The ISMST and the ISMST Newsletter(International Society for Shockwave Therapy)An international platform for communication andknowledge transfer

The XII ISMST Congress will be held in Italy, inSorrento, from the 1st to 4th April 2009. It has beenorganized by Sergio Russo of the University ofNaples “Federico II”, who generously appointed mehonorary President of the Congress both for theaffection and the esteem that join us, and mindful ofour common and continuous activities in the last15 years in order to assert in the medical world thevalue of shock waves in musculoskeletal pathologiesand in other medical areas which step by step aresingled out as possible therapeutic application fieldsof shock waves.

The place for the Congress, the Hilton SorrentoPalace, has been chosen to offer the participants thesight of a magic place well-known around the worldfor its natural beauties. And also to give themembers the opportunity of a short immersion inRoman history, visiting the archaeologicalexcavations of Ercolano which was brought to lightin the last century after its destruction and burialfollowing the catastrophic eruption of Vesuvius in79 a.C. and the serious natural events.

Since years inexorably pass I have theimpression that my soul, growing older and older,has become more sensitive to memories. While I amwriting I can see, as regards the shock wave therapy,the last 15 years during which my closest partners,among whom I want to mention at least SergioRusso, Sergio Gigliotti, Carlo De Durante, have beencollaborating with me since the early experimentsconcerning shock waves in musculoskeletalpathologies and they supported me allowing us all tostudy a therapeutic field that has enriched ourknowledge and spread in all directions.

I remember clearly 1993, when we carried outthe first shock wave application on a patient affectedby pseudoarthrosis of the carpal scaphoid for morethan two years and who, after only two shock waveapplications through an old urologic lithrotriptor,recovered almost by a miracle in a month. Thisthrilled us very much and made us believe more inthe method. I remember the first congresses ofOrthopaedics in Italy and in Europe where weintroduced our early outcomes, causing positiveinterest and also much disbelief in our Italian andforeign colleagues.

I remember with affection Prof. Heinz Kudernaof Vienna with whom and together with otherEuropean scholars such as Prof. Wolfang Schaden,Dr. Richard Thiele and others we met in Vienna, in1997, to establish the European Society of ShockWave Therapy in the musculoskeletal pathologies

(ESMST). Prof. Heinz Kuderna, eminent doctor andscholar, was the first president of the EuropeanSociety and in 1999 I succeeded him during theCongress in London and in Naples in 2000, duringthe III Congress chaired by me, the EuropeanSociety became the International Society (ISMST).

Since 1995 there has been a strong collaborationwith scholars all over the world on the shock wavetherapy and with the increase in experimentalstudies, in clinical experiences, as well as theadjustment of devices to new traumatologicalorthopaedic needs, we achieved in few years thespreading of the method and of the therapeuticaldirections. The positive clinical responses ofthousand of cases around the world bears witness toit, together with the scientific interest that the newmethod caused, promoting the flourishing,especially in Europe, of many scientific Societiesdevoted to this field up to the foundation of theInternational Society for Muskoloscheletal ShockWaves Therapy (ISMST) in 2000, which countsamong its members scholars coming from all overthe world. As it was expected this new therapeuticalsystem could not remain only confined to theorthopaedic traumatological and urological fields.Severalongoing researches make us think that othermedical branches will benefit from the shock wavetherapy. We have been the first scholars to monitorthe system and to experiment it on man as well asto document angiogenesis processes in the tissueshit by shock waves. We also wished to find out thechemical mediators able to turn the mechanic effectinto the biological one. Nowadays we are able tofirmly point out that the main chemical mediatorwhich causes neoangiogenesis is nitrogen monoxide(NO) which originates in the tissues hit by shockwaves in peculiar circumstances. This fundamentaloutcome has also been achieved thanks to thecollaboration between our research group and asimilar research group of the University of Veronachaired by Prof. Hisanori Suzuki.

According to what I have written and to whatI have not reported for the sake of brevity, I mustfirmly reaffirm that the past 15 years of research onshock waves have been the harbinger of reallysatisfactory outcomes.

I am going to conclude this editorial evenmentioning the success of the International Societyfor shock wave therapy (ISMST) which has recordedparticipation and interest beyond the rosiestexpectations giving further value to this newtherapeutical system.

In the end I would like to thank the founder andpublisher Paolo Roberto Dias dos Santos for hiscareful direction of the Newsletter and because heallowed me to write my sincere editorial.

Editorial · Editorial comments

Inside this Issue

Chief Editor• Paulo Roberto Dias dos Santos, MD

(Brazil) - prds@uol.com.br

Associate Editor• Ching-Jen Wang, MD (Taiwan)

w281211@adm.cgmh.org.tw

Editorial Board• Ana Claudia Souza, MD (Brazil)

anaclaudia@cortrel.com.br• Carlos Leal, MD (Columbia)

leal@owc.com.co• Heinz Kuderna, MD (Áustria)

kuderna-unfallchir.wien@aon.ato• Helmut Neuland, MD (Germany)

dr.h.g.neuland@gmx.de• John Furia, MD (USA)

jfuria@ptd.net• Leonardo Jaime Guiloff Waissbluth,

MD (Chile) - lguiloff@davila.ch• Ludger Gerdsmeyer, MD (Germany)

gerdesmeyer@aol.com• Matthias Buch, MD (Germany)

matthiasbuch@aol.com• Paulo Rockett, MD (Brazil)

rockett@terra.com.br• Richard Thiele, MD (Germany)

harthi@t-online.de• Robert Gordon, MD (Canada)

gordon@skockwavedoc.com• Sergio Russo, MD (Italy)

serghiey@hotmail.com• Vinzenz Auersperg, MD (Áustria)

vinzenz.auersperg@akh.linz.at• Weil Lowel Jr, MD (USA)

lweiljr@weil4feet.com • Wolfgang Schaden, MD (Austria)

med.eswt.schaden@aon.at

Veterinary Committee• Scott Mc Clure, DVM (USA)

mcclures@iastate.edu• Ana Liz Garcia Alves, DVM (Brazil)

anaalves@fmvz.unesp.br• Ana Cristina Bassit, DVM (Brazil)

anafbassit@uol.com.br

Editorial OfficeAvenida Pacaembú, 1024CEP 01234-000 - São Paulo - Brazil Fone: 55-11-3825·8699E mail : prds@uol.com.br

website: www.ismst.com

Ezio Maria CorradoDirector of the Department of Surgery,Orthopaedics, Traumatology and Emergency-University of Naples "Federico II"

Editorial comments .............................................................................. 1Extracorporeal Shockwave Treatment for Chronic DiabeticFoot Ulcers .............................................................................................. 2Should we expect similar effects of extracorporeal shockwavetherapy on wounds of different species?......................................... 6Shoulder Rotator Cuff Tendinopathy.Histological, Immunohistochemical and VibrationalSpectroscopy Analysis ....................................................................... 11IVSWT: How can in-vitro shock wave therapy beperformed best? - Preliminary results from cardiac cells .......... 14Brief CommunicationRaman and Surface Enhanced Raman Scattering Applicationsin Shock Wave Therapy Related Research .................................... 18New Guidelines for ESWT ................................................................. 20

Editorial comments .............................................................................. 1Extracorporeal Shockwave Treatment for Chronic DiabeticFoot Ulcers .............................................................................................. 2Should we expect similar effects of extracorporeal shockwavetherapy on wounds of different species?......................................... 6Shoulder Rotator Cuff Tendinopathy.Histological, Immunohistochemical and VibrationalSpectroscopy Analysis ....................................................................... 11IVSWT: How can in-vitro shock wave therapy beperformed best? - Preliminary results from cardiac cells .......... 14Brief CommunicationRaman and Surface Enhanced Raman Scattering Applicationsin Shock Wave Therapy Related Research .................................... 18New Guidelines for ESWT ................................................................. 20

2 3

Background and PurposeDiabetic foot ulcer is caused by

ischemia/hypoxia due to occlusion ofsmall vessel associated withneuropathy and secondary infection.The treatments of diabetic foot ulceresrequire a multidisciplinary approachincluding the control of diabetes,antibiotic, shoe wear, wound care andsurgery in selected cases. The resultsof surgical and non-surgical treatmentsare inconsistent and most areunsatisfactory. Many adjunctivetherapies are designed with theintention to cure the diabetic footulcers. Some showed limited success,but none showed universal results.Extracorporeal shockwave treatment(ESWT) was to shown to induce theingrowth of neovascularizationassociated with increased angiogenicgrowth factors such as eNOS, VEGFand PCNA. Recent studies reportedthe effectiveness of ESWT in acuteand chronic wounds. Othersdemonstrated the antibacterial effectof ESWT in experimental studies. It isreasonable to speculate that ESWTmay be effective in chronic diabeticfoot ulcers. The purpose of thisprospective study was to evaluate theefficacy of ESWT in chronic diabeticfoot ulcers, and to compare the resultswith that of hyperbaric oxygen therapy(HBO), and to investigate theregeneration effects with focus onblood perfusion and molecular changesafter treatment.

MethodsSeventy patients with 72 chronic

diabetic foot ulcers were randomlydivided into two groups. The ESWTgroup consisted of 34 patients with 36ulcers, and 36 patients with 36 ulcersin the HBO group. Both groupsshowed similar demographiccharacteristics. Patients in ESWTgroup received 300 + 100 impulses of

shockwaves at 0.11 mJ energy fluxdensity /cm2 of treatment area onceevery two weeks for 6 weeks. Patientsin HBO group received HBO therapy ina sealed chamber at the pressure of2.5 ATA once a day, 5 days a week fora total of 20 treatments. Local bloodflow perfusion, bacterial culture, andbiopsy were performed before andafter treatment. The evaluationsincluded clinical assessment on thehealing status of the ulcer with photo-documentation, blood flow perfusionscan, bacteriological study,histomorphological examination andimmunohistochemical analysis.

ResultsThe overall results showed

completely healed in 31%, improved in58% and unchanged in 11% for theESWT group; and 22% completelyhealed, 50% improved and 28%unchanged for HBO group in favor ofESWT group (P = 0.001). ESWT groupshowed significantly better local bloodflow perfusion rate (Table 1, Fig. 1-aand Fig. 1-b) and considerably highercell concentration and more activeproliferation and than HBO (Fig. 2-aand Fig. 2-b). The results of bacteriaculture revealed significant decreasesin the bacteria colony counts aftertreatment (Table 2). Onimmunohistochemical analysis, ESWTgroup showed significant increases ineNOS, VEGF and PCNA expressionsand a decrease in TUNEL expressionthan the HBO group (Table 3, Fig. 3-a-1, 3-a-2, Fig.3-b-1, 3-b-2, Fig. 3-c-1, 3-c-2, Fig. 3-d-1 and 3-d-2).

DiscussionThe causes of diabetic foot ulcer

are multi-factorial includingischemia/hypoxia, neuropathy, andinfection, and they often coexist.Management of chronic diabetic skinulcers requires multidisciplinary

approach including the control ofdiabetes, antibiotic, shoe wear, woundcare and surgery in selected cases.The results of the customary standardtreatments are inconsistent and mostare unsatisfactory. Therefore, manyadjunctive therapies are designed withthe intention to cure the diabetic footulcers including hyperbaric oxygentherapy, ultrasound, recombinantplatelet-derived growth factor-BB,vacuum assisted wound closure andacellular matrix. Among them, HBO isthe most commonly employedmodality at our institution. Somestudies showed beneficial effects,however, none showed universalsuccess. The results of the currentstudy showed that ESWT is moreeffective than HBO in chronic diabeticfoot ulcers.

The exact mechanism of ESWTremains unclear. The results of thisstudy demonstrated that clinicalimprovement of the ulcers afterESWT were associated with increasesin angiogenesis and improvement inlocal blood flow perfusion, anddecreases in cell apoptosis andbacteria growth.

ConclusionsESWT is more effective than HBO

in the treatment of chronic diabeticfoot ulcers. It appears that applicationof ESWT results in tissue regenerationwith improvements in blood perfusionand molecular changes in chronicdiabetic foot ulcers.

This paper was selected as theFirst Place Winner in Classification:Tumor/Metabolic Disease at the 75thAnnual Meeting of the AmericanAcademy of Orthopedic Surgeons(AAOS) in San Francisco, CA.

This paper is also accepted forpublication by Journal of SurgicalResearch.

Extracorporeal Shockwave Treatment for Chronic Diabetic Foot UlcersChing-Jen Wang, M.D.

Clinical Professor Department of Orthopedic SurgeryChang Gung University College of Medicine

Chang Gung Memorial Hospital -Kaohsiung Medical Center, TaiwanE-mail: w281211@adm.cgmh.org.tw

Laser Doppler Before treatment After treatment P-value-1

ESWT

Mean±SD 0.64±0.28 0.75±0.19 0.04

(Range) (0.19-1.23) (0.46-1.28)

HBO

Mean±SD 0.50±0.21 0.58±0.11 0.140

(Range) (0.18-0.6) (0.51-0.66)

P-value-2 0.30 0.043

Table 1. Blood Flow Perfusion Rate Before and After Treatment

P-value-1: Comparison of data before and after treatment in the same group.P-value-2: Comparison of data between ESWT and HBO

Mean±SD (Range) Before treatment After treatment P-value-1

eNOS

ESWT 26.62±14.87 (4-57) 48.67±18.82 (6-72) < 0.001

HBO 25.2±17.09 (6-53) 20.08±9.73 (6-30) 0.317

P-value-2 0.438 <0.001

VEGF

ESWT 31.36±22.27 (8-90) 63.69±21.06 (25-91) <0.001

HBO 42.6±12.6 (28-55) 44.40±11.24 (30-56) 0.409

P-value-2 0.086 0.042

PCNA

ESWT 27.0±15.15 (7-53) 55.9±27.86 (8-95) 0.005

HBO 23.0±2.83 (20-26) 26.20±3.11 (23-30) 0.064

P-value-2 0.188 0.004

TUNEL

ESWT 62.42±15.0 (39-82) 31.58±13.44 (14-56) < 0.001

HBO 64.0±25.58 (23-86) 49.4±17.0 (22-65) 0.162

P-value-2 0.451 0.04

Table 3. The Results of Immunohistochemical Analysis

eNOS: Endothelial nitric oxide synthase; VEGF: Vessel endothelial growth factor;PCNA: proliferation cell nuclear antigen; TUNEL: Transference-mediated digoxigenin-deoxy-UTP nick end labeling P-value-1: Comparison of data before and after treatment within the same group.P-value-2: Comparison of data between ESWT and HBO.

Bacteria growth* 0 I II III VI P-value-1

ESWT group

Before treatment 4 3 9 17 3

After treatment 13 4 11 8 0 0.002

HBO group

Before treatment 5 3 9 15 4

After treatment 11 0 12 12 1 0.042

P-value-2 0.984

P-value-3 0.198

Table 2. The Results of Bacteriological Examination

P-value-1: Comparison of data before and after treatment within the same groupP-value-2: Comparison of data between the two groups before treatmentP-value-3: Comparison of data between the two groups after treatment0: No growth; I: Rare growth; II: Light growth; III: Moderate growth; VI: Heavy growth

Tables:

4 5

Figure 1. Laser Doppler scan showedsignificant increases in blood flowperfusion rate after ESWT (Fig. 1-a),whereas the changes were notsignificant after HBO (Fig. 1-b).

Before treatment After treatment

1-a

1-b

Figure 2. Microscopic features of thebiopsy specimen showed higher cellconcentration and more active cellproliferation after ESWT (Fig. 2-a), and lesscell concentration and proliferation afterHBO (Fig. 2-b) (H-E stain x 40).

Before treatment After treatment

2-a

2-b

Figure 3a. Immunohistochemical stainsshowed significant increases in eNOSexpression after ESWT (Fig. 3-a-1),whereas the changes were notsignificant after HBO (Fig. 3-a-2).

Before treatment After treatment

3-a-1

3-a-2

Figure 3b. Immunohistochemical stainshowed significant increases in VEGFexpression after ESWT (Fig. 3-b-1),whereas the changes were notsignificant after HBO (Fig. 3-b-2).

Before treatment After treatment

3-b-1

3-b-2

Figure 3c. Immunohistochemical stainsshowed significant increases in PCNAexpression after ESWT(Fig. 3-c-1), whereas the changes werenot significant after HBO (Fig. 3-c-2).

Before treatment After treatment

3-c-1

3-c-2

Figure 3d. Immunohistochemical stainsrevealed significant decreases inTUNEL expression after ESWT(Fig. 3-d-1), whereas the changes werenot significant after HBO (Fig. 3-d-2).

Before treatment After treatment

3-d-1

3-d-2

Figures:

76

ESWT was shown to induce productionof VEGF and perhaps to modulateexpression of other growth factors.25

ESWT was also shown to decreasetime to re-epithelialization of deep,partial-thickness burns of humanbeings.22

Frequently equine distal limbwounds are expected to heal bysecond intention. It was unknownhow ESWT would affect the woundhealing in the horse.

Study of second intentionhealing of distal limbwounds in horses

In a recent study by the authors ofthis paper26, the effect of ESWT onsecond intention healing of distallimbs wounds of the horse wasevaluated. For this study, a 5-cmdiameter circle was tattooed on thedorsomedial aspect of the mid-metacarpal region of each fore limb ofeach of 6 horses 4 weeks before thestudy began. This allowed for themeasurement of wound expansionand contraction. On day 0, a 4-cmdiameter, circular defect thatincluded skin, subcutis, andperiosteum was created in the centerof each tattoo. At the same time, twosimilar 3-cm diameter wounds weremade on the dorsomedial aspect ofeach metatarsus, one 4 cm proximaland one 4 cm distal to the middle ofthe metatarsus. These wounds werecreated to obtain biopies forimmunohistochemical evaluation.(Figure 3)

On day 1, the wound on onerandomly selected MC was coveredwith ultrasound coupling gel andtreated with ESWTa using 500 pulsesadministered at of 0.11 mJ/mm2. BothMT wounds on one randomly selectedMT were treated with 280 pulseswhich provided an equal number ofpulses per square centimeter ofwound as the larger forelimb wounds.Treatments were repeatedly weeklyuntil the wounds were healed. Duringtreatments, the untreated controlwounds on the contralateral MC andMT were also covered withultrasound coupling gel. At eachbandage change, the wounds weredigitally photographed with a rulerpositioned vertically and horizontallyclose to the wound as reference forthe photographs. The wounds weremaintained under a non-adherentdressing and bandage until healed.

At day 14, full-thickness, full-

width of the wound, rectangularexcisional biopsies were taken fromthe distal wound on each metatarsus,and at day 28, full-thickness biopsieswere taken from each proximalwound on each metatarsus. Eachbiopsy was approximately 6 mm wide,oriented in a transverse plane acrossthe center of the wound, andencompassed adjacent unwoundedtissue on both the medial and lateralaspects of the wound. Each biopsysample was placed into a 10%solution of neutral-buffered formalinand then into 70% isopropyl alcohol.The biopsy was embedded in paraffinusing an automated system. Sampleswere cut into 5-µm thick sections.

Epithelialization andContraction - The digitalphotographs were analyzed usingcomputer softwareb to determine thearea within the tattoo, the area ofepithelialization, and the area of thenon-epithelialized portion of thewound. The percentage ofcontraction was calculated using thefollowing formula:% contraction at Dx = (tatDy-tatDx) x 100%

wndDy,where wndDy denoted the maximumarea of the wound, and tatDy denotedthe maximum area of the tattoo, eachof which was determined after thewound enlarged to its maximumextent after its creation, beforecontraction and epithelializationbegan. Wound contraction wasexpressed as a percentage of thewound's area on day Dy. Dx representedany specific day after Dy.

Granulation tissue - Thequantity of granulation tissue wasscored as: 0 = none; 1 = < 5 mmdepth and < 1 cm2 in area; 2 = < 5mm depth the entire area of thewound; 3 = > 5 mm depth.

Immunohistochemicalstaining for IGF-I, VEGFand TGF-ß1

For immunohistochemical stainingthe IGF-1 antibodiesc were diluted1:10, and the VEGF and TGF-ß1antibodiesc were diluted 1:50 inTris/PBS/BSA solution. Sections werebathed in the primary antibodysolutions for 2 hours at roomtemperature. Endogenous peroxidaseactivity was inhibited by applying 3%H2O2 for 10 minutes. For IGF-1 andVEGF, a multilink, goat, anti-

immunoglobulin secondary antibodywas used at a dilution of 1:80 andwere incubated for 15 minutes atroom temperature. For TGF-ß1, agoat anti-rabbit secondary antibodywas used at a 1:500 dilution. Sampleswere incubated for 15 minutes atroom temperature. All slides werethen exposed to a 1:200 dilution ofHorse-radish Peroxidase-Streptavadinfor 15 minutes then stained withNova Red for 5 minutes and counter-stained with one-quarter strengthShandon's hematoxolin for 2 minutes.Negative controls were incubated inPBS instead of with the primaryantibodies. Normal equine pancreaswas used as the positive control forIGF-1. A section of skin with anextensive focus of granulation tissuewas used for both the VEGF andTGF-ß1 controls.

For each time frame andtreatment group, five fields ofimmature, loose, granulating fibrousconnective tissue were randomlychosen for examination at 600X. Theconnective tissue was subjectivelyevaluated for intensity of cytoplasmicstaining and objectively evaluated forthe density of cells with positivestaining. A field of view was assigneda score of 1 if the uptake of stain waslow in intensity. This corresponded to<15 cells with stain uptake /600Xfield. The field of view was assigned ascore of 2 if the uptake of stain wasmoderate in intensity. Thiscorresponded to 15 -40 cells stained/600X field. The field of view wasassigned a score of 3 if the uptake ofstain was high in intensity. This scorecorresponded to >40 cells stained/600X field.

ResultsEpithelialization and

Contraction -The mean length oftime for wound healing was 76 daysfor wounds treated with ESWT, and90 days for the untreated controlwounds (P = 0.051). The healedtreated and control wounds hadsimilar areas of epithelialization(T = 4.5 cm2; C = 3.9 cm2; P = 0.48)and percentages of contraction(T = 61.3%; C = 61.0%; P = 0.96).(Figures 4 & 5)

Granulation tissue - The meansum of the granulation tissue scoresdid not differ significantly throughoutthe study period (P = 0.52). Whenthe wounds were healed, the sum ofthe granulation tissue scores

Equine distal limb wounds arecommon and often heal slowly bysecond intention. Primary closure ofwounds of the distal portion of the limbis often prevented by the lack of softtissue and immobility of thesurrounding skin.1,2 Wounds of thedistal portion of the limb often heal bysecond intention and healing is ofteninhibited by the formation of exuberantgranulation tissue.3-5 Compared towounds of the trunk, lacerations of thedistal portion of the limb retract more,epithelialize more slowly, and cease tocontract sooner.6,7 Even within theequine species, there are differences inwound healing.

Second-intention healing of woundsoccurs faster in ponies than horses.7

This is the result of a greater and fastercontraction of the wound in the ponies.Wounds in horses fill with granulationtissue faster, however in ponies, thegranulation tissue is more regular witha smooth surface. Horses often developexuberant granulation tissue, howeverthis is less common in ponies.5 (Figure 1)This may be explained by differences inthe inflammatory response betweenhorses and ponies.8 Ponies have agreater initial inflammatory responsethat decreases rapidly after 3 weeks.Horses have less inflammation andfewer neutrophils initially, but theresponse remains for a longer period oftime. During the longer inflammatoryperiod, myofibroblasts are lessorganized in the horse than the pony.Overall, ponies have a more controlledinflammatory stage and greaterorganization of myofibroblasts resultingin the more rapid and greater woundcontraction than the horse.8 In-vitrostudies have shown that there are noinherent differences in fibroblasts andmyofibroblasts of horses and poniestherefore, environmental factors suchas cytokines and the inflammatoryresponse likely account for thedifferences.9

This is where shock wave therapymay be important to help direct the

healing response. Numerous studieshave shown an upregulation of multiplecytokines following ESWT. Theconsistent findings in multiple tissuesare an increase in growth factorsincluding VEGF, TGF-B1, and IGF.10,11,12

TGF-B1 has been documented asimportant in stimulating woundcontraction. The horse has lowerproduction of TGF-B2 than ponieswhich may be on of the reasons for thedifferences in wound contraction ratesbetween horses and ponies.13,14 Anotherpossibility could be oxygen derived freeradicals including superoxide and nitricoxide which have been identified inother tissues following ESWT.15,16

Increased endothelial nitric oxidesynthase has been demonstrated byimmunohistochemistry in tendon andbone following shockwave therapy.10

A nitric oxide releasing gel was shownto increase the rate of epithelializationof burn wounds in rats17 therefore,nitric oxide could be another potentialmechanism for stimulation of woundhealing. Associated with the increasedgrowth factors is a resultant increase inneovascularization which should resultin faster wound healing.10,11,12,18

Undoubtedly, in all species it wouldbe desireable for wounds to healquickly with a return to normalfunction. How this is accomplished ineach species may be by differentmechanisms. In the horse, a mechanismto stimulate the rate and amount ofcontraction would be beneficial.Contraction is usually beneficial forreturn to function of limb wounds inhorses. It usually occurs faster thanepithelialization, and results in a bettercosmetic outcome. Therefore, maximalcontraction is usually desireable in thehorse. Disfiguring and disablingcontracture does not occur on thelower limbs of the horse. Excessivecontracture can occur with injuries tothe lips, muzzle or eyelids, however,even this is not common. (Figure 2)Consequently, there is a largedifference between horses and other

species including humans and dogswhere excessive wound contracture ismore problematic.

Re-epithelialization is an importantstep in wound healing in the horse, butnot important as contraction. Until awound on the distal limb of a horse iscompletely re-epithelialized there is arisk of exuberant granulation tissueformation. Thin, nonpigmentedepithelialized scars can result inrepetitive injuries to the re-epithelialized tissue. Epithelialization isslow and frequently the most prolongedphase of the process with a maximumrate of 1mm/10 days. In horses,epithelialization is limited untilcontraction has subsided, therefore,wounds with greater contraction haveless epithelialization.

Many drugs and devices to stimulatewound healing have been evaluated ondistal limb wounds of horses 3,5,6,19,20 butfew controlled studies document thebenefits of these products.Schumacher, et. al. found no benefit ofisland grafting on the rates ofepithelialization and contraction ofsurgically created wounds on the distalportion of limbs of horses.6 Equine-derived amnion applied as a dressing tofull-thickness wounds on the distal limbof horses significantly spedepithelialization in one study,20 but thisfinding could not be repeated inanother.21 Topical medications,including antimicrobial drugs,3

corticosteroids,5 and variousdressings,19,20,21 have shown little benefitto wound healing. One study diddemonstrate that application of a 1%silver sulfadiazine cream resulted in afaster rate of epithelialization.3

Recent studies, in which EWST wasapplied to skin grafts and epidermalburns in people, demonstrated adecreased time to healing.22 When theeffects of ESWT on the healing ofpartial-thickness wounds of pigs wereevaluated, researchers found that theeffect of ESWT on the speed ofepithelialization was dose related andthat the maximum effect onepithelialization occurred at 10 pulsesat 14kV.13 Recently, the survival ofepigastric skin flaps of rats was shownto be enhanced by the application ofESWT.11,23 Similarly, in a skin flap model,ESWT stimulated healing as much asdid gene therapy using transforminggrowth factor-ß1 (TGF-ß1) or vascularendothelial growth factor (VEGF).24,25

ESWT-treated flaps developed an areaof necrosis of only 2.5%, whereas 17%of the area of flaps that did not receiveESWT was necrotic.24 In another study,

Should we expect similar effects of extracorporealshockwave therapy on wounds of different species?

Scott McClure, DVM, PhD, DACVS;

Dean Morgan, DVM;

Eric Reinertson, DVM, MS;

Michael J. Yaeger, DVM, PhD

98

throughout the study period was 10for the treated wounds and 8 for thecontrol wounds.

Immunohistochemical staining -In study tissues, IGF-1 staining wasobserved in the cytoplasm ofmacrophages, fibroblasts, neutrophils,and plump, immature endothelial cells.Positive staining for VEGF in controland study tissues was identified in thecytoplasm of fibroblasts, endothelialcells, macrophages, and smoothmuscle cells. Staining for TGF-ß1 wasidentified in the fibrinous exudate, andthe cytoplasm of macrophages,fibroblasts, and endothelial cells.

The density of staining of growthfactors varied between regions of thesection examined. Density of stainingwas greatest in areas of wound repairthat were composed of immature,loose, granulating, fibrous connectivetissue. There were no statisticaldifferences in the score for cells withpositive staining for VEGF (P = 1.0and P = 0.37), IGF-1 (P =1.0 andP = 0.31), and TGF-ß1 (P = 1.0 andP = 0.37) between treatment andcontrol wounds at day 14, or day 28,respectively. There was a statisticallysignificant decrease in density scorefor cells staining positive for VEGF(P = 0.03) in control wounds from day14 (mean 3) to day 28 (mean 2.17).A similar trend in VEGF score wasnoted in the treatment wounds(d 14 = 3; d 28 = 2.33; P = 0.06).There was a significant decrease in theIGF-1 density score between the initialand second biopsies among the controlwounds (d 14 = 2.83; d 28 = 1.67;P = 0.015), but not among thetreatment wounds (d 14 = 2.83;d 28 = 2.33; P = 0.25). TGF-ß1 scoresdecreased significantly during the timefrom the first to second biopsy in boththe treatment (d 14 = 3.0; d 28 = 1.5;P = 0.015) and control (d 14 = 3.0;d 28 = 1.67; P = 0.015) wounds.

DiscussionIt is very apparent that we must

look at each study individually Notonly do we need to account for EFD,pulse numbers, waveform, and device,we need to be aware of speciesdifferences. The extrapolationbetween species is only viable asstarting points for research. Thewound type must also be considered.Most burn wounds and skin flaps havesome degree of dermis present. In ourstudy we evaluated the rate of healingof full-thickness, cutaneous defects.

These wounds included all skin,subcutaneoius tissue and periosteum.To our knowledge, similar woundshave not been evaluated in otherspecies.

While the results of this studyindicate that ESWT may speed therate of healing of wounds on the distalportion of the fore limbs of horses, itonly resulted in a 14 day improvement.When healed, the treated and controllimbs had similar percentages ofcontraction and epithelialization. Ourresults show that the increased rate ofhealing was primarily due to anaccelerated rate of epithelializationand to a lesser degree contractionduring the early wound healing period.Clearly there are some benefits forESWT in wounds that must heal bysecond intention in the distal limb ofthe horse. However, from this studyand previous studies, there may beadditional clinical indications.

Chronic non-healing wounds withexuberant granulation tissue arecommon in the horse. In thesewounds, any mechanism to stimulatehealing after debriding the exuberantgranulation tissue is needed. In thestudy presented here, the effect ofESWT was seen predomiantly in thefirst 3-5 weeks after wounding. IfESWT could “restart” the early phasesof wound healing it could be beneficialto these chronic wounds.

Limb wounds in the horsefrequently have flaps of tissue, whichare often lost due to avascularnecrosis. The benefit of ESWT on theepigastric flaps in rats resulted in an14.5% decrease in flap loss. Thiscould be important in these woundswhere there is limited soft tissuecovering. (Figure 6)

The increase in epithelializaitonwas the primary contributor to thedifferences seen in this study. Distallimb wounds in the horse frequentlyrequire skin grafting to achievehealing. The stimulation ofepithelialization from skin grafts wouldgreatly speed the healing of thesegrafted wounds. (Figure 7)

Additionally, ESWT in conjunctionwith other wound therapies mayprovide a way to enhance the responseand further maximize the rate ofwound healing. The topical applicationof platelet-rich plasma has been shownto accelerate epithelialdifferentiation,38 which potentiallycould be synergistic with the ESWT.In equine lower limb injuries wherecontraction is limited and skin grafts

are often required, the value of ESWTon graft take and epithelializationshould be investigated. There were nocomplications seen with the treatmentin this study and no contraindicationswere found involving 208 humanpatients.37 The effects appear to bemost predominant early in the healingprocess, so treatment may best beconcentrated early after injury.

a Equitron, Sanuwave Inc., Marietta, GA.b Image J 1.37v, National Institutes ofHealth

c Antibody source: IGF-I (H-70)-sc-9013, TGFB1 (V)-sc-146, VEGF (A-20)-sc-152: Santa Cruz Biotechnology,Inc., 2145 Delaware Avenue, SantaCruz, CA 95060

References1. Hendrickson DA. Management of

superficial wounds. In: Auer JA, ed. EquineSurgery. 3rd ed. St. Louis, Missouri:Saunders Elsevier Inc., 2006;288-298.

2. Stashak TS. Selected factors that affectwound healing. In: Stashak TS ed. Equinewound management. Malvern, Pa: Lea &Febiger, 1991;22-24, 36-51.

3. Berry DB, Sullins KE. Effects of topicalapplications of antimicrobials andbandaging on healing and granulationtissue formation in wounds of the distalaspect of the limbs in horses. Am J Vet Res2003;64:88-92.

4. Wilmink J, Van Weeren PR, Stolk PWT, etal. Differences in second-intention woundhealing between horses and ponies:histological aspects. Eq Vet J 1999;31:61-67.

5. Bertone AL. Management of exuberantgranulation tissue. Vet Clin North AmEquine Pract 1989;5:551-562.

6. Schumacher J, Brumbaugh GW, HonnasCM, et al. Kinetics of healing of grafted andnongrafted wounds on the distal portion ofthe front limbs of horses. Am J Vet Res1992;53:1568-1571.

7. Wilmink J, Stolk PWT, Van Weeren PR, et al.Differences in second-intention woundhealing between horses and ponies:macroscopic aspects. Eq Vet J 1999;31:53-60.

8. Wilmink JM, Van Weeren PR, Stolk PWT, etal. Differences in second-intention woundhealing between horses and ponies:histological aspects. Eq Vet J 1999;31:61-67.

9. Wilmink JM, Nedorbrost U, Van Weeren PR,et al. Differences in wound contractionbetween horses and ponies: The in vitrocontraction capacity of fibroblasts. Eq Vet J2001;33:499-505.

10. Wang CG, Wang FS, Yang KD. Biologicalmechanisms of musculoskeletalshockwaves. International Society forMusculoskeletal Shockwave Therapy -Newsletter. 2006;1:5-11.

11. Meirer R, Brunner A, Deibl M, et al. Shockwave therapy reduces necrotic flap zonesand induces VEGF expression in animalepigastric skin flap model. J Recon Micro2007;23:231-35.

12. Wang CJ, Huang HY, Pai CH. Shock wave-enhanced neovascularization at the

tendon-bone junction: An experiment indogs. J foot Ankle Surg 2002a;45:16-22.

13. Theoret CL. Update on Wound Repair,Clin Tech Equine Pract 2004;3:110-122.

14. Theoret CL, Barber SM, Moyana TN, et al.Expression of transforming growth factorß1, ß3, and basic fibroblast growth factorin full thickness skin wounds of equinelimbs and thorax. Vet Surg 2001;30:269-277.

15. Park JK, Cui Y, Kim MY, et al. Effects ofextracorporeal shock wave lithotripsy onplasma levels of nitric oxide and cyclicnucleotites in human subtsect. J Urol2002;168:38-42

16. Wang FS, Wan CJ, Sheen-Chen SM,Superoxide mediates shock waveinduction of ERK -dependent ostoegenictranscript factor (CBFA1) andmesenchymal cell differentiation towardosteoprogenitors.J Biol Chem. 2002;277:10931-10937.

17. Zhu H. Ka B, Murad F. Nitric oxideaccelerates the recover from burnwounds. World J Surg 2007;31:624-631.

18. Kersh K, McClure, SR, Van Sickle D. Theevaluation of extracorporeal shock wavetherapy on collagenase inducedsuperficial digital flexor tendonitis VetComp Orthop Trauma 2006;19: 99-105.

19. Gomez JH, Schumacher J, Lauten SD, etal. Effects of 3 biological dressings onhealing of cutaneous wounds on the limbsof horses. Can J Vet Res 2004;68:49-55.

20. Bigbie RB, Schumacher J, Swaim SF, et al.Effects of amnion and live yeast cellderivative on second intention woundhealing in horses. Am J Vet Res1991;52:1376-1382.

21. Howard RD, Stashak TS, Baxter GM.Evaluation of occlusive dressings formanagement of full-thickness excisionalwounds on the distal portion of the limbsof horses. Am J Vet Res 1993;54:2150-2154.

22. Meirer R, Kamelger FS, Piza-Katzer H.Shock wave therapy: An innovativetreatment method for partial thicknessburns. Burns 2005;31:921-922.

23. Meirer R, Kamelger FS, Huemer GM, et al.Extracorporeal shock wave may enhanceskin flap survival in an animal model. BritAssoc Plastic Surg 2005;58:53-57.

24. Huemer GM, Meirer R, Gurunluoglu R, etal. Comparison of the effectiveness ofgene therapy with transforming growthfactor-_ or extracorporeal shock wavetherapy to reduce ischaemic necrosis inan epigastric skin flap model in rats. WndRepair Regen 2005;13:262-68.

25. Meirer R, Huemer GM, Oehlbauer M, et al.Comparison of the effectiveness of genetherapy with vascular endothelial growthfactor or extracorporeal shock wavetherapy to reduce ischaemic necrosis inan epigastric skin flap model in rats. PlastReconstr Surg 2007;60:266-271.

26. Morgan DD, McClure S, Yaeger M, et al.Effects of extracorporeal shockwavetherapy on wounds of the distal portion ofthe limbs of horses. Accepted forPublication: Am J Vet Res

27. Schaden W, Thiele R, Kölpl C, et al. Shockwave therapy for acute and chronic softtissue wounds: A feasibility study. J SurgRes 2007;143:1-12.

28. Carter CA, Jolly DG, Worden CE, et al.Platelet-rich plasma gel promotesdiffentiation and regeneration duringequine wound healing. Exp Mol Pathol2003;74:244-255.

Figure 1. Exuberant granulation tissueon the lateral aspect of a chronic

nonhealing wound.

Figures:

Figure 2. Contracture of the lower lip ofa young horse that was exposed to a

caustic material that created thewounds. The contracture of the wound

on the ventral lower lip resulted in thelip curling downward.

Figure 3. One 4 cm diameter wound on the metacarpus wasrandomly assigned to be treated (green) and the

contralateral metacarpus wound served as the untreatedcontrol (red). Two 3-cm wounds were created on each

metatarsus and they were assigned to treatment or controlthe same as the ipsilateral metacarpus.

1110

IntroductionIncreasing numbers of adult

patients with shoulder tendinopathiesare presenting worldwide and latestadvances in imaging techniques afforda better characterization of thesepatients, however, understanding of thepathophysiology of rotator cuff diseasesremains incomplete. Recent reportshave been focused on the biology ofrotator cuff responses to selectivetreatment with cytokines [1,2,3], whileothers analyze the pathological findingswith better study protocols [4,5,6].

In recent years ExtracorporealShockwave treatment for thiscondition has been applied withincreasingly improved results[7,8,9,10]. Our aim is to gain moreknowledge of the biological responseof the shoulder rotator cuff to thisnew therapy, including vibrationalspectroscopy analysis [11,12].

Patients and MethodsFrom January 2004 to August 2008,

we attended 40 patients (symptomaticrotator cuff tears (38 patients) andcalcified tendinopathy (2 patients)) thatunderwent open surgical treatment. Overthe same time period, shockwave therapywas applied for Calcified ShoulderTendinosis (electro-hydraulic device, 4000pulses, 0.33mJ/mm2, single session,without anesthesia, outpatient procedure)and the same treatment protocol wasoffered to patients with rotator tears whohad not previous surgery. Ten suchpatients accepted the treatment. Patientsreceived full disclosure concerning thedifferent medical and surgical treatmentoptions available to them and informedconsent about technical procedures and

biopsy treatments. Fifty-three biopsies(Group A, all open surgery, 40 initialsurgical patients, 10 patients with SW pre-op, 3 patients who underwent surgicalresolution after SW-failed treatment forCalcified Tendinopathy) were collected,undergoing standard laboratoryprocedures for preservation and staining(haematoxylin-eosin technique), andexamined under light microscope (Nikon-Eclipse E200). We used Riley’shistopathological classification and semi-quantitative analyses for all 53 H-E stains,examining and photographing 3microscopic fields (x10-objective);interesting findings were reviewed withx40 and x100-objectives and also werephotographed.

Twelve biopsies (non-SW treated,Group B) and 13 SW-treated (Group C)underwent immunohistochemicalprocedures (monoclonal antibodies andtechniques for PCNA, cd34+, cd14+, D2-40, Col I, Col III, Tenascin-C) and semi-quantitative analyses were done forcountable stain on formed structures incases of PCNA, D2-40 (lymphaticmarker), cd34+ and cd14+ (endothelialcell marker). We reviewed 5photographed microscopic fields (x10-objective) for each antibody, applying agrille 10x10 (100 chambers), andobtaining a total number and percentage(absent: 0%, low: up to 20%, regular: upto 70%, intense: 80% to 100%).

In the case of Col I, Col III andTenascin-C the photographed fieldsreceived a grille 5x5 (25 chambers),characterizing the stain distributioncomparing intensities over the analyzedarea (low: up to 20% (5 chambers);regular: up to 40% (ten chambers);intense: all the rest). For both groups (B

and C), interestinghistological findingswere reviewed withx40 and x100-objectives and werephotographed.

Biopsies of GroupsB and C receivedspectroscopicprotocols for this kindof analysis. The toolsselected for ourstudies are Ramanspectroscopy and theultrasensitiveanalytical techniqueof Surface-enhancedRaman scattering(SERS). Here wereport structuralinformation obtainedfrom 1016 SERS

spectra of 52 biopsies of tendon tissueson Ag nanoparticles.

ResultsHistopathological analysis

Macroscopic features of biopsiesinclude: (1) the edge of the torn rotatorcuff with 2 to 3 mms of medial portions ina single piece; (2) sample of bone with acartilage border (4-5mms wide, 6-7mmslong), corresponding to the area of normalinsertion of supraspinatus muscle.

For 53 histological observations (GroupA, H-E sections), the distribution accordingto Riley Classification indicates 4 casesgrading type II, 37 cases grading type IIIand 12 cases grading type IV , examined inorder from medial to lateral edge. ForGroup C (SW) the Riley distribution was 7cases type III and 6 cases type IV.

A careful examination of the vascularaspect in tendinopathic non SW-treatedpopulation showed that many vascularbeds comprise damage of pericyte cellsthat envelope endothelial cells innascent neo-angiogenesis and this areatends to develop micro-haemorrhagicinstances (fig.1a, b). Chondroidmetaplasia was seen much more incases of Riley type IV, which occupieszones related to the torn edge. Theseareas evidence profound vascularmetaplasia where non propermorphological features of vessels couldbe identified and also shows manyacellular fields (fig.1c).

Biopsies of patients treated with SWdemonstrates areas of fibroblasticrepairs that tend to appears in clusters(more over the medial portions ofspecimens) and include definite signs ofactive neo-blood vessels withhypertrophic areas corresponding to

Shoulder Rotator Cuff Tendinopathy. Histological,Immunohistochemical and Vibrational Spectroscopy Analysis

Brañes J.A., MD.(1,2), Contreras H., PhD.(2), Aroca R., PhD.(3),

Campos M., PhD.(4), Clavijo A., PhD.(4), Carcamo J. (4),

Brañes M., MD.(1,4)

1. BioSurgery Unit (ShockWave Center), AraucoSalud Clinic,Santiago, Chile.

2. Physiology and Biophysics Program. Institute of Biomedical Sciences. Faculty of Medicine. University of Chile.

3. Department of Chemistry & Biochemistry – Windsor University,Ontario, Canada.

4. Faculty of Sciences, University of Chile. POBox 653 Santiago 21. Chile.

Figure 4. From top to bottom pictures takenon day 0, 30,62 and 90 days after thecreation of the wounds. For this horse theleft limb (on the left) was treated and theright was the untreated control. The darkskin in this horse made it difficult to see thetattoo so the tattoo was marked over with awhite marker for pictures.

Figure 5. The total area of the control (C) and treated (T) wounds increased in size fromday 0 to day 14 when they began to decrease in size from contraction and

epithelialization. There was a greater area of contraction in the control wounds becausethey took longer to heal. The majority of the difference between treated and control

wounds was the difference in the area of epithelialization from 17 through 34 days.

Figure 6. Lower limb injuries in the horsecommonly result in the formation of tissue flapswith poor perfusion that become avascular.Decreasing the area of tissue lost from avascularnecrosis would notably improve healing of thesewounds. Figure 7. This mesh graft was

applied 24 hours previously to alaceration over the pastern region.Shock wave therapy couldpotentially improving the rate ofepithelialization from grafts andshorten healing times.

12 13

Fig. 3

Fig. 3A, SW-treated case in tendinosis type III showing a node of angiofibroblastic response to SW (compare to fig.1A and 1B).; in 3B ,same patient , depict relationship between the neo-angiogenesis area depositing collagen to stabilize the sprout (black arrows) and thenative structural collagen on the tendon (white arrow). 3C, calcified tendinosis SW-treated showing a lymphatic vessel with a macrophage inside (open arrow) containing microgranular calcium salts (black arrows) (Toluidine Blue Stain).

Fig. 3

Fig. 3D, calcified tendinosis SW-treated and immunostaining for D2-40 (lymphatic marker) showing a calcium granuloma underresorption (black arrows) with surrounding lymphatic channels (open arrows) developed in the entire process of resorption. 3E,microgranular calcium inside of lymphatic channel (arrow , D2-40 immunosatining,x100-objective).

Fig. 4

Fig.4A, tendinosis type III ( Group B patient) with immunostaining for PCNA, showing almost absent staining (black arrows) upon 4areas with vessels (open arrows). 4B, example of Group C immunostaining for PCNA, showing intense staining in angiofibroblasticnodes SW-induced (black arrows). 4C, Group C patient with immunostaining for collagen I, showing disposition of the marker in areasassociated to neo-blood vessels SW-induced (black arrows).

References1. Andres B.M., Murrell G. Treatment of

Tendinopathy.Clin Orthop Relat.Res (2008)466:1539-1554.

2. Xu Y., Murrell G.A. The Basic Science ofTendinopathy. Clin Orthop Relat Res(2008) 466:1528-1538.

3. Kovacevic D., Rodeo S. BiologicalAugmentation of Rotator Cuff TendonRepair. Clin Orthop Relat Res (2008)466:622-633.

4. Longo U.G., et al. Light microscopichistology of supraspinatus tendonruptures.Knee Surg Sports TraumalArthrosc (2007) 15:1390-1394.

5. Maffulli N., et al. Movin and Bonar ScoresAsses the same Characteristics of TendonHistology. Clin Orthop Relat Res. 2008Jul;466(7):1605-11. Epub 2008 Apr 25.

6. Riley G. Tedinopathy- from basic scienceto treatment. Nat Clin Pract Rheumatol.2008 Feb;4(2):82-9.

7. Wang L. et al. Extracorporeal Shock WaveTherapy in Treatment of Delayed BoneTendon Healing. Am. J. Sports Med. 2008;36:340-347.

8. Orhan Z., et al. An experimental study onthe application of extracorporealshockwaves in the treatment of tendoninjuries : preliminary report. J Orthop Sci(2001) 6:566-570 .

9. Orhan Z., et al.. The effect ofextracorporeal shock waves on a ratmodel of injury to tendo Achillis. Ahistological and biomechanical study. JBone Joint Surg [B] 2004;86-B:613-8.

10. Brañes M., et al. Tendinosis of theShoulder and Related Entities Treatedwith ESWT. Histopathological and ClinicalCorrelation. ISMST News Letter May 2007vol 3,Issue 1:9-10.

11. Kneipp, K.; Aroca, R.; Kneipp, H.; Wentrup-Byrne, E.; Editors New Approaches inBiomedical Spectroscopy. (Symposiumheld in Honolulu, Hawaii December

2005.) [In: ACS Symp. Ser., 2007; 963],2007.

12. Aroca, R. Surface-enhanced VibrationalSpectroscopy; John Wiley & Sons:Chichester, 2006.

13. Kjaer M. Role of extracellular matrix inadaptation of tendon and skeletal muscleto mechanical loading. Physiol Rev. 2004Apr;84(2):649-98. Review.

14. Shiu Y-T., et al. The role of MechanicalStresses in Angiogenesis. Crit Rev BiomedEng. 2005;33(5):431-510. Review.

15. Kirkpatrick N.D.,et al. Live imaging ofcollagen remodeling duringangiogenesis.Am J Physiol Heart CircPhysiol 292; H3198-H3206, 2007.

16. Seta N., Kuwana M. Human circulatingmonocytes as multipotential progenitors.Keio J Med. 2007 Jun; 56 (2) :41-7. Review.

pericytes envelope (increased in number) (“hypermuscularizedneo-vessels”, fig. 2a,b,c). These “nodes” show areas close tovessels, stain like disorganized new collagen and resemble nativecollagen (fig. 3a,b), but disappear in areas of chondroidtransformation of the tissue (chondroid metaplasia). Biopsies ofpatients with failed-SW treatment in Calcified Tendinosis alsodemonstrated development of lymphatic channels along whichgranular portions of calcium were being removed (fig. 3c,d,e).

Vibrational Spectroscopy analysisThe SERS spectra are dominated by signals corresponding to

the collagen molecular system in the 1300-1200 cm-1 spectralregion. Bands corresponding to the amide III mode shift infrequency and intensity in the tissues before and after shockwavetreatment. This result is interpreted in terms of a conformationalchange in the collagen induced by shockwaves. However theincreased intensity of the band at 1246 cm-1 could be also relatedto an increasing of the amount of the shockwave induced newconformation. This is supported by the spectral behaviour of themost abundant amino acid proline; in fact two of the most intenseproline bands at about840, 1080 and 1410 cm-1 drastically change in intensity. Thecollagen new conformation imposes a different analyte substrateinteraction. The disappearance by shockwave effect of the type IIIcollagen SERS band at 1609 cm-1 could be related to the relativedecreasing of such species by shockwave treatment.

DiscussionIt has been described that 2 to 3% of the volume area of

a normal tendon should be occupied by blood-vessels[13,14]. In our histological review for tendinosis, we foundthat the volume area was enlarged up to 15% in tendinosisgrade II and III. This spontaneous reparative effort includesvessels with damage on the pericytes envelope-sheath withprobabilities for micro-hemorrhages, a significant percentageof which appear to be inactive, non-containing red bloodcells. The total number of vessels and volume areapercentage lessen significantly in tendinosis grade IV, wherechondroid metaplasia predominates in many areas along thetorn edge. Also on those type IV tendinosisSW-treated we did not find neo-blood vessels close tochondroid metaplasia.

In Group C we identified specific features: clusters ofactive neo-vessels occupying up to 23-28% of volume areas,their hypermuscularized aspect correspond to hyperplasia ofpericytes surrounding endothelial cells (probably due toimbalance for more active than inactive forms of PDGF thatexists in tendinopathic matrix), metabolic activity in theseareas shows a precise augmented stain for Col I, Col III andTenascin-C, suggestive of repair matrix behavior [15].

Recovery of PCNA and Tenascin-C from “low” to “regularor intense” suggests an improvement of repair capabilities inthe SW-treated population. Also the recruitment forcd14+/34+ from “low/regular” to “intense/intense” isindicative of activation of blood-supply on those areas ofneo-blood vessels[16].

In summary, according to our results we suggest that SWtreatment induces an improvement of metabolic andintrinsic repair capabilities on tendinopathic rotator cuff ofthe shoulder. A multi-center investigative effort willascertain the real meaning of these findings.

Fig. 1

Fig. 1A, tendinosis type II, zone of initial and spontaneous angiofibroblastic response shows damage of pericyte envelop of nascentvessels (arrows) and hemorrhagic zone (bottom left). 1B, tendinosis type III, similar areas depict hemorrhagic aspect. 1C, tendinosistype IV with chondroid areas showing vascular metaplasic areas, not possible to recognize vascular endothelium (arrows).(H-E stain).

Fig. 2

Fig. 2A, SW-treated case , showing increased number of induced neo-blood vessels (x40 objective). 2B and 2C, both Group C patientsshowing hypermuscularized aspect depending on the development of pericytes ( 2B and 2C , magnification x100-objective, H-E stain).

Figures:

14 15

IntroductionTransthoracic application of shock

waves is recently known to augmentmyocardial vascularization in aporcine model of myocardialinfarction [1, 2], besides it is shownto effect relief of angina symptoms inpatients with severe coronary arterydisease [3]. Nevertheless, pulmonarycontusion causing life-threateninghypoxemia and haemoptysis isdescribed as an adverse event ofshock waves when hitting lung tissue[4, 5]. Therefore transthoraciccardiac shock wave application islimited by lungs partly covering theheart [1-3]. Direct epicardial shockwave therapy (DESWT) may be moresafe, thereby enabling the treatmentof larger myocardial areas and eventhe posterior wall of the heart.

We hypothesized that DESWTduring open heart surgery may serveas an adjunct to surgicalrevascularisation (Coronary arterybypass surgery). Therefore weestablished animal models ofischemic heart failure to show thatDESWT induces myocardialregeneration and improvesventricular function.

In June 2008 our MyocardialRegeneration Research Group fromthe Department of CardiothoracicSurgery under the direction ofProf. Dr. Michael Grimm presentedfirst results from these animaltrials at the 11th InternationalCongress of the ISMST in Juan lesPins, France. Therein DESWTshowed very promising effects [6],although the mechanism remainslargely unknown. Therefore westarted an in-vitro shock wave trial(IVSWT) to learn more about themolecular and cellular mechanismsof shock waves.

BackgroundBy reviewing literature we found

very diverse methods of applying shockwaves onto cell cultures [7-10]. Whilemost research groups have in commonthat they use ultrasound transmissiongel as a contact medium between theshock wave applicator and the targettubes, they all use different methods ofapplying shock waves onto the cells.Some of them are associated withdistinct limitations, especiallydistracting physical effects. Due to thiswe tried to develop an experimentalsetup that would perfectly imitate in-vivo conditions without severedistractions. This resulted in ourbelow-described water bath. However,since results of equal cells treated indifferent ways are not comparable,establishing a standardized model forfuture in-vitro trials was also deemeduseful. A proper in-vitro model may bean important step for intergroupcommunication, which could help all ofus working on IVSWT to learn moreabout the shock waves' mechanism bybeing able to compare our results.

ModelBasically our in-vitro model exists

of a plexiglass built water bath with anadapter for the shock wave applicator(CP-155, DermaGold® from TissueRegeneration Technologies LLC,Woodstock, USA manufactured byMTS Europe GmbH, Konstanz,Germany) [Figure 1]. This adapter canbe customized for all kind of shockwave devices. The water bath is filledwith degassed water to avoidcavitation, a heater at the bottom witha temperature sensor connected to acontrol unit enables to regulatetemperature for imitation of in-vivoconditions. A holder for our cellsamples filled in common cell culture

flasks also serves as a distance controlbar. Its fixation mechanism allows tochange culture flasks easily and quickly[Figure 2].

One of the major reasons to designthe water bath for IVSWT was toavoid reflections caused by thedistinct difference in the impedancebetween culture medium and theambient air. Due to this shock waveswould be reflected, thereby causingnegative pressure onto the cells andalso disturbing upcoming waves. Thewater bath enables propagation ofshock waves far beyond the cellculture flasks, thereby not causing anykind of distraction directly at the celllayer.

Materials & MethodsPrimary cell cultures of endothelial

cells and fibroblasts were establishedfrom native rat hearts. AdditionallyH9C2-cardiomyocytes (American TypeCulture Collection) were used. All celltypes were cultured using DMEMmedium supplement with commonnutrients and growth factors. Adherentcells in common cell culture flasksfilled with culture medium weredunked into the water bath. (Incontrast to cell suspensions adherentcultured cells give the possibility ofanalysing cell communication, e.g. gapjunctions.)

Various energy flux densities ofunfocused SWT were applied indifferent distances to the cells. Non-treated cells were used as a controlgroup. Number of cells and theirvitality then were analysed over aperiod of 7 days.

After the results of several pilottrials we focused on an energy fluxdensity of 0.15mJ/mm2 and a frequencyof 5Hz, since these are the commonlyused parameters in vivo.

IVSWT: How can in-vitro shock wave therapy be performed best? - Preliminary results from cardiac cells

Holfeld J, Kapeller B, Hack F, Tepeköylü C, Dumfarth J, Zimpfer D,

Schaden W, Losert U, Grimm M, Macfelda K.

Department of Cardiothoracic Surgery and Core Unit forBiomedical ResearchMedical University of Vienna, AustriaContact: johannes.holfeld@meduniwien.ac.at

Preliminary ResultsCounting of cells and proving their

vitality are the basic analysis of cellcultures. Vitality was proved usingtrypan blue staining. Trypan blue isnot absorbed in vital cells, just deadcells become blue. This so called DyeExclusion Method showed hardly anyblue cells in the treatment as well asin the control group. Vitality of all cellsamples was about 99%.

Cell counting revealed differentresults in each cell type, especially incomparison to the untreated controlgroup. Shock wave treated cellsobviously proliferated faster. Growthcurves of cells are shown in[Figure 3 A-C].

As a very important parameter forproliferation we calculated the cellduplication time every 24 hours withthe commonly used formulaTc=0.3T/log (A/A0) [Tc...duplicationtime, T...24 hours, A...cell numberafter 24 hrs., A0...initial cell number].The very simple diagram in[Figure 4] shows that the mean valueof duplication in treatment groups isdecreased compared to controls.Especially in a distance of 5cmbetween the shock wave applicatorand the sample the duration of cellduplication is much lower. Inconclusion, each cardiac cell typeneeds less time for proliferation aftershock wave treatment compared to itsuntreated controls. The distancebetween the applicator and thesample has a major impact on thecells' behaviour.

Detailed data interpretation is notyet possible since several analysis,especially concerningimmunohistochemistry and molecularbiology, are still in progress. In thispilot study we only used healthy cellsfrom unharmed myocardium. Fromour previous mentioned in-vivo trialswe already know that healthy cells donot respond that much to SWT thanpathologic cells do [6]. Future trialswith cells from ischemic harmedmyocardium will show the potentialeffect of DESWT in-vitro.

DiscussionBesides the cost-effectiveness and

the reduction of animal experiments,the biggest advantage of IVSWT is thepossibility of studying the specificbehaviour of a certain cell type. Inshock wave mediated tissueregeneration most likely all cells of thetreated tissue are involved, evensystemic effects are discussed.Nevertheless, each cell type plays aspecific role and has its own intrinsicfunction. These we are able to detectby doing IVSWT.

To the best of our knowledge all in-vitro models in literature make effort toelaborate application methods, but donot consider the propagation of wavesafter passing the cell culture.

In our model cell culture flasks aremounted directly into the degassedwater bath, which is connected with theshock wave applicator through acircular opening. As cell culture flasksare filled with culture medium and noother coupling membranes are neededin this system, there is hardly anydifference in acoustic impedancebetween the applicator an the cells.This leads to an undisturbedpropagation of shock waves and avoidsreflection as well as negative pressureand interference with upcoming waves.

Another advantage in doing IVSWTwith this model is the possibility ofvarying the distance between theapplicator and the culture flasks withthe distance control bar fixing thesample.

Although SWT is used for severalclinical indications, its exact molecularmechanism is still not exactlyunderstood. IVSWT can help us to learnmore about the molecular and cellularmechanisms of shock waves. Byunderstanding them new indicationscould be established and moreover ourtoday approved indications could beimproved by knowing more about theinfluence of the different applicationparameters like pressure distribution,energy flux density, number of impulsesand the specific impact of differentshock wave technologies. To addressthis issue we will have to comparefocused with defocused shock wavesand electro-hydraulic with electro-magnetic and piezoelectric waves infuture in-vitro trials.

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H, Uwatoku T, Abe K, Matsumoto Y,Kajihara N, Eto M, Matsuda T, Yasui H,Takeshita A, Sunagawa K. Extracorporealcardiac shock wave therapy markedlyameliorates ischemia-induced myocardialdysfunction in pigs in vivo. Circulation.2004;110(19):3055-3061.

2. Uwatoku T, Ito K, Abe K, Oi K, Hizume T,Sunagawa K, Shimokawa H.Extracorporeal cardiac shock wavetherapy improves left ventricularremodeling after acute myocardialinfarction in pigs. Coron Artery Dis. 2007Aug;18(5):397-404.

3. Fukumoto Y, Ito A, Uwatoku T, Matoba T,Kishi T, Tanaka H, Takeshita A, SunagawaK, Shimokawa H. Extracorporeal cardiacshock wave therapy amelioratesmyocardial ischemia in patients withsevere coronary artery disease. CoronArtery Dis. 2006;17(1):63-70.

4. Malhotra V, Rosen RJ, Slepian RL. Life-threatening hypoxemia after lithotripsy inan adult due to shock-wave-inducedpulmonary contusion. Anesthesiology.1991 Sep;75(3):529-31.

5. Tiede JM, Lumpkin EN, Wass CT, Long TR.Hemoptysis following extracorporealshock wave lithotripsy: a case oflithotripsy-induced pulmonary contusionin a pediatric patient. J Clin Anesth. 2003Nov;15(7):530-3.

6. Zimpfer D, Aharinejad S, Holfeld J,Thomas A, Dumfarth J, Rosenhek R,Czerny M, Schaden W, Gmeiner M, WolnerE, Grimm M. Direct epicardial shock wavetherapy improves ventricular function andinduces angiogenesis in ischemic heartfailure. J Thorac Cardiovasc Surg. in press

7. Delvecchio FC, Brizuela RM, Khan SR,Byer K, Li Z, Zhong P, Preminger GM.Citrate and vitamin E blunt the shockwave-induced free radical surge in an invitro cell culture model. Urol Res. 2005Dec;33(6):448-52.

8. Moosavi-Nejad SF, Hosseini SH, Satoh M,Takayama K. Shock wave inducedcytoskeletal and morphologicaldeformations in a human renal carcinomacell line. Cancer Sci. 2006 Apr;97(4):296-304.

9. Nurzynska D, Di Meglio F, Castaldo C,Arcucci A, Marlinghaus E, Russo S,Corrado B, de Santo L, Baldascino F,Cotrufo M, Montagnani S. Shock wavesactivate in vitro cultured progenitors andprecursors of cardiac cell lineages fromthe human heart. Ultrasound Med Biol.2008 Feb;34(2):334-42.

10. Chao YH, Tsuang YH, Sun JS, Chen LT,Chiang YF, Wang CC, Chen MH. Effects ofshock waves on tenocyte proliferation andextracellular matrix metabolism. UltrasoundMed Biol. 2008 May;34(5):841-52.

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Fig. 1

Fig. 2 Fig. 3

Fig. 4

Figures:

Myofascial Pain Syndrome (ICD 10 - 79.1) –An Excellent Indication for Low Energy Focussed ESWT

During the last years patient studies and clinical trials have revealed new indications for the use of focussedShockwave Therapy (ESWT). Pain conditions of different types caused by diverse lesions of the musculo-skeletal systemhave been often in the centre of the attention. Whereas bony and tendinous structures have been since the beginning ofESWT in orthopaedic diseases literally in the focus of the treatment, muscle tissue has not been considered equally.Recently, along with new scientific studies about the understanding of muscle pain, which is totally different to thenociceptive system of the skin, putting the focus of ESWT on painful spots in the muscular tissue, the so calledMyofascial Trigger Points (MTrP's), a new chapter of understanding and treating pain conditions has been opened.

According to Wheeler (2004) 44 million Americans are estimated to have Myofascial Pain Syndrome (MPS) so it isseen to be one of the most common causes of acute and chronic pain of the musculoskeletal system. It often imitatesother pain conditions e.g. nerval root lesion. MPS is characterized by Myofascial Trigger Points (MTrPs), which arehyperirritable spots in a palpable tense band of skeletal muscle. MTrPs are caused by a dysfunction from involved motorendplates, which is followed by a segmental shortening of groups of sarcomeres. Diagnostic approach is based on thecriteria defined by J.Travell and D.Simons: while palpating an active MTrP a referred and familiar (recognition) pain iselicited. Effective diagnosis and treatment requires clinical experience and diagnostic skills, especially palpation ability.Exact pressure or impulse with minimum irritation or even damage of the collateral tissue is needed to identify andrelease MTrPs.

Focussed ESWT is able to apply an exact mechanical impulse on a small spot to find MTrP's in the muscle, even in thedeeper layers, and while eliciting the patient's typical pain (recognition and referred pain), it can much likely identifyMTrP's as a major source of the patients complaints.

MPS can be treated successfully with focussed ESWT while putting MTrP's exactly in the focus and releasing thesepainful spots.

The use of focussed ESWT is a good method for the diagnosis and treatment of musculo-skeletal pain that is due to MPS.

H. Müller-Ehrenberg, MD, Orthopeadic doctorMünster, Germany

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quality and wehave found theexperimentalconditions thatavoid sampleburning andsampledegradation.We are in theprocess ofrecording now asubstantial amountof new data onthese and othersamples(thousands ofspectra) thatshould give us thestatisticalvalidation for characterizationof the molecular species.We are also beginning to workon the identification of othermolecular components in thetissue.

The preliminary data areencouraging. It can be seenthat there are characteristicwavenumbers that can beassigned to collagen, marked inFigure 3 in all five samples.The experimental work willcontinue as to enhance ourdatabase, and then we willused multicomponent analysis,specially adapted to our needs,to extract the information fromthe spectral maps obtained bySERS of tissue samples. Theaim of the work is to provide aspectral characterization of thetissues before and aftershockwave treatment.

References1. Aroca, R. Surface-enhanced

Vibrational Spectroscopy; JohnWiley & Sons: Chichester, 2006.

2. Kneipp, K.; Aroca, R.; Kneipp, H.;Wentrup-Byrne, E.; Editors NewApproaches in BiomedicalSpectroscopy. (Symposium heldin Honolulu, Hawaii December2005.) [In: ACS Symp. Ser.,2007; 963], 2007.

3. Baker, G. A.; Moore, D. S.Analytical and BioanalyticalChemistry 2005, 382, 1751-1770.

4. Middendorf, H. D.; Hayward, R.L.; Parker, S. F.; Bradshaw, J.;Miller, A. Biophysical Journal1995, 69, 660-673.

The biological applications ofRaman scattering (RS) in its differentforms continues to grow exponentially,and the literature is so extensive thatin a short communication referenceswill not even attempt to do justice tothe field. The fingerprint for molecularstructure provided by vibrationalspectroscopy, and their relation tofunctionality in biochemical systemscan be used for the development of aquantitative technique for biomarkers.These vibrational fingerprints in thespectra are used to track andcharacterize species such as small low-molecular-weight metabolites and alsofollow molecular species in large livingorganisms. Today, researchers aremaking great progress applying RS tounravel the structure/function issues inproteins, nucleic acids, and lipids.Recently, the efforts are devoted tobioanalytical and medical diagnosticapplications. In our group, forbiomedical applications, we integrate afull range of Raman experimentalmethodologies, including Ramanmicroscopy, resonance Ramanscattering microscopy, near-infraredRaman, and the ultrasensitiveanalytical technique surface-enhancedRaman scattering.1 This molecularapproach is then integrated with thebiomedical research in an attempt tounderstand the biological processes.2

Here, we present the first stepstowards the molecular understandingof the important improvements ofrotator cuff supraspinatus tendonsdiseases that have seen aftershockwave treatment. Neo-angiogenesis stimulation andhypercelularization are the result ofshort time periods of treatment. Thebeginning of this work, necessarily,requires an extensive backgroundresearch dedicated to the creation ofthe appropriate database forfingerprint characterization of thebiomolecules present in the tissue.

This is an enormous task that involvesa large group of multidisciplinaryresearchers with a top-down approachof the medical team (the real samples)and a bottom-up approach of thespectroscopist, all helped by thestatistical analysis and modelling of thephysics group. The preliminary results have beenselected from our Raman scatteringand plasmonic driven technique ofSurface-enhanced Raman scattering.3

The background information includedthe studies of the basic amino acidsforming collagen, two different types ofcollagen and 52 biopsies of tendontissues. Briefly, the inelastic Ramanscattering was collected using a micro-Raman system with a spatial resolutionof 1 micron squared and the sample isilluminated with laser lines at 442 nm,514.5 nm, 632.8 nm, or 785 nm,depending on the optimization of theexperimental conditions. SERS wasattained using overlayers of silver andalso colloidal silver nanoparticles.Typical Raman spectra of the aminoacids most commonly found in collagenare shown in Figure 1.

It can be seen, that each moleculeof the amino acid has its owncharacteristic spectral pattern, andcharacteristic wavenumbers can be

identified for each one of them. Theextensive and complete analysis andcomputational work for each moleculewill be published separately.

The collagen detection andcharacterization was demonstratedusing to commercially availablecollagens; the rabbit skin (TR in thespectra), and ox bone (CB in thespectra). The experimental SERS wasobtained by depositing 6 nm massthickness of silver by vacuumevaporation onto the collagen sample.Micro-Raman was recorded usingpoint-by-point-mapping. The mappingshows that a typical pattern repeatitself on the silver coated collagen forboth collagens. These typical spectralpatterns are shown in Figure 2.

It can be said that there are fourcharacteristic wavenumbers in bothcollagen samples. Characteristic heremeans that these Raman bands havesimilar relative intensities and areobserved at approximately the samewavenumber maxima: 799, 1003, 1353and 1637 cm-1. The band at 736 cm-1,clearly marks the difference betweenthe two forms of collagen. Notably, thevibrational spectra of collagen has alsobeen studied using a pulsed sourceneutron spectrometer.4

The final and more challenging partof the work is proveof concept that goodSERS spectra can beobtained form thetissue (biopsies)provided by themedical team. TheSERS spectraobtained for severalof these samples areshown in Figure 3.The technique is thesame applied toobtain the spectra ofcollagen. It can beseen that the spectraare of excellent

BRIEF COMMUNICATIONRaman and Surface Enhanced Raman Scattering Applications

in Shock Wave Therapy Related ResearchAroca R.,PhD.(1,2), Campos M.,PhD.(2), Clavijo A.,PhD.(2), Carcamo J. (2)

1. Department of Chemistry & Biochemistry – Windsor University, Ontario, Canada.2. Faculty of Sciences, University of Chile. POBox 653 Santiago 21. Chile.

Fig. 1

Fig. 3Fig. 2

Since 1994 the ESWT is applied in the field of orthopedic andsurgery and there were no concrete treatment guidelines everpublished.

According to the consensus statement of international board ofexperts from 2008 (see Newsletter 2008) the experts worked outguidelines in that way the AWMF working group of scientificapproved societies in Germany is using.

The expertgroup: (Dr.Auersperg, Dr. Buch, Dr. Gerdesmeyer, Dr.Gleitz, Prof. Maier, Dr. Neuland, Dr. Rädel, Prof. Rompe, Dr.Schaden, Dr. Thiele, Dr. Wille)

There are now guidelines for the approved standardindications for

• chronic tendinopathies as plantar fasciitis with or without heelspur

• Achilles tendon• epicondylopathie (tennis elbow)•o rotator cuff with or without calcification• patella tendon• greater trochanteric pain syndrome

For the impact bone healing function:

• non unions and delayed bone healing• stress fractures• early stage of avasculaer bone necrosis (native X-ray

without pathology)• early stage osteochondritis dissecans (OD postsceletal

maturity)

These guidelines will be published soon.

On the same meeting this board of international experts definedspecial guidelines for the ESWT of skeletal muscles.

ESWT of skeletal muscles

Preamble: Maofascial pain syndromeClassification M62.8 ICD 10

Synonyms

Myogelosis, muscle hardenings, myofascial pain syndrome,pseudo-radicular pain syndrome, trigger points, RSI Syndrome

Etiology

Mainly a subsequent state of an extra muscular pathology e.g. by• static disorders• muscular dysbalance• arthrogenic irritations• visceral irritations• internal diseases• radiculopathies• chronic overload / incorrect weight bearing• acute and chronic injuries of the skeletal muscles

Symptoms

local pressure pain, stretching and tension pain, musclehardening, muscle shortening, strength reduction, motoricdysfunction

Instrument-based diagnostics

ultrasonographylaboratory (inflammation parameter, muscle enzymes)

Differential diagnosisprimary myopathies, neurological diseases, neurogenicdysfunction, rheumatic pains, psychological diseases,neurovegetative syndrome, hormonal disorders (e.g.hyperparathyroidism, hypothyroidism), cardiac diseases, adversereactions

Conservative therapy in alphabetic orderaupuncture, electrotherapy, immobilization, infiltration of localanaesthetics and (or cortisone, needling, neural therapy, non-steroidal antirheumatics, orthesis, strain relief, stretching,thermotherapy, ultrasound

Surgical interventionsdenervationsubcutaneous tenotomy

Shockwave therapyIndication: diagnosis by the MD (physician)

Contraindications: malignant tumor in the focal area, openepiphysis in the focal area, pregnancy

Spatial requirements: requirements for the certification of amedical practice e.g. hygiene plan, emergency plan according toISO 9001:200 available.

Patient preparation: patient positioning in a position withrelaxed muscles to be treated, orientating ultrsonography of thetherapeutic area for local diagnostics and selection of focaldepth, patient information about shockwave therapy andexplicit information about potential hematoma.

MD and assistants: medical treatment, written documentationof the therapy

Therapy procedure:• no local anaesthetics• designation of the shockwave source• designation of the muscles to be treated• 1 - 6 treatments• total energy flux density EFD: 0.05 - 0.35 mJ/mm_• interval 1 week• frequency: focused shockwave therapy FSW: 4 - 8 Hz,

radial shockwave therapy RSW 10 - 30 Hz• maximum of 2000 pulses per muscle per session• ultrasound coupling gel, castor oil or Vaseline when

indicated• localization: patient oriented focusing

Post-therapeutic care: circulatory function monitoring whenindicated

Complications: hematoma, pain increase, nerve irritation

Follow up-care: abstention from sports for 4 weeks (individualadjustment of the sports program)continuation of stretchingclinical evaluation 8 -12 weeks post therapy

ConclusionBased on this guideline the ESWT of skeletal muscles is atreatment only done by physicians. The part of the so calledtriggerpoint-treatment with radioshockwaves could bedelegated to physiotherapists and non-physician-healers.

New Guidelines for ESWTThiele, Richard, MD