Establishment of animal model for studying treatment efficacy of low-intensity pulsed ultrasound stimulations for accelerating bone-tendon repair. Standard partial patellectomy was conducted in the 18-week old rabbits that were then divided into the LIPUS treatment and control groups. The animals were followed for 2, 4, 8, and 16 weeks for various tissue analyses. LIPUS was applied to the experimental animals from postoperative day 3 to 16 weeks. We demonstrated that the healing process of PPT junction was initiated through endochondral ossification. The results showed that the size and length of newly formed bone, and its bone mineral content (BMC), but not its bone mineral density (BMD) were correlated with the failure load, ultimate strength and energy at failure. Using radiographic, biomechanical, histomorphologic and biomechanical methods, it was found that LIPUS had significant accelerating effect on PPT junction repair. We validated our study hypothesis in that LIPUS enhances bone-tendon junction healing by stimulating angiogenesis, chondrogenesis and osteogenesis. / Establishment of in vitro model for mechanism study on effects of low-intensity pulsed ultrasound stimulations. An in vitro model of osteoblast-like cell line (SaOS-2 cells) was studied using cDNA microarray to explore the molecular mechanism mediated by LIPUS. This microarray analysis revealed a total of 165 genes that were regulated at 4 and 24 hours by LIPUS treatment in osteoblastic-like cells. These genes belonged to more than ten protein families based on their function and were involved in some signal transduction pathways. This study has validated the hypothesis that LIPUS can regulate a number of critical genes transient expressions in osteoblast cell line Saos-2. / Keywords. partial patellectomy model; bone-tendon junction repair; low intensity pulsed ultrasound stimulations (LIPUS); gene expression; complementary DNA microarray; rabbit. / This study explored the intact morphology, regular healing and the augmented healing under the effects of low intensity pulsed ultrasound stimulations (LIPUS) on the patella-patella tendon (PPT) junction in a rabbit partial patellectomy model. To probe its possible mechanism, the key genes involved in regulating osteogenesis mediated by LIPUS were identified using the state-of-the-art methods---complementary DNA microarray. / Lu Hongbin. / "June 2006." / Advisers: Ling Qin; Kwok Sui Leung. / Source: Dissertation Abstracts International, Volume: 68-03, Section: B, page: 1548. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2006. / Includes bibliographical references (p. 259-288). / 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, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

Tendonopathy, resulting in the loss of mechanical strength of a tendon, is a serious health problem affecting many people. The common symptom of tendonopathy is pain – patients' daily activities, their participation in sport and exercise, and their ability to work are greatly compromised. Tendonopathy is considered to be a degenerative disorder caused by repetitive injury of the tendon. The most common tendon lesions are Achilles tendon rupture, lateral epicondylitis (tennis elbow) and rotator cuff tear. However, in spite of its clinical significance, our knowledge about tendonopathy is still very poor. This research was undertaken to investigate the pathology of tendonopathy. It is proposed that apoptosis, autophagic cell death and myofibroblasts play a role in the progression of tendonopathy in the rotator cuff; the aim of this study was therefore to determine if this was indeed the case. Tendon tissues were collected from 30 patients suffering from rotator cuff tears. A terminal deoxynucleotidyl transferase biotin-dUTP nick end labelling (TUNEL assay) was performed to detect apoptosis. Autophagic cell death of the tenocytes in the ruptured rotator cuff tendon was detected by immunohistochemical staining for ubiquitin. Myofibroblasts were identified immunohistochemically with anti-alpha-smooth muscle actin (anti--SMA) antibody. The distribution of apoptosis, autophagic cell death and myofibroblasts, as well as the total cell density, were assessed respectively and were correlated using a four-category (i.e. graded from 0-3) degeneration of collagen matrix. – 6 – The results showed that apoptosis, autophagic cell death and myofibroblasts were observed in all of the samples. The highest percentage of autophagic cell death was evidenced in the Grade 2 matrix, while the percentage of apoptosis increased significantly with the increase of matrix degeneration from Grade 0-3; a similar pattern was found for myofibroblasts. The total cell numbers varied among the matrix grades, with the maximum and minimum percentages occurring in Grades 1 and 3, respectively. It can be concluded that apoptosis, autophagic cell death and myofibroblasts might be closely related to the damage of the extracellular matrix (ECM) structure.

Epidemiological studies consistently report that muscular strains are a primary injury type in rugby union with the majority of the strains occurring to the quadricep and hamstring musculature. Recently it has been suggested that poor eccentric muscular strength is a precursor to hamstring and quadriceps strains during intermittent sports that require rapid acceleration and deceleration. Despite the high incidence of these muscle injuries in Rugby Union there has been little research into the possible mechanisms involved. Thus, the purpose of this study was to measure the physiological and perceptual responses during a simulated Rugby Union laboratory protocol and further, to identify changes in muscle recruitment patterns and muscle strength over time by comparing this protocol to a continuous, constant load protocol covering the same distance. The experimental condition (EXP) required university level players to perform 80 minutes of simulated rugby union play in a laboratory setting (on a walkway of 22m) which was compared to that of a control condition (CON) which involved subjects covering the same distance, at a constant speed of 4.2km.h-1 on a treadmill. Physiological, biophysical and perceptual responses were measured pre-, at half-time and post-protocol. Heart rate was significantly (p<0.01) greater as a result of EXP in comparison to the CON. Electromyography (EMG) of the vastus medialis was significantly (p<0.01) greater during the CON protocol. The EXP condition elicited higher iEMG activity in the hamstring musculature at all time intervals. In addition the iEMG of the semitendinosus decreased significantly (p<0.01) as a result of the EXP protocol. Peak eccentric knee extensors (EXT) (-13.19%) and flexors (FLEX) (-12.81%) torque decreased significantly during the experimental protocol. After passive half-time (236.67 + 56.27Nm (EXT) and 173.89 + 33.3NM (FLEX)) and at the end of the protocol (220.39 + 55.16Nm and 162.89 + 30.66Nm) reduced relative to pre protocol (253.89 + 54.54Nm and 186.83 + 33.3Nm). Peak eccentric knee extensors did not change during the control protocol. „Central‟ and ‟Local” Rating of Perceived Exertion values were significantly (P<0.01) greater during the EXP protocol with an increased incidence of hamstring discomfort and perceived pain (5 out of 10). The EXP protocol resulted in significantly (p<0.01) increased incidence of delayed onset muscle soreness (DOMS). In conclusion, a stop-start laboratory protocol elicited increased heart rate, negatively impacted on muscle activity of the hamstrings, decreased eccentric strength in the lower limb musculature, resulted in increased ratings of „Central‟ and „Local‟ exertion and increased pain perception and increased incidence of DOMS. Thus, a stop-start rugby specific laboratory protocol has a negative impact on performance. Due to the specificity of the protocol being designed to match the demands of competitive match play it is expected that these changes in heart rate, muscle activity and strength, particularly eccentric strength, will impact negativity on performance during rugby match play and increase the likelihood of injury

Rotator cuff injuries cause pain, disability, and loss of shoulder function in over 17 million individuals in the United States that result in over 500,000 surgeries performed annually, though with alarming failure rates of 20-94% (Colvin et al., 2012; Galatz et al., 2004; Harryman et al., 1991; Jain et al., 2014; Mather et al., 2013; Oh et al., 2007; Vitale et al., 2007; Yamaguchi et al., 2006). These surgeries involve repair or reconstruction of the damaged rotator cuff tendon(s) along with enlargement of the subacromial space by debriding the overlying bone (acromion) and removing the subacromial bursa (Beard et al., 2018; Burkhart et al., 2016; Dines et al., 2006; Lo & Burkhart, 2003; Rossi & Ranalletta, 2020). The subacromial bursa is a synovial-like tissue that is situated between the acromion and the tendons of the rotator cuff. This tissue has been long understood to serve a primarily mechanical role by providing cushioning and protecting from friction-wear from the acromion on the underlying tendons. More recently, the identification of a robust vascular network within the bursa, a resident population of mesenchymal stem cells, and inflammatory responsiveness to rotator cuff pathology have supported the existence of a biological role of this tissue in addition to its mechanical one (Blevins et al., 1997; Gotoh et al., 1998, 2001; Põldoja et al., 2017; Rathbun & Macnab, 1970; Steinert et al., 2015; Yepes et al., 2007). These observations make surgical excision of the bursa problematic, given our current lack of understanding of the implications of removing the bursa on the biological response to tendon injury. Therefore, the goals of this dissertation were three-fold: (1) to determine the role of the subacromial bursa in the rotator cuff tendon response to injury and healing, (2) to interrogate patterns of cellular crosstalk between the subacromial bursa and the rotator cuff following injury, and (3) to demonstrate therapeutic potential of targeting the subacromial bursa for modulating inflammation and improving tendon healing.Motivated by clinically observed phenotypic changes in the subacromial bursa with rotator cuff pathology, the profiles of human bursa and rotator cuff tendon tissues were assessed using histology, proteomics, and transcriptomics. This data set, analyzed in the context of patient demographics and diagnoses, revealed distinct bursa proteomes according to tissue phenotype (i.e., fibrous, vascular, or fatty), patient age, and presence of a tear in the underlying rotator cuff. These results suggested the presence of crosstalk between the rotator cuff and the bursa that had not been previously appreciated. Employing multiple methods of validation, including histology, microcomputed tomography, gene expression, and flow cytometry, the rat bursa was established as an appropriate animal model of the human bursa. Therefore, we used the rat model to investigate the role of the bursa in tendon injury response and healing; tendon injuries were created surgically with or without a subsequent repair to study healing and responses to injury, respectively. The role of the bursa in the response to injury was assessed using gene expression, transcriptomics, and histology. The bursa promoted inflammatory gene expression in the injured supraspinatus but resolved inflammatory gene expression in the intact infraspinatus. The role of the bursa in tendon healing was assessed using gene expression, histology, microcomputed tomography, and tensile mechanical testing of the cuff tendons. Consistent with responses during the inflammatory phase of healing, the bursa promoted expression of genes related to aberrant, scar-mediated healing in the supraspinatus, whereas it promoted tenogenic and tendon extracellular matrix gene expression in the intact infraspinatus. Mechanical testing demonstrated that the bursa protected the infraspinatus from the inflammatory environment caused by the supraspinatus injury but had a limited functional effect on the healing supraspinatus. Microcomputed tomography also indicated bursa-dependence in cortical and trabecular bone remodeling following tendon injury. Cross-talk between the bursa and the tendon was then studied in a novel tissue explant co-culture platform using gene expression and nitric oxide release as outcome measures. These experiments revealed that the activated bursa engaged in immunomodulation of tendon fibroblast responses to inflammatory stimulus. The in vitro platform also established the glucocorticoid dexamethasone as a viable therapeutic candidate for bursa-targeted treatment based on its capacity to regulate the bursa’s response to an inflammatory stimulus and enhance the bursa’s immunomodulatory potential. Therefore, in the final component of this thesis, dexamethasone was delivered via PLGA microspheres in vivo to the bursa to modulate the post-injury inflammatory response in the supraspinatus and the infraspinatus tendons. Results supported the therapeutic potential of this treatment approach to improve rotator cuff healing outcomes. This body of work demonstrated a robust involvement of the bursa in rotator cuff responses to injury, with distinct roles in the underlying injured and intact tendons. This work also established, for the first time, the immunomodulatory capacity of the activated bursa and provided strong evidence against the clinical practice of bursectomy. Finally, use of sustained-release dexamethasone to dampen the inflammatory responses to rotator cuff injury offers a new direction for harnessing the inherent properties of the subacromial bursa therapeutically for improved rotator cuff tendon healing.

A direct bone tendon junction consists of four zones: tendon, uncalcified fibrocartilage, calcified fibrocartilage, and bone. The uncalcified and calcified fibrocartilage together forms the transition zone. This organization ensures a gradual transition in stiffness and material properties, and protects the junction from failure. Transition zone regeneration during bone tendon junction healing is important to restore this unique protective mechanism. / Bone tendon junction repair is involved in many orthopaedic reconstructive procedures. Healing is observed to be slow. The junction often heals by fibrous tissue formation. Previous attempts to enhance bone tendon junction healing have resulted in increased bone formation. However, fibrocartilage transition zone is not restored. / This thesis describes a series of studies on transition zone regeneration in bone tendon junction healing using two partial patellectomy animal models. The healing process inside a bone trough was first studied and characterized. Little transition zone regeneration was observed except near the articular cartilage cut surface. The possibility of using articular cartilage to stimulate transition zone regeneration was explored. Both articular cartilage autograft and allogeneic cultured chondrocyte pellet implantations resulted in significantly increased fibrocartilage transition zone regeneration. Cell tracking indicated that the regenerated tissue likely originated from host cells. To elucidate the mechanism of stimulation by allogeneic cultured chondrocyte pellet, the role of cellular and matrix component needed to be differentiated. Freezing and rapid freeze thaw cycles permanently devitalized the allogeneic cultured chondrocyte pellet, but retained its structural integrity and matrix contents. Preliminary results indicated that implantation of the devitalized allogeneic cultured chondrocyte pellet could still increase fibrocartilage transition zone regeneration. Cellular activity seemed not to be essential for the stimulatory effect. / With further research and development, it is envisioned that a cartilage-based stimulation method for fibrocartilage transition zone regeneration in bone tendon junction healing will be developed for clinical application. / Wong Wan Nar, Margaret. / Source: Dissertation Abstracts International, Volume: 70-06, Section: B, page: 3423. / Thesis (M.D.)--Chinese University of Hong Kong, 2008. / Includes bibliographical references (leaves 216-231). / 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, [200-] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstracts in English and Chinese. / School code: 1307.

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.

Surgical repair of torn rotator cuff tendons have a high rate of failure and does not address the underlying pathophysiology. Tissue engineering strategies, employing the use of multipotent progenitor cells or growth factors, represent potential therapies to improve the outcome of rotator cuff surgery. The use of glycosaminoglycan-based biomaterials in these therapies may enhance the effectiveness of cell and growth factor delivery techniques. Furthermore, understanding the cellular and molecular mediators in tendon overuse can help elucidate the causes of tendon degeneration. Thus the overall goals of this dissertation were to 1) develop heparin-based biomaterials to enhance cell pre-culture and maintain growth factor bioactivity and 2) characterize the histological and enzymatic changes in a supraspinatus tendon overuse model. To investigate the use of heparin in enhancing dynamic signaling, mesenchymal stem cells (MSCs) were encapsulated in heparin-containing hydrogels and evaluated for differentiation markers when cocultured with a small population of differentiated cells. To probe the effect of sulfation of heparin on the interactions with protein, selectively desulfated heparin species were synthesized and evaluated for their ability to bind and protect proteins. Finally, to develop a tendon overuse model that can become a test bed for testing future targeted therapeutics, an animal model was evaluated for tissue damage and protease activity. Together these studies represent a multi-pronged approach to understanding how tendon tissues become degenerative and for developing technologies to improve the biological fixation of tendon to bone in order to reduce the need for revision surgeries.

Indiana University-Purdue University Indianapolis (IUPUI) / Tendinopathy is a common degenerative pathology that is characterized by activity related pain, focal tendon tenderness, intratendinous imaging changes, and typically results in changes in the histological, mechanical, and molecular properties of the tendon. Tendinopathy is difficult to study in humans, which has contributed to limited knowledge of the pathology, and thus a lack of appropriate treatment options. However, most believe that the pathology is degenerative as a result of a combination of both extrinsic and intrinsic factors. In order to gain understanding of this pathology, animal models are required. Because each tendon is naturally exposed to different conditions, a universal model is not feasible; therefore, an appropriate animal model must be established for each tendon susceptible to degenerative changes. While acceptable models have been developed for several tendons, a reliable model for the Achilles tendon remains elusive. The purpose of this dissertation was to develop an animal model of Achilles tendinopathy by investigating the individual and combined effects of an intrinsic and extrinsic factor on the rodent Achilles tendon. Rats selectively bred for high capacity running and Sprague Dawley rats underwent uphill treadmill running (an extrinsic factor) to mechanically overload the Achilles tendon or served as cage controls. Collagenase (intrinsic factor) was injected into one Achilles tendon in each animal to intrinsically break down the tendon. There were no interactions between uphill running and collagenase injection, indicating that the influence of the two factors was independent. Uphill treadmill running alone failed to produce any pathological changes in the histological or mechanical characteristics of the Achilles tendon, but did modify molecular activity. Intratendinous collagenase injection had negative effects on the histological, mechanical, and molecular properties of the tendon. The results of this dissertation demonstrated that the combined introduction of uphill treadmill running and collagenase injection did not lead to degenerative changes consistent with human Achilles tendinopathy. Intratendiouns collagenase injection negatively influenced the tendon; however, these changes were generally transient and not influenced by mechanical overload. Future studies should consider combinations of other intrinsic and extrinsic factors in an effort to develop an animal model that replicates human Achilles tendinopathy.