M. Tech. / Phototobiomodulation (PBM), also known as low level laser therapy (LLLT) or photobiostimulation, is a non-invasive form of therapy that utilizes low intensity laser light or irradiation to provide healing. However, in order for healing to be successful certain laser parameters need to be taken into consideration i.e. fluence (dosage), wavelength and power density. Laser therapy has been used for various medical applications and fields. Multiple cytokines and growth factors are involved in wound healing including Interleukin (IL)-1, IL-6 and Tumour Necrosis Factor alpha (TNF- a). In diseased state(s) such as diabetes mellitus (DM) or psoriasis, these growth factors or cytokines are either found elevated or decreased depending on various factors and for abnormally prolonged periods. However, inflammatory cytokines are usually elevated. Phototherapy has been reported to accelerate wound healing, attenuate pain and cease inflammation. However, the effect of phototherapy on cytokine modulation has not been explored extensively, especially under various stress mechanisms. Furthermore, the pathway that laser irradiation induces on modulated pro-inflammatory cytokines has not been clearly elucidated as scientists typically report on the up- or down-regulated expression of cytokines. Numerous authors have reported on the efficacy of laser irradiation to enhance the rate of wound healing and proliferation in normal and diabetic cells or tissue; however, literature that has demonstrated the latter on hypoxic insulted cells is inadequate. In this study hypoxic insult was induced as it is one of the factors that usually prolong the healing process in diabetic wounds. Prior to commencing with the main study, a pilot study was done to exclude the effect of osmotic pressure on cells grown in media containing additional glucose, and thus simulating a diabetic model iv in vitro. Mannitol was used as a control since it is not absorbed by the cells. The study involved four groups namely: normal, normal wounded, mannitol wounded and diabetic wounded cells with each group having a non-irradiated control. Mannitol wounded and diabetic wounded cells had a final concentration of 30 mM mannitol and glucose respectively. A wavelength of 636 nm at a fluence of 5 J/cm2 was used on day 1; experiments were repeated four times and all tests were done in duplicate. Cellular responses (Trypan Blue, adenosine triphosphate (ATP) and lactate dehydrogenase (LDH)) and morphological changes were assessed after 1 h incubation post-irradiation in both irradiated and non-irradiated cultures.

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. / 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.

M.Tech. / Stem cells possess self-renewal capacity, long-term viability, and multilineage potential. Stem cells play important roles in normal physiological and disease processes, they also have great therapeutic potential. However, there have been controversies surrounding stem cells in political, religious and ethical arenas. Although the use of certain stem cells (i.e. embryonic stem cells) and the means by which they are obtained contravene certain basic ethical laws, researchers have developed methods with which to ethically obtain and create stem cell lines. Stem cells can be classified as either: totipotent, pluripotent, multipotent, oligopotent and unipotent (Moore, 2007). Totipotent cells have the ability to differentiate into all cell types of an embryo, including the extra-embryonic and post embryonic tissues and organs. Pluripotent cells have the potential to differentiate into almost all tissues found in an embryo (including germ cells), but are not capable of giving rise to supporting cells and tissues. Multipotent stem cells have progeny of several differentiated cell types - but all within a particular tissue, organ, or physiological system. A good example of multipotent cells, are the haematopoietic stem cells that produce blood cell-restricted progenitors, as well as all cell types and elements, such as platelets, that are normal components of blood. Oligopotent stem cells produce two or more lineages within a specific tissue, such as neural stem cells that are able to produce subsets of neurons in the brain. Unipotent cells self-renew, as well as give rise to a single mature cell type, a prime example being the spermatogonial stem cells, that give rise to spermatozoa (Moore, 2007). Adult human subcutaneous adipose tissue contains cells with multilineage developmental plasticity like marrow-derived mesenchymal stem cells (Strem et al., 2005, Tong et al., 2000). Adipose derived stem cells can be obtained in abundance and can differentiate into osteogenic, adipogenic, myogenic and chondrogenic lineages when treated with appropriate growth factors.

published_or_final_version / Medical Sciences / Master / Master of Medical Sciences

A thesis submitted to the Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, in fulfillment of the requirements for the degree of Doctor of Philosophy Johannesburg, 2019 / Background Evidence shows that the global prevalence of knee osteoarthritis (KOA) is high, with limited data on the management of the disease. The use of novel modalities to treat the condition is low due to poor understanding of their clinical effects. Therefore there are gaps in the knowledge on the prevalence and treatment modalities for patients diagnosed with KOA. Aim: The aim was threefold; (i) to determine the prevalence of KOA in South Africa aged 45yrs-75yrs; (ii) to determine the current management of KOA; and (iii) to determine the effect of Low Level Laser therapy (LLLT) on the structural and functional components related to KOA in a South African cohort, aged 45-75yrs. Methods: The methodology will be discussed in terms of the three specified objectives; (i) prevalence study data - a self-reported data collection sheet listing 19 relevant ICD 10 codes; completed by South African medical aid providers. (ii) The treatment paradigm study, which encompassed a deemed KOA management paradigm validated questionnaire sent electronically to 742 general, specialist and allied practitioners, identifying the incidence of KOA and deemed efficacy and compliance of various management tool. These practitioners were identified from a database of medical and allied practitioners in both the private and public sector of South Africa. The questionnaire consisted of two close ended questions indicating the incidence of KOA and bilateral KOA patients consulted at the practice; one choice question indicating the most suggested mode of therapy from a choice of pharmaceutical, surgical, homeopathic, physical exercise therapy and LLLT and finally, 3 Likert type scale questions on the deemed efficacy and compliance of the modes of therapy as stated above. (iii) The intervention study which was a randomized controlled trial (RCT) utilizing pre marked questionnaire sheets on 111 participants. Participants were randomized into one of three intervention groups; (1) exercise group (n=39), (2) LLLT group (n=40), and (3) combined exercise-LLLT group (n=32). Data on knee circumference, the Western Ontario and McMaster Universities Osteoarthritis Index (WOMAC), knee range of motion (ROM) and the one minute timed sit–to-stand test was used. These tests were done at four time points: (T1) baseline, (T2) post-12 session intervention, (T3) one month post intervention and (T4) three months post intervention. Results: The results will be discussed in terms of the three specified objectives; (i) The prevalence of KOA was reported as 17.5%, 28.0% and 38.5% in a South African population over 45yrs. (ii) Four hundred and thirteen clinicians completed the questionnaire, reporting a KOA patient intake of 53%. Pharmacology (36.3%) and physical exercise (35.3%) was the most common management protocols compared to surgical intervention, homeopathy and LLLT. Pharmacotherapy (73%) and physical exercise (92%) were observed as effective treatments. Seventy five percent of all practitioners responded with an answer of “no comment” when asked the deemed efficacy of LLLT. Practitioners viewed patients with KOA to have low compliance with physical exercise and pharmacotherapy (iii) the participant demographic included 86 females and 25 males, the average age reported was 61.8 ± 5.6yrs. At 12-week follow-up, knee circumference decreased significantly in all groups (p<0.05), the effect was highest in the LLLT group. All groups experienced improvements in the WOMAC pain scale, but the LLLT group showed the greatest improvement (p<0.05). Knee ROM values improved significantly across all three groups; however, the effect of the intervention was most significant (p<0.005) in the combined LLLT-exercise group. Physical functionality scores showed a greater improvement in the combined LLLTexercise group at all three data collection points. Conclusions: The estimated prevalence of KOA is 17-35% based on data collected from a specified South African cohort. Pharmacotherapy is a commonly suggested KOA management mode, whilst clinicians view physical exercise as effective. LLLT was not a known tool for the treatment of KOA. In addition to the improved functionality observed, pain was lowered significantly, particularly in the combined exercise-LLLT group. Study results have shown that LLLT used in isolation or in combination with physical exercise is an effective management tool. / MT 2020

published_or_final_version / Anatomy / Doctoral / Doctor of Philosophy

M.Tech. (Chiropractic) / A myofascial trigger point is a hyperirritable point within a tight band of voluntary skeletal muscle. The condition causes levels of mild discomfort to intense pain to patients that usually results in loss of man hours and compulsory pain medication. Dry needling is the treatment of choice and other techniques are seldom considered. Dry needling therapy (DNT) is an effective tool in the chiropractic profession but comes with significant drawbacks, such as patients whom have needling phobias and patients who often experience post needling soreness are challenging to treat with DNT. Further and more serious risks include pneumothorax when needling the muscles over the lung fields, which also limits the treatment scope of DNT. Low level laser therapy (LLLT) is non-invasive and non-threatening to patients and could serve as an alternative to DNT. This study therefore aimed, to determine if LLLT could be an alternative treatment to DNT by comparing DNT to LLLT on a target group that all have active trapezius trigger point two myofascial trigger points. The group consisted of 40 participants with posterior trapezius myofascial neck pain caused by active myofascial trapezius trigger points. Participants were then randomly divided into two groups. Group A (20 participants) would receive DNT to the active myofascialtrapezius trigger point two (TP2) and group B (20 participants) would receive LLLT also to the active myofascial trapezius TP2. Participants would then be treated according to a set protocol, over a two week period with a total of four treatments. Subjective and objective readings were taken and noted on the first, third and fifth visits. Subjective data was collected from the visual analogue scale and the Vernon-Minor neck pain and disability index questionnaires. Whereas the cervical range of motion (CROM) and algometer readings provided the objective data.

M.Tech. (Chiropractic) / Myofascial trigger points (MTrP’s) cause acute discomfort to intense pain and often lead to the use of pain medication as well as loss of man hours (Simons, Travell and Simons, 1999a; Tough, White, Cummings, Richards and Campbell, 2009). Dry needling is very effective and is widely used for the treatment of MTrP’s (Vulfsons, Ratmansky and Kalichman, 2012), but comes with various significant drawbacks, such as the experience of pain during or after treatment (post-needling soreness) or individuals with needle phobias (Unruh, Strong and Wright, 2002). More serious risks also exist, including damage to the viscera (Dommerholt and Fernández-de-las-Peñas, 2013). Low level laser therapy (LLLT) is a non-invasive technique and very little discomfort or pain is experienced by the patient during and after treatment. LLLT is effective in the short- and long-term relief of trigger points and myofascial pain syndrome. Therefore it can easily serve as an alternative to myofacial dry needling (Chow and Barnsley, 2005). This study aimed to determine whether LLLT or myofascial dry needling is more effective in the treatment of active MTrP’s, specifically those of the gluteus medius muscle. It also aimed to determine if LLLT could serve as an alternative treatment to dry needling in cases where dry needling is contraindicated or not desired. Thirty participants who complied with the inclusion criteria were divided into one of two groups. Group 1 (n=15) received dosages of LLLT directly to the active MTrP’s in the gluteus medius muscle and Group 2 (n=15) received myofascial dry needling to active MTrP’s in the gluteus medius muscle. Each participant attended 6 treatment sessions over a course of 2 weeks as well as a 7th measurements-only session.

M.Tech. (Chiropractic) / Purpose: Patients presenting with mechanical cervical spine pain demonstrate myofascial trigger points of the surrounding cervical spine musculature (De Las Penas, Alonso-Blanco, Alguacil-Diego and Miangolarra-Page, 2006). Myofascial trigger points, from specifically the cervical spine musculature, have been seen to be involved to a large extent with not only the local mechanical cervical spine pain but also the accompanying referred pain patterns and symptoms (De Las Penas, Alonso-Blanco and Miangolarra-Page, 2007). The purpose of this study is to compare the efficacy of ischaemic compression and laser therapy respectively, applied to an active myofascial trigger point in participants with acute mechanical cervical spine pain associated with an active trapezius myofascial trigger point TP1, with regards to pain, activities of daily living, pressure pain threshold and cervical spine range of motion. Method: This study consisted of two groups, the ischaemic compression group with fifteen participants and the laser group with fifteen participants. The participants were between the ages of eighteen and forty-five years of age. Prior to becoming a participant of this study, individuals were assessed according to the inclusion and exclusion criteria, a clinical case history, physical examination, cervical spine regional examination and upper trapezius muscle palpation to assess for an active trapezius myofascial trigger point 1. Treatment was applied to the active trapezius myofascial trigger point 1 only, from which the subjective and objective results were based. Procedure: Each participant was treated six times over a period of two consecutive weeks. Prior to initiation of the treatment, each participant was requested to complete the Vernon-Mior Neck Pain and Disability Index questionnaire and the Visual Analogue Scale. Algometer readings were obtained over the trapezius myofascial trigger point 1, bilaterally. The Cervical Range of Motion (CROM) goniometer was used to obtain numerical values for the participant’s active cervical spine ranges of motion: flexion, extension, lateral flexion, and rotation. Ischaemic compression and laser therapy, group 1 and group 2 respectively, then each received treatment of the active trapezius myofascial trigger point 1, for a total of six treatment sessions. Both subjective and objective data readings were obtained before the 1st, 4th, and at the 7th final consultation.