The authors thank Reiner Schultheiss and Wolfgang Schaden for their inspiration for this model. We also thank Christian Dorfm&#252;ller for his all time tremendous efforts to support our research.
Many thanks to Robert G&#246;schl and Hans Hohenegger for careful technical realization of our ideas!

Ethical permission
After obtaining written informed consent of patients, umbilical cords were obtained from Caesarean section at the Department of Gynaecology for isolation of human umbilical vein endothelial cells (HUVECs). Permission was given from the ethics committee of Innsbruck Medical University (no. UN4435).
1. Prepare the IVSWT Water Bath
<ol>
	<li>Prepare 3.5 L&#160;of tap water in an appropriate tank. Water needs to be heated to 37 &#176;C (see Protocol 6)...

<ol>
	<li>Haupt, G., Haupt, A., Ekkernkamp, A., Gerety, B., Chvapil, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Influence+of+shock+waves+on+fracture+healing.">Influence of shock waves on fracture healing.</a> Urology. 39, 529-532 (1992).</li><li>Schaden, W., Fischer, A., Sailler, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracorporeal+shock+wave+therapy+of+nonunion+or+delayed+osseous+union.">Extracorporeal shock wave therapy of nonunion or delayed osseous union.</a> Clin. Orthop. Relat. Res. 387, 90-94 (2001).</li><li>Schaden, W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shock+wave+therapy+for+acute+and+chronic+soft+tissue+wounds:+a+feasibility+study.">Shock wave therapy for acute and chronic soft tissue wounds: a feasibility study.</a> J. Surg. Res. 143, 1-12 (2007).</li><li>Tepek&#246;yl&#252;, C., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Shock+wave+treatment+induces+angiogenesis+and+mobilizes+endogenous+CD31/CD34-positive+endothelial+cells+in+a+hindlimb+ischemia+model:+Implications+for+angiogenesis+and+vasculogenesis.">Shock wave treatment induces angiogenesis and mobilizes endogenous CD31/CD34-positive endothelial cells in a hindlimb ischemia model: Implications for angiogenesis and vasculogenesis.</a> J. Thorac. Cardiovasc. Surg. 146, 971-978 (2013).</li><li>Nishida, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracorporeal+cardiac+shock+wave+therapy+markedly+ameliorates+ischemia-induced+myocardial+dysfunction+in+pigs+in+vivo.">Extracorporeal cardiac shock wave therapy markedly ameliorates ischemia-induced myocardial dysfunction in pigs in vivo.</a> Circulation. 110, 3055-3061 (2004).</li><li>Fukumoto, Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracorporeal+cardiac+shock+wave+therapy+ameliorates+myocardial+ischemia+in+patients+with+severe+coronary+artery+disease.">Extracorporeal cardiac shock wave therapy ameliorates myocardial ischemia in patients with severe coronary artery disease.</a> Coron. Artery Dis. 17, 63-70 (2006).</li><li>Gotte, G., Amelio, E., Russo, S., Marlinghaus, E., Musci, G., Suzuki, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Short-time+non-enzymatic+nitric+oxide+synthesis+from+L-arginine+and+hydrogen+peroxide+induced+by+shock+waves+treatment.">Short-time non-enzymatic nitric oxide synthesis from L-arginine and hydrogen peroxide induced by shock waves treatment.</a> FEBS Lett. 520, 153-155 (2002).</li><li>Wang, F. S., Wang, C. J., Huang, H. J., Chung, H., Chen, R. F., Yang, K. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Physical+shock+wave+mediates+membrane+hyperpolarization+and+Ras+activation+for+osteogenesis+in+human+bone+marrow+stromal+cells.">Physical shock wave mediates membrane hyperpolarization and Ras activation for osteogenesis in human bone marrow stromal cells.</a> Biochem. Biophys. Res. Commun. 287, 648-655 (2001).</li><li>Mittermayr, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Extracorporeal+shock+wave+therapy+(ESWT)+minimizes+ischemic+tissue+necrosis+irrespective+of+application+time+and+promotes+tissue+revascularization+by+stimulating+angiogenesis.">Extracorporeal shock wave therapy (ESWT) minimizes ischemic tissue necrosis irrespective of application time and promotes tissue revascularization by stimulating angiogenesis.</a> Ann. Surg. 253, 1024-1032 (2011).</li><li>Baker, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Use+of+the+mouse+aortic+ring+assay+to+study+angiogenesis.">Use of the mouse aortic ring assay to study angiogenesis.</a> Nat. Protoc. 22, 89-104 (2011).</li></ol>

The significance of the proposed model for in vitro shock wave treatment is the fact that waves can propagate after passing the cell culture in contrast to existing models. Thereby, disturbing physical effects such as tensile forces can be avoided. The model more closely resembles in vivo conditions than that by others applying waves to their cell culture flasks directly.
An additional advantage is the possibility of varying the distance between shock wave source and cells. T...

Shock waves are sound pressure waves arising from a sudden release of energy, e.g. as thunder when lightning. In medicine shock waves have been used for over 30 years&#160;in Lithotripsy for the disintegration of kidney stones. Since the incidental finding of iliac bone thickening in Lithotripsy patients in the early 1980s, first studies were conducted to evaluate the effect of shock wave treatment (SWT) on bone healing1. Impressive results of improved healing of long-bone nonunions could be observed2. Subsequently, indications were expanded to soft tissue wounds3. Basic research findings showed that shock waves do cause a biological stimulus to the target tissue without any subsequent damage. Release of angiogenic growth factors (e.g. VEGF, PlGF, FGF) is followed by significant angiogenesis. This led to a further expansion of indications towards ischemic pathologies. Our group and others showed the positive effect of SWT on ischemic heart disease in animal models as well as in clinical trials4-6.
However, the exact mechanism of how the physical stimulus of SWT is translated into a biological signal (mechanotransduction) remains largely unknown. As the interest in SWT from several fields of medicine increases continuously, the quest for the mechanism is getting more and more intense. Therefore, in&#160;vitro shock wave experiments are gaining importance. Besides reduction of animal experiments and cost-effectiveness, the biggest advantage of in&#160;vitro shock wave treatment (IVSWT) may be the possibility of studying the specific behavior of a certain cell type. In shock wave mediated tissue regeneration most likely all cells of the treated tissue are involved, even systemic effects are discussed. Nevertheless, each cell type plays a specific role and has its own intrinsic function. IVSWT enables us to detect this particular function and thereby gives us better understanding of the complex underlying processes.
Today&#39;s knowledge about shock wave&#39;s effects on cell cultures includes the increase of proliferation, alteration of cell membrane receptors, increase and acceleration of cell differentiation, release of growth factors and chemo-attractants as well as increased cell migration7-9.
Distracting physical effects in most in&#160;vitro models Various methods of applying shock waves onto cell cultures have been described. This fact leads to the problem that it is highly difficult to compare results, as physical conditions of cell stimulation are quite different between these models. In general, all existing models focus on how to best apply shock waves onto cells.
However, this question remains: What happens to the waves after passing the cell culture? The main problem is that the difference of the acoustic impedance of the cell culture medium and the ambient air is that high, that more than 99% of shock waves get reflected Figure 1.
Due to the difference in acoustic impedance of the two media the waves are not only reflected but a phase-shift of 180&#176; occurs resulting in strong tensile forces to the cells Figure 2.
Acoustic impedance is defined as the product of the density of a material and its sound velocity Z=&#961; x c. For water the acoustic impedance is ZWater= 1,440,000 Ns/m3, for air it is only 420 Ns/m3. The large difference of these two values results in reflection and phase shift of shock waves. The phase shift turns a positive pressure pulse into a tensile wave.
Even if this tensile force is not harmful to the cells, it interferes with the idea of mimicking in vivo shock wave effects in vitro. In vivo these tensile forces hardly occur due to large body structures.
Furthermore, the back running waves can even disturb the incoming ones. This may cause interference. Two types of interference are known. Constructive interference means that both waves are added thereby resulting in doubled amplitude&#160;Figure 3. Destructive interference occurs if waves meet diametrically opposed. It causes abolishment of waves (Figure 3). Therefore, IVSWT needs a model that enables shock waves to propagate after passing the cell culture.
IVSWT water bath
Considerations following the above mentioned concerns lead us to design a water bath for avoiding the described problems Figure 4. Basically, it consists of a Plexiglas built container with a membrane to connect every kind of shock wave applicator. For coupling between this membrane and the applicator ultrasound transmission gel has to be used. The water bath is filled with degassed water to avoid cavitation that would occur if gas was soluted within the water. A heater at the bottom with a temperature sensor connected to a control unit enables to regulate temperature for imitation of in vivo conditions and to avoid cell cultures to cool down during the procedure. Temperature can be held stable at 37 degrees centigrade as it is done in an incubator. A holder for the cell samples allows for immersing any kind of culture flask or tube. Thereby, the sample vessel needs to be completely filled with culture medium, as air bubbles would block shock waves! A wedge shaped absorber at the back wall of the bath destructs waves in order not to get reflected and run back to avoid interference.
A further advantage to other IVSWT models is the possibility of varying the distance between the applicator and the culture flasks. Findings of our group and others who use this model clearly show that every cell type reacts very specifically to different treatment parameters. Moreover, defining the distance between the source of the waves and the sample is crucial as it enables us to control the cells to be at a specific position in relation to the focus of the shock wave applicator.

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	<tbody>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">Orthogold shock wave device</td>
			<td colspan="2" style="width:756px;">Tissue Regeneration Technologies, Woodstock, GA &#8211; manufactured by MTS-Europe GmbH, Konstanz, Germany</td>
			<td style="width:827px;"></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">IVSWT Water Bath V2.0</td>
			<td style="width:537px;">Johann Hohenegger - Technical Products</td>
			<td style="width:219px;">-</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;">EBM-2 Basal Medium 500 m +EGM-2 SingleQuot Suppl.&#38;Growth Factors</td>
			<td style="width:537px;">Lonza</td>
			<td style="width:219px;">CC-3156 &#38; CC-4176</td>
			<td>This medium was used for the shown experiments with HUVECs to fill the cell culture flask. For other cell types, use the recommended medium.</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;"><a href="http://www.labplanet.com/pechiney-parafilm-m-pm996.html">Pechiney&#160;Parafilm M PM996</a></td>
			<td style="width:537px;">Pechiney Plastic Packaging</td>
			<td>PH-LF-PM996-EA at labplanet.com</td>
			<td>for sealing flasks</td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">Falcon Serological pipets 25ml</td>
			<td style="width:537px;">Becton Dickinson Labware</td>
			<td style="width:219px;">357525</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">CellMate II Serological Pipette&#160;</td>
			<td style="width:537px;">Matrix Technologies</td>
			<td style="width:219px;">-</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">Skintact Ultrasonic Gel</td>
			<td style="width:537px;">Skintact</td>
			<td style="width:219px;">UL-01 250 ml</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">T25 cell culture flasks</td>
			<td style="width:537px;">COSTAR</td>
			<td style="width:219px;">3056</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">mikrozid disinfectant</td>
			<td style="width:537px;">Sch&#252;lke</td>
			<td style="width:219px;">-</td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">3,5l degassed water</td>
			<td style="width:537px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
		<tr height="17">
			<td height="17" style="height:17px;width:420px;">paper towels</td>
			<td style="width:537px;"></td>
			<td style="width:219px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

shock wave application to cell cultures

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to target cells or tissue without any subsequent damage. Therefore, in vitro experiments are of increasing interest. Various methods of applying shock waves onto cell cultures have been described. In general, all existing models focus on how to best apply shock waves onto cells.
However, this question remains: What happens to the waves after passing the cell culture? The difference of the acoustic impedance of the cell culture medium and the ambient air is that high, that more than 99% of shock waves get reflected! We therefore developed a model that mainly consists of a Plexiglas built container that allows the waves to propagate in water after passing the cell culture. This avoids cavitation effects as well as reflection of the waves that would otherwise disturb upcoming ones. With this model we are able to mimic in vivo conditions and thereby gain more and more knowledge about how the physical stimulus of shock waves gets translated into a biological cell signal (&#8220;mechanotransduction&#34;).

Using the described method we applied shock waves to human umbilical vein endothelial cells (HUVECs) that we afore isolated from umbilical cords. Umbilical cords were obtained from elective caesarian sections.
HUVECs were treated at a confluence of 90% in a T25 cell culture flask with an electrohydraulic shock wave therapy system. Treatment parameters were an energy flux density of 0.1 mJ/mm2 and a frequency of 5 Hz. 300 impulses were applied from a distance of 5 cm from the shock w...

Watch this Scientific Journal Video about Shock Wave Application to Cell Cultures at JoVE.com

Shock Wave Application to Cell Cultures

Shock waves nowadays are well known for their regenerative effects. Therefore in vitro experiments are of increasing interest. We therefore developed a model for in vitro shock wave trials (IVSWT) that enables us to mimic in vivo conditions thereby avoiding distracting physical effects.

Shock waves nowadays are well known for their regenerative effects. Basic research findings showed that shock waves do cause a biological stimulus to ...

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12.6K 조회수.  Innsbruck Medical University.  충격파는 요즘 그들의 재생 효과가 알려져있다. 따라서 생체 외 실험은 증가 관심입니다. 따라서 우리는 이에 산만 물리 효과를 방지 생체 내 조건에서 모방하기 위해 우리를있게 체외 충격파 시험 (IVSWT)에 대한 모델을 개발했다. 