England

FIGURE 1. Schematic drawing of the ultrasound irradiation model used during the assays. The transducer was kept in motion to avoid standing wave formation and inserted into the culture medium above the cells. These were plated into six well plates that were placed floating over the water tank (37°C). The whole system was placed in a sterile air flow cabinet, and insonation was applied for 5 minutes for each well.

412 IN VITRO EFFECTS OF THERAPEUTIC ULTRASOUND

mL was removed immediately after the US treatment, and the plates were cultured for a further 18 hours at 37°C in 5% CO*, 95% air.

CELL PROLIFERATION ASSAY (DNA SYNTHESIS)

This assay was performed for fibroblasts and osteoblasts. After the l&hour period following insonation, 1.5 mL of the medium was removed and stored at -70°C for the cytokine assays. The cells were radiolabeled with 5-3H thymidine (Radiochemical Centre; Amersham, Buckinghamshire, United Kingdom) to a final concentration of 0.5 pCi/mL in 1.5 mL culture medium. The cells were reincubated for another 6 hours, and the medium was then removed and 1 mL 5% trichloroacetic acid (TCA) was added, stopping the culture, and left at 4°C for at least 2 hours. The TCA was then removed and the cells were washed three times with PBS. Then 300 I.IL of sodium hydroxide (NaOH) 0.5 mol/L was added to each well and left for 20 to 30 minutes at 4°C. This was removed and transferred to scintillation vials (Mini- tubes, Hughes and Hughes Ltd, London, England) contain- ing 200 IJL of acetic acid 0.5 mol/L. Scintillation fluid (Unisolve 1; Koch-Light; London, United Kingdom) was added to each tube (4 mL), and radioactivity measured with a Beta counter (Rackbeta, LKB, Wallac, Finland), with external standardization expressed in disintegrations per minute.

COLLAGEN/NON-COLLAGENOUS PROTEIN SYNTHESIS ASSAYS

This assay was also performed on the fibroblasts and osteoblasts. After the l&hour period after insonation, 1.5 mL of the medium was removed for the cytokine assays. The cells were radiolabeled with 5-‘H proline (Radiochemical Centre, Amersham) to a final concentration of 2 yCi/mL in 1.5 mL of culture medium. The cells were reincubated for another 6 hours, and thereafter 700 ILL of the medium was transferred to Eppendorf tubes containing 700 uL of 10% TCA (final concentration: 5% TCA) and left at 4°C for at least 2 hours. The tubes were centrifuged at 4°C (2,500g for 30 minutes) to remove unbound isotope and small peptides from the cells, and the supernatant was discarded. The pellets were dissolved in 1 mL of 0.5 mol/L acetic acid containing pepsin (0.5 mg/mL; EC 3.4.4.1, Sigma) and left at 4°C overnight (16 hours). In this way, pepsin extracted the collagen by digesting the noncollagenous protein. Rat acid- soluble collagen was added (100 uL of collagen at 5 mg/mL in 0.5 mol/L acetic acid) to act as a carrier for the newly formed collagen. Collagen was precipitated over 3 hours at 4°C by the addition of sodium chloride (NaCl) to a final concentration of 5% (w/v) in 0.5 mol/L acetic acid. The tubes were then mixed gently and centrifuged at 4,000 g for 30 minutes at 4°C. The supernatant containing the noncollag- enous protein (NCP) was stored in scintillation vial inserts (Minitubes, Hughes and Hughes Ltd., England). The pellets were redissolved’m 1 mL of 0.5 mol/L acetic acid, and the collagen was reprecipitated with NaCl, as described, for 2 to 3 hours. The tubes were then centrifuged again at 5,000 to 8,000 g for 30 minutes at 4°C and the second supernatant (NCP) was stored in the same scintillation vial used for the first supernatant. The final precipitates of purified collagen were resuspended in 400 I-IL of 0.5 mol/L acetic acid and transferred into another disposable scintillation vial insert. Each scintillation vial was filled with 4 mL scintillation fluid (Unisolve 1, Koch-Light), and the radioactivity was mea- sured as for the proliferation assays.

ELISA ASSAYS FOR INTERLEUKIN-1 f3 (IL-1 p), IL-6, IL-8, AND TUMOR NECROSIS FACTOR a, (TNF-CY)

These measurements were performed for fibroblasts, osteoblasts, and monocytes. The medium from the fibro- blasts and osteoblasts was obtained in the previous experi- ments before radiolabeling. The monocyte medium was obtained in separate experiments. The following antibodies and standards were used:

Coating antibodies (diluted in bicarbonate coating buffer, pH 8.2 to 8.3) used were IL-18-immunoaftinity-puritied polyclonal antibodies from sheep anti-IL-lp serum S77/BM, diluted to 2 pg/mL; IL-6-immunoaftinity-purified polyclonal antibodies from goat anti-rh IL-6 serum G15O/BM, diluted to 1 pg/mL; IL-8-immunoaffinity-purified polyclonal antibodies from sheep anti-human IL-S serum S333/BM, diluted to 2 I.lg/mL; tumor necrosis factor alpha (TNFo)-fast protein liquid chromatography (FPLC)-purified monoclonal mouse anti-human TNFol 101-4, diluted to 2 pg/mL.

Detecting antibodies (biotinylated immunoaftinity puri- fied antibodies) used were IL-18 goat anti-IL-18 serum G102/BM (diluted l:l,OOO); IL-6-goat anti-rh IL-~ serum GlSO/BM (diluted 1:500); IL-S-sheep anti-human IL-8 serum S333/BM (diluted l:l,OOO); TNFor-biotinylated FPLC-purified polyclonal antibodies from sheep anti-human TNFa serum H/34 or H/91 (diluted 1:200).

Standards (human recombinat standards) were IL-1B (IS. 86/680, 1 pg/mL) and IL-6 (I.S. 89/548, 1 ug/mL), at a concentration range of 8,000 to 1.0 pg/mL; IL-8 (NIBSC 89/520, 1 ug/mL), and TNF-cx (NIBSC 87/650), at a concentra- tion range of 10,000 to 1.0 pg/mL.

Technique Microtiter plates were coated with 100 FL/well of coating

antibody, and the plates were incubated overnight at 4°C. Unbound coating antibody was removed by washing the plates three times with wash/dilution buffer, pH 7.2 to 7.4 (NaCI, 0.5 mol/L; NaHaP04, 2.5 mmol/L; NaaHP04, 7.5 mmol/L; and Tween 20, 0.1% v/v). Standards of the cyto- kines and the supernatants to be tested were added to the remaining wells (100 ltL volumes). Plates were incubated for 4 hours (3 hours for IL-8, and 2 hours for IL-6 and TNFo() at room temperature and washed three times with wash/ dilution buffer. Detecting antibody (100 uL) was added to each of the wells and incubated for a further 1 hour at room temperature. Plates were washed three times with wash/ dilution buffer, and 100 FL avidin horseradish peroxidase (Avidin-HRP, Dako Ltd) diluted 1:4,000 in wash-dilution buffer was added into each well. Plates were incubated for 15 minutes at room temperature before washing three times with wash/dilution buffer. Wells were developed with 100 uL of color reagent (0.4 mg orthophenylenediamine and 0.4 ltL of 30% hydrogen peroxide [H202] in 1 mL of 0.1 mol/L citric acid phosphate buffer, pH 5.0) and incubated for 15 to 30 minutes at room temperature. The reaction was terminated by the addition of 150 uL of 1 mol/L sulfuric acid (HaSO& and the absorbance was measured at 492 nm on a Titertek Multiscan spectrophotometer (Flow, Irwing, Scot- land). A standard curve was plotted of the absorbance (optical density) versus the concentration of the standards.

BASIC FIBROBLAST GROWTH FACTOR (BFGF) and VASCULAR ENDOTHELIAL GROWTH FACTOR (VEGF) IMMUNOASSAY

These proteins were assayed using a quantitative sand- wich enzyme immunoassay technique (R&D Systems; Abing- don, Oxon, United Kingdom). Briefly, a monoclonal anti-

DOAN ET AL

body specific for bFGF or VEGF was precoated on 96well plates. Standards and samples (assayed in duplicates or triplicates) were pipetted into the wells and left to bind to the antibody. After washing away any unbound substances, an enzyme-linked polyclonal antibody specific for bFGF or VEGF was added to the wells. After a wash to remove any unbound antibody-enzyme reagent, a substrate solution was added to the wells and color developed in proportion to the amount of the protein bound in the first step. The color development was stopped and the intensity measured at 570 nm. Standard curves were obtained as usual.

STATISTICAL ANALYSIS

Each cell insonation experiment was repeated at least twice. The number of observations for controls and for each intensity evaluated was five (n = 5). The medium was assayed by enzyme-linked itnmunosorbent assay (ELISA) at least in duplicate. The results obtained were analyzed in Microsoft Excel (Seattle, WA), using ANOVA single factor and Student’s t-test for unpaired samples. Significance was accepted at the P < .05 level or higher.

Results

CELL PROLIFERATION (DNA SYNTHESIS)

The cell proliferation assays showed an increase in DNA synthesis with both ultrasound machines. The fibroblast group treated with 45 kHz (Pig 2A) showed an increase of 30% and of 43% with 15 and 50 mW/cm*, respectively (P < .Ol). With fibroblasts treated with 1 MHz ultrasound (Fig 2B), the most significant results were an increase of 47% at 0.7 W/cm2 (P < .Ol) and of 37% at 1.0 W/cm2 (P < .05).

When the osteoblasts were treated with 45 kHz

FIGURE 2. Cell proliferation as- says [incorporation of 3H thymi- dine into the DNA] show the results for fibroblasts treated with A, 4.5 kHz ultrasound, and 5, 1 MHz ultrasound. The graphs show the results for osteoblasts treated with C, 45 kHz ultrasound and D, 1 MHz ultrasound. Controls received the same treatment, but with the US generator switched off. Values

are given as percentages (%) of the controls + SEM. Significance level as compared with controls (sham insonated): *P < .05, **P < .Ol, ***PC ,001.

413

ultrasound (Pig 2C), a more uneven distribution oc- curred, showing an increase of 32% at 5 mW/cm* and of 35% at 30 mW/cm* (P < .05 and P < .Ol). With 1 MHz ultrasound (Fig 2D), again an increase in DNA synthesis was observed at the higher intensities. This was in the order of 34% at 0.7 W/cm2 (P < .Ol) and of 52% at 1.0 W/cm* (P < .OOl).

COLtAGEN/NONCOLtAGENOUS PROTEIN SYNTHESIS

The fibroblast group treated with 45 kHz ultrasound (Fig 3A) showed increases in collagen ranging from 37% to 44%, although this was significant only at 15 and 50 mW/cm* (P < .Ol and P < .05). When using l-MHz ultrasound there was a clear tendency for increased collagen production at the lower intensities (Fig 3B), with increases of 48%, 57%, and 52%, at 0.1, 0.4, and 0.7 W/cm2, respectively (P < .Ol, .05, and .Ol, respectively).

The collagen/NCP production by osteoblasts was the most significant, because these are the target cells in the repair in osteoradionecrosis. With the 45-kHz ultrasound regimen, the increased production of colla- gen was significantly higher than with the 1 MHz regimen, of the order of 112% at 30 mW/cm* (P < .05) (Fig 3C). Furthermore, the noncollagenous protein synthesis was significantly increased at all intensities evaluated (P < .Ol) ranging from 59% to 88%. The l-MHz-treated cells also showed increased collagen production at the lower intensities (Fig 3D), with increases in collagen synthesis of 55% and 38% at 0.1

A Penentagrd ccmds B

IN VITRO EFFECTS OF THERAPEUTIC ULTRASOUND

and 0.4 W/cm2, respectively (P -=c .05>; the increase in noncollagenous protein synthesis was only significant at 0.4 W/cm2 (P < .05>.

CYTOKINE PRODUCTION

The induction of IL-lp synthesis by the three cell types was produced by the 1 MHz ultrasound, but it was only induced in monocytes by the 45 KHz ultrasound. However, the levels of stimulation, al- though significantly different from controls, were still very low for osteoblasts and fibroblasts. Monocytes showed the highest level of IL-lfi synthesis using 45 kHz, followed by osteoblasts with 1 MHz (Fig 4).

The production of IL6 was not stimulated in any of the three cell types, and TNFol production in fibro- blasts was low and only reached significance with the 5 mW/cm2 and 45 KHz (data not shown).

The angiogenesis-related cytokines, IL-S, bFGF, and

FIGURE 3. Collagen and noncol-

lagenous protein synthesis assays showing the results for fibroblasts treated with A, 4.5 kHz ultrasound and with 5, 1 MHz ultrasound. C,

ihe graph (C) shows the results for osteoblasis treated with 45 kHz ultrasound and D, with 1 MHz ultrasound. Controls received same treatment, but with the US genera-

ior switched off. Values are given as percentages 1%) of the controls + SEM. Significance level as com- pared with controls (sham in-

sonated): *P < .05; **P < .Ol, ***p<.oo1.

VEGF, were significantly stimulated in the following manner: 1) The production of IL-S was enhanced in the osteoblasts treated with both ultrasound machines (Fig 5), but at higher levels with 1 MHz ultrasound. However, IL-S production in monocytes and fibro- blasts did not differ from controls; 2) bFGF production was significantly elevated in osteoblasts (Fig 6) with both US machines, and 3) VEGF was significantly stimulated at higher levels than bFGF in all three cell types (Fig 7). Once again, this occurred with both machines.

In summary, the angiogenesis-related cytokines, IL-S and bFGF, were significantly stimulated in osteo- blasts, and VEGF was significantly stimulated in osteo- blasts, fibroblasts, and monocytes. Both ultrasound machines produced significant results, and the best intensities were 15 and 30 mW/cm2csA) with 45 kHz US, and 0.1 and 0.4 W/cm2(sApA) with 1 MHz US.

FIGURE 4. A, IL-1 p production

by monocytes stimulated by 5 min- utes of 45 kHz continuous ultra-

sound. Medium of the cells was collected 18 hours after stimula- tion and assayed by ELISA. 5, It-1 p production by osteoblasts stimulated by 5 minutes of 1 MHz pulsed 1 :4 ultrasound. Medium of

the cells was collected 18 hours after stimulation and assayed by ELISA. Controls on A and B were sham-insonated. Bars show mean values + SEM. Significance level as compared with controls (sham insonated]: *P < .05, **P < .Ol, ***p< ,001.

DOAN ET AL 415

IL8 @@nl, 1200

FIGURE 5. Ii-8 production by ,100 osteoblasts stimulated by A, 45 kHz and b B, 1 MHz ultrasound. “” Medium o the cells was collected Y 9w 18 hours after stimulation and as- sayed by ELISA. Controls in A and *On 5 were sham-insonated. Bars show mean values + SEM. Significance

,oo

level as compared with controls 600 [sham insonated): *P < .05, **p< .Ol, ***p< ,001. 500

400

Discussion

Wound healing proceeds through a complex series of events involving inflammatory, proliferative, and remodeling phases.s6 The proliferative phase of wound healing consists of rapid fibroblast growth and in- creased synthesis of collagen/NCP in response to chemotactic factors released during the inflammatory phase. Thus, the release of inflammatory cytokines and the increase in osteoblast and fibroblast prolifera- tion observed in this study simulate the inflammatory and proliferative phases of wound healing. Wound healing is marked by angiogenesis through which ingrowth of capillaries accompanies fibroblast and osteoblast (in the case of bone) proliferation to form granulation tissue. Finally, fibroblasts maintain colla- gen production, which accumulates during the remod- eling phase.

Wound healing and tissue regeneration can be impaired by underlying medical factors such as diabe- tes mellitus, connective tissue diseases, chronic ve- nous insufficiency, cachexia, smoking, previous radia- tion therapy, cytotoxic therapy, and infection. Compromised angiogenesis is a major reason for delayed healing or nonhealing in most of these cases. In nonhealing wounds such as ORN, a perceived problem is a nonstimulatory level of hypoxia resulting from inadequate perfusion. Moderate levels of hyp-

oxia may facilitate wound healing by inducing the synthesis of collagen precursors and activating macro- phages to stimulate angiogenesis.” However, with increased levels of hypoxia, a reduction in fibroblast migration and lower collagen synthesis, with impaired hydroxylation of lysine and proline, can be ob- served.3’ Fibroblasts synthesize an intracellular pep- tide collagen precursor but fail to release it. Matura- tion and cross-linking of collagen is inversely proportional to the degree of hypoxia but responds to a small increase in oxygen concentration.

Therapeutic angiogenesis is the term used to de- scribe the controlled induction or stimulation of neovascularization and neocellularization for the treat- ment or prevention of pathologic clinical situations characterized by local hypovascularity.35 Healing and tissue regeneration can be improved or accelerated by therapeutic angiogenesis. Traditionally, surgical meth- ods have been used to achieve therapeutic angiogen- esis, that is, the transposition of autologous tissues with uncompromised vasculature and high angio- genie potential such as muscle flapss5 as was used originally by Harris. l1 The classic surgical ways of producing therapeutic angiogenesis might be supple- mented in the near future by the local application of angiogenic factors and the implantation of autologous capillary endothelial cells cultured ex vivo. Consider-

FIGURE 6. FGFb production by osteoblasts stimulated by A, 45 kHz and b 5, 1 MHz ultrasound. Medium o Y the cells was collected 1 8 hours after stimulation and as-

sayed by ELISA. Controls in A and 6 were sham-insonated. Bars show mean values + SEM. Significance level as compared with controls [sham insonated): *P < .05, **p< .Ol, ***p< ,001.

416 IN VITRO EFFECTS OF THERAPEUTIC ULTRASOUND

T **

MGF IwhW C 135 /-~-------- --.-.. 1

FIGURE 7. VEGF production af- ter ultrasound stimulation in (A and

5) monocytes and osteoblasts (C and D). The graphs on the left (A, C) show cells stimulated with 45 kHz ultrasound, and the graphs on

the right (6, 0) show cells stimu- lated with 1 MHz ultrasound. Me- dium of the cells was collected 1 8 hours after stimulation and as-

able experimental data, as well as some preliminary clinical data, exist to support the usefulness of angio- genie factors for therapeutic angiogenesis.

Ultrasound therapy is the simplest way of produc- ing therapeutic angiogenesid. Young and Dyson33 reported the induction of angiogenesis by US, ob- served in rat skin lesions. There is considerable clinical evidence to support Young and Dyson’s work, as shown by the several US effects mentioned in the literature.*1J4J7-s2 Osteoradionecrosis is specifically an area that benefits from therapeutic angiogenesis. The tissues have a complex metabolic/homeostatic defi- ciency, bordering on ischemic necrosis, and are prone to breakdown, leading to a chronic nonhealing wound. Therefore, the treatment or prevention of this compli- cation aims to restore the mandibular blood supply as well as restore the normal soft tissue and bone homeostasis.

The proliferation assays (DNA synthesis) showed that both US machines were able to induce prolifera- tion in fibroblasts and osteoblasts (Fig 2). This is a crucial effect, because osteoradionecrosis is a hypocel- lular tissue and because these assays are a measure of connective tissue formation. Our data showed an increase around 30% to 50% in sH-thymidine incorpo- ration, which was similar to that observed with HBO used to stimulate fibroblasts cell proliferation.38 How- ever, this also can be interpreted as a deleterious affect, because the cells may be involved in cellular division and not in the production of collagen and other physiologic proteins. This was observed when

sayed by EllSA. Controls were sham-insonated. Bars show mean

values + S.E.M. Significance level as compared with controls (sham

insonated]: *P < .05, **f < .Ol, ***PC ,001.

the cells were stimulated with the l-MHz machine, when increased proliferation was observed at intensi- ties that caused reduced collagen production (Fig 2D and Fig 3D). The 45-kHz US showed similar observa- tions in fibroblasts; however, with osteoblasts the proliferation was high at the same intensities that increased collagen synthesis (Fig 2C and Fig 3C).

The collagen/NCP synthesis assays showed that both ultrasound machines induced collagen produc- tion. However, in osteoblasts the 45-kHz US machine produced much higher increases in collagen synthesis (up to 112%) than the l-MHz machine (maximum of 55%) (Fig 3A, 30 With the l-MHz machine, this was more evident at lower intensities, both in fibroblasts and in osteoblasts (Fig 3B, 3D). These results were higher than those observed by Harvey et a1,22 who found an increase of both collagen and NCP synthesis, which was intensity dependent, using human skin fibroblasts insonated in suspension and subsequently cultured in vitro. Fibroblasts exposed to continuous US (0.5 W/cm2 @*‘> showed a 20% increase in collagen secretion, which was increased to 30% when the US was pulsed (0.5 W/cm2 csApA)). Webster et al24 also observed smaller increases in protein synthesis by fibroblasts, 29% using a ~-MHZ signal at 0.5 W/cmZ. We showed that 3 MHz pulsed US stimulates bone forma- tion (collagen and NCP production) using a mouse calvarium model, with the best results at 0.1,0.25, and 0.5 W/cm2 c2@. The NCP synthesis was also stimulated, but it could not be correlated to the collagen synthesis in most of the assays. Furthermore, the NCP synthesis

DOAN ET ;u. 417

increase reached significance only in osteoblasts, and mainly with the 45-kHz US (Fig 3C), where all intensi- ties significantly stimulated the cells. This may be an important observation because the NCP contains many cytokines, growth factors, angiogenic factors, and enzymes that may enhance healing and angiogen- esis.

The intensity of US previously used in the clinical treatment of osteoradionecrosis” was relatively high (3 MHz, pulsed 1:4, 1.0 W/cm2(SAPA)). The favorable results observed could therefore be explained in terms of US promoting angiogenesis33 rather than to the effects on collagen protein synthesis, which is higher at lower intensities. This was supported by the use of near-infrared spectroscopy scans of irradiated mandibles,39 which showed higher levels of deoxyhe- moglobin concentration in the osteoradionecrotic mandibles of patients after treatment with this ultra- sound regimen. This suggests significant improve- ment in the metabolic activity of the mandibular tissue, probably due to neo-angiogenesis.

This finding led us to study the stimulation of cytokine and angiogenic factors. The role of cytokines and angiogenic factors in wound healing and tissues regeneration is now well documented in the literature (Table 1).40-67 Cytokines are considered to be intercel- lular signals that regulate local and systemic inflamma- tory and anti-inflammatory responses. In comparison with endocrine hormones, cytokines are produced by a variety of cells rather than by a specialized group of cells3s These substances vary widely in origin, molecu- lar weight, chemical composition, and biological activ- ity. Most are only partly characterized.s* Enzyme- linked immunosorbent assays (ELISA) have been used extensively for identifying a variety of cytokines, in particular those that are angiogenic factors.

The results presented in this report suggest that the

Endothelial Endothelial Cell Cell

Name Migration Proliferation

JJZGF*O*~ Yes Yes TVPF44 Yes Yes PD-ECGF45x46 Yes Yes E&Q?*‘-49 Yes Yes Angiotropin50~51 Yes Yes Angiogenin52-54 No effect No effect/yes bFGF%-5’ * Yes Yes IL-85659 Yes Yes IGF-160 Yes Yes TGF@ Yes Yes TGF136264 * No (inhibits) No (inhibits) T~~~6566 * Yes No (inhibits) PDGFG7 Yes Yes

*These factors also promote tube formation.65

healing effect of US on soft tissues, fractures, and osteoradionecrosis may all be the result of the stimula- tion of angiogenic factors such as IL-S, bFGF, and VEGF. As can be observed in the ELISA results, particularly VEGF, a potent angiogenic factor,43 was significantly stimulated in all three cell types. Mechani- cal stress can induce a significant increase in the synthesis of IL-l-like factors in deformed osteoblast cultures.@ However, no study on the effect of US on cytokine production has been documented in the literature. It was notable that the production of IL-lp was stimulated at low levels, and this may enhance the production of IL-S, because it has general immunopo- tentiating activities. However, IL-~ and TNFo were not significantly stimulated, suggesting that the angiogen- esis stimulation may be a mechanism distinct from inflammation.

These cytokines could have been released into the insonated media either by disruption of the cell membrane or by an increase in either exocytosis or in permeability of the membrane.33 All cell types used in the experiment were counted, and postirradiation viability tests were performed (cell proliferation). These indicated that the osteoblasts, fibroblasts, and monocytes were not lethally damaged. Therefore, it seems more likely that there was a change in either exocytosis or membrane permeability.

The exact cellular mechanism underlying the thera- peutic action of US is still unknown. From the current literature, Yang et al’* proposed several possibilities. First, the compression of microtubules, or cavitation, could produce oscillatory movement of microbubbles and acoustic streaming, and have a direct effect on the permeability of the cell membrane and second messen- ger adenylate cyclase activity.@ Such changes in ion or protein transport could consequently modify intracel- lular signals for gene expression.24,69-71 Second, the effects of mechanical pressure at the cell surface could activate the “stretch receptor” type of cation channel proposed by Sachs,72 and changes in cation concentra- tion also could modify intracellular signals regulating gene expression. Third, the mechanical energy trans- ferred by the US might activate changes in the attachment of the cytoskeleton to the extracellular matrix. Wang et al73 showed in their “tensegrity model” that the application of mechanical forces to the cytoskeleton affects cell metabolism and gene expression. This was also confirmed by Sandy,@ who showed that mechanical stress can induce a significant increase in the synthesis of IL-l -like factors compared with nonstressed osteoblast cultures. Fourth, electri- cal currents in bone may be potentiated by exposure to US energy. Investigators have reported increased potentials as a function of US intensity, frequency, and burst pattern.74z75 Finally, a rise in temperature during ultrasonic exposure may have an effect on cell metabo-

418 IN VITRO EFFECTS OF THERAPEUTIC ULTRASOUND

lism. The use of low-intensity US reduces tissue heating and also reduces the possibility of cavitation phenomena, that is, the pulsation of gas or vapor-filled voids in a sound field.76 We have shown a maximum temperature rise of 1.8”C at 2.0 W/cm2, but no measurable rise was observed at the best stimulatory dose of 0.1 W/cm2(sApA).26 The use of 45-kHz US machines has been shown to produce less heating than the l-MHz machine.”

Comparing the effects on angiogenic factor produc- tion the two US machines (45 kHz and 1 MHz), one concludes that both produced good results, and that each has a particular intensity range where it is more effective. When using 45 kHz, the best angiogenic response should be obtained by using 15 or 30 mW/cm2cSA). With the l-MHz machine, the choice would be 0.1 to 0.4 W/cm2@APA,. These recommenda- tions are in agreement with the studies on cell proliferation and collagen/noncollagenous synthesis, which also showed these intensities as the most effective. There are several advantages of using the long wave ultrasound, such as a higher penetration depth, less heat production, reduced treatment time, and a spherical head applicator giving a bigger effec- tive treatment area. Because its in vitro effects are similar to or even better then those of the l-MHz machine, we recommend it for treatment of mandibu- lar osteoradionecrosis.

HBO has been recommended for the treatment of osteoradionecrosis, but it usually is used as adjunctive preparation for resection and reconstruction.1,7 How- ever, treatment with HBO has several disadvantages, not the least being availability and expense. The use of US seems to be superior because it is accessible, quicker, cheaper, and safer. We have shown that US addresses the main problems of osteoradionecrosis, hypocellularity by the stimulation of cell proliferation, the enhancement of healing and bone formation through the increase of collagen/NCP synthesis, and the hypovascularity and hypoxia through the stimula- tion of angiogenesis. This work also shows that US most likely produces angiogenesis through the produc- tion of IL-S, bFGF, and VEGF, all known angiogenic factors. Prospective clinical trials for the treatment and prevention of osteoradionecrosis using low- frequency (long wave) therapeutic US are indicated.

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