O objetivo neste estudo foi avaliar a velocidade de movimentação dentária no fechamento dos espaços durante a fase de retração em pacientes ortodônticos, submetidos a tratamento com extração dos primeiros pré-molares. Foram selecionados 18 pacientes sendo 9 homens e 9 mulheres, com idades entre 13 e 18 anos, alocados sequencialmente em dois grupos: LED (GLED) composto por dez pacientes (5 H, 5 M) que fizeram auto aplicação com aparelho de LED, durante 10 minutos diários, durante a fase de retração ortodôntica, por um período de 12 semanas e Controle (GC), composto por oito pacientes (4 H, 4 M) que não aplicaram o LED durante a mesma fase. O fechamento dos espaços das extrações foi obtido com mecânica de deslize com arcos de aço com calibre 0.019\" x 0.025\" e molas de níquel-titânio com 200g de força, ativadas a cada 4 semanas. A mecânica escolhida permitiu-nos analizar 36 quadrantes e não 18 pacientes e isto justifica o tamanho final da nossa amostra: GLED 20 quadrantes superiores e 16 quadrantes inferiores e GC 16 quadrantes superiores e 12 quadrantes inferiores. Na arcada superior a retração foi ancorada em mini-implantes ortodônticos, e na arcada inferior sem acessórios de ancoragem e por este motivo foram avaliadas separadamente. Para avaliar o fechamento dos espaços, após calibragem, um único operador realizou dois tipos de medições: clínicas e em modelos de estudo, com compasso de ponta seca e paquímetro digital modificado, considerando quatro variáveis consistentes em distância entre os dois dentes vizinhos ao espaço da extração. Os resultados mostraram quedo início (T0) ao término do período de observação (T3) não houve diferença estatísticamente significante entre os grupos, exceto na medição dos modelos da arcada inferior, nas variáveis 1,3 e 4, com maior redução dos espaços no GLED: GLED > GC. Observou-se também diferença significante entre os grupos apenas na variável 1 do GLED na medição dos modelos da arcada inferior, no sentido de maior velocidade entre T1 e T2. Não houve diferença estatística entre os tipos de medidas realizadas clinicamente e nos modelos de estudo. Independentemente da aplicção ou não do LED obdservou-se uma significativa redução dos espaços ao longo do tempo em ambos os grupos, tanto da arcada superior, como na arcada inferior, que permitiu o sucesso do tratamento ortodôntico. Com base nos resultados pôde-se concluir que o protocolo de aplicação do LED utilizado na casuística estudada não caracterizou diferencialmente os grupos estudados. Porém, por ser um método novo na avaliação da influência sobre a movimentação dentária, sem dados na literatura para comparar os nossos, faz-se necessário a realização de estudos futuros que testem protocolos de mecânica ortodôntica e aplicação de LED. / The aim of this study was to evaluate the speed of tooth movement in space closure during the retraction stage, in orthodontic patients submitted to treatment with first premolar extractions. Eighteen (18) patients undergoing orthodontic treatment were selected, of whom 9 were men, and 9 women, age-range between 13 and 18 years, sequentially allocated to two groups. LED (GLED) composed of ten patients (5 M, 5 W) who performed self-application with a LED appliance, for 10 minutes daily, during the stage of orthodontic retraction, for a period of 12 weeks, and the Control Group (GC), composed of eight patients (4 M, 4 W) who did not apply LED during the same stage. Space closure of the extractions was obtained with sliding mechanics with steel archwires, caliber 0.019\" x 0.025\" and nickel-titanium springs with 200 g of force, activated every 4 weeks The mechanics chosen allowed the authors to analyze 36 quadrants and not 18 patients, and this justified the final size of our sample: GLED 20 maxillary quadrants and 16 mandibular quadrants; and GC 16 maxillary quadrants and 12 mandibular quadrants. In the maxillary arch, retraction was anchored on orthodontic mini-implants and in the mandibular arch, no anchorage accessories were used; and for this reason, they were evaluated separately. To evaluate space closure, after calibration a single operator performed two types of measurements: clinical, and in study models, by using a dry tip compass and modified digital pachymeter, considering four variables of distances between the two neighboring teeth, compatible with the extraction space. The results showed that from the beginning (T0) up to conclusion of the observation period (T3) there was no statistically significant difference between the groups, except for the measurements of the mandibular arch models, in the variables 1,3 and 4, showing greater reduction of the spaces in GLED: GLED > GC. The authors also observed significant difference between the groups only in variable 1 of GLED, in the measurements of the mandibular arch models, showing greater velocity between T1 and T2. There was no statistical difference between the types of measurements performed clinically and those in the study models. Irrespective of the application, or no application of LED, a significant reduction was observed in the spaces over time in both groups, both in the maxillary and mandibular arches, which allowed the orthodontic treatment to be successful. Based on the results, the authors could conclude that the LED application protocol used in the casuistic studied did not characterize the groups studied in a differential manner. However, since this was a new method for evaluating the influence on tooth movement, without data in the literature to compare with those of the authors, it is necessary to conduct future studies that test the orthodontic mechanics and LED application protocols.

A poiquilodermia de Civatte é uma condição caracterizada por hiperpigmentação, telangiectasia e atrofia que acomete predominantemente o pescoço e a face de forma simétrica, poupando a área encoberta pelo mento. A sua ocorrência é associada à predisposição genética, à exposição cumulativa ao sol, ao processo de envelhecimento, à hipersensibilidade de contato e a fatores hormonais. Os tratamentos atualmente preconizados baseiam-se na utilização de sistemas de luzes que atuam através do princípio da fototermólise seletiva, isto é, dano térmico confinado a alvos específicos no tecido. Estudos clínicos mostram bons resultados no tratamento da poiquilodermia de Civatte com a luz intensa pulsada, porém não descrevem se estes resultados são acompanhados por alterações histopatológicas correspondentes. O objetivo deste estudo foi avaliar os achados clínicos e histopatológicos nas lesões de poiquilodermia de Civatte, antes e após o tratamento com a luz intensa pulsada. O estudo avaliou 16 pacientes portadores de poiquilodermia de Civatte na região cervical, submetidos a 5 sessões mensais de aplicação de luz intensa pulsada, utilizando-se o filtro de 560 nm. Os pacientes foram fotografados e biopsiados antes do tratamento e 30 dias após o término da quinta sessão. A avaliação clínica foi feita através da comparação das fotografias por 5 avaliadores independentes. Os resultados mostraram que o componente vascular responde melhor do que o componente pigmentar ao tratamento. A avaliação histopatológica foi feita de forma cega em relação aos cortes pré e pós-tratamento, sendo ao final das leituras revelados e comparados. Após cinco sessões não houve diferença na densidade das fibras elásticas na derme superficial e profunda. Não houve redução no número de vasos e na média da área do lúmem dos vasos marcados pelo anticorpo anti-CD34 na derme superficial e profunda. A quantificação do pigmento melânico mostrou redução significante na epiderme, porém não na derme. / Poikiloderma of Civatte is a dermatosis in which there is hyperpigmentation associated with telangiectasia and atrophy. It occurs predominantly on the neck and face in a symmetric fashion, sparing shade-protected areas under the chin. Many factors are included in its etiology, including genetics, chronic excessive sun exposure, aging, hormonal modifications, and contact hypersensitivity. Currently advocated treatment methods are based on the use of selective photothermolysis via optic radiation, in other words, thermal damage confined to specific targets in the tissue. Clinical studies report promising results with treatment of poikiloderma of Civatte with intense pulsed light, however, they don\'t describe whether or not the results are accompanied by histopathological alterations. The objective of this study was to assess clinical and histopathological findings in the lesions of poikiloderma of Civatte before and after treatment with intense pulsed light. Sixteen patients with poikiloderma of Civatte in the neck area were treated with five monthly sessions of intense pulsed light, using the 560 nm cutoff filter. The patients were photographed and biopsies were obtained before the first session and 30 days after the fifth session. Clinical evaluation was performed through comparison of the photographs by five independent observers. Results demonstrated that the vascular component responded better to treatment than the pigmentary component. Histopathological analysis was carried out blindly as to pre- and post-treatment samples, which were unveiled and compared at the end of the readings. After five sessions, in the superficial and deep dermis, there was no difference in the density of elastic fibers nor was there a reduction in vessel number or in medium luminal area of vessels tagged with anti-CD34 antibodies. Quantification of melanin pigment showed a significant reduction within the epidermis, however, this did not occur in the dermis.

A poiquilodermia de Civatte (PC) é uma alteração dermatológica caracterizada por atrofia, pigmentação macular ou reticulada e telangiectasias na face, pescoço e tórax anterior. Várias tentativas de tratamentos não foram bem sucedidas por se mostrarem ineficazes e/ou com efeitos colaterais indesejáveis. Estudos clínicos mostram bons resultados clínicos no tratamento da PC com luz intensa pulsada (LIP). A terapia fotodinâmica (TFD) é uma modalidade terapêutica que envolve a administração tópica de um composto fotossensibilizante seguido de irradiação seletiva da lesão com luz visível. O propósito deste trabalho consiste na avaliação clínica e histopatológica da pele com PC, no tórax anterior, em 12 pacientes do sexo feminino, antes e após o tratamento com a TFD. Foram realizadas 3 sessões de tratamento com intervalos de 30 dias entre as sessões. A TFD foi realizada com a utilização prévia de cloridrato de aminolevulinato de metila 16% em creme (Metivix) 1h e 30min (oclusivo) antes da aplicação da LIP (Lime light). A avaliação clínica mostrou melhora em 84,6% no componente vascular, 80,8% no componente melanodérmico, 82,9% na textura, 77,1% nas rítides e 58,3% na flacidez de pele. Na histopatologia notou-se após o tratamento aumento estatisticamente significativo da espessura epidérmica, diminuição estatisticamente significativa do pigmento melânico e da área dos vasos marcados pelo fator VIII. Não houve nenhum efeito colateral permanente, mostrando que a TFD-MAL é uma nova opção terapêutica segura e eficaz para o tratamento da PC no tórax anterior / Poikiloderma of Civatte (PC) is the dermatological modification characterized by atrophy, macular or reticulate pigmentation and telangiectasias on the face, neck and anterior thorax. Several treatment attempts were unsuccessful, revealing to be ineffective and/or causing adverse effects. Clinical studies indicate good clinical results in the treatment of poikiloderma of Civatte with intense pulsed light (IPL). Photodynamic Therapy (PDT) is a therapeutic modality that involves the topical administration of a photosensitizing compound, followed by a selective irradiation of the lesion with visible light. The objective of this study consisted in the clinical and histopathologic evaluations of the skin with poikiloderma of Civatte (PC) on the anterior thorax of 12 female patients, before and after being treated with photodynamic therapy (PDT). Three treatment sessions were given at 30-day intervals. Photodynamic therapy was performed with methyl aminolevulinate hydrochloride 16% (Metivix) cream applied 1h and 30min under occlusion prior to LIP (Lime light) exposure. The clinical evaluation indicated an 84.6% improvement of vascular component, 80.8% of melanodermic component, 82.9% of texture, 77.1% of wrinkles and 58.3% of skin laxity. In the histopathology, a statistically significant increase of epidermal thickness was observed after the treatment, as well as a statistically significant reduction in the number of melanin pigment and of blood vessels area marked for Factor VIII. There were no permanent side effects, showing that the PDT-MAL is a new, safe and effective technic for treating PC on the anterior thorax

La synthèse d’une nouvelle famille d’ènediynes comportant un motif perfluoré a été réalisée en 6 à 7 étapes. Leur activité anticancéreuse a été étudiée sur un panel de lignées cellulaires provenant de différents tissus ; des IC50 de l’ordre du µM ont été obtenus. Leur réponse en IRM du fluor 19 a aussi été évaluée, dans le but d’impliquer ces composés en théranostique (association de la thérapie et du diagnostic). Une étude mécanistique a été menée afin de comprendre le mécanisme biologique en jeu au cœur des cellules.Les ènediynes ont souvent vu leur développement stoppé en raison d’effets secondaires trop importants. Afin d’anticiper cette problématique, des stratégies de ciblage ont été développées pour nos composés. Dans un premier temps, la réponse à l’irradiation UV de nos composés a été mesurée. Dans un second temps, leur encapsulation dans des micelles polymériques a été réalisée.Enfin, dans un projet plus fondamental, la réactivité de substrats ènediynes et diène-ynes a été étudiée. Des cycloisomérisations procédant avec double transfert de chiralité ont été développées, permettant l’accès à des benzofulvènes et indènes. Deux variantes d’une réaction tandem d’Alder-Ene conduisant à des allyl-indènes ont aussi été décrites. / Synthesis of a new family of enediynes bearing a perfluorinated moiety was reached in 6 to 7 steps. Their anticancer activity was studied on a cell line panel from different tissues; IC50 values in the µM range were obtained. Their 19 fluorine MRI response was also evaluated, in order to involve them in a theranostic strategy (association of therapy and diagnosis). A mechanistic study was conducted to understand the biological mechanism at stake inside cells.Enediynes often saw their development stopped because of harmful side effects. To anticipate this problem, targeting strategies were developed for our compounds. First, their response to UV irradiation was measured. We then succeeded in their encapsulation inside polymeric micelles.Finally, in a more fundamental project, reactivity of enediyne and diene-yne substrates was studied. Cycloisomerisations proceeding with a double chirality transfer were developed, allowing access to benzofulvenes and indenes. Two variations of a tandem Alder-Ene reaction leading to allyl-indenes were also described.

M. Tech. / Stem cells are regarded as undifferentiated cells that are capable of selfrenewal, proliferation, production of a great number of differentiated progeny, and regeneration of tissues (Blau et al., 2001). The therapeutic potential of multilineage stem cells for tissue engineering (TE) applications is vast. Two general types of stem cells are potentially useful for this application: embryonic stem cells (ESCs) and adult (autologous) stem cells (Zuk et al., 2001). Traditionally, ESCs are isolated from the inner cell mass (ICM) of blastocysts, however harvesting of these cells results in the death of the embryo, which has led to ethical, religious and political issues (Moore, 2007). In contrast, adult stem cells, by virtue of their nature, are immunocompatible and have no ethical issues associated with their use (Zuk et al., 2001). Subcutaneous adipose tissue is an active and highly complex tissue composed of several different cell types, and is derived from the mesodermal germ layer and contains a supportive stromal vascular fraction (SVF) that can be easily isolated. This SVF contains a heterogeneous mixture of cells including preadipocytes (Raposio et al., 2007; Schäffler and Büchler, 2007; Jurgens et al., 2008). The preadipoctyes are considered as the multipotent stem cells termed adipose derived stem cells (ADSCs) that have similar properties to bone marrow mesenchymal stem cells (BM-MSCs) (Fraser et al., 2006). ADSCs are idyllic for cellular therapy applications due to various factors: they can be harvested, multiplied and handled easily, efficiently and noninvasively, they have a pluripotential and proliferative capacity comparable to BM-MSCs, and morbidity to donors is considerably less, requiring only local anaesthesia and a short wound healing time. Human ADSCs (hADSCs) can be expanded in an undifferentiated state and have multipotential differentiation capacity along the classical mesenchymal lineages of adipogenesis, osteogenesis, chondrogenesis and myogenesis (de Villiers et al., 2009).

Light therapy is a general term used for all therapies that utilise different frequencies of light (colours) for therapeutic purposes. The use of light as a healing agent dates back into antiquity to ancient Rome, Greece, China and Egypt, where colour was used in worship and as a healing agent (Leven, 2000). In the year 1892, Niels Finsen of Denmark received the Nobel Prize for successfully treating skin tuberculosis lesions with ultra-violet light. Today, there are many modalities of light therapy of which laser therapy is the best known and researched. The medical profession utilises certain frequencies of light for conditions such as neonatal jaundice, improved healing of surgical wounds, sterilization of blood (externally) and certain types of skin cancer (Liberman, 1991). Extensive research into light and its effects on the human body have given rise to other, not commonly known, forms of light therapy such as Heliotherapy, Spectro-Chrome Therapy, Colourpuncture, Syntonics and the Homoeopathic light and colour remedies. Within this dissertation, the most successful and prevalent light therapies will be discussed in enough detail to give the reader a basic introduction into each modality. The potentially valuable information regarding these healing modalities is widely scattered and therefore effectively out of the reach of the general health practitioner. Bringing this information together in a comprehensive and accessible format would serve to inform health practitioners of the possible alternative therapies available to help prevent/treat disease and deteriorative conditions. The aim of this study is to investigate, compile and organise information regarding the various healing modalities of light and colour therapy, and to determine treatment effectiveness in terms of research and clinical findings. The study aims to create an easily accessible, comprehensive database of pertinent information. Data, pertaining to the different light and colour therapies, will be collected from sources which include books, journals, articles, clinical trials, the internet and lecture notes. The information will be analysed according to the origin, development, application and existing clinical research, if any. From this information the efficacy each therapy can be explored. This information will be written up in the form of a literary survey. Possible outcomes will include increased awareness of therapeutic alternatives to conventional medicine, a more complete and easily accessible information base on each modality, possible inclusion into homoeopathic and allopathic practice, and to stimulate further research. / Dr. Solomon

Prof. H. Abrahamse

M.Tech. / Low level laser therapy, commonly known as LLLT or biomodulation, is a form of phototherapy which involves the application of low power monochromatic and coherent light to injuries and lesions to stimulate healing. In the medical field, lasers are classified as high power or surgical lasers and low level lasers which are used to stimulate cellular responses. Phototherapy has been successfully used for pain attenuation and induction of wound healing in non healing defects. Even though phototherapy has been found to be beneficial in a wide variety of therapeutic applications, it has been shown that phototherapy can induce DNA damage; however this damage appears to be repairable (Houreld and Abrahamse, 2008). DNA repair is vital to cells to avoid mutation. Literature reports show that red light or phototherapy up or down regulates genes involved in DNA repair (Zhang et al., 2003). N-methylpurine DNA glycosylase (MPG) is involved in DNA repair by catalysing the excision of a variety of modified bases. The exact mechanism by which phototherapy works is still poorly understood. Several authors have demonstrated that phototherapy enhances cell proliferation and migration. However, these cellular responses seem to confuse scientists as to whether wound healing is due to cell proliferation or migration or both. To determine the effect of phototherapy on cell proliferation or migration, a mini project was conducted (Zungu et al., 2008). Thus, cell proliferation was arrested using 5 mM hydroxyurea (HU) which is an antiproliferative drug. Wounded (W) human skin fibroblast cells (WS1, ATCC iii CRL 1502) were irradiated with 5 J/cm2 using a Helium-Neon (He-Ne) laser with a wavelength (λ) of 632.8 nm on day 1 and 4. Cell morphology, viability and proliferation were measured 24 h post irradiation. Reports indicate that several cell culture studies have used HU to control proliferation (Cai et al., 2000; Hamuro et al., 2002). Thereafter, the main study which was aimed at determining the effects of phototherapy on DNA damage and gene activation related to repair using 5 or 16 J/cm2 on W human skin fibroblast (WS1) cells was performed. Both studies involved growing WS1 cells aseptically in complete minimum essential medium (MEM) with Earle’s balanced salt solution and incubated at 37 °C in 5% CO2 and 85% humidity. Normal (N) and W cell cultures were irradiated with 5 or 16 J/cm2 30 min and 72 h (day 1 and 4) post wounding. Non irradiated cells (0 J/cm2) served as controls, while irradiated cells were the experimental groups. A wound was simulated by creating a central scratch across a monolayer of cells using a sterile 1 ml pipette. A 3 mW/cm2 He-Ne laser, λ 632.8 nm, was used to irradiate cells. After a repair time of 1 or 24 h on day 4, cell morphology (microscopy), cell viability (Trypan blue exclusion test and ATP luminescent assay), proliferation (XTT assay) and DNA integrity (alkaline comet assay with and without Formamidopyrimidine glycosylase [Fpg]) were assessed. The up or down regulation of the DNA repair gene, MPG, and regulation of three reference genes namely; beta Actin (ACTB), Glyceraldehyde 3 phosphate dehydrogenase (GPDH) and Ubiquitin c (UBC) were assessed by real time reverse transcriptase polymerase chain reaction (real time RT-PCR). iv Non irradiated HU treated cells had a reduced number of cells in the central scratch compared to non irradiated non treated cells, suggesting that HU inhibited cellular proliferation. Irradiated HU treated cells showed an increased number of cells in the central scratch compared to non irradiated treated cells. This observation proved that this increase was due to the stimulatory effect of irradiation with 5 J/cm2. The addition of HU had no significant effect on cell viability. The Trypan blue exclusion test showed no significant difference in percent viability between treated and non treated cells. Irradiated non treated cells showed a significant increase in the formazan dye, which is as a result of cleavage of XTT by the mitochondrial succinate dehydrogenase in actively proliferating cells, compared to non irradiated non treated cells (P=0.01). W cells, which were not irradiated, showed incomplete wound closure at both 1 and 24 h, while W cells irradiated with 5 J/cm2 showed complete wound closure. Similarly, W cells irradiated with 16 J/cm2 showed incomplete wound closure at 1 and 24 h. Cell viability, proliferation and DNA integrity assays showed that irradiated and non irradiated N cells were not significantly affected at both 1 and 24 h post irradiation. W cells (1 h) irradiated with 5 J/cm2 showed a significant increase in percentage cell viability and ATP compared to non irradiated W cells (1 h), (P=0.05 and P=0.04 respectively), while irradiation with 16 J/cm2 showed a significant decrease (P=0.014 and P=0.02 respectively). W cells (24 h) irradiated with 5 J/cm2 also showed a significant increase in percentage cell viability and ATP when compared to non irradiated W cells (24 h), (P=0.006 and P=0.04 respectively). Contrary, irradiation with 16 J/cm2 showed a significant decrease (P<0.001 and P=0.003 respectively). v Cell proliferation results showed that irradiation with 5 J/cm2 was stimulatory while 16 J/cm2 was inhibitory. The comet assay demonstrated that N cells irradiated with 5 or 16 J/cm2 exhibited an insignificant change in DNA damage at both 1 and 24 h when compared to their respective controls. This finding is in agreement with Karu et al., (2003) who observed that phototherapy does not alter the biological activity of cells which at the time of irradiation are functioning normally. W cells (1 and 24 h) irradiated with 16 J/cm2 showed a significant increase in DNA damage compared to their respective controls. However, there was a significant decrease in damage at 24 h compared to 1 h incubation due to the activation of DNA repair mechanisms. Though not significant, comet assay with Fpg (modified comet assay) showed more DNA damage compared to comet assay without the enzyme (conventional comet assay). It can be explained that the modified comet assay detected and cleaved oxidised bases in addition to single strand breaks, which the conventional comet assay detected, suggesting that the modified comet assay is more sensitive than the conventional comet assay. After validation of the three reference genes, ACTB was chosen to be the gene with which to normalise MPG expression in WS1 cells. It was found to be the least variable; its expression was consistent in W cells as well as cells exposed to a He-Ne laser at a fluence of 5 or 16 J/cm2. It produced an acceptable correlation coefficient (R2 >0.999) and PCR efficiency (94%). Conversely, other primers like GAPDH produced a low PCR efficiency (82%), while UBC produced a low R2 (0.898). Wang et al., (2006) recommends the value of R2 to be more than 0.995 and a PCR efficiency of between 90 and 100% for PCR results to be reliable. Other researchers have not supported the use of ACTB as a reference gene, stating that it is highly regulated (Wang et al., 2006), however this study showed that ACTB was not regulated by laser irradiation (632.8 nm at 5 or 16 J/cm2). The cell culture conditions and vi laser irradiation in this study did not induce MPG expression; perhaps an alternative repair pathway might have been induced, and hence repaired the DNA damage. In conclusion, the mini project demonstrated that HU is able to inhibit cell proliferation through its cytostatic effect without affecting the viability of W WS1 cells. This study also showed that irradiation of W cells with 5 J/cm2 using the correct parameters enhances cell migration and proliferation as evidenced by the presence of more cells in the central scratch in HU treated cells, and a significant increase in cell proliferation as shown by the XTT assay in non treated cells respectively. Thus, migration and proliferation are the direct result of phototherapy as both are involved in wound closure. This study further confirmed that irradiation of W cells with 5 J/cm2 stimulated ATP production, and hence cellular viability, as well as cell proliferation and migration. Irradiation of cells with higher fluences such as 16 J/cm2 is damaging to DNA and inhibitory to cell proliferation, migration and possibly to MPG expression. The study further showed that N cells are not stimulated by phototherapy, supporting the notion that lasers stimulate compromised cells. Thus, if they are growing normally there is nothing to stimulate. This understanding helps to clarify why N cells irradiated with 5 or 16 J/cm2 had insignificant responses. Cell culture conditions, fluence and duration of exposures are important parameters that can affect gene expression, and hence documentation of all experimental conditions needs to be emphasised and published if reproducibility is to be achieved.

M.Tech. / Low Level Laser Therapy (LLLT), also known as photo-biostimulation or simply phototherapy, has widely been used in the treatment of wounds, with its history dating back to the early 1960s (Ohshiro and Calderhead, 1991). Despite some literature reporting negative and non-existent cellular responses to LLLT, a growing body of literature reports the positive and beneficial effects of LLLT. LLLT has proved to be efficient in speeding and improving the quality of wound healing. Stressed cells respond more favourably to LLLT by recovering to their most natural state and functional capability (Bernett, 1998; Karu, 1998). When healing appears to be impaired, these tissues respond positively to the appropriate doses of light, especially light that is within 600 to 1,000 nm wavelengths (Enwemeka et al., 2004). Cellular responses to LLLT include changes in mitochondrial intracellular calcium ion (Ca2+) levels, Mitochondrial Membrane Potential (MMP), Adenine Triphosphate (ATP) concentration, and cyclic 5’, 3’ Adenosine Monophosphate (cAMP) (Karu, 1998). The mitochondrion is the power house of a cell and the major location of cellular ATP synthesis (Bayens and Dominiczak, 1999). ATP is an energy rich molecule that drives processes responsible for cell growth or proliferation (Klug et al., 2003). LLLT alters intracellular pH which is related to activation of ATPase leading to an increase in ATP production in the mitochondria of the cell (Alexandratou et al., 2002; Karu, 1998). However the mechanisms by which the beneficial effects are attained by cells in stress or injury state are not clear.

M. Tech. / Stem cells are defined as undifferentiated cells that can proliferate indefinitely and have the capacity of both self-renewal and differentiation to one or more types of specialised cells. Traumatic tissue injury and age-related degenerative diseases are a major problem in South Africa and worldwide. Stem cells could be used for tissue engineering and reconstructive surgery. In treating these conditions, the main principle of stem cell therapy is the replacement of damaged and dead cells in injured tissues and organs with new healthy ones expanded in vitro from stem cells (Orlic et al., 2002). These cells can be isolated from adipose tissue in significant numbers and exhibit stable growth and proliferation kinetics in culture and could be differentiated into bone, fat, cartilage and muscle when treated with established lineage-specific factors (Zuk et al., 2002). Low Level Laser Therapy (LLLT) is currently applied in the treatment of numerous diseases and pathological conditions (Gasparyan et al., 2004). LLLT produces positive effects on irradiated cells and tissues such as proliferation of cells, capillary growth and adenosine triphosphate (ATP) activation (Schindl et al., 1998). Low level laser radiation at different intensities has been shown to stimulate as well as to inhibit cellular processes (Moore et al., 2005). Epidermal growth factor (EGF) is a growth factor that plays important roles in the regulation of cell growth, proliferation and differentiation. This study investigated the effect of low level laser radiation alone as well as in combination with EGF on adult adipose derived stem cells (ADSCs) isolated from human adipose tissue. ADSCs were isolated from human adipose tissue through collagenase digestion and cultured in DMEM-F12 containing 10% FBS and antibiotics and incubated at 37°C in a humidified atmosphere of 5% CO2 (Zuk et al., 2001). iii Semi-confluent monolayers of ADSCs were exposed to low level laser at 5 J/cm2 using 636 nm diode laser with a power density of 12.1 mW/cm2 at room temperature in the dark. Cell morphology was monitored at 0, 24 and 48 h using an inverted light inverted microscope. Cell viability was evaluated at 0, 24 and 48 h using the Trypan Blue exclusion test and an adenosine triphosphate (ATP) luminescence assay. bFGF (basic fibroblast growth factor) indirect ELISA and optical density assays were used to monitor cell proliferation at 0, 24 and 48 h post irradiation. In addition the expressions of stem cell markers, β1-integrin and Thy-1, were monitored by immunocytochemical live cell surface labelling and Western blot analysis. Cells were incubated with EGF to enhance proliferation and differentiation and the cell morphology, viability and proliferation were monitored as well as the expressions of stem cell markers, β1-integrin and Thy-1. Morphology of the cells was not altered by irradiating them with 5 J/cm2 using diode laser at 0, 24 and 48 h. Cell viability and proliferation showed an increase at 24 and 48 h post irradiation. At 0 h, there was no significant difference between irradiated and non-irradiated cells in cell viability and proliferation. There was an increase in the expression of β1-integrin and Thy-1 after irradiation as shown by Western blot analysis and immunocytochemical live cell surface labelling. Cell viability and proliferation showed a significant increase at all time points post irradiation with the addition of EGF. There was no noticeable change in cellular morphology at any time point. Low level laser irradiation of human ADSC’s at 636 nm with 5 J/cm2 and 12.1 mW/cm2 increased the viability and proliferation of these cells in vitro. Furthermore, low level laser irradiation appeared to increase the expression of stem cell markers, β1-integrin and Thy-1. In addition, laser irradiation did not alter the morphology of the cultured cells. The addition of EGF to the cells also increased their viability and proliferation as well the expression of the markers, β1-integrin and Thy-1. The study showed that laser irradiation stimulates two important cellular responses namely cell viability and proliferation which indicates that ADSCs may be suitable for tissue engineering and future cell differentiation studies.