To investigate the interactions of ultrasonic waves with biological tissues, we developed and standardized several in vitro models. Using these systems - artificial stones, human erythrocytes, L1210 mouse leukemia cells, multicellular spheroids, cavitation assay - we are able to elucidate the mechanisms of interaction as well as the cause of clinically observed side effects

Bachleitner, Christoph

Brümmer, Franz

Hülser, Dieter F.

Irmer, Ulrich

Suhr, Dierk

Online Publikationen der Universität Stuttgart

BIOPHYSICAL INVESTIGATIONS OF THE IN VITRO EFFECTS OF 
SHOCK WAVES AND ULTRASOUND 
Franz BrGmmer, Dierk Suhr, Uwe Irmer, Christoph Bachle~ner, and Dieter F. HGlser 
Unlversltiit Stuttgart. BloIoglsches Inst~ut, Abtenung Blophyslk, 
Pfaffenwaldring 57, 7000 Stuttgart 80, Germany 
Abstract: To Investlgale Ihelnleractlonl of ultrasonic 
WIves with biological tissue., we developed and 
ltandardlzed leveral In vitro models. Ullng Ihese 
systems - artificial stones, human erythrocytes, 
L 1210 mouse leukemla cells, muhlcellular spheroid., 
cavHallon a .. ay - we are able 10 elucidate Ihe 
mechanisms of Interaction 8S well I. the cause of 
clinically observed side effects. 
I. INTRODUCTION 
Side effects of ultrasonic waves on organs, tissues, and 
cell suspensions have been Investigated for several 
decades. Since the advent of extracorporeal shock 
wave lithotripsy of kktney and gall stones, accompanied 
by tissue damage and obvious Impairment of organ 
function [3J, the Importance of quant~atlve analysis of 
biological effects Increased. Physical characterization of 
focused high energy shock waves Including quantHatlve 
measurements of the peak negative pressure needs 
highly sophisticated technical equipment [5,B[. We, 
therefore, characterized the acoustic output as well as 
the biological effects of different shock wave sources In 
clinical use for the therapy of kidney stones and 
experimental ultrasound devices for tumour therapy. 
11. IN-VITRO-MODELS 
Artnlclal stones, 15 mm In diameter, 1.46 - 1.54 g In 
weight, made of a mixture of dental cement and glass 
microspheres were shock wave treated until 
disintegration into fragments smaller than 2 mm. The 
number 0' shock waves needed for this approach Is a 
measure of the fragmentation efficacy. 
Human erythrocytes obtained by venous puncture from 
volunteers were washed and suspended in phosphate 
buffered saline. Cell suspensions (3·5 '10' cells/ml) 
were transferred In polyethylene pipettes and treated 
with shock waves. After determination of the number of 
Intact cells as well as the amount of free haemoglobin In 
the supernatant, the resulting dose-response·curves 
were calculated. 
L1210 cells, a lymphocytic mouse leukemla cell line, 
were concentrated to 2-3'105 cells/ mI and treated wtth 
shock waves. The percentage of geometrically 
destroyed and intact cells was measured, and the latter 
fraction was Investigated for physiologically viable and 
dead cells using a double staining technique with 
flUorescent dyes and flow cytometry. A LD50 was 
calculated from the dose·response·curves [4) . 
Multicellular spheroids, three-dImensional growing cell 
aggregates wfth numerous cell-cell and ceU-matrtx 
Interactions. are a model for tissue In vivo. For the 
In~latlon of multicellular spheroids, single cell 
suspensions were seeded Into plastic dishes. and within 
three days small cell aggregates were formed. These 
aggregates were transferred Into spinner flasks and can 
be cultured up to several 100 ",m In diameter. Shock 
wave treated spheroids were Investigated by light and 
electron mlcroscoplcal histology. 
Spheroids Immobilized In 12% gelatine were recovered 
by liquefying the gel at 37'C, and processed In the 
same way [IJ. 
The occurrence of acoustic cavitation, accompanied by 
free radicals and chemical oxidation products, was 
investigated by our cavitation assay. This assay uses 
the radical-sensitive fluorescent dyes dihydroethldium 
and dlchlorofluorescln-dlacetate which are suitable for 
extra- and intracellular measurements. After sonication 
of dye solutions and stained cells, the increase In 
fluorescence of the oxidation products ethldlum and 
dichlorofluorescein was determined by fluorometry and 
flow cytometry [9J . 
129 
Ill. RESULTS AND DISCUSSION 
Using our standardized In vi/ra-models, we 
demonstrated the Influence of various parameters on 
fragmentation efficacy and on biological as well as on 
chemical effects. 
We examined the fragmentation efficacy of different 
types of Ikhotrlpters wkh regard to emitted acoustic 
energy, temperature, gas content, and posklon of the 
surface of the waterbath. Under the same experimental 
condklons, we compared two Identical mhotrlpters 
(Domler XL-I In Stuttgart and Munich) and determined 
fragmentation efficlencles of 1 00 ~ 1 and 1 05 ~ 1 shock 
waves. This reveals the high reliability and 
reproducibility of this stone model. 
A simple and well established model for biological 
effects are human erythrocytes. The occurrence of free 
haemoglobin In the urine of patients after Ikhotrlpsy 
Indicates the clinical relevance of this model. The 
number of destroyed erythrocytes after sonication of 
erythrocytes In vitro accounts only for half of the 
released amount of free haemoglobin, Indicating a 
transient Increase In membrane permeability In Intact 
erythrocytes. By combining experiments wkh artKlclal 
stones and erythrocytes, we characterized several 
electrodes for electrohydraullc Ikhotrlpters wHh regard 
to their efficiency and tissue damage. 
After shock wave treatment, viable and dead mouse 
leukemla L 1210 cells were distinguishable, resulting in 
different dose-response-curves related to experimental 
condklons. Degasslng «2 mg 0,/1), heating (41'C vs. 
3rC vs. 21'C) and lowering the surface (2 cm vs. 
10 cm behind the focal region) of the water In the tank 
of an electrohydraullc Ikhotrlpter resulted In an Increase 
of destroyed and dead cells. 
Whh this experimental approach we Investigated several 
cell lines of different origin for the senskivlty of normal 
and tumour cells to shock waves. Our resutts Indicate 
that under carefully controlled and constant 
experimental conditions, cells have different sensitivities 
to shock waves, but no general difference between 
normal and malignant cells can be seen [6] . 
Long-term Investigations using growth curves or the 
MTT -cell prolKeration assay [2] resulted In delayed 
proliferation of cells after shock wave treatment for a 
lag-phase of 72 hours. 
Shock wave treated multicellular spheroids exhlbhed 
severe damage In light- and electron mlcroscoplcal 
histology, e.g., fragmentation of spheroids, alterations of 
organelle ultrastructure, perinuclear cisterns, and 
transient membrane ruptures. Immobilizing spheroids In 
12% gelatine leads to a tissue phantom wkh acoustic 
properties known for biological tissues and organs, e.g., 
sound velocity of 1530 m/s vs. 1520 m/s for blood and 
1570 m/s for muscle [7]. With Immobilized spherOids 
we were able to separate direct shock wave effe~ 
from secondary mechanIsms, e.g., cavitation. and 
detected differences In the pattern of damage after 
treatment wnh shock waves or high energy pulSed 
ultrasound. 
A closer view on the mechanisms of cellular damage 
using the cavitation assay revealed the occurrence 01 
extra- and Intracellular cavkatlon-generated free 
radicals. We found a dose- and repetftlon frequency_ 
dependent Increase In fluorescence of the oxidation 
products after shock wave treatment. This Increase was 
higher for 8 Hz than for 1 Hz. The contribution of these 
free radicals to the shock wave Induced cell damage 
was demonstrated using antioxidants. 
Our standardized in vitro models (artKlclal stones, now 
cytometry of single cell suspensions, histology 01 
suspended and Immobilized multicellular spheroids, 
cavitation assay) are reliable and reproducible test 
systems for detailed characterization of shock waves 
and ultrasound and their Interactions wfth biological 
tissues. 
REFERENCES 
(1) Th. Briuner. F. Brilmmer. D.F. HUlser: Histopathology of shock 
wave treated tumor cell suspensions and multicell tumor sph.rolds, 
Ultrasound Med. Bioi. 15, 451-460, 1989. 
[2] F. Briimmer, Ch. Bachleltner, D.F. Hulser: MTI.cell prolifiration 
assay optimized for single cell suspensions and adhefently growing 
cell eullures, EUf. J. Cell BioI. 57 (Suppl. 36), 12, 1992. 
(3) F. Brummer, Th . Briiuner, D.F. HUlser: Biological ettects of shock 
wave1, World J. Urol. 8, 224-232, 1990. 
(4) F. Brummer, J. Brenner, rh. Briiuner, D.F. Hulser: Effect of shock 
waV81 on suspended and Immobilized L 1210 cells, Ultrasound Med. 
BioI. 15,229-239, 1989. 
(5) F. Briimm.r. J. Staud.nraus, M, Nosper, D, Suhr, W, 
Eisenmenger, D.E HUlser: Biological ettects and physical 
characterization of shock waves generated by an XL.1 experimental 
lithotrlpter, Ultrasonics Intemational 89 Conference Proceedings, 
Bunerworth, Guildford, 1130-1135, 1989. 
(6) F. Brummer, D. $uhr, D.F. HUlser: Sensitivity of normal and 
malignant cells to shock waves, J. Stone Dis. 4, 243-248, 1992. 
(7) M. Petzold , H. Pein, R. MilIner, H . .J. Heln: Gewebeiquivalente 
Ultraschall·Phantomsubstanz, medizintechnik 26, 11~113, 1986. 
[8] J. Staudenraus, W. Eisenmenger; Fiber optic probe hydrophOfle 
for ultrasonic and shock wave measurements In water, Ultrasonics, 
In pre", 1993. 
(9) D. Suhr, F. Blummer, D.F. Hulser: Cavitatlon-generated free 
radicals during shock wave exposure: investigations with cell·fr .. 
solutions and suspended cells, Ultrasound Moo. BioI. 17, 761·768, 
1991. 


Biophysical investigations of the in vitro effects of shock waves and ultrasound

http://elib.uni-stuttgart.de/bitstream/11682/1980/1/huel86.pdf

Biophysical investigations of the in vitro effects of shock waves and ultrasound

Abstract

Similar works

Full text

Available Versions

Online Publikationen der Universität Stuttgart