The purpose of this study was to determine the effects of varying periods of immobilization on ligamentous healing and strength in a rat experimental model. Sixty-one mature male Wistar rats were used. The left knee medial collateral ligament was surgically exposed, divided, and repaired. The rats were randomly placed into one of four groups: Group A, no immobilization, Group B, 2 weeks' immobilization, Group C, 6 weeks' immobilization, and Group D, 10 weeks' immobilization of the operated limb. The right knee served as a control. The ligaments were studied histologically and biomechanically at 2 weeks, 6 weeks, 10 weeks and 20 weeks post-operatively. Histologic samples were objectively evaluated with the light microscope using a Maturity Index Score and Scale that were devised based on the numbers and orientation of the fibroblasts and the amount and orientation of the collagen fibres. Ligament-bone preparations were studied using an Instron material testing machine to determine the biomechanical properties of the ligament until failure. Utilizing the Maturity Index Score and Scale, it was shown that Group A, with no immobilization, matured more rapidly than the other groups, and achieved full maturity at 20 weeks post-operatively. The other groups all showed a retarded rate of healing while immobilized. The electron microscopic study supported this data by demonstrating the level of metabolic activity of the fibroblasts which decreased with increasing maturity and by demonstrating that the size, amount and orientation of the collagen fibers increased with mobilization. The biomechanical testing showed that at 2 weeks post-operative, Group A had achieved a strength which was 46% of controls while Group B was only 29% of controls (p = 0.055). At 6 weeks Group A was 65% of controls, Group B was 56% of controls and Group C was 39% of controls (p = 0.0004). At 20 weeks Group A was 83% of controls, Group B was 71% of controls, Group C was 66% of controls and Group D was 48% of controls (p = 0.0005). Group A was 71% stronger than Group D at this time, indicating that the healing medial collateral ligament attained a greater strength and histologically matured more rapidly if mobilization is begun immediately. / Science, Faculty of / Botany, Department of / Zoology, Department of / Graduate

Doctor of Philosophy / The aim of the work presented in this thesis was to examine the associations between the kinematics of the knee characterised by the tibiofemoral contact pattern, and degenerative change, in the context of anterior cruciate ligament (ACL) injury. While the natural history of degenerative change following knee injury is well understood, the role of kinematics in these changes is unclear. Kinematics of the knee has been described in a variety of ways, most commonly by describing motion according to the six degrees of freedom of the knee. The advantage of mapping the tibiofemoral contact pattern is that it describes events at the articular surface, important to degenerative change. It was hypothesised that the tibiofemoral contact pattern would be affected by injury to the knee. A model of ACL injury was chosen because the kinematics of the knee have been shown to be affected by ACL injury, and because the majority of chronic ACL-deficient knees develop osteoarthritis, the associations between kinematics and degenerative change could be explored. A technique of tibiofemoral contact pattern mapping was established using MRI, as a quantifiable measure of knee kinematics. The tibiofemoral contact pattern was recorded from 0º to 90º knee flexion while subjects performed a leg-press against a 150N load, using sagittal magnetic resonance imaging (MRI) scans. The technique was tested and found to be reliable, allowing a description of the tibiofemoral contact pattern in 12 healthy subjects. The tibiofemoral contact patterns of knee pathology were then examined in a series of studies of subjects at a variety of stages of chronicity of ligament injury and osteoarthritis. Twenty subjects with recent ACL injury, 23 subjects with chronic ACL deficiency of at least 10 years standing, and 14 subjects with established osteoarthritis of the knee were recruited. The 20 subjects with recent ACL injury were examined again at 12 weeks and 2 years following knee reconstruction. The tibiofemoral contact patterns were examined for each group of subjects and the associations between changes in the contact patterns and evidence of joint damage explored. Evidence of joint damage and severity of osteoarthritis were recorded from xrays, diagnostic MRI, operation reports and bone densitometry at the tibial and femoral condyles of the knee. Each of the three groups with knee pathology exhibited different characteristics in the tibiofemoral contact pattern, and these differences were associated with severity of joint damage and osteoarthritis. The recently ACL-injured knees demonstrated a tibiofemoral contact pattern that was posterior on the tibial plateau, particularly in the lateral compartment. Those with chronic ACL deficiency demonstrated differences in the contact pattern in the medial compartment, associated with severity of damage to the knee joint. Osteoarthritic knees showed reduced femoral roll back and longitudinal rotation that normally occur during knee flexion. Two years following knee reconstruction there was no difference between the contact pattern of the reconstructed and healthy contralateral knees. This technique of tibiofemoral contact pattern mapping is sensitive to the abnormal characteristics of kinematics in ligament injury and osteoarthritis. This is the first time the tibiofemoral contact characteristics of chronic ACL-deficient and osteoarthritis knees have been described and links examined between tibiofemoral contact patterns and degenerative change.

Doctor of Philosophy / The aim of the work presented in this thesis was to examine the associations between the kinematics of the knee characterised by the tibiofemoral contact pattern, and degenerative change, in the context of anterior cruciate ligament (ACL) injury. While the natural history of degenerative change following knee injury is well understood, the role of kinematics in these changes is unclear. Kinematics of the knee has been described in a variety of ways, most commonly by describing motion according to the six degrees of freedom of the knee. The advantage of mapping the tibiofemoral contact pattern is that it describes events at the articular surface, important to degenerative change. It was hypothesised that the tibiofemoral contact pattern would be affected by injury to the knee. A model of ACL injury was chosen because the kinematics of the knee have been shown to be affected by ACL injury, and because the majority of chronic ACL-deficient knees develop osteoarthritis, the associations between kinematics and degenerative change could be explored. A technique of tibiofemoral contact pattern mapping was established using MRI, as a quantifiable measure of knee kinematics. The tibiofemoral contact pattern was recorded from 0º to 90º knee flexion while subjects performed a leg-press against a 150N load, using sagittal magnetic resonance imaging (MRI) scans. The technique was tested and found to be reliable, allowing a description of the tibiofemoral contact pattern in 12 healthy subjects. The tibiofemoral contact patterns of knee pathology were then examined in a series of studies of subjects at a variety of stages of chronicity of ligament injury and osteoarthritis. Twenty subjects with recent ACL injury, 23 subjects with chronic ACL deficiency of at least 10 years standing, and 14 subjects with established osteoarthritis of the knee were recruited. The 20 subjects with recent ACL injury were examined again at 12 weeks and 2 years following knee reconstruction. The tibiofemoral contact patterns were examined for each group of subjects and the associations between changes in the contact patterns and evidence of joint damage explored. Evidence of joint damage and severity of osteoarthritis were recorded from xrays, diagnostic MRI, operation reports and bone densitometry at the tibial and femoral condyles of the knee. Each of the three groups with knee pathology exhibited different characteristics in the tibiofemoral contact pattern, and these differences were associated with severity of joint damage and osteoarthritis. The recently ACL-injured knees demonstrated a tibiofemoral contact pattern that was posterior on the tibial plateau, particularly in the lateral compartment. Those with chronic ACL deficiency demonstrated differences in the contact pattern in the medial compartment, associated with severity of damage to the knee joint. Osteoarthritic knees showed reduced femoral roll back and longitudinal rotation that normally occur during knee flexion. Two years following knee reconstruction there was no difference between the contact pattern of the reconstructed and healthy contralateral knees. This technique of tibiofemoral contact pattern mapping is sensitive to the abnormal characteristics of kinematics in ligament injury and osteoarthritis. This is the first time the tibiofemoral contact characteristics of chronic ACL-deficient and osteoarthritis knees have been described and links examined between tibiofemoral contact patterns and degenerative change.

Ligament tears are more prevalent than all other knee injury pathologies, and contribute significantly to musculoskeletal joint pain and disability reported worldwide. Despite current soft tissue reconstruction techniques, the injured ligament fails to regenerate due to dysregulated cell-extracellular matrix (ECM) interactions that culminate in scar formation. Hallmarks of scar formation, or fibrotic healing include disorganized ECM, pathological stiffness or tissue rigidity, and the accumulation and persistence of myofibroblasts. A primary driver of fibrosis, myofibroblasts are characterized by high contractility, excessive deposition of collagen type I, coupled with inflammatory and fibrotic signaling. Notably these cells are critical early on in the response to injury, by aiding in the contracture of the wound bed and depositing collagen to repair the injury site. However, myofibroblasts are not capable of fully regenerating the native ligamentous matrix, and resolution of the phenotype is necessary in order to cue surrounding cells, prevent chronic inflammation and aberrant tissue remodeling. Persistence of the myofibroblast phenotype thus leads to a ligament scar that is functionally weaker than the healthy tissue matrix, characterized by significantly different histological, biochemical, and biomechanical properties. The consequential instability of this scar disrupts load distribution within the knee joint and increases the risk of subsequent injury, osteochondral degeneration, and ultimately, the development of post-traumatic osteoarthritis. Therefore, there is a critical need for strategies that target the inflammatory and fibrotic myofibroblast phenotypes for soft tissue healing. It follows that the overarching goal of this thesis is to engineer an immunomodulatory matrix to regulate myofibroblast activation and downstream fibrogenic signaling. To this end, models of soft tissue fibrotic repair are explored in order to test the central hypothesis that cues from the repairing ECM play an important role in regulating myofibroblast activation and persistence. Specifically, this thesis will compare myofibroblast differentiation and signaling in three in vitro models of tissue repair: 1) 2D on tissue culture polystyrene (TCPS), and two 3D models namely 2) collagen hydrogel and 3) electrospun collagen fiber matrices. As expected, on the 2D model, a persistent myofibroblast phenotype could be generated over time with an optimized transforming growth factor beta 1 (TGF-β1) stimulation protocol. To create repair-relevant 3D matrix models, we engineered collagen hydrogels with controlled mechanical properties, as well as electrospun fiber platforms that isolate key matrix factors including, collagen content, stiffness, fiber diameter, and alignment. These models emulate the connective tissue repair process via recapitulating the increasing matrix stiffness and fiber assembly of the early (granulation tissue), proliferative, and remodeling stages of the repair. Myofibroblast differentiation potential, parallel inflammatory and fibrotic cytokine secretion, as well as matrix remodeling potential were observed to be dependent on matrix model parameters. Moreover, single-cell resolution RNA sequencing revealed heterogenous myofibroblast populations within the context of response to engineered collagenous substrates. Specifically, myofibroblast accumulation was observed on hydrogel substrates that recapitulate the pathologically stiff mechanics and disorganization of fibrotic scar tissue while architectural cues of engineered fiber substrates prevented myofibroblast differentiation in a diameter and alignment-dependent manner. Moreover, nanoscale fibers elicited the greatest anti-fibrotic and anti-inflammatory properties compared to microscale fibers and stiff collagen-based hydrogels. Throughout, this thesis also explores the contribution of NF-κB signaling to myofibroblast plasticity and persistence using engineered collagen-based platforms, highlighting the dynamic role of myofibroblasts as critical immunoregulating cells. The NF-κB signaling pathway is implicated in a broad array of fibrotic and chronic inflammatory conditions, and more recently has been associated with survival of persistent myofibroblast populations in soft-tissue fibrosis and tendon degeneration models. In this thesis, NF-κB activation was seen to be related to the persistent myofibroblast phenotype and increase over time in both 2D TCPS and 3D collagenous hydrogel matrices that mimic pathologically stiff scar tissue, while a temporally dependent activation pattern was observed in electrospun collagen fiber-based models. At the transcriptional level, NF-κB survival signaling was significantly enriched in myofibroblast populations supported by TCPS and stiff collagen-based hydrogels but downregulated on soft hydrogels and fibers with decreasing fiber diameter that prevented robust myofibroblast differentiation at single cell resolution. Building upon these new insights regarding matrix cues that drive myofibroblast activation, we designed an immunomodulatory matrix that mediates small molecule release targeting NF-κB inhibition. The immunomodulatory matrix achieved robust amelioration of the myofibroblast phenotype as well as reduced the secretion of key inflammatory and fibrotic cytokines by these cells. Moreover, a similar anti-fibrotic response was seen for human ligament fibroblasts treated with these matrices. Collectively, this thesis work presents a systematic evaluation of myofibroblast plasticity and persistence within the context of 2D (TCPS), 3D (collagen-based hydrogels), and finally 3D with defined microarchitectural cues (electrospun collagen-based fibers) that recapitulate the progressive stages of scar-mediated healing, and reveals NF-κB as a promising target for reducing myofibroblast persistence. Moreover, the immunomodulatory control of myofibroblast plasticity and persistence via matrix cues coupled with NF-κB inhibition informs future strategies for true ligament healing.

Lam, Mak Ham. / Thesis (Ph.D.)--Chinese University of Hong Kong, 2011. / Includes bibliographical references (leaves 110-131). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Abstract also in Chinese.