R e s e a r c h A r t i c l e T h e E f f e c t o f G a l l i u m A l u m i n i u m A r s e n i d e L a s e r o n F i b r o b l a s t A c t i v i t y : An In Vitro Dosimetry Study A B ST R A C T : The effect o f different doses o f low intensity laser ther­ apy (L.I.L.T.) on human fibroblasts was investigated to determine the optimal dose required to stimulate fibroblast proliferation. Human fibroblasts were cultured in vitro and irradiated with different energy densities o f 83Onm continuous output infra-red laser using a Gallium Aluminium Arsenide laser. The fibroblasts were irradiated on three consecutive days at energy densities, ranging from 0.2 to 5 J.cm2, delivered at an average radiant pow er o f 30 mW, and at a constant distance o f lc m from the fibroblasts. Fibroblast activity was assessed on the fourth day using a calorimetric M TT (tetrazolium) cleavage assay. There was a significant increase in fibroblast proliferation at laser treatment energy densities o f 0.4 J.cm2 and 5 J.cm2. Difficulties associated with in vivo and in vitro studies o f the effect o f laser treatment are discussed. KEY WORDS: LASER THERAPY, FIBROBLASTS, CELL CULTURE, DOSE SUSAN MARS, ANIL CHUTURGOON, DHAMARAI PILLAY AND MAURICE MARS INTRODUCTION Therapeutic lasers used in m edicine can be classified as either high or low energy lasers. H igh energy lasers provide discretely focused and localised therm al energy to coagulate or vaporise tissue, w hile low energy “atherm ic” lasers are used in low inten sity la ser therap y (L.I.L.T.) w hich has becom e a popular form o f non-invasive treatm ent to p ro ­ m ote w ound healing. (B asford, 1989). Low intensity laser therapy is thought to p ro v id e a b io stim u la to ry effe ct, enhancing the natural process o f w ound healing (Brom , 1994). M ester, et al., (1985), dem onstrated that laser prom oted w ound healing by increasing collagen form ation, vasodilatation and D N A syn­ thesis. Favourable responses to w ound healing have been reported follow ing L .I.L .T . (M este r and Jaszsag i-N a g y , 1973; D yson & Young, 1985, 1986; E nw em eka, et al., 1990). D espite a large body o f in vitro and in vivo research on the use o f LILT, the clinician is faced w ith the problem that there is no general­ ly agreed or scientifically validated dose for laser therapy in hum ans (Enw em eka, 1988) and the dose related efficacy o f c o r r e s p o n d e n c e M rs S M ars D epartm ent o f Physiology U niversity o f N atal M edical School Pvt Bag 7 C ongella 4013 Fax +31 260 4455 e-m ail m ars@ m ed.und.ac.za L.I.L.T. rem ains elusive. (K itchen & Partridge, 1991). This is due in part to inadequate reporting o f dosage in terms o f laser power, area o f application, d u ra­ tion and frequency o f treatm ent and the use o f d ifferen t m ethods, lasers and ex p erim en tal m odels. (B axter, 1994). Further research in the form o f thorough­ ly docum ented and controlled studies are necessary. (M iller & D yson, 1996). A dequately controlled clinical studies on the efficacy o f L.I.L.T. on w ound healing are difficult to conduct because o f the large num ber o f variables involved in the healing process. Som e control can be achieved by studying the com ponents o f the healing process in isolation in vitro, fo r exam ple, the fibroblast w hich plays an im portant role in w ound heal­ ing. T he triphasic m odel o f w ound heal­ ing describes sequential, partially over­ lapping, inflam m atory, proliferative and re m o d e llin g p h ases. (C lark, 1988). D u ring the in flam m a to ry phase, fibronectin and other cytokines recruit fib ro b la sts to the in ju red area and en co u rag e fibroplasia. (Silver, 1984). The fibroblasts w hich proliferate during granulation m ay transform into actin rich m yofibroblasts w hich have the ability to contract and m ove over each other, thus co n trib u tin g to w o u nd co n trac tio n . (Clark, 1988; G abbiani, et al., 1972). M y o fib ro b la sts sy n th esise co lla g en w hich increases the tensile strength o f the healing tissue. As fibroblasts are rela­ tively easily grow n in cell culture, they m ake a suitable m odel fo r the investiga­ tion o f the effects o f laser therapy. The m ajority o f studies o f L.I.L.T. on fibroblast function have used the H elium N eon (H e-N e) laser. Follow ing He-N e laser treatm ent, fibroblasts have been sh ow n to p ro life ra te (B o u lto n & M arshall, 1986), transform into m y o fi­ broblasts w ith in 24 hours (P ourreau- Schneider, et al., 1990) and increase their co lla g en p ro d u c tio n (B o satra, et al., 1984). T he m ost com m only used laser for L .I.L .T in clinical practice is how ev­ er the in fra-red G a lliu m A lum iniu m A rsenide (G a-A l-A s) laser. (Baxter, et al., 1991). It is a com m ercially available, “atherm ic” , diode-based laser em itting light at a w ave length o f 830nm and an average pow er o f 30mW. L ittle is know n o f its effects on fibroblasts as previous studies reporting on the use o f G a-A l-A s laser have not specifically looked at its effect on fibroblast proliferation. Young, et al. (1989) treated cultured m acro­ phages w ith different doses o f G a-A l-A s laser, and added the supernatant o f the culture m edium to a culture o f fibroblasts to show that a factor released from treat­ ed m acrophages can stim ulate fibroblast p ro life ratio n . W h ile A b erg el, et al. (1 987) sh ow ed that G a-A l-A s laser increased collagen gene expression and S k in n er, et a l. (1 9 9 6) d e m o n strate d in c rease d p ro c o lla g e n p ro d u c tio n by m onitoring 3 hydroxyproline synthesis. T he aim o f this study w as to investi­ gate the effect o f infra-red G a-A l-A s laser therapy on a com ponent o f the w ound healing process, hum an fibroblast activity, and to determ ine the optim al do se req u ire d to stim u late fib ro b la st activity in vitro. METHODS Cell Culture. H um an fibroblasts were 18 SA J o u r n a l o f P h y s io th e r a p y 1997 V o l 54 No 1 R ep ro du ce d by S ab in et G at ew ay u nd er li ce nc e gr an te d by th e P ub lis he r (d at ed 2 01 3. ) mailto:mars@med.und.ac.za o b tain ed from th e D ep artm e n t of Virology, U niversity o f N atal M edical School and cultured according to the standard laboratory m ethod o f Freshnay (1988), in 25ml flasks. Flasks that co n ­ tained cells w ith a 20 to 30% confluency required a routine m edium change sup­ plem ented w ith 5% foetal ca lf serum (FCS). C om m ercially available Eagle's M inim um Essential M edium (EM EM ) supplem ented w ith Earle's Salts, Hepes, non-essential am ino acids and L-gluta- m ine was routinely used. In addition heat inactivated FCS, antibiotics (penicillin, streptom ycin and fungizone), H ank's bal­ anced salt solution (HBSS) and trypsin w ere used. The cells w ere routinely passaged as follows: C u ltured flasks co ntaining m edium w ith depleted nutrients w ere aspirated to g eth er w ith loo sely attach ed cells. These flasks w ere then rinsed w ith HBSS (10ml) at 37C to rem ove cellular debris and further w ashed twice in H BSS. All cell culture procedures w ere carried out under strict aseptic conditions using a lam inar flow. Cell detachm ent was detected m icro ­ scopically or view ed w ith the naked eye (d eta ch ed cells gave the m e diu m a cloudy appearance). Five m illilitres o f culture m edium containing 5% antibi­ otics was then added and the flasks w ere returned to the incubator (37C). The fibroblasts w ere confluent after 3 days o f subculture. T he m edium from cu ltu re flasks co n ta in in g co n flu en t fibroblasts was discarded and the cells w ere w ashed tw ice w ith HBSS. A 1ml trypsin solution (0.25% ) was added to the confluent flasks and trypsin digestion was allow ed to proceed for ap p ro x im a tely 1 to 5 m in u te s until m icro sco p ic o b se rv atio n show ed the cells to have rounded off slightly but not to have becom e dislodged from the sur­ face. The trypsin was then rem oved with a sterile Pasteur pipette. Culture m edium (1.5m l) containing 10% FCS was added to the flask and the cells w ere dispersed in the solution by tapping the flask firm ­ ly against the hand. The resultant suspen­ sion was repeatedly pipetted w ith a ster­ ile pipette to separate clum ps o f cells. To the 0,5ml cell suspension was added 4m l o f m edium fo r a 1:3 split. Culture flasks (50ml) w ere then stoppered and incubat­ ed at 37C w ithout further disturbance until the cells attached to the substrate. C o n flu en t fib ro b la st cells w ere trypsinized and resuspended in culture m edium using a haem ocytom eter, to give a cell count o f 11 x l0 4 cells/m l. This cell suspension was used to fill each o f the wells o f a 96 w ell m icrotiter plate with 100 pi o f cell suspension per well. Two hundred m icrolitre culture m edium was added to each well. Control wells also contained 100 pi o f cell suspension and 200 pi o f culture m edium . The m icrotiter plate was incubated fo r 24 hours at 37 C. C ell Irradiation. E ach w ell was then irrad iated three tim es on consecutive days, i.e. at 2 4 ,4 8 and 72 hours p ost p lat­ ing o f the fib ro b lasts. A G a-A l-A s E ndolaser 476, infrared (C lass3B), con­ tinuous, low intensity laser was used. The w avelength was 830nm and the average po w er output o f the laser was 30 mW. The laser probe was sw abbed with absolute alcohol and aseptic conditions w ere strictly adhered to w hen treating the cell culture. The laser probe was held p erpendicular to, and steady at, the "mouth" o f each well at a constant distance o f 1cm from the fibroblast layer on the base o f each well. E ach colum n (2-11) was irradiated w ith a pre-set dose m easured in J.cm 2. Colum ns 1 and 12 served as controls and w ere not irradiated. The E ndolaser probe is fitted w ith a target light to clearly indi­ cate the treatm ent area and protective eye goggles w ere worn. E ach dose was preset on the E ndolaser, with the laser unit auto­ m atically setting the correct tim e require­ m ent according to the selected output. A continuous, 100% output was selected. O nly 1 well was exposed at a time, the other wells w ere screened with tinfoil to p revent accidental irradiation. Culture m edium was renew ed daily from each w ell, and the plate was incubated at 37C. E ach o f the 3 irradiations was perform ed at the same tim e daily. The doses used w ere 0.2, 0.4, 0.6, 0.8, 1, 1.5, 2.3, 3, 4, and 5 J.cm 2 fo r each colum n o f 8 wells respectively. The doses w ere calculated on a well area o f 0.3318 cm 2. F ib ro b la st V iab ility A ssay. C ell V iability w as m easured using the M TT [ 3 - ( 4 . 5 - d i m e t h y l t h i a z o l - 2 - y l ) - 2 . 5 - diphenyl tetrazolium brom ide] reduction assay. (Hanelt, et al., 1994). The princi­ ple o f this assay is that the tetrazolium salt (yellow ) is reduced to a form azan (blue) dye by the succinate dehydroge­ nase enzym es o f the m itochondria of viable cells. The w ells w ere decanted and 100 pi o f fresh culture m edium was added together with 10 pi o f M TT (101/1001) and the plates w ere further incubated fo r 4 hours at 37C. Supem atants were then aspirated and 100 pi o f D M SO (dim ethylsulfoxide) w as added to each well to solubilise the form azan crystals and incubated fo r a further h o ur at 37C. E ach w ell was dilut­ ed by a facto r o f 10 and the optical d en­ sity o f each well was m easured spec- trophotom etrically using a B io-R ad spec- troreader at a w avelength o f 595 nm, w ith a reference w avelength o f 630 nm. T h e sp e ctro p h o to m etric assay was repeated three tim es fo r each sam ple and the average recorded. T he absorbance readings w ere m ade w ith the operators blinded to the dose received by the sample. RESULTS Cell Culture. The fibroblast cells grew rapidly and after three days in culture the cells appeared scattered on the substrate. The cells w ere m onolayered, agranular and w ere tightly adherent to one another. A fter a w eek in culture, cells appeared tig h tly p ac k ed together. M an y cells (+ 80% ) d isp lay ed p ro m in en t n u clei, w hich w ere observed by phase contrast m icroscopy (x l6 0 0 ). Cell Viability. The results o f the M TT assay using the 96 well plates are show n in table 1. The cells w ere diluted 10 tim es to ensure that absorbance readings were w ithin the range o f the spectrophotom e­ ter. A n aly sis o f v arian c e (A N O V A ) show ed a significant difference betw een the controls and the treated cells (F = 2.827 and P=0.004). Post hoc testing u sin g u n p a ire d T tests w ith the Bonferonni correction show ed only the doses o f 0.4 J.cm 2 and 5 J.cm 2 to differ significantly from the controls. The co ­ efficient o f variance ranged from 21.9% to 39.8% . DISCUSSION T he nom enclature used to describe LILT dosage has caused m uch confusion, and m akes interpretation o f the literature difficult. D osage has been reported inde­ pendently in term s o f tim e (s), pow er output (W ), energy (J = W.s), pow er den­ sity (W .cm2.s) or energy density (J.cm 2). Ideally dosage should be described in term s o f the total dose o f energy supplied per unit area to the tissue, the energy d en­ sity, expressed in Joules per square cen ­ tim etre (J.cm 2). To determ ine energy density, the area o f the light beam incident on the surface o f the skin or w ound (the spot size), the av erag e rad ian t p o w e r o f the laser source, the duration o f irradiation, the SA J o u r n a l o f P h y s io th e ra p y 1997 V o l 54 No 1 19R ep ro du ce d by S ab in et G at ew ay u nd er li ce nc e gr an te d by th e P ub lis he r (d at ed 2 01 3. ) dep th o f the targ et tissu e and the absorbance and scattering characteristics o f the tissues through w hich the light is passing m ust be know n. C om parison o f the literature is further com plicated by the observation that the results obtained at an energy density produced by a laser o f one w avelength, m ay not be valid or reproducible w hen using a laser o f a dif­ ferent wavelength. M ost previous studies on fibroblast proliferation have used the H e-N e laser (6 3 2 .8nm ). (M ester, et al., 1978; A bergel, et al., 1984; Tocco, et al., 1985; B oulton and M arshall, 1986; H allm an, et al., 1988; Pourreau-Schneider, et al., 1990; Zugaro, et al., 1991; Lubart, et al., 1992; Van Brugel & Bar, 1992). The He- N e laser usually em its a non pulsed con­ tinuous w aveform . T he sem iconductor G a-A l-A s laser (830nm ) used in this study is currently the m ost popular in the clinical situation (Baxter, et al., 1991) and m ay be used in continuous or pulsed m ode. The three studies reporting the use o f the G a-A l-A s laser investigated its effect on m acro p hage stim u latio n o f fibroblasts (Young, et al., 1989), on gene e x p ressio n o f co lla g en fo rm atio n (A bergel, et al., 1987) and on collagen p ro d u c tio n and D N A re p lic atio n (Skinner, et al., 1996). We could find no previous studies on the use o f continuous m ode G a-A l-A s laser on fibroblasts. U sing the H e-N e laser Van Breugel and B ar (1992), felt that pow er output as well as irradiation tim e was important. They found that irradiation tim es o f 0.5 - 2 m inutes to be m ost stim ulatory, and that pow er outputs of 0.55 - 5.98 m W en h an c ed cell p ro liferatio n . U n fo r­ tunately the surface area irradiated was not stated, so the energy density is not know n. In the present study, the treat­ m ent tim e was calculated fo r each dose to ensure that the stated energy density was delivered. Based on H e-N e laser w ound healing stu d ies, M e ste r and Jasz sag i-N a g y (1973) stated that the therapeutic treat­ m ent w indow was betw een energy densi­ ties o f 1 - 4 J.cm 2 and that above 4 J.cm 2 a saturation effect occurred. Bolton, et al (1990; 1991) found m axim al fibroblast proliferation at energy densities o f 2.4 - 7.2 J.cm 2. This is in accordance with the A m d t-S c h u ltz L aw w hich states that there is, theoretically, an optim al thresh­ old dose to achieve a biostim ulatory effect in the target tissue, but that at a cer­ tain point saturation w ill occur and a plateau effect w ill occur. (B a x te r , 1994). In the present study, no saturation effect was noted and significant differences in fibroblast proliferation w ere seen at 0.4 and 5 J.cm 2. It is unusual to find reports o f fibroblast stim ulation at a dose as low as 0.4 J.cm 2. Betw een energy densities of 0.6 J.cm 2 to 4 J.cm 2 there was no statisti­ cal ev id e n c e o f an in c rease in cell turnover. A t 1 J.cm 2 and 3 J.cm 2 the m ean absorbance values noted w ere less than that o f the controls. There are very few studies to have used a dose as low as 0.4 J.cm 2 and the stim ulatory effect at this dose is unusual and requires further investigation. The d ifferen ces n o ted in d o se resp o n se b etw een H e-N e and G a-A l-A s m ay be due to the different w avelength o f light u tilised or experim ental differences. It could be argued that the assay used to dem onstrate cell viability was inappro­ priate or not sensitive enough fo r this type o f study. It w as chosen because laser irradiation causes increased ATP produc­ tion in the ferric sulphide redox system in the m ito c h o n d ria and the resu lta n t increase in energy production increases cell function. (K aru, 1987). T he M TT assay, w hich m easures the activity of succinate dehydrogenase in the m ito ­ chondria has been used successfully in m any o th e r cell c u ltu re stu d ies and should be sufficiently sensitive to identi­ fy changes in cell proliferation. A nother possible explanation o f the findings m ay be that the M TT assay should have been delayed until the latent biostim ulatory Table 1. Absorbance readings for each of the 96 wells following MTT assay. Columns 1 and 12 were not lasered and served as controls. Column 1 2 3 4 5 6 7 8 9 10 11 12 Dosage J.cm-2 0 0.2J 0.4J 0.6J 0.8J 1.0J 1.5J 2.3J 3J 4J 5J 0 Row A 0.71 1.00 0 .7 7 1.06 1.09 0.66 0.96 0.63 1.05 1.25 1.29 0.93 B 1.12 1.46 0.92 1.11 1.11 0.87 1.18 0.44 0.52 0.95 1.84 0.97 C 0.69 0.77 1.04 1.22 0.25 0.85 0.83 1.27 0.57 0.55 1.35 0.99 D 0.61 1.44 1.71 0.96 1.16 0.44 1.27 1.04 0.42 0.95 1.66 0.9 E 0.71 1.07 1.71 0.81 0.81 0.5 0.62 0.77 0.48 0.88 0.86 1.24 F 0.62 0 .87 1.04 0.94 1.06 1.11 0.31 0.65 0.58 0.72 0.53 1.03 G 0.38 0.98 1.06 0.65 1.06 1.08 0.64 0.91 0.66 1.22 1.16 0 .4 7 H 0.73 0.72 1.05 0.68 1.05 0.56 0.63 1.11 0.97 0 .7 7 0.92 0.82 Avg 0.70 1.04 1.16 0.93 0.95 0.76 0.81 0.85 0.66 0.91 1.20 0.92 Std 0.21 0.28 0.35 0.20 0.30 0.26 0.32 0.28 0.23 0.24 0.43 0.22 COV 29.5 26.9 30.2 21.9 31.7 33.9 39.8 32.8 35.1 26.3 35.8 23.8 20 SA J o u r n a l o f P h y s io th e ra p y 1997 V o l 54 No 1 R ep ro du ce d by S ab in et G at ew ay u nd er li ce nc e gr an te d by th e P ub lis he r (d at ed 2 01 3. ) response noted by R ajarath an , et al. (1994) occurred. They noted no differ­ ences in proliferation at 72 hours post treatm ent, but significant changes at 120 hours. T he few clinical studies on non-heal- ing ulcers w hich report L.I.L.T. dosage in term s o f energy density, suggest that energy densities o f 4 J.cm 2 (M ester and M ester, 1989), 4.5 J.cm 2 (K hurshudian, 1989) are optim al fo r prom oting healing. This is in keeping with the observations o f the in vitro increase in fibroblast pro­ liferation at 5 J.cm 2. A lim itation to this and other in vitro cell cu ltu re m o d els is the d istan ce b etw een the probe and the base o f the cell culture wells, w hich in this study was 1cm. W hat is not know n is the absorbance o f light and hence energy by the cell culture m edium . As different m edia m ay have different absorbencies, dose in cell culture m ay well then be also m edium dependent. A nother uninvesti- REFERENCES A bergel RP, Lyons R F & C astel JC. 1987. B iostim u lation o f w ound h ealing by lasers: experim ental approaches in animal m odels and fibroblast cu ltu res. J o u r n a l D e r m a to lo g ic a l S u rg ery O n co lo g y , 13:127- 133. A bergel RP, M eeker C, Lam T, D w yer R M , L esa v o y M A & Vitto J. 1984. Control o f c o n ­ n ectiv e tissue m etabolism by laser. R ecent d e v elo p m en ts and future p ro sp ects. D e r m a to lo g ic S u rgery, 11:1142-1150. B asford JR. 1989. L ow -en ergy laser therapy: c o n tr o v er sies and n ew research fin d in g s. L a s e r s in S u rg ery a n d M e dicin e, 9:1-5. B ax ter G D . 1 99 4. T h e ra p e u tic L a s e r s : T h eo ry a n d P ra c tic e . Churchill L ivin gston e, London. B axter G D , B e ll AJ & A llen JM. 1991. L ow le v e l laser therapy. Current clin ical practice in Northern Ireland. P h y sio th e ra p y, 77: 171- 178. B o lto n P, Y oung S & D y s o n M . 199 1. M acrophage resp on siveness to ligh t therapy w ith varying p ow er and energy densities. L a s e r T h era p y, 3:105-111. B o lto n P, Y oung S & D y s o n M . 1 99 0. M acrophage resp on siveness to light therapy - a d ose response study. L a s e r T h era p y, 2 :101- 106. Bosatra M , Jucci A , O lliano P, Q uacci P & S acch i S. 1984. In vitro fibroblast and dermis fibroblast activation by laser irradiation at lo w energy. D e r m a to lo g ic a , 168 :1 57 -1 6 2. Boulton M & M arshall J. 1986. H e-N e laser stim ulation o f human fibroblast proliferation and attach m ent in vitro. L a s e r s in L ife S cien ces, 1:125-134. gated variable is the am ount o f reflection and refraction o f the laser light by the m edium . This affects the energy reaching the cells. A lthough every attem pt was m ade to isolate each well during treat­ m ent by screening the other wells with tin foil, som e energy m ay have been transm itted through the plastic o f the 96 well plate. CONCLUSION R esearch into the use o f laser in w ound healing is beset w ith m any problem s. The issues o f differences in dose (continuous or pulsed), treatm ent tim e, pow er output, treatm ent area and treatm ent depth m ake com parison o f studies difficult. In vitro there are further problem s such as the o f the lack o f the skin layer w hich will influence delivered dose, no inform ation on the absorbance, refraction, reflection and scattering capacity o f culture media, and the attem pt to infer from the activity o f one cell line in isolation its activity in a m ultifaceted repair process. In the pre- Brom B . 1994. L ow energy Laser Treatment in General Practice. South A frica n J ou rn a l o f F am ily P ra c tic e , 15:226-235. Clark RAF. 1988. O v erv iew and general consideration s o f w ou n d repair. In Clark RAF, H enson PM , (ed s.) T he M olecu lar and C ellular B io lo g y o f Wound Repair, pp 3 -33. Plenum Press, N ew York. D y so n M & Young S. 1985. T he effe ct o f la ser therapy on w ou n d co n tractio n . I n te r n a tio n a l C o n g r e s s L a s e r M e d ic in e S u rgery, pp 2 1 5 -2 1 9 . D y so n M & Young S. 1986. T he effe ct o f laser therapy on w ound contraction and cellu- larity in m ice. L a s e r s in M e d ic a l S cien ce, 1:125-130. E n w em ek a C S, R odriquez O & G all N G . 1990. C orrelative ultrastructural and b io m e­ chanical ch anges induced in regenerating ten­ d on s e x p o s e d to la ser p h oto -stim u la tio n . L a s e r s in S u rg ery a n d M edicin e, Suppl. 2:12. E n w em ek a C S. 1988. L aser biostim ulation o f healing wounds: S p ecific effects and m ech a­ nism s o f action. J o u rn a l o f O rth o p a e d ics a n d S p o rts P h y sio th e ra p y, 9 :3 3 3 -3 3 8 . Freshnay RI. 1988. C u lture o f a n im al c e lls: a m anu al o f b a s ic tech n iqu es. 2nd edition, L iss, N ew York. Gabbiani G, H irschel BJ, Ryan G B , Statkov PR & M ajno G. 1972. Granulation tissue as a contractile organ. A study o f the structure and function. J o u rn a l o f E xp erim en ta l m ed icin e, 1 3 5 :7 1 9 -7 3 4 . H allm an H O , B asford JR, O ’Brien JF & C um m ings L A . 1988. D o e s lo w energy H e- N e laser irradiation alter in vitro replication o f hum an fibroblasts? L a s e r s in S u rg ery an d M e d icin e 8 :1 25 -1 29 . sent study G a-A l-A s laser delivered in three consecutive daily doses o f 0.4 J.cm 2 and 5 J.cm 2 induced increased human fibroblast proliferation in vitro. Review o f the literature also suggests that there is sufficient evidence to support the bene­ fits o f laser. The exact nature o f its bene­ ficial action and the optim al dosages still aw ait elucidation. Further studies investi­ gating laser induced cytokine expression in m acrophages and fibroblasts and their effects on the com ponents o f the w ound healing process are required. A ck n o w le d g em e n t. T h is w o rk was funded by the U niversity o f N atal, Sm ith and N ephew W ound H ealing R esearch G roup. This paper was presented in p art at the South A frican S p o rts M ed icin e A ssociation C ongress - 1997. Table 1. A bsorbance readings fo r each o f the 96 wells follow ing M TT assay. Colum ns 1 and 12 w ere not lasered and served as controls. Hanelt M , G areis M & K ollarczik B . 1994. C ytotoxicity o f m y co to xin s evaluated by the M T T c e ll culture assay. M y c o p a th o lo g ia , 128 :1 67 -1 7 4. Karu TI. 1987. P hotob iological fundam entals o f lo w -p o w e r la ser therapy. J o u r n a l o f Q uantum E le ctro n ics, 2 3 :1 7 0 3 -1 7 1 7 . K hurshudian A G . 1989. U se o f H e-C a lasers in the co m p lex treatment o f suppurative d is­ e a se in patients w ith d ia b etes m ellitu s. K h iru rg iya , 6 :3 8-42 . K itchen SS & Partridge CJ. 1991. A rev iew o f lo w le v e l la ser therapy. P h y s io th e r a p y , 7 7 3 :1 6 1 -1 6 3 . Lubart R , W ollm an Y, F riedm ann H , R ochkind S & Laulicht I. 1992. E ffects o f v is ­ ib le and near - infrared lasers on c e ll cultures. J o u r n a l o f P h o to c h e m is tr y a n d P h o to b io lio g y , 12:305-310. M ester E, Jaszsagi & N ag y E. 1973. E ffect o f laser radiation on w ound healing and co lla g en syn th esis. S tu d ies in B io p h y sic s, 3 5 :2 2 7 -2 3 0 . M ester E , M ester A E & M ester A . 1985. The b iom ed ical effe ct o f laser application. L a sers S u rg ery a n d M e d icin e , 5 :31-39. M ester A F & M ester A . 1989. W ound h ealing. L a s e r T h era p y, 1:7-15. M ester E, N ag y lu csk ay S, T isza S & M ester A. 1978. Stim ulation o f w ound h ealing by m ea n s o f la ser rays. A c ta C h iru r g ic a A c a d e m ia e S c ie n tific a r u m H u n g e ric a e 19:163-170. M iller M & D y so n M. 1996. P rin c ip le s o f w o u n d C are. A P rofessional N urse p u blica­ tion. M acm illan M agazines Ltd. London. Pourreau-Schneider N , A hm ed A , Sondry M, Jacquem ier J, K opp F, Franquin JC & ^ Martin PM . 1990. H eliu m - neon laser SA J o u r n a l o f P h y s io th e r a p y 1997 V o l 54 No 1 21 R ep ro du ce d by S ab in et G at ew ay u nd er li ce nc e gr an te d by th e P ub lis he r (d at ed 2 01 3. ) B o o k ^ treatment transforms fibroblasts into m yofibroblasts. A m erican ^ J o u rn a l o f P a th o lo g y , 137:171-178. Rajarathan S, B olton P & D y so n M. 1994. M acrophage resp o n siv e­ n ess to laser therapy with varying pu lsin g freq u en cies. O riginal arti­ cle, T issue Repair Research unit, U nited M edical and D ental S ch o ols, Guy's H ospital Cam pus, London. Silver 1A. 1984. O x yg en and T issue repairs. In Ryan TJ (ed .) An en v i­ ron m en t f o r h ea lin g : the role o f o cclu sio n . R oyal S o c ie ty o f M edicin e International C ongress and S ym p osium S eries, 1: 88:18. Skinner SM , G age JP, W ilce PA & Shaw RM . 1996. A preliminary study o f the effects o f laser radiation on co lla g en m etabolism in ce ll culture. A u stra lia n D e n ta l J ou rn a l, 4 1 :1 8 8 -1 9 2 , 1996. T occo G , L e Borgue de K a ov el C & Aubert C. 1985. H e N e and I.R. m id laser in flu en ces in skin ce lls in vitro; prelim inary results. P roceedings o f the International C ongress o f L asers in M edicin e and Surgery, 175-182. Van B reugel HH & Bar PR. 1992. P ow er density and exposure tim e o f H e-N e laser irradiation are more important than total energy clo se in photo-biom odulation o f human fibroblasts in vitro. L a s e r s in S u rg ery a n d M e d icin e , 1 2:528-537. Young S, B olton P, D yso n M , H arvey W & D iam antopoulis C. 1989. M acrophage resp on siveness to light therapy. L a s e r s in S u rg ery an d M e d icin e , 9 :4 9 7 -5 0 5 . Zugaro A , R izzo G L, P iccin n i M , C ontinenza M A & R icciardi G. 1991. A pplication o f infrared lasers on in vitro culture o f fibroblasts: effe cts o f the duration o f treatment parameter. A n n a li Ita lia n i d i C h iru rgia, 6 2:5 7 7 -5 8 0 . EXCELLENT WEEKLY PAY+BONUSES SHORT/LONG TERM POSITIONS NATIONWIDE UK ENTRY CLEARANCE ADVICE FREE INDEMNITY INSURANCE UK CPSM REGISTRATION REFUND FREE PERSONAL TAX ADVICE ACCOMMODATION OFTEN ARRANGED PERSONAL AND FRIENDLY SERVICE - ALWAYS FREEPHONE UK from SOUTH AFRICA 0800 990 767 or fax 09 44 181 554 9900 o r c o n ta c t o u r R ep re se n ta tiv e f o r S o u th A fric a DANIEL KOTZE BDIAC Social Work/BA(SW) a t o u r o ffic e s e ve ry M o n d a y PS. We also help O T s . Speech Pathologists Nursery Nurses and Social Workers V is it o u r W e b s ite h ttp :/ / w w w .a sa -locu m s.d em .on .co.u k Em ail lo c u m s @ a s a *lo c u m s . d e m o n , co.u k T h e C h a r te r H o u se • C h a r ie r M e w s - 18a B e e h iv e L a n e - Ilfo rd • E s se x - IG1 3 R D • U K R e v i e w CYRIAX'S ILLUSTRATED MANUAL OF ORTHOPAEDIC MEDICINE 2 n d edition E d ite d b y : Y v o nn e R B u rn s an d J u lie M a c D o n a ld Pub. WB Saunders Company Ltd (1996) ISBN: 0-7020-1942-9 Eds. J H C yriax and P H Cyriax. Butterworth and H einneman, Scotland, 1996. ISB N 0 7506 3274 4 R eview ed by Jessica Fortune This is the revised edition o f C yriax Illustrated M anual of O rthopaedic M edicine, w idely used by undergraduate students as part o f th e ir m usculo-skeletal course. T he focus o f the 2nd edition rem ains on the assessm ent, diagnosis and treatm ent o f m ainly soft tissue lesions and jo in t pathology. T he bo ok em phasizes a holistic approach to the assessm ent o f soft tissues including the join ts and spine. In the assessm ent o f a painful area, the structure and function o f each soft tissue is follow ed through logically to determ ine the possib le diagnosis. T h ese assessm ents are clearly illustrated. T he m ethod o f treat­ m ent used by C yriax is that o f deep m assage, intra-articular m anipulation and injections o f the painful areas. T hese are clearly illustrated using colour p hotographs and line draw ings. In the preface, C yriax acknow ledges that alternative form s o f treatm ent to that prescribed in the text exist. T he 2nd edition includes discussion about m o d e m eq uipm ent and diagnostic aids, w hich include M R I and diagnostic ultrasound. In ad d i­ tion, use o f alternative techniques to treat som e o f the soft tis­ sue pathology is m entioned, for exam ple, acupuncture. A lim i­ tation though is that there is very little discussion encom pass­ ing n ew er diagnostic m odalities and alternative form s o f treat­ m ent, w ith lim ited references. N o references are included to support or substantiate C yriax's techniques The publishers have im proved the layout, w ith a larger, easy- to-read type. T his brings the captions in closer proxim ity to their subject m atter on each page. M ost o f the illustrations used have been refined. Photographs have been replaced w ith new line draw ings; close-up photographs are enlarged m aking the exam ination sequences easy to follow. T he chapters are w ritten in a style w hich is sim ple and easy to follow. In addition, the chapters describing each jo in t assessm ent, differential d iagn o ­ sis and treatm ents are follow ed w ith a tabulated synopsis o f the m ost salient points. T hese synopses are enclosed as fact sheets in the appendix and ensure that the bo ok is w ell sum m arized. T he book is w ell indexed, m aking it very user friendly. Both the novice and experienced p h ysio th erap ist w ill find the text easy to read and the superb illustrations co m p lim en t the text very w ell. H ow ever, the m anual should n o t be exclusively relied on regarding the treatm ent o f soft tissue orthopaedic co n ­ ditions, as alternative techniques, have not been adequately addressed. In conclusion, this is a valuable reference fo r the assessm ent o f m ost soft tissue and jo in t pathologies. 22 SA J o u r n a l o f P h y s io th e r a p y 1998 V o l 54 No 1 R ep ro du ce d by S ab in et G at ew ay u nd er li ce nc e gr an te d by th e P ub lis he r (d at ed 2 01 3. ) http://www.asa-locums.dem.on.co.uk