O exercício excêntrico tem sido utilizado na prevenção/reabilitação de lesões e em programas de treinamento de força para melhorar o condicionamento físico de indivíduos saudáveis. O entendimento das adaptações causadas pelo treinamento excêntrico nos músculos flexores plantares se justifica: 1) pela importância desta musculatura na manutenção de posturas e no ciclo da marcha; 2) pela alta incidência de lesões do tendão de Aquiles; e 3) pelo uso sistemático deste tipo de treinamento em programas de prevenção e reabilitação do tríceps sural. Assim, a presente tese de doutorado busca verificar os efeitos do treinamento excêntrico nas propriedades neuromecânicas e morfológicas dos músculos flexores plantares. No capítulo I foram compiladas informações acerca das adaptações neuromusculares dos flexores plantares e do tendão de Aquiles de indivíduos saudáveis submetidos à programas de treinamento excêntrico. Os estudos encontrados indicam que o treinamento excêntrico pode aumentar a produção de força e ativação muscular, especialmente em testes excêntricos. No entanto, resultados conflitantes e lacunas identificadas na literatura motivaram a realização de dois estudos originais. Os objetivos dos estudos originais foram: 1) determinar a temporalidade das adaptações na ativação e massa muscular de flexores plantares, bem como sua contribuição para os ganhos de força em contrações excêntricas, isométricas e concêntricas ao longo do programa de treinamento (Capítulo II); e 2) avaliar os efeitos de 12 semanas de treinamento excêntrico nas propriedades morfológicas, mecânicas e materiais do tendão de Aquiles de indivíduos saudáveis (Capítulo III). Vinte participantes do sexo masculino realizaram um programa de treinamento excêntrico isocinético (duas vezes por semana, 3-5 séries de 10 repetições máximas). As avaliações das propriedades neuromecânicas e morfológicas dos flexores plantares foram realizadas a cada quatro semanas. Ao final de 12 semanas, o programa de treinamento excêntrico aumentou a produção de torque máximo excêntrico, isométrico e concêntrico; aumentou a atividade eletromiográfica máxima excêntrica e isométrica; e aumentou a espessura muscular. Além disso, os ângulos do pico de torque excêntrico e concêntrico foram deslocados para posições em que os músculos estavam mais alongados. O torque máximo e a espessura muscular aumentaram progressivamente até a oitava semana de treinamento. A ativação neural durante contrações excêntricas e isométricas aumentou após quatro semanas de treino e permaneceu constante até o final do treinamento, enquanto que a ativação neural durante contrações concêntricas permaneceu inalterada durante todo o período de treinamento. Além disso, houve aumento da área de secção transversa, da rigidez e do módulo de Young do tendão de Aquiles. Os incrementos na rigidez e no módulo de Young foram observados após quatro semanas de treinamento, enquanto que o aumento significativo da área de secção transversa tendínea ocorreu após oito semanas de treinamento. Quando tomados em conjunto, estes resultados nos possibilitam entender de que forma as adaptações neuromecânicas e morfológicas dos flexores plantares ocorrem. O aumento da força isométrica e excêntrica nas primeiras quatro semanas de treinamento parece ocorrer devido a adaptações neurais, musculares e tendíneas. No entanto, após maiores períodos de treinamento (i.e. acima de quatro semanas), o aumento da força ocorre devido a incrementos na massa muscular e na rigidez tendínea. Além disso, a ausência de adaptações neurais evidencia que os ganhos de força concêntrica podem estar relacionados apenas com adaptações musculares e tendíneas. / Eccentric exercises are commonly used in prevention, rehabilitation and conditioning training programs. Understanding the adaptations caused by eccentric training on the plantar flexor muscles is justified by: 1) the importance of these muscles in maintaining posture and during gait cycle; 2) the high incidence of Achilles tendon injuries; and 3) the systematic use of this type of training in triceps surae prevention and rehabilitation programs. Thus, the present PhD thesis aims to verify the effects of eccentric training in neuromechanical and morphological properties of the plantar flexor muscles. Chapter I compiled information about the neuromuscular adaptations of the plantar flexors and Achilles tendon of healthy subjects undergoing eccentric training programs. The studies found indicate that eccentric training can increase the production of muscle strength and muscle activation, especially in eccentric tests. The studies found indicate that eccentric training can increase muscle strength and muscle activation, especially in eccentric tests. The purposes of the original studies were: 1) to determine the adaptations time course in plantar flexors activation and muscle mass, as well as their contribution to the strength gains in eccentric, isometric and concentric contractions during the training program (Chapter II); and 2) to evaluate the effects of 12 weeks of eccentric training on Achilles tendon morphological, mechanical and material properties in healthy subjects (Chapter III). Twenty male subjects performed an eccentric isokinetic training program (twice a week, 3-5 sets of 10 maximal repetitions). Plantar flexor neuromechanical and morphological evaluations were performed every 4 weeks. The 12-week training program led to increases in maximum eccentric, isometric and concentric torques; maximum eccentric and isometric electromyographic activity; and muscle thickness. The angles of peak torque in eccentric and concentric tests were shifted towards longer muscle lengths. Maximum torque and muscle thickness increased progressively until the 8th training week. Eccentric and isometric activation increased up to the 4th training week and remained constant until the 12th training week, while no change was found in concentric activation. In addition, Achilles tendon cross-sectional area, stiffness and Young's modulus were increased. The increases in stiffness and Young's modulus were observed after four weeks of training, while the significant increase in tendon cross-sectional area occurred after eight weeks of training. Taken together, these results allow us to understand how the neuromechanical and morphologic adaptations occur in the plantar flexors muscles subjected to a 12-week eccentric training program. The increase in isometric and eccentric strength in the first four weeks of training seems to be related to neural, morphological and tendinous adaptations. However, after longer training periods (i.e. up to four weeks), the strength increase is due to increases in muscle mass and tendon stiffness. Moreover, the absence of evidence in terms of neural adaptations during concentric contractions suggest that the concentric strength gains seem to be related only with muscle and tendon adaptations.

O desenvolvimento de novos recursos terapêuticos capazes de acelerar o processo de reparo tem proporcionado avanços significativos no tratamento de lesões tendinosas. O objetivo do presente estudo foi analisar a influência da radiação LASER HeNe em cicatrização de tendões. Foi desenvolvido um modelo experimental de lesão de tendão de Aquiles através de um procedimento minimamente invasivo em coelhos adultos. Foram utilizados 40 animais distribuídos em quatro grupos de 10 animais (I, II III IV). No grupo I a lesão do tendão foi unilateral (pata direita) e o tendão lesado não recebeu estímulos com LASER. No grupo II a lesão foi unilateral (pata direita) e o tendão lesado recebeu estímulos com LASER. No grupo III a lesão do tendão foi bilateral e apenas a pata direita foi estimulada com LASER. No grupo IV foi realizada \"Sham Operation\" bilateralmente e apenas o tendão da pata direita recebeu estímulo com LASER. Todos os animais foram tratados com dose a 5 J/cm2 de forma pontual com contato direto durante 8 dias consecutivos, diariamente. Após este período os animais foram sacrificados e os tendões de Aquiles submetidos a ensaios mecânicos de tração em máquina de ensaio mecânico. Foram ensaiados 71 tendões, sendo 09 descartados. Embora não tenhamos observado diferença estatística significante nas comparações feitas entre os grupos, podemos notar que existiu uma tendência apontando que os tendões estimulados com LASER apresentaram resistência menor que os grupos controles. Estes resultados sugerem que o LASER diminuiu a resistência dentro das condições estudadas. Relevância Clínica: O presente trabalho mostra o conceito de que a irradiação LASER pode diminuir a resistência mecânica da cicatrização tendinosa em fases precoces do processo. / The development of new therapeutic resources addressing accelerating the repairing process has proportioned a meaningful advance in the treatment of tendon lesions. The purpose of the present study was to analyse the influence of He-Ne LASER radiation on tendon healing. An experimental model of Achilles tendon lesion was developed through a minimally invasive procedure in adults rabbits. We used 40 animals divided in groups of 10 animals each. In group I the tendon lesion was unilateral (right-paw) and the injured tendon did not receive LASER stimulation. In group II the lesion was unilateral (right-paw) and only, the injured side received LASER stimulation. In group III the tendon lesion was bilateral and only the right-paw was stimulated by LASER. In group IV bilateral sham operation was performed and only the right - paw received LASER stimulation. All the animals were treated with 5 J/cm2 dosis in a punctual way with direct contact for 8 successive days. After this period the animals were killed and the Achilles tendons were submitted to mechanical tests in traction on a mechanical testing machine. Seventy-one tendons were tested and 9 were discarded. No statiscally significant difference was observed in the comparison done between groups, however we could notice a tendency suggesting that the tendons stimulated by LASER presented a lesser mechanical resistance. These results suggest that LASER probably reduces the resistance in the studied conditions. Clinic Relevance: The present study supports the concept that LASER irradiation may induce an attenuation of the mechanical properties of the healing tendon in a very early phasis of the proccess.

A maioria dos casos de pé torto congênito tratados pelo método de Ponseti requer a secção do tendão calcâneo para correção do equino residual. Evidências clínicas sugerem que há completa cicatrização entre os cotos tendíneos, mas este processo reparativo ainda não foi suficientemente estudado. Esta investigação teve como objetivo avaliar o processo de reparação que ocorre após a secção percutânea do tendão calcâneo para a correção do equino residual no pé torto congênito idiopático tratado pelo método de Ponseti. Por meio de estudo prospectivo, foram analisadas 37 tenotomias em 26 pacientes com pé torto congênito idiopático tratados pelo método de Ponseti, com seguimento mínimo de um ano após a secção. A tenotomia foi realizada percutaneamente com agulha biselada de grosso calibre, sob sedação e anestesia local. O exame ultrassonográfico foi feito logo após a secção tendínea para assegurar que ela tenha sido completa e mensurar o afastamento entre os cotos. A reparação foi estudada por meio da ultrassonografia realizada três semanas, seis meses e um ano após a tenotomia. A ultrassonografia, realizada imediatamente após o procedimento, mostrou que, em alguns casos, feixes tendíneos residuais persistiam entre os cotos, mas foram completamente seccionados, em seguida, sob controle ultrassonográfico. Houve afastamento médio de 5,65 mm ± 2,26 (2,3 a 11,0 mm) entre os cotos tendíneos logo após a secção. Em um caso ocorreu sangramento maior que o habitual, que foi controlado com pressão local e não provocou interferência no tratamento. Após três semanas, a ultrassonografia mostrou regeneração tendínea com preenchimento do espaçamento entre os cotos por tecido hipoecoico com ecotextura irregular e com restituição da continuidade entre os cotos demonstrada dinamicamente pela transmissão de movimentos do músculo tríceps sural para o calcanhar. Seis meses após a tenotomia, o exame ultrassonográfico evidenciou que o tecido de reparação apresentava ecotextura de aspecto fibrilar e, quando comparado ao tendão normal, havia leve ou moderada hipoecogenicidade e espessamento cicatricial. Um ano após a tenotomia, o exame ultrassonográfico mostrou estrutura fibrilar na região de reparação, com ecogenicidade semelhante ao tendão normal, mas ainda apresentando espessamento tendíneo cicatricial. Em termos gerais, ocorreu rápida cicatrização após a secção percutânea do tendão calcâneo, que restabeleceu a continuidade entre os cotos. Ao final do período de observação, o tecido de reparação tendínea apresentou aspecto ultrassonográfico semelhante ao lado normal, exceto por leve espessamento, o que sugere um mecanismo de reparação predominantemente intrínseco. / Most cases of congenital clubfoot treated by the Ponseti technique require percutaneous Achilles tenotomy in order to correct the residual equinus. Clinical evidences suggest that complete healing occurs between the cut tendon stumps, but there have not yet been any detailed studies investigating this reparative process. This study was performed to assess the Achilles tendon repair after percutaneous sectioning to correct the residual equinus of clubfoot treated by the Ponseti method. A prospective study analyzed 37 tenotomies in 26 patients with clubfoot treated by the Ponseti technique, with a minimum follow-up of one year after the section. The tenotomy was performed percutaneously with a large-bore needle bevel with patient sedation and local anesthesia. Ultrasonographic scanning was performed after section to ascertain that the tenotomy had been completed and to measure the stump separation. In the follow-up period, the reparative process was followed ultrasonographically at three weeks, six months and one year post-tenotomy. The ultrasonography performed immediately after the procedure showed that in some cases, residual strands between the tendon ends persisted, and these were completely sectioned under ultrasound control. A mean retraction of 5.65 mm ± 2.26 (range, 2.3 to 11.0 mm) between tendon stumps after section was observed. Unusual bleeding occurred in one case and was controlled by digital pressure, with no interference with the final treatment. After three weeks, ultrasonography showed tendon repair with the tendon gap filled with irregular hypoechoic tissue, and also with transmission of muscle motion to the heel. Six months after tenotomy, there was structural filling with a fibrillar aspect, mild or moderate hypoechogenicity, and tendon scar thickening when compared to a normal tendon. One year after tenotomy, ultrasound showed a fibrillar structure and the echogenicity at the repair site that was similar to a normal tendon, but with persistent mild tendon scarring thickness. It was observed that there was a fast reparative process after Achilles tendon percutaneous sectioning that reestablished continuity between stumps. The reparative tissue evolved to tendon tissue with a normal ultrasonographic appearance except for mild thickening, suggesting a predominantly intrinsic repair mechanism

Introdução: O alongamento dos músculos isquiotibiais (ISQ) tem sido utilizado com frequência para a correção da contratura em flexão dos joelhos na paralisia cerebral (PC), porém o aumento da anteversão da pelve (AP) e a recidiva da deformidade podem ocorrer a longo prazo. Objetivos: O objetivo deste trabalho foi avaliar se a transferência do semitendíneo para o tubérculo dos adutores (TXST) está relacionada a uma menor taxa de recidiva e a um menor aumento da AP no período pós-operatório, quando comparada ao alongamento deste músculo. Métodos: Foi realizado um estudo tipo coorte retrospectivo. Pacientes com PC diparesia espástica, Gross Motor Function Classification System (GMFCS) I-III, sem cirurgias prévias nos joelhos, submetidos ao alongamento dos ISQ mediais ou à TXST, e com completa documentação no laboratório de marcha foram incluídos no estudo. Foram excluídos aqueles que receberam de forma concomitante a osteotomia extensora do fêmur distal e o encurtamento patelar. Trinta e nove pacientes preencheram os critérios de inclusão e foram divididos em dois grupos de acordo com os procedimentos cirúrgicos realizados: Grupo A (22 pacientes / 44 joelhos), composto por aqueles que receberam o alongamento dos ISQ mediais; Grupo B (17 pacientes / 34 joelhos), formado por aqueles que receberam a TXST ao invés do alongamento do semitendíneo (AST), em conjunto com o alongamento dos demais ISQ mediais. Parâmetros clínicos e de cinemática foram avaliados nos dois grupos antes e após as cirurgias. Resultados: Os grupos não exibiram diferença quanto à distribuição por gênero, idade na cirurgia e tempo de seguimento. A deformidade irredutível em flexão estava presente em 9,1% dos joelhos no Grupo A e em 50% no Grupo B (p < 0,001) antes do tratamento, e em 25% dos joelhos do Grupo A e 20,6% no Grupo B (p=0,647) após os procedimentos cirúrgicos. O número de joelhos com deformidade irredutível em flexão aumentou no Grupo A (p=0,047) e reduziu no Grupo B (p=0,011) após a intervenção. Houve redução significativa da deformidade média em flexão dos joelhos ao exame físico (de 7,3° para 4,4°, p= 0,04) e da flexão dos joelhos durante a fase de apoio da marcha (de 34,2° para 20,2°, p < 0,001) apenas no Grupo B. A AP aumentou nos Grupos A e B após a correção cirúrgica da deformidade em flexão dos joelhos. Conclusão: O aumento da AP foi observado nos dois grupos após o tratamento efetuado. A redução da deformidade em flexão dos joelhos ao exame físico e o aumento da extensão dos joelhos na fase de apoio foram observados apenas nos pacientes submetidos à TXST / Introduction: Hamstrings surgical lengthening has been frequently used for the correction of knee flexion contracture in cerebral palsy (CP), however the increase of anterior pelvic tilt and the recurrence of the deformity can occur in a long-term follow-up. The aim of this study was to evaluate if semitendinosus transfer to distal femur (STTX) is related to less increase of anterior pelvic tilt and less recurrence of knee flexion deformity after treatment than semitendinosus surgical lengthening (STL). Methods: A retrospective cohort study was conducted. Patients with diplegic spastic CP, GMFCS levels I to III, without previous surgical procedures at knee, undergone to bilateral medial hamstrings surgical lengthening or STTX, and with complete documentation at gait laboratory were included in this study. Patients with concomitant distal femur extension osteotomy and patellar tendon shortening were excluded. Thirty-nine patients matched the inclusion criteria and they were divided in two groups according surgical procedures at knees: Group A (22 patients / 44 knees), including patients who received medial hamstrings surgical lengthening as part of multilevel approach; Group B (17 patients / 34 knees), represented by patients who underwent orthopedic surgery including a STTX instead of STL. Clinical and kinematic parameters were evaluated at baseline and at follow-up for all groups. Results: The two groups matched at gender distribution, age at surgery and follow-up time. Fixed knee flexion deformity (FKFD) before surgery was observed at 9.1% of knees in Group A and at 50% in Group B (p < 0.001). At final follow-up, 25% of knees in Group A and 20.6% in Group B shown FKFD (p=0.647). FKFD increased in Group A (p=0.047) and decreased in Group B (p=0.011) after treatment. The reduction of mean FKFD (from 7.3° to 4.4°, p= 0.04) and of knee flexion during gait stance phase (from 34.2° to 20.2°, p< 0.001) were observed only in Group B after surgical procedures. The anterior pelvic tilt increased at both groups after treatment. Conclusion: The increase of anterior pelvic tilt occurred at both groups after correction of knee flexion deformity. Patients who received STTX exhibited less fixed knee flexion deformity and better knee extension during stance phase after surgical treatment than those whom undergone to STL

In conclusion, the present study firstly addressed the relationship between graft incorporation and peri-graft bone quality and quantity after ACL reconstruction in a rabbit model. The findings suggested that the non-invasive measurement of peri-graft bone would be useful to predict graft incorporation. Peri-graft bone loss was region-specific after surgery, which might be associated with stress shielding in the specific region after tunnel creation. The use of Brushite CPC might be a promising way to augment peri-graft bone and enhance graft incorporation. (Abstract shortened by UMI.) / In the fourth part, brushite CPC was successfully applied to augment the peri-tendon bone volume and connectivity. It was revealed under mechanical testing that the ultimate strength and stiffness of graft fixation in bone tunnel on experimental side was higher than that of the control by 117% and 102% respectively at 6 weeks, postoperatively (p&lt;0.05 for both). The use of brushite CPC caused a paradigm shift in failure mode from intra-tunnel to intra-articular portion at 12 weeks postoperatively (p=0.013). / Keywords. anterior cruciate ligament reconstruction, bone mass, microarchitecture, tendon-to-bone healing, graft incorporation, brushite calcium phosphate cement / The first part of this thesis compared the histological characteristics of T-B healing interface tissue in femoral and tibial tunnels following ACL reconstruction in rabbits. Results revealed that less cartilaginous interface tissue was formed in tibial tunnel than in femoral tunnel. Such cartilaginous tissue was gradually mineralized during reestablishment of a direct T-B integration. T-B healing in tibial tunnel was inferior to that in femoral tunnel. The disparity of T-B healing in various osseous milieus suggested the potential association in between. / The second part of this original work further explored the numeric relationship between the strength of T-B attachment and peri-graft bone mass and connectivity. Results of Micro-computed tomography (micro-CT) showed that peri-graft bone mass and connectivity was significantly lower on tibial side than those on femoral side. It was found under biomechanical evaluation that grafted tendon was prone to be pulled out from tibial tunnel with the bone attachment; the weakest point of the complexes shifted from the healing interface at time zero to peri-graft bone at week 6 after operation. With reverse of peri-graft bone at week 12 postoperatively, the weakest point shifted to intra-osseous tendinous portion. The stiffness of graft fixation correlated with peri-graft BV/TV (r2=0.68, p=0.001) and connectivity (r2=0.47, p=0.013) at week 6 after operation. / The third part addressed the changes of peri-graft bone in spatial and temporal manners using high resoluation multiple-slice peripheral quantitative computed tomography (pQCT) and micro-CT. Under pQCT evaluation, a decrease in BMD was present in specific regions, medial region of femoral tunnel by 26% and posterior and lateral regions of tibial tunnel by 22% and 42%, respectively at week 12 postoperatively than the baseline (p&lt;0.05 for all). It was accompanied by a decrease in trabecular number and increase in trabecular spacing, the shift of plate-like to rod-like trabeculae and loss of anisotropy under micro-CT evaluation. It was echoed by histological findings showing increased osteoclastic activities and poor T-B healing in these specific regions. The postoperative bone loss and associated poor T-B healing was region-specific. / by Wen, Chunyi. / Adviser: Kai-ming Chan. / Source: Dissertation Abstracts International, Volume: 71-01, Section: B, page: 0217. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2009. / Includes bibliographical references (leaves 148-168). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Electronic reproduction. Ann Arbor, MI : ProQuest Information and Learning Company, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese.

肌腱修復一直是一個難題，因為依靠現在的治療很難將肌腱功能恢復到正常水平，近年來肌腱幹細胞的分離和發現為肌腱修復提供了新的策略。但是在利用肌腱幹細胞修復肌腱之前，我們應該瞭解肌腱幹細胞的哪些方面呢？ / 不同來源的成體幹細胞雖然具備相似的幹細胞特性，但是他們仍然具有組織特異性和功能的差異。這就意味選擇合適的細胞來源對於肌腱再生和肌腱組織工程有特殊意義。所以我們認為與骨髓間充質幹細胞相比，肌腱幹細胞具備特殊的幹細胞特性。迄今為止，還沒有研究比較肌腱幹細胞和骨髓間充質幹細胞的幹細胞特性。臨床應用要求幹細胞在體外增殖培養，體外的微環境也會影響幹細胞的幹性和治療潛能，所以我們還並不清楚肌腱幹細胞的幹性在體外培養中維持多久。成功的幹細胞治療需要深入理解組織特異性幹細胞的體內特徵和他們在組織修復中的作用。肌腱幹細胞的体内特徵还有没详细研究过，而且也不知道這些內源性幹細胞是否參與肌腱修復。 / 所以為了更好地利用肌腱幹細胞進行肌腱修復，本研究的總體目標是比較肌腱幹細胞和骨髓間充質幹細胞的幹細胞特性，同時從臨床角度考慮研究肌腱幹細胞體外幹性的維持。進一步研究鑒定肌腱幹細胞的體內特徵，並且探索他們在肌腱癒合中的作用。本研究將會探討我們應該瞭解關於肌腱幹細胞的體內和體外特性。 / 在第一部分研究中， 我們從同一隻GFP大鼠中分離出肌腱幹細胞和骨髓間充質幹細胞。經過比較，我們發現肌腱幹細胞与骨髓間充質幹細胞相比具备更高的克隆形成能力，增殖速度，更強的多向分化能力和更高的肌腱相关的基因表达。所以肌腱幹細胞表現出更好的幹性，可能是比骨髓间充质干细胞更好的用于肌腱再生的细胞来源。 / 在第二部分研究中，我們發現肌腱幹細胞伴隨體外傳代培養細胞衰老β-半乳糖苷酶活性增高，而同時間充質幹細胞標誌物和多向分化能力降低，所以研究人員和臨床醫生在利用肌腱幹細胞進行組織工程時需要考慮在體外傳代培養中他們的幹性的變化。 / 在第三部分研究中，IdU標記滯留細胞方法用於在體內標記幹細胞。我們發現休眠的幹細胞以IdU標記滯留細胞的形式存在於肌腱中，相比肌腱本體更多標記滯留細胞位於和肌腱腱鞘和肌腱骨結合部位。其中我們發現在肌腱腱鞘中的標記滯留細胞位於血管周圍的微環境血管，所以血管周圍的微環境可能是肌腱幹細胞來源之一。肌腱損傷后，位於損傷區域的標記滯留細胞的數量，增殖標誌物，肌腱相關標誌物， 多能性標誌物，和微血管相關標誌物都有明顯增加，意味著標記滯留細胞可能通過遷移，增殖和分化參與肌腱修復。 / 綜上所述，我們的結果為理解肌腱幹細胞的體外幹性特徵和在體外培養中的幹性變化以及体内肌腱幹細胞的鑒定提供了新的解釋，這有利于未來促進肌腱幹細胞的組織工程應用於肌腱修復。 / Tendon repair remains a great challenge due to current therapies cannot restore normal tendon function. Tendon-derived stem cells (TDSCs) have been isolated from tendon tissues and characterized in vitro in recent studies and provide new strategies for tendon repair. But what should we know about tendon stem cells before we use them to repair injured tendon? / Although stem cells that originate from different tissues share some common stem cell characteristics, they might also exhibit some tissue unique properties and hence functional differences. Therefore, we hypothesized that TDSCs have unique stemness properties compared with bone marrow-derived stem cells (BMSCs). There has been no study to compare the stemness properties of TDSCs and BMSCs. Clinical applications often require the in vitro expansion of stem cells. In vitro microenvironment also affects the stemness properties and therapeutic potential of stem cells. It is not clear if the stemness properties of TDSCs can be maintained and how long that they can be preserved during in vitro expansion. Moreover, successful stem cell-based repair therapies will require an understanding of tissue specific stem cells in vivo and their roles in the tissue repair. Tendon stem cells have not been described in details in vivo and it is unknown whether these endogenous stem cells participate in the tendon healing. / Therefore, in order to better make use of TDSCs for tendon repair, the objective of this study is to characterize the stemness properties of TDSCs compared with BMSCs and also to investigate the stemness limitation of TDSCs during culture in vitro for clinical use purpose. Furthermore, this study aims to identify the putative tendon stem cells in vivo and their role in tendon healing. This study would tell how much we should know about tendon stem cells in vitro and in vivo. / In the first part of the study, TDSCs and BMSCs were isolated from the same GFP Sprague-Dawley rat. TDSCs showed higher mensenchymal and pluripotent stem cell makers; clonogenicity; proliferative capacity; and tenogenic, osteogenic, chondrogenic, and adipogenic differentiation markers and multi-lineage differentiation potential than BMSCs. Compared with BMSCs, TDSCs shows great stemness properties and might be an alternative cell source for tendon regeneration. / In the second part of this study, the senescence-associated β-galactosidase activity of TDSCs increased while their stem cell-related marker expression and the multi-lineage differentiation potential decreased during in vitro passaging. It suggests that researchers and clinicians need to consider the changes of stemness properties of TDSCs when multiplying them in vitro for tissue engineering. / In the third part of the study, IdU label-retaining method was used for the labeling of stem cells in vivo. We have identified quiescent stem cells as IdU label retaining cells (LRCs) at the peritenon, tendon mid-substance and tendon-bone junction. More LRCs were found at the peri-tenon and tendon-bone junction compared to the mid-substance. Some LRCs could be identified in the peri-vascular niche in the peri-tenon, suggesting that peri-vascular niche is one source of tendon stem cells. After injury, The LRC number and the expression of proliferative, tendon-related, pluripotency and pericyte-related markers in LRCs in the window wound increased, indicating that LRCs might be involved in tendon repair via cell migration, proliferation and differentiation. / In conclusion, our results have provided new findings about the understanding of tendon-derived stem cells including their stemness properties and their changes during the in vitro culture, as well as in vivo identity of tendon stem cells, which might facilitate the application of TDSCs in tissue engineering for tendon repair in the future. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Detailed summary in vernacular field only. / Tan, Qi. / Thesis (Ph.D.) Chinese University of Hong Kong, 2014. / Includes bibliographical references (leaves 130-162). / Abstracts also in Chinese.

Tang Yin Nei. / Thesis (M.Phil.)--Chinese University of Hong Kong, 2001. / Includes bibliographical references (leaves xv-xxv). / Abstracts in English and Chinese. / ACKNOWLEDGEMENT --- p.i / ABBREVIATIONS --- p.ii-iii / INDEX FOR FIGURES --- p.iv-v / INDEX FOR TABLES --- p.vi / ABSTRACT (Chinese and English) --- p.vii-xi / TABLE OF CONTENTS --- p.xii-xiv / Chapter CHAPTER I 226}0ؤ --- INTRODUCTION --- p.1 / Chapter 1.1 --- Background --- p.2 / Chapter 1.2 --- Tendon / Chapter 1.2.1 --- Structure and function --- p.3 / Chapter 1.2.2 --- Tendon fibroblast --- p.6 / Chapter 1.2.3 --- Components of the extracellular matrix --- p.7 / Chapter 1.2.3.1 --- Collagen --- p.8 / Chapter 1.2.3.2 --- Proteoglycan --- p.9 / Chapter 1.2.3.3 --- Non-collagenous structural glycoprotein --- p.10 / Chapter 1.3 --- Inflammation disorders of tendon / Chapter 1.3.1 --- Inflammation --- p.11 / Chapter 1.3.2 --- Treatment --- p.12 / Chapter 1.3.2.1 --- Glucocorticoid as an anti-inflammatory agent --- p.12 / Chapter 1.3.2.2 --- Dexamethasone --- p.14 / Chapter 1.3.3 --- Clinical occurrence of tendon rupture --- p.15 / Chapter 1.3.4 --- Animal research related to glucocorticoids and tendon rupture --- p.18 / Chapter 1.4 --- Platelet-derived growth factor isoform B (PDGFBB) / Chapter 1.4.1 --- Structure and function --- p.21 / Chapter 1.4.2 --- PDGFbb effects on connective tissue --- p.22 / Chapter CHAPTER II 226}0ؤ --- AIM OF THE STUDY --- p.23 / Chapter 2.1 --- Limitations of the past researches --- p.24 / Chapter 2.2 --- Hypothesis of this study --- p.25 / Chapter 2.3 --- Objectives --- p.26 / Chapter 2.4 --- Long term significance --- p.26 / Chapter CHAPTER III 226}0ؤ --- METHODOLOGY --- p.27 / Chapter 3.1 --- Chemicals and materials used / Chapter 3.1.1 --- Chemicals --- p.28 / Chapter 3.1.2 --- Materials --- p.28 / Chapter 3.2 --- Specimen collection and preparation / Chapter 3.2.1 --- Collection --- p.29 / Chapter 3.2.2 --- Preparation and isolation --- p.30 / Chapter 3.2.3 --- Cell culture --- p.31 / Chapter 3.3 --- Reagent preparation / Chapter 3.3.1 --- Charcoal-stripped serum --- p.32 / Chapter 3.3.2 --- Phenol-red free DMEM --- p.33 / Chapter 3.3.3 --- MTT --- p.33 / Chapter 3.3.4 --- Dexamethasone --- p.34 / Chapter 3.3.5 --- PDGFbb --- p.34 / Chapter 3.3.6 --- Trypan blue --- p.35 / Chapter 3.3.7 --- TCA/Tannic acid --- p.35 / Chapter 3.3.8 --- Collagenase buffer --- p.35 / Chapter 3.4 --- Morphology / Chapter 3.4.1 --- Inverted phase contrast light microscopy --- p.36 / Chapter 3.4.2 --- Scanning electron microscopy --- p.36 / Chapter 3.5 --- Biological assays / Chapter 3.5.1 --- "MTT (3-[4,5-Dimethylthiazol-2-yl]2,5-diphenyltetrazolium bromide) assay" --- p.38 / Chapter 3.5.1.1 --- Correlation between MTT assay and trypan blue dye method --- p.38 / Chapter 3.5.1.2 --- Growth kinetics for tendon fibroblasts --- p.41 / Chapter 3.5.1.3 --- Cell viability --- p.43 / Chapter 3.5.2 --- Brdu (5-bromo-2'-deoxyuridine) assay --- p.44 / Chapter 3.5.3 --- Flow cytometry --- p.45 / Chapter 3.5.4 --- Apoptosis --- p.47 / Chapter 3.5.5 --- 3H-Proline incorporation assay --- p.48 / Chapter 3.5.6 --- 35Sulfate incorporation assay --- p.51 / Chapter 3.5.7 --- Immunocytochemistry (PDGF-β receptor) --- p.54 / Chapter 3.6 --- Statistical analysis / Chapter 3.6.1 --- Dose-response curve of dexamethasone on cell viability and proliferation --- p.55 / Chapter 3.6.2 --- Comparison among various treatments of fibroblasts --- p.55 / Chapter CHAPTER I´Vؤ --- RESULTS --- p.56 / Chapter 4.1 --- In vitro effect of dexamethasone on rat tendon fibroblasts / Chapter 4.1.1 --- Viable cell number between two sexes --- p.57 / Chapter 4.2 --- In vitro effect of dexamethasone and PDGFBB on human tendon fibroblasts / Chapter 4.2.1 --- Gross morphology --- p.58 / Chapter 4.2.2 --- Cell cycle --- p.60 / Chapter 4.2.3 --- Apoptosis --- p.61 / Chapter 4.2.4 --- Viable cell number / Chapter 4.2.4.1 --- Effect of dexamethasone --- p.62 / Chapter 4.2.4.2 --- Effect of PDGFBB --- p.63 / Chapter 4.2.5 --- Cell proliferation / Chapter 4.2.5.1 --- Effect of dexamethasone --- p.65 / Chapter 4.2.5.2 --- Effect of PDGFbb --- p.67 / Chapter 4.2.6 --- Collagen synthesis --- p.68 / Chapter 4.2.7 --- Proteoglycan synthesis --- p.72 / Chapter 4.2.8 --- PDGF-rβ expression --- p.74 / Chapter CHAPTER V 226}0ؤ --- DISCUSSION --- p.75 / Chapter 5.1 --- Dexamethasone and PDGFBB induced change of cell morphology --- p.77 / Chapter 5.2 --- Dexamethasone retarded cell growth of human tendon fibroblast --- p.80 / Chapter 5.3 --- Dexamethasone inhibited collagen synthesis --- p.82 / Chapter 5.4 --- Dexamethasone inhibited proteoglycan synthesis --- p.86 / Chapter 5.5 --- PDGFbb could counteract the inhibitory effects of dexamethasone --- p.88 / Chapter 5.6 --- Expression of PDGF-(3 receptor is regulated by dexamethasone and PDGFBB --- p.90 / Chapter 5.7 --- Limitations of this study / Chapter 5.7.1 --- Not enough sample to differentiate different between two sexes --- p.92 / Chapter 5.7.2 --- Small sample size and few assays --- p.92 / Chapter 5.7.3 --- Limitations of the cell culture model --- p.93 / Chapter 5.7.4 --- Difficult to further in vivo study on human --- p.93 / Chapter 5.8 --- Contributions of this study / Chapter 5.8.1 --- Improve the limitation of the past research --- p.94 / Chapter 5.8.1.1 --- Human tendon specimen --- p.94 / Chapter 5.8.1.2 --- In vitro system --- p.94 / Chapter 5.8.2 --- Understand the effect of dexmaethasone on human tendon fibroblasts --- p.95 / Chapter 5.8.3 --- Counteract the deleterious effects of dexamethasone by PDGFBB --- p.95 / Chapter CHAPTER VÍؤ --- CONCLUSION & FUTURE STUDY --- p.96 / Chapter 6.1 --- Conclusion --- p.97 / Chapter 6.2 --- Future study --- p.98 / Chapter 6.2.1 --- Study the balance between matrix synthesis and degradation --- p.98 / Chapter 6.2.2 --- Determine collagen typing --- p.99 / Chapter 6.2.3 --- Further explore the effect of glucocorticoid in organ culture model --- p.100 / Chapter 6.2.4 --- Investigate molecular mechanism of dexamethasone and PDGFBB --- p.100 / REFERENCES --- p.xv-xxv / APPENDIX --- p.xxvi

Este trabalho investiga a influência de polimorfismos na região promotora do gene das metaloproteases 1, 3 e 8 na fisiopatogenia da insuficiência primária do tendão tibial posterior. A amostra de 150 pacientes selecionados é dividida em grupo-teste: 50 pacientes com diagnóstico clínico e anatomopatológico de tendinopatia do tendão tibial posterior e grupo-controle: 100 pacientes com tendão tibial posterior íntegro. O DNA dos voluntários é obtido a partir de células epiteliais da mucosa bucal mediante a extração com acetato de amônia. As técnicas de PCR e RFLP são utilizadas para análise dos genótipos. A análise estatística dos resultados é realizada pelo teste do qui-quadrado com nível de significância de 5%. Os resultados mostram que os polimorfismos -1607 da MMP-1 e -799 da MMP-8 estão relacionados com risco maior para tendinopatia primária do tendão tibial posterior, enquanto o polimorfismo -1612 da MMP-3 parece não influenciar essa tendinopatia / The aim of this study was to investigate the influence of polymorphisms in the promoter region of the gene of metalloproteinases 1, 3 and 8 in physiopatology of primary posterior tibial tendon insufficiency. The sample of 150 selected patients was divided into test group: 50 patients undergoing surgical procedures and pathological diagnosis of degenerative lesions of the posterior tibial tendon, and control group: 100 patients with posterior tibial tendon intact and no signs of degeneration. The DNA of the volunteers was obtained from oral mucosa epithelial cells, by extraction with ammonium acetate. PCR and RFLP were used for analysis of genotypes. Statistical analysis of results was performed by Chi-squared test with significance level of 5%. The results show that polymorphisms -1607 of MMP-1 and -799 of the MMP-8 are associated with increased risk for primary tendinopathy of the posterior tibial tendon, while the -1612 polymorphism of MMP-3 does not influence this tendinopathy

A estimativa das forças musculares durante o movimento humano tem inúmeras aplicações na área da Biomecânica. Nesse trabalho descrevo a aplicação de métodos de estimativa das forças musculares a dois diferentes problemas. O primeiro consiste em se quantificar as cargas a que os músculos e as articulações do membro inferior de adultos jovens estão sujeitos durante o andar em ambiente aquático e terrestre. Já o segundo consiste na investigação do efeito do aumento da complacência do tendão de Aquiles (TAq; tendão do triceps sural) na dinâmica de produção de força dos músculos gastrocnêmio e sóleo (G e S respectivamente; ambos músculos do triceps sural), visando compreender se o aumento da complacência, observado com o envelhecimento, poderia contribuir para as alterações na biomecânica da marcha de idosos. A hipótese no primeiro estudo era de que as forças desenvolvidas pelos músculos do membro inferior durante o andar em ambiente aquático não seriam menores em relação ao ambiente terrestre. Especificamente, esperávamos observar um aumento nas forças geradas nos flexores e extensores de quadril e joelho. No segundo estudo, nossas hipóteses eram de que o aumento na complacência do TAq faria com que as fibras musculares operassem mais distantes do seu comprimento ótimo, aumentando os níveis de ativação e o consumo metabólico dos músculos G e S. Com o objetivo de verificar as hipóteses levantadas nos dois estudos propostos utilizamos o software OpenSim 3.1 e dados experimentais dos movimentos estudados, para realizar simulações do andar humano e estimar as forças nos músculos do membro inferior durante essa tarefa. Em ambos os estudos os dados experimentais foram adquiridos através de sistemas de análise do movimento humano compostos por câmeras, para filmar os movimentos corporais dos voluntários, e plataformas de força, para medir as forças de reação do solo. Os resultados obtidos no primeiro estudo confirmaram nossas hipóteses, uma vez que indicam que durante determinadas fases do ciclo da marcha as forças geradas pelos músculos flexores e extensores de joelho e quadril, tais como os músculos isquiotibiais e o gluteo máximo, podem exceder as forças geradas em ambiente terrestre. Esses resultados corroboram a idéia de que o andar em ambiente aquático pode ser efetivo no ganho de força muscular. As hipóteses para o segundo estudo também foram confirmadas. Adicionalmente, nossos resultados previram que, para o triceps sural gerar a potência necessária para manter o indivíduo andando com velocidade confortável, os tendões dos músculos G e S devem se movimentar de maneira independente. A diferença no movimento dos feixes do TAq é tanto maior quanto maior a sua complacência. Além disso, verificamos que há um mínimo no consumo metabólico do gastrocnêmio em um nível específico de complacência do TAq, enquanto para o sóleo o consumo aumenta sistematicamente com o aumento da complacência. Esses resultados indicam que um aumento na complacência do TAq pode comprometer o desempenho dos músculos do triceps sural e aumentar o seu consumo energético, contribuindo para as alterações na biomecânica da marcha de idosos. As informações fornecidas nos dois estudos aqui apresentados podem auxiliar profissionais de área da saúde no planejamento de programas de treinamento e reabilitação para adultos e idosos, tanto no ambiente terrestre quanto no ambiente aquático. / Muscle force estimation during human motion has numerous applications in Biomechanics. In this work, we describe the application of methods of muscle force estimation to solve for two different problems. The first problem is to quantify lower limb muscle and joint loads that young adults are subjected to when walking in the aquatic and terrestrial environment. The second problem is to understand the effect of increased Achilles tendon compliance (AT; the triceps surae tendon) in the gastrocnemius and soleus contractile dynamics (G and S respectively; both triceps surae muscles), aiming at understanding if increased AT compliance, that occurs with aging, could play a role in the elderly gait alterations. Our hypothesis for this first study was that the forces developed by the lower limb muscles in water are not always lower than on land. Specifically, we hypothesized that the forces developed by the hip and knee flexors and extensors would be grater in water than on land. For the second study our hypothesis was that the compliant AT would cause the fibers to operate far from its optimal length resulting in higher levels of activation in both G and S, as well as higher specific metabolic consumption. In order to verify our hypotheses for both studies we used the software OpenSim 3.1 together with experimental data of volunteers walking in aquatic and terrestrial environments to simulate human walking and to estimate the forces developed by the lower limb muscles during this task. In both studies, experimental data were acquired through human movement analysis systems composed of cameras, to record the movements of the volunteers\' body, and force plates, to measure ground contact forces. We confirmed our hypotheses to the first study since our results showed that in certain periods of the gait cycle the forces developed in the knee and hip flexors and extensors, such as the hamstrings and the gluteus maximus, inside water exceeded the forces in corresponding periods of land walking. Those results corroborate the idea that walking inside water may be effective in muscle strengthening. We also confirmed our hypotheses to the second study. Additionally, we predicted that to generate the necessary power to walk with comfortable speed the triceps surae G and S tendon must move independently. This differential behavior becomes larger the greater the AT compliance. In addition, we also observed that G metabolic energy consumption was minimized for a specific level of AT compliance while S systematically increased. Those results suggest that increased AT compliance can compromise the triceps surae performance and increase metabolic consumption, contributing to the alterations on the elderly gait biomechanics. The results of our two studies may assist health professionals to better plan training and rehabilitation programs for adults and elderly in both, terrestrial and aquatic environment.

O crescente aumento do interesse no desenvolvimento em técnicas cirúrgicas menos invasivas, como nas reconstruções tendíneas e ligamentares, tem levado ao aumento das pesquisas referentes ao uso de aloenxertos esterilizados por radiação ionizante. O processamento por radiação ionizante é um método seguro e não deixa resíduos, sendo utilizado como esterilização final. O presente estudo teve como proposta avaliar os efeitos da aplicação de radiação ionizante, produzida por fonte de 60Co, em amostras de tendões humanos pré processados de doadores multiorgãos obtidas por meio de colaboração com Bancos de Tecidos. O pré-processamento das amostras deu-se por métodos químicos e preservação por congelamento em -80 °C. As doses aplicadas no processamento por radiação foram de 12,5 kGy, 15,0 kGy e 25,0 kGy, cada uma com seu respectivo controle não irradiado. As amostras foram avaliadas por meio de testes histológicos, ópticos e biomecânicos, com o objetivo de analisar possíveis modificações morfológicas e estruturais. Os resultados apresentados demonstraram que o processamento por ultrassom e peróxido de hidrogênio causaram alterações na morfologia dos tecidos, o que ocasionou danos à sua estrutura, inviabilizando as amostras. Nas amostras processadas por álcool e antibiótico não foram observados danos na rede de colágeno pela aplicação da radiação. Os resultados dos testes biomecânicos apresentaram diferenças significativas entre os métodos aplicados. As amostras processadas com álcool e antibiótico apresentaram perda pouco significativa no módulo de elasticidade, comparadas às amostras processadas por ultrassom e peróxido de hidrogênio que mantiveram a propriedade viscoelástica. Contudo na dose de 12,5 kGy foi observado um aumento no módulo elástico e na viscoelasticidade. Com base nas análises, podemos concluir que o método de processamento com álcool, antibiótico e irradiação demonstrou menor dano, tanto na biomecânica quanto na esterilização, sendo que as amostras irradiadas a 15,0 e 25,0 kGy apresentaram características semelhantes ao controle não irradiado. / The increasing interest of development in less invasive surgical techniques, such as reconstructions of ligament tendon, has led to the increase of the research concerning the use of Allografts sterilized by ionizing radiation. Processing by ionizing radiation is a safe method and leaves no residues, being used as final sterilization. The present study was to evaluate the effects of proposed application of ionizing radiation, produced by 60Co source in human tendon pre-samples processed multiorgans donors obtained through collaboration with tissue banks. The pre-processing of samples given by chemical methods and preserved by freezing at -80 °C. The doses applied in radiation processing were 12.5, 15.0 and 25.0 kGy, each with your corresponding non-irradiated control. The samples were evaluated by means of histological and biomechanical testing, with the purpose of analyzing possible structural and morphological changes. The results showed that the ultrasound processing and hydrogen peroxide caused changes in the morphology of the tissues, which caused damage to the structure, making your samples. In the samples processed by alcohol and antibiotics were not observed damage on the network of collagen by the application of radiation. The results of biomechanical tests showed significant differences between the methods used. The samples processed with alcohol and antibiotics showed negligible loss in modulus of elasticity compared with the samples processed by ultrasound and hydrogen peroxide which kept the viscoelastic property, however in 12.5 kGy dose was observed an increase in elastic modulus and viscoelasticity. Based on the analysis we can conclude that the method of processing with alcohol, antibiotics and irradiation showed less damage, both in biomechanics and sterilization, in the samples irradiated with 15.0 and 25.0 kGy, showing results similar to the non-irradiated control.