Flexor tendonitis is a common cause of lameness and wastage in the equine athlete. Current techniques for tendonitis therapy provide limited success, and horses that do recover tend to return at a decreased level of performance. Current treatment techniques have begun to focus on regenerative medicine to improve tissue healing. Investigations of new treatments are made difficult by the lack of reliable in vitro models that allow for accurate comparison of treatment protocols. New techniques are often implemented into the clinical setting prior to thorough investigation for safety and efficacy. In vitro testing is an important step in the development of new therapeutic agents. However, results of in vitro tests should only be deemed as useful if the model used is one that is reliable and mimics the clinical situation that the reseachers are attempting to investigate. Equine flexor tendonitis is believed to be the result of microdamage caused by cyclic loading of tendons. Cyclic loading of fibroblasts results in increased production of the inflammatory cytokine prostaglandin E2 (PGE2). Thus the exposure of tendon fibroblasts to exogenous PGE2 may induce metabolic changes in the cells similar to what is seen in clinically affected animals making this a useful model for the investigation of therapeutic techniques. Currently a variety of techniques exist for treatment of flexor tendonitis; however, no single treatment has separated itself as superior. A new technique using autogenous conditioned serum (ACS) in humans for treatment of muscle injury has been shown to speed tissue regeneration. ACS produced from human blood has been shown to contain significantly increased levels of III growth factors that may improve tendon fibril formation and strength. We propose to investigate the effect of ACS on cellular metabolism in equine tendon fibroblast monolayers. This will involve cell culture, PGE2-induced cellular injury, and analysis of the cellular response to injury when treated with ACS. Controls will include fetal bovine serum, normal equine serum, and ACS without PGE2-induced cellular injury. The cellular response will be investigated biochemically by quantification of DNA, glycosaminoglycan, and soluble collagen levels and by real time PCR to assess gene expression for matrix metalloproteinases (MMP)-1, MMP-3, and MMP-13, collagen types I and III, and the non-collagenous proteins cartilage oligomeric matrix protein (COMP) and decorin. Data will be analyzed by analysis of variance and post-hoc comparisons. Significance will be set at p<0.05. We hypothesize that the addition of exogenous PGE2 to culture media for monolayers of equine tendon fibroblasts will insight alterations in cellular metabolism that will generate a suitable model for the in vitro study of fibroblast response to novel therapies. We then hypothesize that the addition of ACS to PGE2-treated fibroblasts will result in increased gene expression for collagen types I and III, cartilage oligomeric matrix protein, and decorin. ACS will also stimulate increased protein production of collagen and glycosaminoglycans, and stimulate increased cell proliferation. The use of ACS will decrease gene expression of inflammatory molecules important in tendon degradation, namely matrix metalloproteinases -1, -3, and -13. / Master of Science

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Submitted by Nadir Basilio (nadirsb@uninove.br) on 2015-07-21T15:29:23Z No. of bitstreams: 1 Carla Helrigle Silva.pdf: 884644 bytes, checksum: 450ed90ece95b161e2e9976cead3aea4 (MD5) / Made available in DSpace on 2015-07-21T15:29:23Z (GMT). No. of bitstreams: 1 Carla Helrigle Silva.pdf: 884644 bytes, checksum: 450ed90ece95b161e2e9976cead3aea4 (MD5) Previous issue date: 2013-09-09 / The aim of this experimental study was to investigate the effects of LED phototherapy on the inflammation process in the calcaneal tendon of ovariectomized rats (OVX) through the involvement of inflammatory mediators IL-6, IL-10 and TNF-α. Thirty-five female Wistar rats were divided into 4 groups: 3 ovariectomized groups, untreated (NTTA); treated (TTA) and control subdivided into experimental periods of 3 , 7 and 14 days, totaling 30 rats and one group (n = 5) not ovariectomized (not OVX). Tendinitis was induced by trauma using a 208g mass placed at 20 cm from the right tendon of each animal with energy of 0.70 J. The animals were treated 12 hours after tendonitis with LED therapy ( 945nm , power 32mW, energy of 3.84 J, power density of 0.06 W / cm ², energy density of 7.68 J / cm ², beam output area of 0.5 cm ² and time of 120 s) and every 48 hours at 3, 7 and 14 days. Then, euthanasia was performed in a CO2 chamber 24h after the last treatment. The tendons were dissected and stored in liquid nitrogen at -196ºC, thawed only at the time of immunoenzymatic test (ELISA). After analysis of results, groups treated with LED showed decrease in the number of pro-inflammatory cells, IL- 6 and TNF- α (p < 0.05) and increased number of anti-inflammatory IL -10 ( p <0.05) even when compared to the not OVX group (p < 0.05). It was concluded that low-intensity LED in the studied parameters has repairing effect on the tendinopathy process (tendonitis) caused by trauma in female rats (OVX). / Este estudo experimental tem objetivo de analisar os efeitos da fototerapia LED, no processo inflamatório em tendão de Aquiles de ratas ovariectomizadas (OVX), pelo envolvimento dos mediadores inflamatórios IL-6, IL-10 e TNF-α. Foram utilizados 35 ratas Wistar fêmeas, divididas em 4 grupos: sendo 3 grupos ovariectomizados, não tratados (NTTA); tratados (TTA) e controle subdivididos nos períodos experimentais de 3, 7 e 14 dias, totalizando 30 ratos; e um grupo (n=5) não ovariectomizados (Não OVX). A tendinite foi induzida por trauma, utilizando uma massa de 208g posicionada a 20 cm do tendão direito de cada animal, com energia de 0,70J. Os animais foram tratados 12h após a tendinite, com terapia LED (945nm, potência de 32mW, energia de 3,84J,densidade de potência 0,06W/cm²,densidade de energia 7,68J/cm²,área de saída do feixe 0,5 cm² e tempo de 120s) e a cada 48h nos períodos de 3 , 7 e 14 dias. Em seguida, a eutanásia decorreu em câmara de CO2, 24h ao último tratamento. Os tendões foram dissecados e acondicionados em nitrogênio líquido a 196ºC negativos, descongelados apenas no momento da realização do teste imuenzimátioco (ELISA). Após a analise dos resultados os grupos tratados com LED apresentaram diminuição no número de células pró-inflamatórias, IL-6 e TNF-α (p<0,05), e aumento no número da IL-10 (p<0,05) anti-inflamatória mesmo quando comparado como grupo não OVX (p<0,05). Assim conclui-se que a LED de baixa intensidade, nos parâmetros estudados tem efeito reparador em processo de tendinopatia (tendinite) traumática em ratas (OVX).

This project investigated the effects of Kinesio® Tape on pain, kinesiophobia, and proprioception in participants with patellar tendonitis. Thirty participants with patellar tendonitis were divided into three groups, the first received a supportive Kinesio® Tape application at the knee, the second received a facilitative application at the hip, and the third received both. A Visual Analog Score, Tampa Scale for Kinesiophobia score, and a proprioceptive score quantified through the Biodex Balance System were obtained both immediately after application, and 24 to 36 hours following. Statistically significant improvement in VAS scores and in proprioceptive ability with eyes closed 24 to 36 hours following Kinesio® Tape application was observed under all interventions. Kinesio® Tape application for pain, cause of pain and proprioception may assist patients with patellar tendonitis.

Introdução: A tendinopatia de Aquiles caracteriza-se pela presença de sinais clássicos da resposta inflamatória, como o edema e a dor, além de alterações estruturais no tecido. Neste estudo avaliamos o processo de reparação e reorganização tecidual de tendões de ratos submetidos a tendinopatia por colagenase, sendo posteriormente tratados com laserterapia de baixa intensidade, anti-inflamatório esteroidal e anti-inflamatório não-esteroidal. Materiais e Método: Ratas Wistar fêmeas foram submetidas a injeção transcutânea de colagenase (100<font face=\"Symbol\">mg/tendão) na região peritendínea das patas e posteriormente divididas e tratadas nos seguintes grupos: laser (3J), diclofenaco potássico (1,1mg/kg) e dexametasona (0,02mg/kg). Foram realizadas análises histomorfológicas, incluindo o score de achados, quantidade de colágeno presente no tecido e nível de agregação/organização das fibras colágenas. Resultados e Discussão: A colagenase produziu o aumento de células inflamatórias, extravasamento plasmático e hemorragia. Entre os tratamentos testados, o laser mostrou-se a melhor opção, atenuando a resposta inflamatória, aumentando a concentração de colágeno e mantendo a organização das fibras colágenas. / Introduction: Achilles tendinopathy is characterized by presence of classical signs of inflammatory response such as edema, pain, and structural changes in tissue. In this study we evaluated the repair process and tissue reorganization of rats tendons submitted to collagenase induced tendinopathy, being posteriorly treated with low-level laser therapy, steroidal anti-inflammatory and non-steroidal anti-inflammatory. Materials and Methods: Female Wistar rats were submitted to transcutaneous collagenase injection (100<font face=\"Symbol\">mg/tendon) at paws peritendinous site and posteriorly divided and treated in following groups: laser (3J), potassium diclofenac (1.1mg/kg), and dexamethason (0.02mg/kg). Histomorphological analyses were made including findings score, tissue collagen amount and collagen fibers aggregation/organization levels. Results and Discussion: Collagenase produced enhancement of inflammatory cells, edema and hemorrhage. Laser therapy showed be the better option between tested treatments, decreasing inflammatory response, increasing collagen concentration and mantaining collagen fibers organization.

Introdução: A tendinopatia de Aquiles caracteriza-se pela presença de sinais clássicos da resposta inflamatória, como o edema e a dor, além de alterações estruturais no tecido. Neste estudo avaliamos o processo de reparação e reorganização tecidual de tendões de ratos submetidos a tendinopatia por colagenase, sendo posteriormente tratados com laserterapia de baixa intensidade, anti-inflamatório esteroidal e anti-inflamatório não-esteroidal. Materiais e Método: Ratas Wistar fêmeas foram submetidas a injeção transcutânea de colagenase (100<font face=\"Symbol\">mg/tendão) na região peritendínea das patas e posteriormente divididas e tratadas nos seguintes grupos: laser (3J), diclofenaco potássico (1,1mg/kg) e dexametasona (0,02mg/kg). Foram realizadas análises histomorfológicas, incluindo o score de achados, quantidade de colágeno presente no tecido e nível de agregação/organização das fibras colágenas. Resultados e Discussão: A colagenase produziu o aumento de células inflamatórias, extravasamento plasmático e hemorragia. Entre os tratamentos testados, o laser mostrou-se a melhor opção, atenuando a resposta inflamatória, aumentando a concentração de colágeno e mantendo a organização das fibras colágenas. / Introduction: Achilles tendinopathy is characterized by presence of classical signs of inflammatory response such as edema, pain, and structural changes in tissue. In this study we evaluated the repair process and tissue reorganization of rats tendons submitted to collagenase induced tendinopathy, being posteriorly treated with low-level laser therapy, steroidal anti-inflammatory and non-steroidal anti-inflammatory. Materials and Methods: Female Wistar rats were submitted to transcutaneous collagenase injection (100<font face=\"Symbol\">mg/tendon) at paws peritendinous site and posteriorly divided and treated in following groups: laser (3J), potassium diclofenac (1.1mg/kg), and dexamethason (0.02mg/kg). Histomorphological analyses were made including findings score, tissue collagen amount and collagen fibers aggregation/organization levels. Results and Discussion: Collagenase produced enhancement of inflammatory cells, edema and hemorrhage. Laser therapy showed be the better option between tested treatments, decreasing inflammatory response, increasing collagen concentration and mantaining collagen fibers organization.

Thesis (PhD (Physiological Sciences))--University of Stellenbosch, 2007. / SUPERFICIAL digital flexor tendon injury has a serious negative impact on the competitive horse industry. Injured horses require up to a year of rest for recovery and likelihood of re-injury upon return to normal activity is as high as 80 %. Tendon healing requires (a) production of collagen by fibroblasts, to provide tensile strength and elasticity to the tendon, (b) minimisation of restrictive fibrosis, which compromises tendon gliding function and (c) minimisation of peritendinous adhesions. We review conventional treatments for tendon healing before exploring stem cell application as a therapeutic alternative. We promote the use of hematopoietic and mesenchymal stem cells derived from adult peripheral blood - as opposed to bone marrow-derived stem cells or embryonic stem cell sources - and review published research output in this regard. In conclusion, we outline our research objectives and present and discuss our results in the chapters that follow. Mononuclear cells - consisting of hematopoietic stem cells, mesenchymal stem cells and leucocytes – were isolated from the peripheral blood of sheep and horses through red blood cell lysis and blood plasma extraction. Cell counts and propidium iodide dye exclusion viability tests were conducted on the cell pellets. Sheep sub samples were tested for CD45 expression and horse sub samples for CD4 and CD11a/18 cell surface markers by flow cytometry for characterisation purposes. In both cases, separate sub samples were incubated with matched immunoglobulin (IgG) isotypes, conjugated to fluorescein isothiocyanate (FITC), to serve as controls. For the culture of mononuclear cells, 4.5 x 106 cells were selected for autologous sheep injections, 3 x 106 CD45- cells for allogeneic sheep injections (the latter excluding leucocytes that may induce an immune response) and 72 x 106 cells for horse injections. These cells were incubated with bromo-deoxyuridine (BrdU), cultured and subsets were extracted for a second round of cell counts and viability tests before being resuspended in blood plasma. For the horse samples an additional 1 x 106 mononuclear cells were incubated until reaching 60 % confluence and tested for myogenic differentiation. Low cell mortality and lack of fluorescence from IgG-FITC controls reflected effective protocols and a lack of false positive results. The fact that the equine cell population differentiated into myotubes verified the presence of mesenchymal stem cells in injections. We tested whether surgical incisions or collagenase injections best mimicked naturally occurring tendon injuries and compiled macroscopic and microscopic descriptions of tendon injury sites at seven weeks post-injury. The superficial digital flexor tendons of 27 sheep received an incision, a collagenase injection or a saline control injection. After one week a number of sheep were sacrificed while the remainder received further saline treatment and were sacrificed after another seven weeks. Tendons were examined through clinical observations, image analysis of maximum tendon diameter, mechanical testing and histological sectioning of affected tissues. Collagenase-induced injury resembled tendonitis more closely than surgically-induced injury. Collagenase-injured tendons (a) induced lengthier lameness in affected limbs, (b) were more swollen and difficult to palpate, (c) assumed the bow appearance characteristic of natural injury, (d) experienced extensive haemorrhage due to collagen lysis, (e) had decreased elasticity and capacity to carry loads and stress, (f) displayed decreased stiffness due to collagen fibre disruption and (g) developed severe inflammation. After seven weeks injured tendons displayed increased vascularisation in the areas of haemorrhage and in the adjacent collagen matrix. High inflammation rates and low collagen levels however still persisted. Collagenase injections were used to induce tendonitis in the superficial digital flexor tendons of 27 sheep. After one week these tendons received treatment with a control saline solution, autologous peripheral blood mononuclear cells (MNCs) or allogeneic peripheral blood CD45- MNCs. Healing rates were compared after a further seven week period by conducting ultrasonographic evaluations, clinical observations, image analyses of maximum tendon diameter, mechanical tests and histological investigations. Tendons treated with MNCs displayed an improvement in echogenicity and fibre linearity, higher and more organised collagen levels, stronger mechanical properties and less swelling. Although these improvements were not always significant, they provided strong evidence to suggest marked healing benefits over a longer time period. Collagenase injections were used to induce tendonitis in the superficial digital flexor tendons of four horses. After one week these tendons received treatment with either a control saline solution or autologous peripheral blood mononuclear cells (MNCs). Healing rates were compared after a further seven week period by conducting ultrasonographic evaluations, clinical observations, image analysis of maximum tendon diameter and histological investigations. Tendons treated with MNCs displayed significant improvements in fibre linearity in the direct vicinity of the lesion, as well as recovery rate thereof, and experienced less swelling when compared with their untreated counterparts. Healing trends suggested that, given a longer period of observation post-injury, more significant improvements may become apparent. Human adipose tissue is known be an easily accessible and high yielding source of multipotent mesenchymal stem cells. These stem cells could potentially be used for therapeutic advancement of tendon regeneration. Our first goal was to examine the in vitro myogenic differentiation potential of adipose-derived, adherent mononuclear cells (MNCs) from six adult sheep. The second goal was to characterise the population of cells isolated through various available ovine specific, non-mesenchymal stem cell surface markers, namely, CD1, CD31, CD34 and CD45. After incubation, only four of the six MNC cultures started to proliferate. These four cultures all exhibited high myogenic differentiation ability. The isolated cell populations did not express any of the non-mesenchymal stem cell specific cell surface markers. In conclusion, our data suggests that peripheral blood stem cells and adipose-derived stem cells are important candidate cell types for therapeutic application to improve tendon repair in horses and sheep. Sufficient time must be allowed following injury and prior to stem cell treatment (at least one month) and a controlled exercise program should be followed posttreatment. A larger sample size is required and at least six months of recovery before macroscopic and histological repair can be analysed more accurately and conclusively. Ultrasonography should be carried out on a continuous basis, as it is a non-invasive method of monitoring change over time.