Browsing by Author "Krawetz, Roman J."
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Item Open Access Cardiac extracellular matrix gel for ESC-derived cardiomyocyte maturation(2020-08-31) Thompson, Madalynn Jade; Rancourt, Derrick E.; Greenway, Steven C.; Krawetz, Roman J.; Ungrin, Mark D.The need for precision medicine has been established and the use of organoids to test individual drug efficacy and develop individual disease models is creating an opportunity for innovation. Human embryonic stem cells (hESCs) are commonly used for these applications but differentiation often results in immature phenotypes that may not be physiologically relevant. While hESCs can be differentiated into any cell type using small molecules, emerging concepts suggest that additional exogenous factors influence differentiation. In situ, stem cells exist in an organ-specific niche which contains extracellular matrix (ECM) proteins that have important paracrine effects to promote the maturity. This project aimed to investigate whether either of two alternative preparations of organ-specific ECM matrices support would offer improved support for tissue-specific differentiation and maturation. ECM matrices from porcine hearts (i.e. cardiogel) were prepared and decellularized either completely or partially. These cardiogel preparations were then studied using mass spectrometry and cytochemical staining before being used to support the maturation of hESCs that had been differentiated into cardiomyocytes (CMs). Moderate decellularization of porcine heart was achieved as shown by histochemical staining and the cardiogels contained variable amounts of important ECM components. However, neither cardiogel preparations appeared to increase the maturity of hESC-derived CMs, as assessed by cellular gene expression after 4 days of culture on cardiogel following differentiation. In conclusion, a protocol to generate cardiogels from porcine heart was achieved but significant batch variability was found. Further optimization may lead to a substrate which promotes cellular maturation and which can be used in conjunction with other technologies to increase cellular yield and homogeneity of resulting cardiac lineages.Item Open Access The Characterization of Proteoglycan 4 and Hyaluronan Composition in the Vitreous Humour and Their Potential Contributions to Steady Shear Viscosity(2020-01-27) Alarifi, Abdulaziz A.; Schmidt, Tannin A.; Hart, David A.; Krawetz, Roman J.; Stell, William K.; Moritz, Orson L.Background and aim: The vitreous humour is a connective tissue in the eye that contributes to several physiological and pathological processes. The full details of the complex vitreous humour composition and the extent of its functionality remain elusive. Proteoglycan 4 (PRG4), a mucin-like O-linked glycosylated protein, has recently been identified in human vitreous humour. PRG4 is known to act synergistically with hyaluronan (HA) in synovial fluids, where it functions as a boundary lubricant, contributes to viscosity, and more. Studying PRG4 in this newly discovered site might advance knowledge about its rheological properties (specifically within the eye) and whether or not PRG4 is involved. The aim of this thesis was to analyze the presence, source and potential function of PRG4 in vitreous humour. The objectives were: 1) to quantify the PRG4 and HA (including its molecular weight (MW) distribution) content, 2) to immunolocalize PRG4 in the eye globe, and examine its local synthesis, and 3) to characterize the viscosity of vitreous humour samples with varying PRG4 and HA concentrations. Methods: Human vitreous humour samples were collected from post-mortem human patients. Alpha amplified luminescent proximity homogeneous assay (AlphaLISA) and enzyme-linked immunosorbent assay (ELISA) tests were performed to measure the concentration of PRG4 and HA, respectively. The MW distribution of HA was determined by agarose gel electrophoresis. Vitreous humour cells were cultured from porcine to examine the synthesis of PRG4. PRG4 was immunolocalized in eye globes from wild type (WT) and PRG4-KO mice. Steady shear viscosity was characterized for human vitreous humour using a rheometer, before and after adding exogenous recombinant human PRG4 (rh PRG4). Results: The mean (± standard deviation) concentration of PRG4 in human vitreous humour (N=36) was 25 ± 32 µg/mL. For HA, the mean concentration in human vitreous humour was 344±317 µg/mL, and 71% of the overall HA in vitreous humour was below 459 kDa. Increasing age was positively correlated with concentration of PRG4. PRG4 was synthesized by cultured porcine vitreous humour cells. Using wild type and PRG4-KO (knockout) mice, PRG4 was immunolocalized in the ciliary body, cornea, sclera, vitreous cortex, and some parts of the retina. Viscosities for human vitreous humour samples showed minor variations, and were negatively correlated with HA MW. Conclusion: This is the first investigation of both the presence and potential function of PRG4 in vitreous humour. The findings serve to expand understanding of vitreous humour composition, and how that might be a factor in ocular pathology and potential therapies.Item Open Access The Contribution of Endogenous and Exogenous Stem Cells in Fracture Repair(2019-12-17) Ferrie, Leah Elizabeth; Duncan, Neil A.; Krawetz, Roman J.; Matyas, John Robert; Rancourt, Derrick E.Throughout the lifespan, bone remodels in response to damage, such as fracture. However, diseases such as osteoporosis can cause impaired bone healing, increasing the risk of progression to non-repairing defects called fracture non-unions. Promoting the healing of fracture non-unions is a promising target for bone tissue engineering due to the limited success of current clinical treatment methods. There has been significant research on the use of stem cells with and without biomaterial scaffolds to treat bone fractures due to their promising regenerative capabilities. However, the relative roles of transplanted stem cells (exogenous stem cells) and stem cells found naturally in the body (endogenous stem cells) and their overall contribution to in vivo fracture repair is not well understood, thereby delaying the translation of new tissue engineering therapies to the clinic. The purpose of this research was to determine the interaction between exogenous and endogenous stem cells and biomaterials during bone fracture healing. This study was conducted using a burr-hole fracture model in a mesenchymal stem cell lineaging-tracing mouse. Burr-hole fractures were treated with collagen-I biomaterial loaded with and without green fluorescent protein tagged induced pluripotent stem cells. Using lineage-tracing, the roles of exogenous and endogenous stem cells during bone fracture repair could be elucidated. It was determined that in both a normal and impaired model of fracture healing treatment with exogenous stem cells did not result in improved bone formation and did not promote the recruitment of endogenous stem cells. However, treatment with exogenous stem cells in an impaired model of healing may offer healing advantages compared to in a normal model of healing. The outcomes of this study provide fundamental knowledge required for developing more effective stem cell and biomaterial therapies to treat bone fractures.Item Open Access Endogenous Articular Cartilage Regeneration After Injury(2019-01-21) Jablonski, Christina Lynn; Krawetz, Roman J.; Cobb, John A.; McCafferty, Donna Marie; Salo, Paul T.; Manske, Sarah LynnOnce injured, articular cartilage cannot regenerate, and a consequence of this inadequacy is osteoarthritis (OA), a chronic, degenerative joint disorder. Mesenchymal stem cells (MSCs) have shown promise in the treatment of cartilage injuries as they possess immunomodulatory properties and can differentiate into chondrocytes (e.g. cartilage cells). However, the outcomes of MSC-based therapies to date have been highly-variable, illustrating our incomplete understanding of how MSCs function in the joint. Superior cartilage healing post-injury has been observed in the MRL ‘super-healer’ mouse and has been linked to a deficiency in the cell cycle regulator, p21. Therefore, the purpose of this thesis was to determine the cell type(s) and/or mechanism(s) involved in endogenous cartilage regeneration in p21-/- mice. To accomplish this, we lineage traced Prx1+ (transcription factor specific to mesenchymal cells) MSC/progenitors in vivo after cartilage injury in p21-/- and C57 wild-type mice. We further examined the inflammatory cytokine profile of mice in the presence/absence of p21 and functionally tested the role of the CCL2/CCR2 signalling axis, which was found to be altered in p21-/- mice in response to cartilage injury. While deletion of p21 resulted in endogenous articular cartilage regeneration, Prx1+ MSC/progenitors did not differentiate into the new cartilage and/or subchondral bone observed in p21-/- mice. Furthermore, few differences in cell number (e.g. MSCs/stem cells, macrophages, proliferating cells) were observed between p21-/- mice and C57 wild-type mice suggesting that secreted factors or additional cell types may be responsible for the cartilage regeneration observed. To support this hypothesis, we further demonstrated that the cartilage regeneration ability of p21-/- mice could be recapitulated through deletion of the chemokine receptor CCR2, yet if its ligand CCL2 was deleted, cartilage regeneration ability was lost. Future studies using additional lineage reporter mice in the context of p21 deletion will be necessary to determine the cell(s)/factor(s) responsible for cartilage regeneration in these mice and to determine if targeting the cell cycle has potential as a safe and effective therapeutic strategy for treating cartilage injuries and/or OA.Item Open Access Exercise and dietary interventions in a rat model of metabolic knee osteoarthritis(2019-09-05) Rios, Jaqueline Lourdes; Herzog, Walter; Hart, David D.; Reimer, Raylene A.; Krawetz, Roman J.; Beier, FrankOsteoarthritis is a debilitating chronic disease which has no cure or effective treatment. If no changes are made in prevention and treatment, osteoarthritis will continue to represent a significant economic burden to patients and society. The goal of this thesis was to determine the effects of moderate aerobic exercise and prebiotic fibre supplementation on the onset and progression of the metabolic knee osteoarthritis phenotype in rats exposed to a high-fat/high-sucrose diet. This study was divided into three phases. Phase 1: we evaluated the effects of a 12-week aerobic exercise program of varying duration on healthy rat knee cartilage. We determined that moderate, high and extra-high duration treadmill exercise has no detrimental effects on knee joint health, function and integrity. Therefore, we concluded that treadmill exercise at any tested duration was a safe exercise for rats in terms of knee osteoarthritis-like damage, and therefore, could be used as a safe exercise intervention in a pre-clinical rat model of knee osteoarthritis. Phase 2: we evaluated the effects of moderate aerobic exercise, prebiotic fibre supplementation, and the combination of exercise and fibre over a 12-week intervention in rats exposed to a high-fat/high-sucrose diet that has been shown to cause knee joint damage. Our findings indicated that prebiotic fibre and aerobic exercise prevented knee joint damage in this model. Phase 3: we evaluated if the damaging effects of the high-fat/high-sucrose diet could be delayed/reversed. We determined that neither prebiotic fibre supplementation nor aerobic exercise were able to stop the progression of existing knee osteoarthritis-like damage induced by a high-fat/high-sucrose diet. In summary, this thesis provides insight into two different approaches to prevent the development of metabolic osteoarthritis phenotype. However, the studies presented in this thesis were not able to show an effective way to stop the progression of the disease.Item Open Access Inflammatory Profiling in Early Osteoarthritis(2018-09-20) Ren, Guomin; Krawetz, Roman J.; Emery, Carolyn A.; De Koning, A. P. JasonOsteoarthritis (OA) is one of the most common chronic diseases worldwide which can lead to disability. There is a desperate need for the efficient and reliable detection of OA at the early stage when patients are likely to benefit most from disease interventions. It has been shown in previous studies that inflammation plays important roles in cartilage degeneration, synovitis, remodeling of the subchondral bone and pain. The purpose of this thesis was to determine if a panel of inflammatory cytokines were distinct within individuals with pre-radiographic OA and/or an increased risk of developing OA. Serum inflammatory profiles were analyzed within a number of patient cohorts [i.e., radiographic OA patients (hip and knee), youth with a history of intra-articular knee injury, corresponding controls]; and it was found that inflammatory profiles were distinct between knee vs. hip OA patients. Additionally, a computation method was developed which identified a coordinated change in cytokine profiles in the youth knee injury cohort. This computational methodology highlighted a number of candidate biomarkers that contributed to this observed difference, including C-C motif chemokine 22 (CCL22)/macrophage derived chemokine (MDC) which was selected for further study. In a pre-clinical rat OA model, it was found that CCL22 plays a functional role in chondrocyte apoptosis and cartilage degeneration. Further, it was found that CCL22 treated synovial fibroblasts demonstrated altered expression of inflammatory factors. These results suggested that CCL22 may be a biomarker and potential drug target in early OA. These results also suggested that CCL22 may be associated with OA pain, yet this was not examined directly and an in vivo model where CCL22 expression could be regulated would be required to test this hypothesis. While it was observed that CCL22 is expressed in damaged cartilage and acts on human chondrocytes and synovial fibroblasts, additional studies are required to determine how CCL22 triggered these changes in synovial fibroblasts as these results suggest this is CCR4 independent. Furthermore, it would be essential to validate these findings in an independent cohort to examine the sensitivity and/or specificity of CCL22 as an early OA biomarker.Item Open Access Inflammatory Profiling in Early Osteoarthritis(2018-09-20) Ren, Guomin; Krawetz, Roman J.; Emery, Carolyn A.; De Koning, A. P. JasonOsteoarthritis (OA) is one of the most common chronic diseases worldwide which can lead to disability. There is a desperate need for the efficient and reliable detection of OA at the early stage when patients are likely to benefit most from disease interventions. It has been shown in previous studies that inflammation plays important roles in cartilage degeneration, synovitis, remodeling of the subchondral bone and pain. The purpose of this thesis was to determine if a panel of inflammatory cytokines were distinct within individuals with pre-radiographic OA and/or an increased risk of developing OA. Serum inflammatory profiles were analyzed within a number of patient cohorts [i.e., radiographic OA patients (hip and knee), youth with a history of intra-articular knee injury, corresponding controls]; and it was found that inflammatory profiles were distinct between knee vs. hip OA patients. Additionally, a computation method was developed which identified a coordinated change in cytokine profiles in the youth knee injury cohort. This computational methodology highlighted a number of candidate biomarkers that contributed to this observed difference, including C-C motif chemokine 22 (CCL22)/macrophage derived chemokine (MDC) which was selected for further study. In a pre-clinical rat OA model, it was found that CCL22 plays a functional role in chondrocyte apoptosis and cartilage degeneration. Further, it was found that CCL22 treated synovial fibroblasts demonstrated altered expression of inflammatory factors. These results suggested that CCL22 may be a biomarker and potential drug target in early OA. These results also suggested that CCL22 may be associated with OA pain, yet this was not examined directly and an in vivo model where CCL22 expression could be regulated would be required to test this hypothesis. While it was observed that CCL22 is expressed in damaged cartilage and acts on human chondrocytes and synovial fibroblasts, additional studies are required to determine how CCL22 triggered these changes in synovial fibroblasts as these results suggest this is CCR4 independent. Furthermore, it would be essential to validate these findings in an independent cohort to examine the sensitivity and/or specificity of CCL22 as an early OA biomarker.Item Open Access The influence of maximal and submaximal cyclic concentric and eccentric exercise on chondrocyte death and synovial fluid proteins in the rabbit knee(Elsevier, 2020-08) Abughazaleh, Nada; Abusara, Ziad; Krawetz, Roman J.; Herzog, WalterMechanical stimulation of joints regulates the biosynthetic activity of chondrocytes. It has been argued that excessive loading might cause chondrocyte death, leading to degeneration of cartilage and cause osteoarthritis. The aims of this study were to apply a high, short-term loading, and a low intensity, long-term loading protocol to intact joints in life animals and determine changes in synovial fluid and the percentage of dead cells in rabbit knee cartilageItem Open Access Key cytoskeletal proteins are upregulated in 2D cell culture in comparison to 3D culture(2020-01) Aslam, Saba; Ungrin, Mark D.; Krawetz, Roman J.; Nezhad, Amir SanatiThe cytoskeleton is the structural framework of the cell, and plays an important role in cell motility, signalling pathways, and cell behaviour. It is composed of various proteins that form a dynamic network of filaments and tubules to mechanically support the cell. Commonly, cytoskeletal proteins such as β-actin are used as controls or references in many laboratory techniques because they are assumed to remain stable across various conditions. However, most of the research to date has been performed on 2D monolayers of cells adhered to plastic. Recently, 3D cell culture techniques have become more feasible and increasingly popular as they better represent the physiological microenvironment found in the body. As more research shifts from 2D to 3D culture, the changes in cytoskeleton due to culture conditions need to be better characterized. To study gene expression, eleven human cell lines were cultured in 2D and 3D, and reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) was performed. For analysis, ten control genes were evaluated for stability, and the geometric mean of the three most stable genes was used for normalization. The results show a significant downregulation of several cytoskeletal genes such as β-actin, vimentin, and keratin in 3D compared to 2D. Similarly, key cytoskeletal proteins were also found to be downregulated in 3D using Western blots normalized to total protein. These results demonstrate that the microenvironmental differences between culture conditions can considerably influence cytoskeletal component levels across various cell types. Integrating these findings with the published literature, we suspect that the supra-physiological stiff surface in plastic-adherent culture leads to overexpression of cytoskeletal proteins – presumably also impacting many other pathways and cell behaviour. These findings challenge the assumption that β-actin provides a consistently stable reference gene/protein, emphasize the importance of physiologically relevant culture systems, and highlight the need to select appropriate controls for specific experimental conditions.Item Open Access Mesenchymal progenitor cells from non-inflamed versus inflamed synovium post-ACL injury present with distinct phenotypes and cartilage regeneration capacity(2023-06-25) Krawetz, Roman J.; Larijani, Leila; Corpuz, Jessica M.; Ninkovic, Nicoletta; Das, Nabangshu; Olsen, Alexandra; Mohtadi, Nicholas; Rezansoff, Alexander; Dufour, AntoineAbstract Background Osteoarthritis (OA) is a chronic debilitating disease impacting a significant percentage of the global population. While there are numerous surgical and non-invasive interventions that can postpone joint replacement, there are no current treatments which can reverse the joint damage occurring during the pathogenesis of the disease. While many groups are investigating the use of stem cell therapies in the treatment of OA, we still don’t have a clear understanding of the role of these cells in the body, including heterogeneity of tissue resident adult mesenchymal progenitor cells (MPCs). Methods In the current study, we examined MPCs from the synovium and individuals with or without a traumatic knee joint injury and explored the chondrogenic differentiation capacity of these MPCs in vitro and in vivo. Results We found that there is heterogeneity of MPCs with the adult synovium and distinct sub-populations of MPCs and the abundancy of these sub-populations change with joint injury. Furthermore, only some of these sub-populations have the ability to effect cartilage repair in vivo. Using an unbiased proteomics approach, we were able to identify cell surface markers that identify this pro-chondrogenic MPC population in normal and injured joints, specifically CD82LowCD59+ synovial MPCs have robust cartilage regenerative properties in vivo. Conclusions The results of this study clearly show that cells within the adult human joint can impact cartilage repair and that these sub-populations exist within joints that have undergone a traumatic joint injury. Therefore, these populations can be exploited for the treatment of cartilage injuries and OA in future clinical trials.Item Open Access Muscular loading of joints triggers cellular secretion of PRG4 into the joint fluid(Journal of Biomechanics, 2013-04-26) Abusara, Ziad; Krawetz, Roman J.; Steele, Bridgett L.; DuVall, Michael M.; Schmidt, Tannin A.; Herzog, WalterWe developed a novel testing system that allows quantification of joint loading and permits analysis of changes in total protein and PRG4 contents in joint fluid of intact knees in live mice. A sequence of 15 repeat, isometric muscular contractions of "low" intensity (less than 50% of the maximal isometric muscular force), and "high" intensity (greater than 55% of maximal) were applied repeatedly (up to five times with a 15 min rest between contractions) to the mouse knee. Increases in knee joint loading were accompanied with significant increases in total protein (p<0.0001) and PRG4 concentrations in the synovial fluid. Total protein and PRG4 concentrations decreased with repeated "high" intensity loading. However, the addition of cell secretion inhibitors to the knee prior to muscular loading resulted in PRG4 levels that remained below the detection limit for all loading conditions. These results suggest that changes in synovial fluid proteins and PRG4 concentrations upon joint loading are mediated by cells within the joint, and that these changes may be used as quantitative indicators for the intensity and duration of acute joint loading, and might serve as a powerful clinical tool to assess the effectiveness of rehabilitation and prevention exercise programs.Item Embargo STING Activation as an Immunotherapeutic Strategy for Soft Tissue Sarcoma(2020-06-02) Marritt, Kayla Lynn; Monument, Michael J.; Jirik, Frank Robert; Krawetz, Roman J.; Yipp, Bryan G.Immunotherapy is an emerging field of cancer treatment that is transforming the management of numerous human cancers. However, there remains a substantial proportion of cancer subtypes that are unresponsive to many modern immunotherapy strategies. Soft tissue sarcomas (STS) are aggressive, connective-tissue derived solid cancers and are notoriously resistant to systemic therapies including immunotherapy. Immunologically, sarcomas are frequently characterized by a paucity of lymphocytic infiltrates, an immune suppressive microenvironment, and resistance to immunotherapies such as immune checkpoint inhibition and oncolytic viruses. Activation of the STING (stimulator of interferon genes) pathway can induce potent innate and adaptive anti-tumour immune responses within immunogenic solid tumours. However, this approach has never been tried in immune-inert sarcomas. Herein, STING activation in STS was analyzed to determine if STING activation could induce therapeutic anti-tumour effects and promote anti-tumour immunity. The long-term therapeutic responses of STING activation via 5,6-dimethylxanthenone-4-acetic acid (DMXAA) injection were assessed using a syngeneic murine model of undifferentiated pleomorphic sarcoma (UPS). Intratumoural DMXAA resulted in a durable cure in 50-60% of UPS-bearing mice. Flow cytometry was used to quantify immune infiltration after STING activation treatment and a higher proportion of CD8+/CD3+ cells was observed seven days post DMXAA treatment compared to the vehicle control treatment. Surviving mice all rejected UPS re-challenge in both the extremity and lung, and the therapeutic effects of DMXAA were mitigated by lymphocyte deficiency suggesting adaptive immunologic pathways are integral to the therapeutic response. This data suggests modulation of the STING pathway can elicit local and systemic anti-tumour immune responses in UPS and deserves further consideration as a novel local and systemic treatment for sarcomas.