Intermittent Applications of Continuous Ultrasound on the

Viability, Proliferation, Morphology, and Matrix Production

of Chondrocytes in 3D Matrices

SANDRA NORIEGA, M.S.,1 TARLAN MAMEDOV, Ph.D.,1 JOSEPH A. TURNER, Ph.D.,2

and ANU SUBRAMANIAN, Ph.D.1

ABSTRACT

Chondrocytes, the cellular component of the articular cartilage, have long been recognized as strain-sensitive cells, and have the ability to sense mechanical stimulation through surface receptors and intra-cellular signaling pathways. This strain-induced biological response of chondrocytes has been exploited tofacilitate chondrocyte culture in in vitro systems; examples include the application of hydrostatic pres-sure, dynamic compression, hydrodynamic shear (i.e., rotating bioreactors), and low-intensity pulsedultrasound (US). While the ability of US to influence chondrogenesis has been documented, the precisemechanisms of US-induced stimulation continue to be investigated. There remains a critical need to eval-uate the impact of US on chondrocytes in 3D culture, which is a necessary microenvironment for main-taining the chondrocyte phenotype. In this study, a continuous US wave for predetermined time intervalswas employed, as opposed to pulsed US used in previous studies, to stimulate chondrocytes seeded in 3Dscaffolds. The chondrocytes (n= 6) were subjected to US stimulation as follows: 1.5MHz for 161 seconds,5.0MHz for 51 seconds, and 8.5MHz for 24 seconds, and the US signal was applied twice in a 24-hourperiod. Scaffolds that are not stimulated by US served as the control. Both the control and the US-stimulated groups were maintained in culture for 10 days, and at the conclusion of the culture period,chondrocytes were assayed for total DNA content, morphology, and cartilage-specific gene expression byreverse transcriptase polymerase chain reaction. Our results show that chondrocytes when stimulatedwith continuous US for predetermined time intervals possessed higher cellular viability (1.2 to 1.4 times)and higher levels of type II collagen and aggrecan mRNA expression when compared to controls.

INTRODUCTION

THOUGH ARTICULAR CARTILAGE is essentially avascular,

alymphatic, and aneural, the chondrocytes that popu-

late the extracellular matrix (ECM) are strain-sensitive cells,

and have the ability to sense mechanical stimulation through

surface receptors and intracellular signaling pathways, such

as protein phosphorylation and calcium cascade. The ECM

interacts with chondrocytes through a variety of receptors to

modulate metabolism, phenotype, and response to mechan-

ical stimuli. In vivo, articular cartilage is loaded frequently

as the joint performs its normal functions. In vivo studies

have shown that restrictions in joint mobility results in de-

generative changes characterized mainly by loss of proteo-

glycan production.1 Conversely, the in vivo application of

mechanical force can influence the metabolic response of

joint cartilage2–4 and can actually improve cartilage repair

following induced injury.5 This strain-induced biological

response of chondrocytes has been exploited to facilitate

chondrocyte culture in in vitro systems, with the aim of

1Department of Chemical and Biomolecular Engineering, University of Nebraska, Lincoln, Nebraska.2Department of Engineering Mechanics, University of Nebraska, Lincoln, Nebraska.

TISSUE ENGINEERINGVolume 13, Number 3, 2007# Mary Ann Liebert, Inc.DOI: 10.1089/ten.2006.0130

611

generating tissue-engineeredneocartilage. Examplesof stim-

ulation to facilitate cartilage matrix formation in vitro in-

clude the application of hydrostatic pressure,6,7 dynamic

compression,8,9 hydrodynamic shear (i.e., rotating bioreac-

tors),10 and low-intensity pulsed ultrasound (US).11–13

A 3D environment is apparently necessary for the produc-

tion of type II collagen, and for the maintenance of the phe-

notype by chondrocytes cultured in vitro,14 as chondrocytes

maintained in monolayer cultures tend to dedifferentiate into

their fibroblast form. When the cell/polymer constructs are

of the same dimensions as the in vivo tissue, they must be cul-

tured in a well-mixed environment to overcome limitations

in the diffusion of nutrients to cells in the center of the con-

structs.15 While mixed systems support growth of tissue con-

structs with many of the same biochemical characteristics as

articular cartilage, additional stimuli may be needed to de-

velop mechanically functional biological grafts. The effect of

dynamic compression, intermittent hydrostatic pressure, and

rotating shear stress on the biosynthetic behavior of chon-

drocytes cultured in 3D scaffolds has been evaluated. When

pressurized dynamically, chondrocytes increase production of

proteoglycan,16–18 which plays the important role of main-

taining tissue hydration and resistance to compression. The

benefits of applying dynamic forces at physiological levels,19

and over long durations,20 to chondrocytes cultured in a 3D

environment21 have been demonstrated; however, a system

that combines all of these conditions has only recently been

developed.7 Chondrocyte-seeded scaffolds when cultured in

low-shear, rotating-wall bioreactors developed biochemical

and mechanical properties that were superior to scaffolds cul-

tured in static and mixed cultures,10,15,22 and under select

conditions approximated the properties of natural cartilage.

Thus, it can be concluded that mechanical stimuli modulates

the morphology, biochemical composition, and mechanical

properties of the cartilage constructs.

Low-intensity US has been shown to promote healing

and mechanical strength of fracture callus in clinical studies

and to shorten the period of treatment in human and animal

models.23–26 Particularly, studies in the last 10 years have

shown that chondrocytes maintained in in vitro culture can

be stimulated by US,11–13,27,28 but the mechanism of me-

chanotransduction mediated by US stimulus remains largely

uncharacterized. Interestingly, the response to US stimulation

was found to differ between monolayer cultures and cells

maintained in alginate or collagen hydrogels. For example,

when in vitro monolayer cultures of rat chondrocytes, origi-

nating from the cartilaginous femoral condyles of the neonatal

rat, were stimulated, with two different 1.0MHz US sig-

nals with spatial and temporal average intensities of 50 or

120mW/cm2, aggrecan mRNA and proteoglycan protein

synthesis were found to be upregulated. However, no signif-

icant differences in cell proliferation was observed.11 As

chondrocytes used in this particular study11 were develop-

ing toward calcification, this study may be more relevant to

endochondral ossification and less relevant to chondrocytes

in articular cartilage. Additionally, the chondrocytes were

maintained in a monolayer culture, where they tend to de-

differentiate into fibroblasts.

The effects of low-intensity US (1.5MHz with a 200mstone burst repeated at 1.0 kHz, also known as Exogen�signal) on articular chondrocytes embedded in Atelecolla-

gen� gel and alginate gel were also evaluated.12,13,27 These

studies employed US at intensities of 2 and 30mW/cm2,

respectively. US promoted the synthesis of aggrecan but did

not significantly enhance cell proliferation in chondrocytes

seeded in Atelecollagen gel.27 For chondrocytes seeded in

alginate gel, there was no enhancement in aggrecan mRNA

or proteoglycan synthesis, but an increase in type II collagen

expression was observed. Interestingly, both studies used the

same Exogen signal. One possible explanation for this anom-

aly may be that the ultimate energy delivered to the cells,

which is likely to impact cellular functions, depends on the

geometry of the chamber and composition of the material, as

well as the coupling of the transducer head to the sample. In

summary, while US has been shown to impact cartilage func-

tion at the cellular level, there is still a need to better un-

derstand the effect of US stimulation on chondrocytes seeded

and maintained in 3D scaffolds, which are better representa-

tives of chondrocytes in vitro culture. The purpose of the

present study is to (1) evaluate the effect of US on chon-

drocytes seeded in 3D scaffolds and (2) develop a correlation

between US signal and chondrocyte morphology, biosyn-

thetic activity, and cartilage-specific gene expression in US-

stimulated chondrocytes.

MATERIALS AND METHODS

Isolation of chondrocytes

Chondrocytes were obtained from healthy human cartilage

(approved by the University of Nebraska-Institutional Re-

view Board) harvested from amputated limbs of young ostoe-

chondrosis patients. Only nondiseased, nonarthritic cartilage,

distal from the required amputation site and not in anyway af-

fected by the disease, was used. Chondrocytes were isolated

by digestion in pronase and collagenase for 22 hours and

plated at 104 cells/cm2 in RPMI containing 10% fetal bovine

serum (FBS), 1mM sodium pyruvate, 100mM HEPES buf-

fer, 100U/mL penicillin, 100mg/mL streptomycin, 0.29mg/

mL L-glutamine, and various growth factors.

Scaffold preparation

A 2% w/v solution of chitosan (81.7% deacetylated, MW

276 kDa, lot #01-CISQ-1702; VansonHaloSource, Redmond,

WA) in 1% acetic acid was freshly prepared prior to scaffold

preparation. For the preparation of scaffolds via freeze-dry

and lyophilization (FDL), 2mL of the chitosan solution was

pipetted into each well of a 24-well tissue culture polystyrene

plate (TCP, Falcon, Fisher Scientific, St. Louis, MO). The

samples were then frozen at�208C and lyophilized for 24 to

36 hours.The scaffoldswere thenneutralizedwith 1Msodium

612 NORIEGA ET AL.

hydroxide (NaOH) and rinsed with multiple washings of ster-

ile phosphate buffered saline (PBS). In order to preserve the

shape (pores) of the scaffolds, the scaffolds, before air drying,

were gradually dehydrated with a series of ethanol solutions

(20%–70%). The scaffold sample used in the cell-seeding

experiments was 0.4 – 0.5 cm in diameter and 0.3–0.5 cm in

thickness.

Cell seeding

Articular chondrocytes were grown in the presence of

10% fetal calf serum (GIBCO, Gaithersburg, MD), 1% non-

essential amino acids (GIBCO), 2mM L-glutamine, and 1%

penicillin-streptomycin(SigmaChemicalCompany,St.Louis,

MO). All cells were grown at 378C under a 5% carbon dioxide

(CO2)–humidified atmosphere. Cells from third passage were

collected by trypsinization and resuspended in complete me-

dium to a cell density of 2.5�105 cells/mL. Scaffold discs

were seeded with human chondrocytes at a seeding density of

105 cells/cm2 and maintained in 12-well tissue culture plates

(Fig. 1), with six scaffolds per treatment (n¼ 6). Control

treatments did not include US treatment. Disks were trans-

ferred to TCP wells with fresh complete media per well and

placed in the incubator. Media was changed every alternate

day. Unseeded disks were included as controls.

Ultrasound stimulation

The US was introduced into the wells using nonfocused

immersion transducers (e.g., Panametrics, V300, 0.5 inch

(1.27 cm) diameter, 5MHz center frequency), schemati-

cally represented in Figure 1. Three levels of US were ap-

plied to the plates: 1.5, 5.0, and 8.5MHz. The US transducer

was placed directly over the medium above the scaffolds,

and the application time was varied so as to have the same

number of cycles in all the scaffolds. Control plate was kept

without US. Duration of the application was 161 seconds

for 1.5MHz, 51 seconds for 5.0MHz, and 24 seconds for

8.5MHz, and US signal was applied twice over a 24-hour

period. The probe was sterilized with ethanol and rinsed in

medium before each application.

Cell proliferation

Cultured chondrocytes were released from control poly-

styrene and test scaffolds by adding 0.25% trypsin with 0.1%

ethylene diamine tetra acetate followed by incubation at 378Cwith 5% CO2. Medium was added to the trypsinized cells to

bring thefinal volume to2mL.Cell concentrationwas counted

using a hemocytometer. In a parallel experiment the cell

viability was also determined by (4,5-dimethyl-thiazol-2yl)-

2,5-diphenyltetrazolium bromide (MTT) assay.29 Scanning

electron microscopy (SEM) analysis was used to determine

the morphology of the chondrocytes cultured on test surfaces.

DNA quantification

At the conclusion of the experiment (i.e., 10 days after

cell seeding), five replicate samples of each specimen were

harvested to quantify the total amount of DNA. The DNA

content (mg/sample) was measured using a commercially

available kit (PicoGreen; Molecular Probe, Eugene, OR). A

reference DNA standard was used for extrapolation. Total

DNA at each time-point was used as a baseline.

De novo synthesis of total collagen

and total glycosaminoglycan content

Scaffolds were first digested with papain at 608C for

12 hours. The digest was then assayed for total sulfated

GAG content by dimethylmethylene blue (DMMB) assay,30

with shark chondroitin sulfate used as a standard. In a sep-

arate experiment, the total collagen content was determined

from the hydroxyproline content after acid hydrolysis (6N

hydrochloric acid at 1308C for 3 hours) and reaction with p-

dimethylaminobenaldehyde and chloramine-T, using a hy-

droxyproline to collagen ratio of 0.1.31

Morphology studies

Cell and cytoskeletal morphology was assessed using

rhodamine-conjugated phalloidin to visualize filamentous

actin. The cell-seeded surface was washed twice with PBS

(pH 7.4) solution before fixing with 3.7% formaldehyde

(20minutes) and permealizing with 0.1% Triton-X 100 (3–5

minutes). Following extensive rinsing, the cell-seeded sur-

faces were exposed to rhodamine-conjugated phalloidin (Mo-

lecular Probe). The cell-seeded surfaceswere visualized under

FIG. 1. Schematic for the US stimulation of chondrocyte-seeded

scaffolds. Chondrocyte-seeded scaffolds were placed in 12-well tis-

sue culture plates, with six scaffolds per group per well. The dimen-

sions of the wells are shown. The probe was positioned 8.5mm

above the well’s bottom and the height of the medium in the well was

kept at 10.2mm.

EFFECT OF CONTINUOUS ULTRASOUND ON CHONDROCYTES 613

a fluorescent microscope, equipped with a digital camera,

upon proper excitation (Biotechnology Center, Lincoln, NE).

RNA analysis by reverse transcriptase

polymerase chain reaction

Cellular total RNA extracted with Trizol# (1mg) was

subjected to reverse transcriptase polymerase chain reaction

(RT-PCR) at 428C for 1 hour with oligo (dt18) primers. The

transcripts were then amplified by RT-PCR. Primers specific

for every gene of interest were used. Sample cDNA was

amplified for ribosomal RNA subunit S14 as a housekeeping

gene marker for equal loading. Two mL of cDNA template

was used in each PCR reaction. Primers (forward and reverse)

were used for amplification. These were obtained from the

Biotechnology Center (Lincoln, NE) and Clontech (Palo Alto,

CA). PCR reactions were conducted in a thermal cycler, and

RT-PCR products were analyzed on 1.5% agarose gels. The

design for PCR primers for chondrocyte-specific ECMmark-

ers (type II collagen, aggrecan, type I collagen) as well as

glyseraldehyde-3-phosphate dehydrogenase (GAPDH) (in-

ternal control for mRNA loading) was based on information

provided in the literature andGENBANK, and are provided in

Table 1.

RESULTS

In the present study, US stimulation was provided twice

in a 24-hour period and the excitation was at least 6 hours

apart. Both control (no US) and US-stimulated scaffolds

were maintained in culture for a maximum of 10 days. At

the conclusion of the treatment, the chondrocytes on the

scaffolds were assayed for total DNA, cell viability, bio-

synthetic activity, mRNA expression, and cell morphology

by SEM analysis. The US excitation was sinusoidal at 2.5V

at 1.5, 5.0, and 8.5MHz, respectively. The total time for

each ultrasonic treatment was chosen such that the number

of cycles (*4.3�106) was approximately constant and of

the same order as in a previous work.11 Thus, for the US

signal at 1.5MHz, the fluid medium containing the sus-

pended scaffolds was excited for 160 seconds; at 5.0MHz

the excitation was for 51 seconds, and at 8.5MHz the ex-

citation was for 30 seconds. The estimated intensity was

less than 30mW/cm2.

By design, the initial seeding density was similar for all the

study groups. The effect of US on the total DNA content (ng/

mL) is shown in Figure 2. We found the US to influence the

total DNA content and cell-seeded scaffolds stimulatedwhere

the 5.0MHz US treatment yielded the highest amount of total

DNA (245ng/mL). Control treatments (no US), and samples

treated with a US signal of 1.5MHz had similar amounts of

DNA (in the range of 155 to 175 ng/mL). Chondrocytes

stimulated byUS (1.5, 5.0, and 8.5MHz) had viabilities 1.2 to

1.4 times higher, as determined by the MTT assay (data not

included).

Figure 3 shows fluorescence images of chondrocyte cells

cultured on 3D scaffolds and stimulated by US as detailed

earlier. Live cells, stained green, appeared to adhere well and

exhibited a normal morphology under US stimulation. These

cells appeared to be substantially more in number than those

on unstimulated scaffolds, and exhibited a more uniform dis-

tribution.Dead cells, stained red,were foundonmost surfaces;

however, they were observed more on control scaffolds and

scaffolds stimulated with the 8.5MHz US signal.

Cell adhesion and spreading were studied by SEM. SEM

images of US-stimulated and control chondrocytes on scaf-

folds on day 10 are shown in Figure 4. The chondrocytes

from the control remain spherical and nebulous. The cells

TABLE 1. FORWARD AND REVERSE PRIMERS USED FOR RT-PCR ANALYSIS

Gene Primers

Size

Base pairs (bp)

Gene accession

number

Collagen type II 50-GGCTCCCAGAACATCACCTA-30 (forward) 356 BC007252

50-TGCAACGGATTGTGTTGTTT-30 (reverse)

Aggrecan 50-AGACTTGAGGGGGAGGTGTT-30 (forward) 310 BC036445

50-GCATACCTCGGAAGCAGAAG-30 (reverse)

GAPDH 50-GCATACCTCGGAAGCAGAAG-30 (reverse) 323 BT006893

50-GTCTTCTGGGTGGCAGTGAT-30 (reverse)

FIG. 2. Effect of US stimulation on total DNA content. Cell-

seeded scaffoldswere subjected toUS stimulation and the totalDNA

was assayed after 10 days. An average value from the analysis of six

scaffolds per study group is reported. The standard error measure-

ment was then less than 5%.

614 NORIEGA ET AL.

subjected to 1.5MHz US signal also show little change.

However, those subjected to 5MHz and 8.5MHz US treat-

ment show a change in morphology (Fig. 4B, C). It is also

known that a rise in temperature during US exposure may

have an effect on cell metabolism. However, in our exper-

iments we observed a temperature increase only of the order

of 0.5 to 18C. In addition to the positive cell structure ob-

served for the 5MHz treatment, production of total collagen,

as measured by hydroxyproline content, was significantly

higher when compared to other treatments. No increase in

hydroxyproline content was observed in cells stimulated

by 1.5MHz US signal, whereas cells stimulated by US at

5.0MHz and 8.5MHz had 1.2 to 1.5 times higher hydro-

xyproline, respectively, when compared to controls.

To assess the ability of US stimulation to promote chon-

drogenic redifferentiation, chondrogenesis was assessed by

RT-PCR for cartilage-specific markers (collagen type I and

type II and aggrecans) and gene expression was normalized to

GAPDH.We have analyzed the expression of select cartilage-

specific genes at day 10 in human articular chondrocytes cul-

tured on3D scaffolds subjected toUS stimulation. ThemRNA

expression levels of collagen type II and aggrecans are shown

in Figure 5. At day 10, the values were significantly higher in

US-stimulated samples than in the control group; in addition,

gene expression was significantly higher in samples stimu-

lated with 5.0MHz and 8.5MHz.

DISCUSSION

Our research approach seeks to better understand how US

stimulates chondrocytes seeded on scaffolds, and is very dif-

ferent from previous studies11–13,27,28,32 for several reasons.

First, while previous studies have mainly used monolayer

cultures or chondrocytes embedded in hydrogels, we have

used a 3D culture environment that is required to maintain the

chondrocyte phenotype as monolayer cultures are less impor-

tant for tissue-engineering applications. Additionally, we hy-

pothesize that an US stimulation that can excite cells with an

appropriate level of strain to stimulate growth and prolifera-

tion is necessary. Further, the use of continuous, diffuse US

(i.e., incoherent) is plausible if the frequency is chosen in the

neighborhood of a cell resonance. Thus, we have used a con-

tinuous USwave for predetermined time intervals, as opposed

to pulsed US used in previous studies. We estimate impor-

tant resonances associated with chondrocytes seeded on scaf-

FIG. 3. Live (stained green) and dead (stained red) staining of

chondrocytes cultured for 10 days: (A) no US, (B) 1.5MHz, (C)

5.0MHz, and (D) 8.5MHz. Scale bar is 50 mm in all panels. Color

images available online at www.liebertpub.com/ten.

FIG. 4. SEM of chondrocyte-seeded scaffolds stimulated by

US: (A) no US, (B) 5.0MHz, and (C) 0.5MHz. Scale bar: (A)

25mm and (B & C) 20mm.

FIG. 5. RT-PCR analysis. Total RNA was extracted from liquid-

nitrogen-frozen chondrocytes using Trizol method and treated with

DNase. DNA-depleted RNA preparations were used for synthesis

of the first strand cDNA with random hexamers primer using

the SuperScript First-Strand Synthesis System kit (Invitrogen). PCR

amplification was performed using primers specific to the sequences

of genes of interest and GAPDH gene used as loading control.

EFFECT OF CONTINUOUS ULTRASOUND ON CHONDROCYTES 615

folds to be in range of several megahertz.33 Results associated

with this hypothesis have shown promise and are presented

in this paper. Finally, while most of the previous studies have

focused on the use of US for fracture and callus healing, the

focus here is on the effect of US stimulation on chondrocytes

maintained in 3D scaffolds. Fracture healing requires mini-

mal proliferation of cells and minimal maintenance of tissue

maturation, and the Exogen US signal has been shown to

achieve these in some reported studies.11,12 However, for

tissue-engineering approaches that seek to develop neocarti-

lage in in vitro cultures, the mechanical stimuli are expected

to influence cell proliferation, along with biosynthetic activity

to produce type II collagen and proteoglycan, and to achieve

tissue maturation that is in the range of articular cartilage.

Thus, we hypothesize that cells on scaffolds have to be stim-

ulated optimally, to impact cell metabolism and biosynthetic

activity.

Our proposed approach to stimulate chondrocytes may be

put in context with previous ultrasonic studies by examining

the input power in the region near 5MHz. The time domain

signal used in previous studies is shown in Figure 6. Figure

6A shows a pulsed 1.5MHz signal with a 200 ms pulse du-

ration and 1 kHz repeat rate (20% duty cycle). Figure 6B

shows the square wave that was used recently to influence

chondrocyte growth in adherent monolayers.34 Although the

time domain content of these signals is of interest, we be-

lieve that it is the frequency content that is important for

understanding the matching of the input power with chon-

drocyte resonances. The interest here is to drive the cells

with sufficient power near resonance such that they are

sufficiently stimulated.

The power spectra associated with the signals in Figure

6A and B are shown in Figure 7A and B, respectively, in

the region near 5MHz. Figure 7C is the power spectrum of

a continuous wave signal at 5MHz, which we have used in

this study. The signals represented in Figure 6 might appear

to be similar in the time domain due to the 1 kHz repeat

rate. However, it is clear that the square wave (pulse alone,

Fig. 7B) contains more power at higher frequencies than

the pulsed 1.5MHz signal (Fig. 7A). This result may ex-

plain those described in Argadine et al.,34 associated with

the response of chondrocytes: The square wave is a better

stimulant than the pulsed wave, yielding a higher level of

synthesis of cartilage-specific biomolecules like aggrecan

and collagen type II. The square pulse has a fairly uniform,

although low-power, response at high frequencies. Thus, it

may be used to stimulate cells comparably to a 1.5MHz

pulse. Cell stimulation could be greatly enhanced if the

power near the resonance is increased. Figure 7C shows the

expected result—the power content of a 5MHz continuous

wave signal is significantly higher at 5MHz compared to

either of the signals shown in Figure 6. Power spectrum

results at other frequencies, such as 8.5MHz, are similar to

that shown in Figure 7C.

Our present study has demonstrated that intermittent

applications of continuous US impact biosynthetic activity

and cartilage-specific mRNA expression of chondrocytes

maintained in 3D cultures. In future experiments, we will

continue to investigate the effect of duration and frequency

of US stimulation on chondrogenesis.

FIG. 6. Time domain signals associated with previous ultra-

sonic work with chondrocytes: (A) 1.5MHz pulse (200 ms) at

1 kHz repeat rate (the inset shows the 1.5MHz sine wave at higher

resolution); (B) 1 kHz square wave.

FIG. 7. Power spectra associated with the ultrasonic signals of

interest for chondrocytes in the region near 5MHz: (A) spec-

trum for the 1.5MHz signal pulsed at 1 kHz; (B) spectrum for the

1 kHz square wave; and (C) spectrum for the 5MHz continuous

wave.

616 NORIEGA ET AL.

REFERENCES

1. van Kampen, G.P.J., and van de Stadt, R.J. Cartilage and

chondrocyte responses to mechanical loading in vitro. In: Hel-

minen, H.J., Kiviranta, I., Saamanen, A-M., Tammi, M., Pau-

konen, K. and Jurvelin, J., eds. Joint Loading, Biology and

Health of Articular Structures. UK: Butterworth Kent, 1987, pp.

112–125.

2. Caterson, B., and Lowther, D.A. Changes in the metabolism of

the proteoglycans from sheep articular cartilage in response to

mechanical stress. Biochim. Biophys. Acta 540, 412, 1978.

3. Helminen, H.J., Kiviranta, I., Saamanen, A.M., Jurvelin, J.S.,

Arokoski, J., Oettmeier, R., Abendroth, K., Roth, A.J., and

Tammi, M.I. Effect of motion and load on articular cartilage

in animal models. In: Articular Cartilage and Osteoarthritis.

New York: Raven Press, 1992, pp. 503–510.

4. Kiviranta, I., Tammi, M., Jurvelin, J., Saamanen, A.M., and

Helminen, H. Moderate running exercise augments glycos-

aminoglycans and thickness of articular cartilage in the knee

joint of young beagle dogs. J. Orthop. Res. 6, 188, 1988.

5. Todhunter, R.J., Minor, R.R., Wootton, J.A.M., Krook, L.,

Burton-Wurster, N., and Lust, G. Effects of exercise and

polysulfated glycosaminoglycan on repair of articular cartilage

defects in the equine carpus. J. Orthop. Res. 11, 782, 1993.

6. Carver, S.E., and Heath, C.A. Increasing extracellular matrix

production in regenerating cartilage with intermittent physi-

ological pressure. Biotechnol. Bioeng. 62, 166, 1999.

7. Carver, S.E., and Heath, C.A. Semi-continuous perfusion

system for delivering intermittent physiological pressure to

regenerating cartilage. Tissue Eng. 5, 1, 1999.

8. Sah, R.L., Doong, J.H., Grodzinsky, A.J., Plaas, A.H.K., and

Sandy, J.D. Effects of compression on the loss of newly

synthesized proteoglycans and proteins from cartilage ex-

plants. Arch. Biochem. Biophys. 286, 20, 1991.

9. Bonassar, L.J., Grodzinsky, A.J., Frank, E.H., Davila, S.G.,

Bhaktav, N.R., and Trippel, S.B. The effect of dynamic com-

pression on the response of articular cartilage to insulin-like

growth factor. I. Journal of Orthop. Res. 19, 11, 2001.

10. Freed, L.E., Vunjak-Novakovic, G., and Langer, R. Cultiva-

tion of cell-polymer implants in bioreactors. J. Cell. Biochem.

51, 257, 1993.

11. Parvizi, J., Wu, C., Lewallen, D.G., Greenleaf, J.F., and Bo-

lander, M.E. Low intensity ultrasound stimulates proteogly-

can synthesis in rat chondrocytes by increasing aggrecan gene

expression. J. Orthop. Res. 17, 487, 1999.

12. Zhang, Z., Huckle, J., Francomano, C.A., and Spencer, R.G.S.

The influence of pulsed low-intensity ultrasound on matrix

production of chondrocytes at different stages of differentia-

tion: an explant study. Ultrasound. Med. Biol. 28, 1547, 2002.

13. Zhang, Z., Huckle, J., Francomano, C.A., and Spencer, R.G.S.

The effects of pulsed low-intensity ultrasound on chondrocyte

viability, proliferation, gene expression and matrix produc-

tion. Ultrasound Med. Biol. 29, 1645, 2003.

14. Benya, P.D., and Shaffer, J.D. Dedifferentiated chondrocytes

reexpress the differentiated collagen phenotype when cultured

in agarose gels. Cell 13, 215, 1982.

15. Vunjak-Novakovic, G., Martin, I., Obradovic, B., Treppo, S.,

Grodzinsky, A.J., Langer, R., and Freed, L.E. Bioreactor

cultivation conditions modulate the composition and mechan-

ical properties of tissue-engineered cartilage. J. Orthop. Res.

17, 130, 1999.

16. Veldhuijzen, J.P., Huisman, A.H., Vermeiden, J.P.W., and

Prahl-Anderson, B. The growth of cartilage cells in vitro and

the effect of intermittent compressive force. A histological

evaluation. Connect. Tissue Res. 16, 187, 1987.

17. Parkkinen, J.J., Ikonen, J., Lammi, M.J., Laakkonen, J.,

Tammi, M., and Helminen, H.J. Effects of cyclic hydrostatic

pressure on proteoglycan synthesis in cultured chondrocytes

and articular cartilage explants. Arch. Biochem. Biophys.

300, 458, 1993.

18. Burton-Wurster, N., Vernier-Singer, M., Farquhar, T., and

Lust, G. Effect of compressive loading and unloading on the

synthesis of total protein, proteoglycan and fibronectin by

canine cartilage explants. J. Orthop. Res. 11, 717, 1993.

19. Torzilli, P.A., Grigiene, R., Huang, C., Friedman, S.M., Doty,

S.B., Boskey, A.L., and Lust, G. Characterization of cartilage

metabolic response to static and dynamic stress using a me-

chanical explant test system. J. Biomech. 30, 1, 1997.

20. Heath, C.A., and Magari, S.R. Mini-review: mechanical fac-

tors affecting cartilage regeneration in vitro. Biotechnol.

Bioeng. 50, 430, 1996.

21. Buschmann, M.D., Gluzband, Y.A., Grodzinsky, A.J., and

Hunziker, E.B. Mechanical compression modulates matrix

biosynthesis in chondrocyte/agarose culture. J. Cell Sci. 108,

1497, 1995.

22. Dunkelman, N.S., Zimber, M.P., LeBaron, R.G., Pavelec, R.,

Kwan, M., and Purchio, A.F. Cartilage production by rabbit

articular chondrocytes on polyglycolic acid scaffolds in a

closed bioreactor system. Biotechnol. Bioeng. 46, 299, 1995.

23. Heckman, J.D., Ryaby, J.P., McCabe, J., Frey, J.J., and Kil-

coyne, R.F. Acceleration of tibial fracture-healing by non-

invasive low-intensity pulsed ultrasound. J. Bone Joint Surg.

Am. 76, 26, 1994.

24. Rubin, C., Bolander, M., Ryaby, J.P., and Hadjuargyrou, M.

The use of low-intensity ultrasound to accelerate the healing

of fractures. J. Bone Joint Surg. 83(A), 259, 2001.

25. Wang, N., Butler, J.P., and Ingber, D.E. Mechanotransduction

across the cell surface and through the cytoskeleton. Science

260, 1124, 1993.

26. Yang, K.-H., Parvizi, J., Wang, S.-J., Lewallen, D.G., Kin-

nick, R.R., Greenleaf, J.F., and Bolander, M.E. Exposure

to low-intensity ultrasound increases aggrecan gene expres-

sion in a rat femur fracture model. J. Orthop. Res. 14, 802,

1996.

27. Nishikori, T., Ochi, M., Uchio, Y., Maniwa, S., Kataoka, H.,

Kawasaki, K., Katsube, K., and Kuriwaka, M. Effects of low-

intensity pulsed ultrasound on proliferation and chondroitin

sulfate synthesis of cultured chondrocytes embedded in Atelo-

collagen gel. J. Biomed. Mater. Res., Part B: Applied Bio-

materials 59, 201, 2002.

28. Ebisawa, K., Hata, K.I., Okada, K., Kimata, K., Ueda, M.,

Torii, S., and Watanabe, H. Ultrasound enhances transform-

ing growth factor (-mediated chondrocyte differentiation of

human mesenchymal stem cells. Tissue Eng. 10, 921, 2004.

29. Subramanian, A., Lin, H., Vu, D., and Larsen, G.F. Prepara-

tion and evaluation of chitosan-based scaffolds for potential

use in cartilage tissue engineering. J. Biomat. Sci. Polymer

Ed. 16, 861, 2005.

EFFECT OF CONTINUOUS ULTRASOUND ON CHONDROCYTES 617

30. Farndale, R.W., Buttle, D.J., and Barrett, A.J. Improved

quantitation and discrimination of sulfated glycosaminogly-

cans by use of dimethylmethylene blue. Biochim. Biophys.

Acta 883, 173, 1986.

31. Reddy, G.K., and Enwemenka, C.S. A simplified method for

the analysis of hydroxyproline in biological tissue. Clin.

Biochem. 29, 225, 1996.

32. Duda, G.N., Kliche, A., Kleemann, R., Hoffmann, J.E., Sittin-

ger, M., and Haisch, A. Does low-intensity pulsed ultrasound

stimulate maturation of tissue-engineered cartilage? J. Biomed.

Mater. Res., Part B: Applied Biomaterials 68B, 21, 2003.

33. Kalyanam, S. Quantitative aspects of cell membrane inves-

tigations using atomic force microscopy, M.S. Thesis, Uni-

versity of Nebraska, Lincoln, 2005.

34. Argadine, H.M., Kinnick, R.R., Greenleaf, J.F., and Bolander,

M.E. 1 kHz vibration increases proteoglycan production in

ATDC5 chondrocytes. 149th meeting of the Acoustical So-

ciety of America (paper 3aBBa5),Vancouver, Canada, May

2005.

Address reprint requests to:

Anu Subramanian, Ph.D.

207L Othmer Hall

UNL-Lincoln, NE 68588-0643

E-mail: asubramanian2@unl.edu

618 NORIEGA ET AL.