Understanding the origins of the multiaxial material properties of soft tissues is crucial for quantifying the tissue material properties that govern the material behavior for acurate predictive modeling of the biological systems. Unlike many engineering structural materials, biological materials exhibit complex material behavior due to structural anisotropy and various interactions between each of the microstructrual units of the tissue. Therefore, this study aims to quantify the shear material behavior of rabbit patellar tendon under simple shear loading along the fiber direction and perpendicular to the fiber direction and to understand the role of tissue shearing and its contribution to the overall mechanical behavior of the tendon. It was hypothesized that tendon demonstrates anisotropic material response under simple shear. Results suggests that tendon exhibits direction-dependent viscoelastic shear properties, reflecting structural anisotropy. The data obtained from our present study could be used for development of constituent models using internal state variable theory.

The ultrastructural mechanism for strain rate sensitivity of collagenous tissue has not been well studied at the collagen fibril level. The objective is to reveal the mechanistic contribution of the collagen fibril to strain rate sensitivity. Collagen fibrils underwent significantly greater fibril strain relative to global tissue strain at higher strain rates. A better understanding of tendon mechanisms at lower hierarchical levels would help establish a basis for future development of constitutive models and assist in tissue replacement design. High rate mechanical property of tendon was also studied. Tendon was compressed under high strain rate (550 /s) using a polycarbonate split Hopkinson pressure bar (PSHPB). The objectives are to investigate the tissue behavior of porcine tendon at high rates. Tendon’s high rate behavior was compared with brain and liver at both hydrated and dehydrated states to investigate how water content and ultrastructural affect high rate responses of soft tissues.

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<p> Although most muscle spindle investigations have used the cat model and mvasiVe surgical measurement techniques, several investigators have used microneurography to record from the Ia and II fibres in humans during tendon vibration. In these studies the muscle spindle primary (Ia) endings are stimulated using transverse vibration of the tendon at reflex sub-threshold amplitudes. Others have used low amplitude vibration and the H-reflex (monosynaptic electrical response) to determine reflex properties during both agonist and antagonist voluntary contractions. Both of these methods explore only certain parts of the monosynaptic reflex arc; microneurography focus on the properties and firing characteristics of the muscle spindles themselves, whereas the H-reflex response to vibration is a representation of the response of the spinal cord as well as the muscle spindles. </p> <p> In the past we have developed a PC based instrument that uses Lab VIEW and a linear servomotor to study tendon reflex properties by recording H-reflexes (or stretch reflexes for mechanical stimuli) from single tendon taps or electrical stimuli to the afferent nerve. In this thesis we describe a further development of this system to provide precise vibrations of the tendon at up to 55 Hz with amplitudes up to 4 mm. The resultant vibration stretch reflex train is extracted from 2 major background noise sources, 60 Hz power line noise, and vibration artifact noise, of the EMG recording via phase coherent subtractive filtering. </p> <p> To demonstrate the versatility and efficacy of this system in studying the monosynaptic reflex arc, test results from several pilot studies are presented, using the system to vibrate the human distal flexor carpi radialis tendon: (i) whether stretch reflexes could be entrained with high frequency vibration, as contrary to H-reflexes, (ii) whether the responses were affected by low levels of agonist or antagonist contraction, in agreement with the existing pool of work on the subject using the H-reflex, (iii) whether a separation of the Ia (primary) and II (secondary) ending pathways is observable as individual but delayed responses at low vibration frequencies due to different activation characteristics, and axon diameters, of each ending. Possible physiological mechanisms that explain the resultant behaviour are also discussed. </p> / Thesis / Master of Applied Science (MASc)

Anterior cruciate ligament (ACL) ruptures and tears are significant orthopedic problems that result in discomfort and limited mobility. Fully functional tissue engineered ligament replacements are promising alternatives to current graft choices for repair of ACL disruptions. The cell-based approach to construct engineered ligament grafts presented herein involves the culture of mesenchymal stem cells (MSC) on biodegradable, fibrous polymeric scaffolds to promote tissue formation. Multipotent MSCs are advantageous because of their in vitro proliferative capacity and ease of harvest; however; the promotion of MSC differentiation into mature fibroblasts and subsequent extracellular matrix (ECM) development is unknown. The proposed studies utilized three complementary methods to promote differentiation of MSCs: scaffold architecture, mechanical stretch and over-expression of the transcription factor, scleraxis. First, elastomeric scaffolds were fabricated by electrospinning a segmented poly(esterurethane urea) with variations in fiber diameter and fiber alignment. Primary mesenchymal stem cells and the mesenchymal stem cell line, C3H10T1/2, were seeded on these scaffolds and assumed spindle-shaped morphologies and oriented with the direction of fiber alignment. Fiber diameter affected cellular responses, including the expression of ECM genes (e.g. collagen type 1 and decorin) which were elevated on smaller mean fiber diameter scaffolds initially. However, scleraxis gene expression was greatest on larger mean fiber diameter scaffolds at the end of two weeks. Second, cyclic stretch was applied to C3H10T1/2 cells on semi-aligned scaffolds using a novel bioreactor. Cell attachment was verified during and after the application of mechanical stress by confocal microscopy. Cyclic stretch induced cells to assume a highly elongated morphology; however ECM gene expression changes were moderate. Third, forced constitutive expression of scleraxis was accomplished by nucleofection of C3H10T1/2 cells. Transient mRNA expression, accumulation of the gene product in the cell nucleus, and cell death were observed. Future work will seek to refine the experimental methods, including the development and testing of an inducible scleraxis transgene and the application of longer periods of mechanical stimulation. Finally, these complementary approaches may be combined to further extend this work in pursuit of directed differentiation of stem cells and the ensuing generation of a robust tissue graft. / Ph. D.

The adipose-derived stromal vascular fraction (SVF) is a promising new therapy for equine flexor tendonitis. This heterogeneous population of cells may improve tendon healing via the production of growth and chemotactic factors capable of recruiting endogenous stem cells and increasing extracellular matrix production by tendon fibroblasts (TFBL). The purpose of this study was to evaluate the ability of adipose-derived cells (ADC) culture expanded from the SVF to act as trophic mediators in vitro. We hypothesized that ADCs would produce growth and chemotactic factors important in tendon healing and capable of inducing cell migration and matrix protein gene expression. Superficial digital flexor tendons and adipose tissue were harvested from eight adult horses and processed to obtain SVF cells, ADCs and TFBLs. Adipose-derived cells and TFBLs were grown in monolayer culture for growth factor quantification, to produce conditioned media for microchemotaxis, and in co-culture for quantification of matrix protein gene expression by TFBLs. Growth factor gene expression by SVF cells was significantly greater than in ADCs or TFBLs. Co-culture of TFBLs and ADCs resulted in modest up-regulation of matrix protein expression (collagen types I and III, decorin, and cartilage oligomeric matrix protein) by TFBLs. Media conditioned by ADCs induced ADC migration in a dose dependent manner. These findings support the role of both SVF and ADCs as trophic mediators in tendon regeneration. The differences detected in gene expression between SVF cells and ADCs indicate that additional studies are needed to evaluate the changes that occur during culture of these cells. / Master of Science

An attempt to reproduce tendon sheaths using autologous venous grafts has been undertaken in Cercopithecus aethiops (Blue Vervet Monkey). Ten venous grafts were tested. Five were placed around sutured tendons in paratenon, and the remainder around tendons in sheath formation. In no instance did synovial-like sheaths form. In the latter series, the experiment was controlled, and the results obtained expressed in terms of function. The functional results of the venous ensheathed tendons were worse than those of the controlled tendons.The controlled experiment was confined to the digital sheath, an area notoriously liable to adhesion formation, and the one which offered the most critical test of operative technique.A method for the evaluation of function, following the repair of divided tendons in the experimental animal has been presented. Voluntary movement of joints in the experimental animal was obviously impossible, but the method employed in this experiment, namely, the electrical stimulation of muscles, and the photographic recording of the range of joint movement, presented no disadvantages.The experimental animal used in this study was ideal, in that anatomical studies of the hand of this species, revealed features both structurally and functionally comparable to those in man.The anatomical and physiological aspects of tendon action, the mechanics of tendon gliding, and the healing processes in divided tendons have been studied.The fate of human autologous venous grafts has been investigated.A review of previous methods employed to prevent peritendinous adhesions has been presented.

Tendon and ligament injuries are common orthopedic problems that have an enormous impact on the quality of life of affected patients. Despite the frequency at which these injuries occur, current treatments are unable to restore native function to the damaged tissue. Because of this, reinjury is common. It is well known that mechanical stimulation is beneficial for promoting tendon and ligament development and tissue homeostasis; however, the specific mechanisms remain unclear. The transcription factor scleraxis (Scx) is an interesting candidate for mediating the tendon and ligament mechanoresponse, as it has been shown that Scx expression is induced by cyclic mechanical strain in tenocytes and is required for mechanically-induced stem cell tenogenesis. Moreover, Scx expression is increased in adult tendons following exercise. The studies described in this dissertation therefore focus on the combined role of Scx and mechanical stimulation in two contexts: 1) influencing ligament cell differentiation and 2) regulating adult tenocyte behavior. In the first study, transient Scx overexpression combined with mechanical strain in a 3D collagen hydrogel model was investigated as a means of deriving mature ligament cells from stem cells for use in ligament tissue engineering. Scx overexpression in C3H10T1/2 cells cultured in collagen hydrogels under static strain resulted in increased construct contraction and cell elongation, but no concurrent increase in the expression of ligament-related genes or production of glycosaminoglycans (GAG). When combined with low levels of cyclic strain, Scx overexpression resulted in increased mechanical properties of the tissue constructs, increased GAG production, and increased expression of ligament-related genes compared to cyclic strain alone. Together, these results demonstrate that Scx overexpression combined with cyclic strain can induce ligament cell differentiation and suggest that Scx does so by improving the mechanosensitivity of cells to cyclic strain. In the second study, the role of Scx in adult tenocyte mechanotransduction was explored using RNA-sequencing (RNA-seq) and small interfering RNA (siRNA) technologies. Equine tenocytes were exposed to siRNA targeting Scx or a control siRNA and maintained under cyclic mechanical strain prior to being submitted for RNA-seq. Comparison of the resulting transcriptomes revealed that Scx knockdown decreased the expression of several genes encoding important focal adhesion adaptor proteins. Correspondingly, Scx-depleted tenocytes showed abnormally long focal adhesions, decreased cytoskeletal stiffness, and an impaired ability to migrate on soft surfaces. This suggests that Scx regulates the tenocyte mechanoresponse by promoting the expression of focal adhesion-related genes. Combined, the results of these studies support a role for Scx in tendon and ligament cell mechanotransduction and identify the regulation of genes related to maintaining the cell-extracellular matrix connection and cytoskeletal dynamics as a potential mechanism. These findings enhance our understanding of how mechanical stimulation influences cell behavior and provide new research directions and methodologies for future studies of tendon and ligament mechanobiology. / Ph. D. / Tendon and ligament injuries are very common, but current treatments are unable to completely repair the damaged tissue. We know that exercise plays an important role during the development of tendons and ligaments and in keeping them healthy during adulthood. Despite this, we do not understand exactly how either of these processes occur. A tendon and ligament related protein called scleraxis (Scx) appears to be important in translating mechanical strain into a cellular response. As such, Scx could be useful for making ligament cells from stem cells, which could then be used in custom-engineered, patient-specific tissue grafts for surgical repair of torn ligaments. Studying what Scx does in adult tendon cells could also help us to understand how tendon cells sense and respond to exercise or physical changes in their environment. To explore whether or not Scx can promote stem cell differentiation, we generated stem cells with increased levels of Scx and put them into 3D collagen constructs. When the constructs were held under static tension, cells with increased Scx became longer and were better able to organize the collagen compared to normal cells. When we exercised the constructs, cells with increased Scx also had higher levels of ligament-related genes and resulted in stiffer constructs compared to normal cells. Increasing Scx expression in combination with mechanical strain could therefore be a useful way to make ligament cells that can be used in engineered replacement tissues. To explore the function of Scx in adult tendon cells, we created tendon cells with decreased levels of Scx and exposed them to mechanical strain. We then generated a database of all the genes affected by the decrease in Scx. Analysis of this database revealed that decreasing the levels of Scx also decreased the expression of genes associated with several pathways involved in linking the internal skeleton of the cell to the extracellular environment. This suggested that Scx helps facilitate the ability of a tendon cell to sense and respond to its surroundings. To evaluate this, we examined the ability of tendon cells with decreased Scx to migrate on different surfaces, the stiffness of the internal skeleton, and the structure of the protein complexes responsible for anchoring cells to a surface. As predicted by our gene database, we found that decreasing Scx levels also decreases the stiffness of the cell’s internal skeleton, changes the shape of the anchoring complexes, and impairs the ability of tendon cells to migrate on soft surfaces. These results show that Scx governs tendon cell function by affecting the cell’s ability to interact with its local environment.