Browsing by Author "Krawetz, Roman"
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- ItemOpen AccessAbsence of p21 Expression Sensitizes iPSCs to iCaspase9 Induced Apoptosis While Not Impacting Ability to Participate in Exogenous Cartilage Repair(2022-11-01) Larijani, Leila; Rancourt, Derrick; Krawetz, Roman; Childs, Sarah J.; Bob, Argiropoulosp21 is a multifunctional protein that is critical to the control cellular proliferation and plays a number of other functions in the cell. In the nucleus, p21 functions as a tumour suppressor, while in the cytoplasm, phosphorylated p21 functions as a proto-oncogene and can also suppress apoptosis. Because p21 deficiency has been linked to increased tissue regeneration, in this thesis, I sought to investigate if p21 mutations can improve the regenerative capacity of induced pluripotent stem cells in a cellular therapy approach for cartilage injuries in mice. However, because p21 is a tumor suppressor and its loss can result in tumorigenesis, I employed the inducible Caspase9 cell suicide system to purge iPSCs through forced apoptosis. In vitro, iCaspase9-mediated cell death resulted in a statistically significant increase in the apoptosis rate in p21-/- iPSCs compared to p21+/+ iPSCs (wild-type). RNA sequencing was undertaken to determine which pathways are involved in p21 mediated apoptosis. Increased expression of 41 apoptotic and 6 healing-related genes were observed in p21-/- iPSCs compared to wildtype. In vitro chondrogenesis of p21-/- iPSCs showed an increase in chondrogenic genes relative to the wild-type cells. When these iPSCs were transplanted into a focal cartilage injury in mice, ectopic cartilage formation was observed. Neither tumors, nor ectopic cartilage formation was observed in mice that were treated with CID drug to induce iCasp9 mediated apoptosis. Finally, I performed a drug screen to discover compounds that promote apoptosis with iCasp9. In addition to identifying several epigenetic drugs that promoted iCasp9 function in vitro, I also demonstrated that p21 inhibitors could phenocopy the enhanced iCasp9 mediate cell killing seen previously in the p21 mutant iPSCs. Overall, my findings indicate that p21 does play a role in protecting iPSCs from apoptosis and its downregulation can be exploited to increase cell-suicide approach effectiveness. I was also able to demonstrate that iPSCs can induce robust cartilage repair in mice regardless of the expression of p21. Therefore, modulation of p21 warrants further research to determine if exploiting this protein can be used in iPSC clinical trials safely in the future.
- ItemOpen AccessAssessment of the Efficacy of MRI for Detectionof Changes in Bone Morphology in a MouseModel of Bone Injury(Wiley, 2013-07-11) Taha, May A; Manske, Sarah; Kristensen, Erika; Taiani, Jaymi; Krawetz, Roman; Wu, Ying; Ponjevic, Dragana; Matyas, John; Boyd, Steven; Rancourt, Derrick; Dunn, Jeffrey F.Purpose To determine whether magnetic resonance imaging (MRI) could be used to track changes in skeletal morphology during bone healing using high-resolution micro-computed tomography (μCT) as a standard. We used a mouse model of bone injury to compare μCT with MRI. Materials and Methods Surgery was performed to induce a burr hole fracture in the mouse tibia. A selection of biomaterials was immediately implanted into the fractures. First we optimized the imaging sequences by testing different MRI pulse sequences. Then changes in bone morphology over the course of fracture repair were assessed using in vivo MRI and μCT. Histology was performed to validate the imaging outcomes. Results The rapid acquisition with relaxation enhancement (RARE) sequence provided sufficient contrast between bone and the surrounding tissues to clearly reveal the fracture. It allowed detection of the fracture clearly 1 and 14 days postsurgery and revealed soft tissue changes that were not clear on μCT. In MRI and μCT the fracture was seen at day 1 and partial healing was detected at day 14. Conclusion The RARE sequence was the most suitable for MRI bone imaging. It enabled the detection of hard and even soft tissue changes. These findings suggest that MRI could be an effective imaging modality for assessing changes in bone morphology and pathobiology.
- ItemOpen AccessCell Surface Receptors Expression Profile Of Human Synovial Mesenchymal Stem Cells In-Situ And In-Vitro From Normal And Osteoarthritic Knee Joints(2016) Al-Jezani, Nedaa; Krawetz, Roman; Rancourt, Derrick; Duncan, NeilOsteoarthritis is a chronic disease that results in the degeneration of the articular cartilage, eventually affecting the whole joint. Due to the lack of regenerative capacity in the articular cartilage, synovial mesenchymal stem cells, having chondrogenic capacity and derived from the synovium (SMSCs) are an attractive source for articular cartilage regeneration. However, it is important to identify the optimal SMSCs that have tri-lineage potential, which will help the regeneration of the whole joint. Unfortunately, MSC populations are phenotypically heterogeneous, in terms of their cell surface expression. In this project, SMSCs derived from OA joint with the optimal differentiation capacity display a marker profile of CD90+, CD44+, and CD73+. However, the same profile was expressed on clones that didn’t present with multi-potential differentiation capacity. Therefore, additional markers are necessary to accurately purify SMSCs that would may have increased regenerative capacity for use in cell therapies for patients with OA.
- ItemOpen AccessDeveloping a novel biomimetic bioreactor for bone graft engineering with murine embryonic stem cells(2012) Lee, Poh Soo; Rancourt, Derrick; Krawetz, RomanFollowing blood, bone graft transplantation is the second most common tissue transplant. Although tissue engineering holds great potential to fulfill demands for better treatment outcomes, it remains technologically challenging to produce bone grafts with normal physiological properties. During skeletal development, endochondral ossification initiates long bone formation and fracture healing. In this study, I aimed to build an in-vitro biomimetic bioreactor to recapitulate physiological niches and processes essential for endochondral ossification to grow bone tissues with anatomical and mechanical properties similar to the native tissues. Here, I have built a prototype capable of generating a dynamic cultivation environment and producing an ivory-toned construct with a stiff texture. The engineered construct greatly resembles a hyaline cartilage model undergoing initial stages of endochondral ossification during skeletal development. In the future, a possible engineered vasculature system may be integrated into the existing bioreactor design to enhance further maturation of constructs to form compact bone.
- ItemOpen AccessElucidating the Relationship Between Macrophages and Synovial Mesenchymal Progenitor Cell Chondrogenesis(2014-10-02) Fichadiya, Akash; Krawetz, RomanOsteoarthritis (OA) is a complex disease affecting articular cartilage and joint tissues. Macrophages in OA synovium contribute to the inflamed joint environment by releasing inflammatory mediators promoting cartilage loss. What remains unknown is how macrophages interact with synovial mesenchymal progenitor cells (sMPCs) in the joint. sMPCs are able to differentiate into cartilage but for unknown reasons, sMPC chondrogenic capacity is lost in OA. This thesis elucidates the relationship between macrophages and sMPC using synovial explants obtained from OA and normal patients. I found that altered macrophage activity (through cytokine stimulation and/or macrophage depletion) differentially regulates the chondrogenic capacity of sMPCs in a patient-specific manner. Also, the secretory profile of the explant is different between normal and OA patients in response to treatment. Taken together, there is heterogeneity within the responses of the OA patients, highlighting the need for personalized approaches to repair the cartilage within the OA joint.
- ItemOpen AccessHip Derived Synovial Mesenchymal Progenitor Cell Surface Markers In Situ as Indicators for Differentiation Potential(2016) Affan, Asmaa; Krawetz, Roman; Sen, Arindom; Powell, JimThe degeneration of articular cartilage observed in patients with osteoarthritic (OA) joints coupled with the lack of regenerative abilities of cartilage play a major role in causing disability in OA patients. The synovial membrane within the joints is home to synovial mesenchymal progenitor cell (sMPC) populations that have the ability to undergo chondrogenesis (in vivo and in vitro). However, it remains unknown if these sMPCs express any markers in vivo/in situ that give information as to which of the specific MPC sub-populations have pro-chondrogenic capacity. In the patient cohort examined in this study, the most common cell surface marker profile on MPCs was determined to be CD90+/CD44+/CD73+, and though it included cells that had chondrogenic capacity, it also included cells that did not. Additional markers are therefore required to further discriminate the heterogeneous populations of MPCs and identify synovial MPCs that are enriched for chondrogenic capacity.
- ItemOpen AccessImpact of Homeostasis Disruption on the Structure and Function of Murine Articular Cartilage(2023-09-12) Oliveira Masson, Anand; Krawetz, Roman; Edwards, William Brent; Biernaskie, Jeffrey AllanArticular cartilage plays a vital role in facilitating pain-free movement and load distribution in synovial joints, such as the knee. Owing to its complex structure-functional requirements and limited regenerative capacity, articular cartilage is particularly vulnerable to deterioration triggered by intrinsic and extrinsic insults. For instance, abnormal loading, trauma and aging can disrupt cellular, anatomical, and functional homeostasis within the knee joint and/or articular cartilage microenvironment, contributing to the pathogenesis of tissue degradation and degenerative joint diseases, particularly osteoarthritis (OA). In this context, it is essential to understand how cartilage and the joint microenvironment respond to differential levels of tissue homeostatic disruption and the resulting implication on remodeling and repair outcomes. In this multifaceted study, I employed several transgenic mouse models in conjunction with histological, imaging, and mechanical testing modalities to deepen our understanding of structural and functional changes associated with degeneration and regeneration of murine articular cartilage.
- ItemOpen AccessInvestigating the mechanisms involved in producing limb length variation in mice(2017) Marchini, Marta; Rolian, Campbell; German, Rebecca Z.; Jirik, Frank; Kurrasch, Deborah; Krawetz, Roman; Cobb, JohnThe genetic and developmental mechanisms involved in outgrowth, patterning and elongation of the vertebrate limb during ontogeny are relatively well documented. However, how these mechanisms contribute to continuous variation in bone length remains unknown. Limb bone length is a complex quantitative trait, which involves the expression and interaction of many genes and gene pathways, as well as interactions with the environment. Understanding how phenotypic variation in limb bone length can be produced through development can help to understand how limb length diversity across mammal evolution. Limb bones are patterned during embryonic development, and in postnatal ontogeny they undergo elongation and remodeling to reach their adult size and shape. The elongation of the bone occurs by endochondral ossification within the growth plate, a cartilaginous structure in one or both ends of a long bone that is composed of distinct cellular zones reflecting different stages in the life cycle of chondrocytes: resting, proliferating and hypertrophic. This chondrocyte life cycle plays a key role in the ossification and elongation of long bones. Changes in the mechanisms of proliferation and/or hypertrophy can produce changes in bone apposition that cause variation in bone size and shape. Similarly, perturbation of either embryonic and/or postnatal development can cause severe effects on skeletal development, producing a range of disorders classified as skeletal dysplasias. Since 2010, our research group has selectively bred mice, known as Longshanks, for increases in relative tibia length, achieving a 15% increase at generation F20. Using artificial selection in mice in a controlled environment, and comparing selectively bred mice with unselected controls, provides the resolution necessary to study the developmental and genetic processes involved in producing continuous phenotypic differences in tibia length between individuals. Specifically, large differences in tibia length between selected and control mice can be used to identify where, when and how cell and molecular differences in growth plate chondrocyte function in the tibia contribute to continuous variation in long bone length. This dissertation aims to identify and quantify how cellular dynamics and gene expression in the growth plate contribute to limb bone length variation in Longshanks mice. We also investigate a novel dysmorphic phenotype identified during the Longshanks experiment that exhibits a severe but viable skeletal dysplasia-like phenotype (Shorty mice), in order to better understand how the Shorty mutation relates to broader categories of skeletal disorder. Using histomorphometry (Chapter 2) I discovered that Longshanks mice have a thicker proliferative zone with more chondrocytes per column compared to Controls, but the hypertrophic zone does not show differences in numbers and size of its chondrocytes. Proliferation assays further showed that there were no differences in the number of mitotic cells per proliferating chondrocytes in a column, suggesting that proliferative chondrocytes in Longshanks do not undergo mitosis faster than Control mice. Using RNAseq and qPCR (Chapter 3) I compared the epiphyseal and growth plate transcriptome between juvenile Longshanks mice and Control. My data suggest that the cellular differences we have found using histomorphometry (Chapter 2), may be due to the downregulation of Fxyd2, a γ-subunit of the NA+/K+ ATPase pump. Two other genes, Sox9 and Dlk1 may also play important roles in generating the underlying cellular differences between Longshanks and Control mice. In Chapter 3, I also investigate (Chapter 3) the role of the NA+/K+ ATPase pump in bone growth using embryonic limb and tibia culture using ouabain, a pharmacological inhibitor of the pump. The limbs and tibiae cultured with ouabain grew less than the controls. We also noticed a decrease of proliferative chondrocytes and using flow cytometry, and a parallel increase of programmed cell death in hypertrophic chondrocytes. These data suggest that regulation of the NA+/K+ ATPase pump is important in regulating chondrocyte differentiation and hypertrophic zone chondrocyte turnover. In Chapter 4, I studied the Shorty mice phenotype using µCT scanning and histology. My data suggest that the mutation that causes this unique skeletal dysplasia-like phenotype does not interfere with limb development in a manner comparable to common genetically-characterized skeletal dysplasias. The phenotype appears before embryonic stage E14, and may appear during limb outgrowth and patterning. This study provides a strong phenotypic foundation for the study of the underlying genetic causes of the Shorty mutation, and identifies a novel variant that may provide insight into underappreciated aspects of the limb development program. Overall, the research presented in this dissertation provides important insight into aspects of limb development and growth plate biology that have been previously difficult to identify. In particular, this dissertation used a novel approach to answer one of evolutionary developmental biology’s long-standing questions: the developmental origins of phenotypic variation.
- ItemOpen AccessIsolation and Characterization of an Adult Stem Cell Population from Human Epidural Fat(2019-03-17) Al-Jezani, Nedaa; Cho, Roger; Masson, Anand O.; Lenehan, Brian; Krawetz, Roman; Lyons, Frank G.Study Design. Isolation and characterization of human epidural fat (HEF) stem/progenitor cells. Objective. To identify a progenitor population within HEF and to determine if they meet the minimal criteria of a mesenchymal stem cell (MSC). Summary of Background Data. The biological function, if any, has yet to be determined for HEF. The presence of MSCs within HEF may indicate a regenerative potential within the HEF. Methods. HEF was isolated from 10 patients during elective spinal surgery. HEF cells were differentiated along osteo-, adipo-, and chondrogenic lineages, with differentiation analyzed via qPCR and histology. The cell surface receptor profile of HEF cells was examined by flow cytometry. HEF cells were also assayed through the collagen contraction assay. Prx1CreERT2GFP:R26RTdTomato MSC lineage-tracking mice were employed to identify EF MSCs in vivo. Results. HEF cell lines were obtained from all 10 patients in the study. Cells from 2/10 patients demonstrated full MSC potential, while cells from 6/10 patients demonstrated progenitor potential; 2/10 patients presented with cells that retained only adipogenic potential. HEF cells demonstrated MSC surface marker expression. All patient cell lines contracted collagen gels. A Prx1-positive population in mouse epidural fat that appeared to contribute to the dura of the spinal cord was observed in vivo. Conclusions. MSC and progenitor populations are present within HEF. MSCs were not identified in all patients examined in the current study. Furthermore, all patient lines demonstrated collagen contraction capacity, suggesting either a contaminating activated fibroblast population or HEF MSCs/progenitors also demonstrating a fibroblast-like phenotype. In vivo analysis suggests that these cell populations may contribute to the dura. Overall, these results suggest that cells within epidural fat may play a biological role within the local environment above providing a mechanical buffer.
- ItemOpen AccessLubricin Binds to and Regulates Toll-like Receptor (TLR) Activity(2015-10-01) Iqbal, Shah; Krawetz, Roman; Schmidt, TanninIn this study, lubricin, a protein involved in the lubrication of joints, was discovered to bind to and activate toll-like receptors (TLR). Since TLRs are a major component of the innate immune system, this suggests that lubricin may also play a role in the regulation of the inflammatory response. We attempted to elucidate whether lubricin has a pro- or anti-inflammatory role in the joint following TLR binding. To determine if lubricin-TLR interaction could be playing a role in Osteoarthritis (OA), we examined the cytokine secretions of OA fibroblasts treated with lubricin and demonstrated a potentially anti-inflammatory effect. Furthermore, in vivo studies demonstrated lubricin’s ability to mitigate pain, joint damage, and inflammation following an injury in a rat OA model. The experiments presented in this thesis suggest that lubricin could have anti-inflammatory effects when binding to TLRs at the cellular and tissue level.
- ItemOpen AccessMatrix metalloproteinase protein expression profiles cannot distinguish between normal and early osteoarthritic synovial fluid(BioMed Central, 2012-07-23) Rattner, Jerome B.; Heard, Bryan J.; Martin, Liam; Frank, Cyril B.; Hart, David A.; Krawetz, Roman
- ItemOpen AccessMesenchymal Progenitors in the Epidural Fat and Dura Mater Participate in Tissue Homeostasis and Wound Healing(2021-07-12) Shah, Sophia; Krawetz, Roman; Salo, Paul; Mitha, AlimMesenchymal progenitor cells (MPCs) are adult cells capable of self-renewal and differentiation into cells that make up mesodermal tissues such as bone, cartilage, and fat. MPCs are believed to play a significant role in tissue maintenance and repair. MPCs are present in many adult connective tissues but are typically found in higher quantities in adipose tissues for yet unknown reasons. Recently, our research group identified MPC populations within epidural fat and the adjacent dura mater. Clinically, epidural fat is frequently considered a space-filling, biologically inert tissue; therefore, it is common practice for spine surgeons to discard it during surgical procedures. As the development and cellular origins of both epidural fat and the dura mater remain unclear, I hypothesized that epidural fat MPCs contribute to the maintenance of dural integrity throughout growth and post-injury. Using Paired related homeobox 1 (Prx1) and Hypermethylated in cancer 1 (Hic1) transgenic lineage tracing mice, the localization of epidural fat MPCs were identified during normal maturation and at skeletal maturity. This lineage tracing revealed an overlap between Prx1+ and Hic1+ populations, indicating a potential hierarchical relationship between the two MPC populations. When Prx1+/ Hic1+ MPCs were ablated, the expression of the dural marker α-SMA was lost in adjacent dura mater suggesting these cells are required for tissue homeostasis. Both MPC populations were observed to respond to dural injuries by homing to the lesion site. The process by which epidural fat MPCs maintain the dura mater through growth and after injury was accelerated in p21-/- mice (known for increased tissue regeneration/ cell proliferation). While MPCs have been identified and characterized in other adipose tissues, the role in epidural fat remained elusive. This study contributed to our knowledge of the role of epidural fat MPCs in vivo in aspects of growth, homeostasis, and repair of dural tissue. This thesis emphasizes the importance of revisiting the prevalent notion of epidural fat as biologically insignificant and the process of discarding it during surgery.
- ItemOpen AccessMicro-environmental factors directing differentiation of murine embryonic stem cells down osteogenic and chondrogenic lineages(2013-02-15) Hazenbiller, Olesja; Duncan, Neil; Krawetz, RomanThe field of bone tissue engineering (BTE) aims to develop graft substitutes for diseased or difficult to heal fractures. We used a BTE construct made of collagen type I and murine embryonic stem cells (mESCs) which has been shown to trigger mESCs differentiation into osteoblasts, and successfully contribute to fracture repair in vivo within a mouse model system. Bone healing is a complex process involving the interplay of biochemical and biomechanical cues. Therefore, this project aimed to systematically emulate the roles of chemical and mechanical cues present during fracture repair on the differentiation of mESCs in vitro in order to optimize treatment strategies for BTE. Further characterization of this cell/gel construct revealed that mESCs differentiate into a heterogeneous cell population of chondrocytes and osteoblasts, replicating the process of endochondral ossification that normally occurs during fracture repair. To study the effect of biomechanical cues, a loading system was specifically designed and characterized to apply confined compressive load to a soft, viscoelastic cell/gel construct. Mechanical stimuli enhanced chondrogenic differentiation but had no effect on osteogenic differentiation. Moreover, the role of integrins in directing mESCs differentiation and transducing mechanical signals was evaluated. Finally, the synergistic effect between extracellular matrix mediated differentiation, mechanical stimulation and BMP-2 delivery to the system using nano-particles was studied.
- ItemOpen Accessp21−/− mice exhibit enhanced bone regeneration after injury(2017-11-09) Premnath, Priyatha; Jorgenson, Britta; Hess, Ricarda; Tailor, Pankaj; Louie, Dante; Taiani, Jaymi; Boyd, Steven; Krawetz, RomanAbstract Background p21(WAF1/CIP1/SDI1), a cyclin dependent kinase inhibitor has been shown to influence cell proliferation, differentiation and apoptosis; but more recently, p21 has been implicated in tissue repair. Studies on p21(−/−) knockout mice have demonstrated results that vary from complete regeneration and healing of tissue to attenuated healing. There have however been no studies that have evaluated the role of p21 inhibition in bone healing and remodeling. Methods The current study employs a burr-hole fracture model to investigate bone regeneration subsequent to an injury in a p21−/− mouse model. p21−/− and C57BL/6 mice were subjected to a burr-hole fracture on their proximal tibia, and their bony parameters were measured over 4 weeks via in vivo μCT scanning. Results p21−/− mice present with enhanced healing from week 1 through week 4. Differences in bone formation and resorption potential between the two mouse models are assessed via quantitative and functional assays. While the μCT analysis indicates that p21−/− mice have enhanced bone healing capabilities, it appears that the differences observed may not be due to the function of osteoblasts or osteoclasts. Furthermore, no differences were observed in the differentiation of progenitor cells (mesenchymal or monocytic) into osteoblasts or osteoclasts respectively. Conclusions Therefore, it remains unknown how p21 is regulating enhanced fracture repair and further studies are required to determine which cell type(s) are responsible for this regenerative phenotype.
- ItemOpen AccessProduction of Adult Human Synovial Fluid-Derived Mesenchymal Stem Cells in Stirred-Suspension Culture(2018-03-27) Jorgenson, Kristen D.; Hart, David A.; Krawetz, Roman; Sen, ArindomThe chondrogenic potential of synovial fluid-derived mesenchymal stem cells (SF-MSCs) supports their use in cartilage regeneration strategies. However, their paucity in synovial fluid necessitates their proliferation in culture to generate clinically relevant quantities. Here it was determined that 125 mL stirred suspension bioreactors utilizing Cytodex-3 microcarrier beads represent a viable platform for the proliferation of these cells. During the inoculation phase, a bead loading of 2 g/L, an inoculation ratio of 4.5 cells/bead, and continuous agitation at 40 rpm in a medium with 5% serum resulted in high cell attachment efficiencies and a subsequent overall cell fold expansion of 5.7 over 8 days. During the subsequent growth phase, periodic addition of new microcarriers and fresh medium increased culture longevity, resulting in a 21.3 cell fold increase over 18 days in the same vessel without compromising the defining characteristics of the cells. Compared to static tissue culture flasks, a bioreactor-based bioprocess requires fewer handling steps, is more readily scalable, and for the same cell production level, has a lower operating cost as it uses approximately half the medium. Therefore, stirred suspension bioreactors incorporating microcarrier technology represent a viable and more efficient platform than tissue culture flasks for the generation of SF-MSCs in culture.
- ItemOpen AccessProteolytic Regulation of Proteoglycan 4 in Inflammation(2023-05-05) Das, Nabangshu Shekhar; Dufour, Antoine; Schmidt, Tannin; Krawetz, Roman; Rezansoff, AlexanderProteoglycan 4 (PRG4) is an extracellular macromolecule that is synthesized and secreted by cells lining surfaces of multiple tissues. While it has traditionally been studied and described as a boundary lubricant, recent evidence suggests that it can bind to and affect downstream signaling of several cell surface receptors, including toll-like receptors (TLRs) that are involved in regulating inflammatory responses. Although previous studies have shown that proteolysis of PRG4 reduces its boundary lubricating ability in vitro compared to intact PRG4, the effect of proteolysis on inflammatory signaling remained uncharacterized. Furthermore, while differential levels of PRG4 expression have been associated with various inflammatory conditions, such as osteoarthritis, the role of PRG4 in maintaining inflammatory signaling during normal aging under homeostatic conditions is still unclear. The objectives of this thesis were to 1- Determine expression of PRG4 in global proteomes during inflammation, and 2- Characterize of proteolytic processing of PRG4 and the mechanism and effects in OA and 3- Examine the effects of PRG4 on the global proteomes during age-related joint inflammation. Global proteome analysis revealed PRG4 expression levels changed locally during inflammation as demonstrated by a reduction in the PRG4 expression in the tears while an increase in the saliva of Sjogren’s syndrome patients comparing to healthy individuals. These differential levels of PRG4 expression were associated with differential expression profiles of different signaling molecules (i.e., proteases) associated with multiple inflammatory pathways including neutrophil degranulation both of the tissue environment. With respect to proteolytic susceptibility of PRG4 during inflammation, this study revealed that the proinflammatory serine protease, tryptase β, cleaves PRG4 altering its functional properties with respect to lubrication and inflammation: it reduces the ability to provide boundary lubrication and increases the ability to activate NF-κB-mediated inflammation through the TLR pathway. In a destabilization of medial meniscus (DMM) model of osteoarthritis (OA) in rat knee joint, differential colocalization of tryptase β and PRG4 in knee joints was associated with the development of OA and disease progression. Treatment with an intra-articular injection of exogenous PRG4 was able to resolve joint inflammation and OA phenotypes in the post-DMM rat knee by restoring the expression of PRG4 on articular cartilage and tryptase β by cartilage chondrocytes. In the absence Prg4, chondrocytes in murine knee cartilage express elevated level of proteases, particularly tryptase β and Htra-1, along with concurrent development of neutrophil-like phenotypes during aging. The neutrophil-like phenotype of chondrocytes is associated with loss of joint integrity in Prg4-/- mice. Overall, this thesis demonstrates the importance of expression levels and structural integrity of PRG4 in maintaining homeostasis through regulating inflammation, provides greater insights into the complex interplay between PRG4, proteases and inflammation, and provide the foundation and motivation for the development of new treatments for inflammatory diseases.
- ItemOpen AccessRegulating p21 Expression to Increase Chondrogenic Potential in Human Mesenchymal Progenitor Cells(2016) Bertram, Karri; Krawetz, Roman; Rinker, Kristina; Biernaskie, JeffCartilage does not regenerate in humans, and therefore cartilage degeneration is a problem that affects a significant percentage of the population, including those with diseases such as Osteoarthritis (OA). The p21 knockout (p21-/-) mouse contains the only known single mutation in mammals that can induce a cartilage regenerative phenotype. Work in this thesis aims to identify p21 expression inhibitors for use in humans and to characterize their effects on human synovial mesenchymal progenitor cells (MPCs) during culture and chondrogenesis. I have identified one putative p21 expression inhibitor (acting through HSP90), that induces human synovial MPCs to display phenotypic properties similar to fibroblasts from p21-/- mice. Additionally, this inhibitor promotes cartilage formation in a mouse cartilage injury model. These results indicate that p21 inhibition through HSP90 may be a potential pharmaceutical target for stimulating chondrogenic regeneration for the treatment of cartilage defects or in cartilage degenerating diseases such as OA.
- ItemOpen AccessScaling up Production of Pluripotent Stem Cells in Stirred Suspension Bioreactors for Regenerative Medicine(2022-01-10) Borys, Breanna; Kallos, Michael; Krawetz, Roman; Rancourt, DerrickThis thesis focused on overcoming engineering challenges of using stirred suspension bioreactors for optimized PSC expansion and production scale-up. This objective was achieved through a combination of computational modeling and biological testing. Computational fluid dynamic modeling of various scale horizontal-blade stirred suspension bioreactors and single-use vertical-wheel bioreactors mapped out a hydrodynamic comparison that could be used for scale-up and scale-out predictions. Mouse embryonic stem cells were first cultured as aggregates in traditional horizontal-blade bioreactors to establish an understanding between hydrodynamic distributions, cell growth kinetics, aggregate sizes and distributions, and pluripotency maintenance. Bioprocess bottlenecks surrounding cryopreservation, inoculation density, and 3D serially passaging were addressed before beginning work with hiPSC stem cell cultures. hiPSC culture was optimized in vertical-wheel bioreactors, a scalable platform designed specifically for use with shear sensitive stem cell. Studies were completed to optimize inoculation methods, agitation rates, oxygen availability and nutrient availability. The vertical-wheel bioreactor was used as an effective tool in the design of single-cell inoculation methods and the first published protocol for in-vessel aggregate dissociation. Finally, results from these studies were compiled to better understand the relationships between the bioreactor hydrodynamic environments and PSC biological outputs. From here, effective scale-up correlation equations were derived allowing operators to define a working range of hydrodynamic variables at one scale and calculate corresponding agitation rates at other modeled scales. These equations were derived to maintain the CFD calculated variable of volume average energy dissipation rate. This variable was found to greatly impact cell quantity and quality through the hydrodynamic control of aggregate size and homogeneity. A suggested operating range of agitation rates was set and biologically validated for the successful culture and scale-up of iPSCs aggregates. Taken together, this thesis provides a set of tools and protocols for the robust expansion and scale-up of defined high-quality PSCs in stirred suspension bioreactors as a critical step in advancing cell therapy towards clinical cures in the field of regenerative medicine.
- ItemOpen AccessShear Stress Modulates Gene Expression in Normal Human Dermal Fibroblasts(2017) Zabinyakov, Nikita; Rinker, Kristina; Rinker, Kristina; Xilong, Zheng; Krawetz, Roman; Rancourt, Derrick; Bertram, JohnApplied mechanical forces, such as those resulting from fluid flow, trigger cells to change their functional behavior or phenotype. However, there is little known about how fluid flow affects fibroblasts. The hypothesis of this thesis is that dermal fibroblasts undergo significant changes of expression of differentiation genes after exposure to fluid flow (or shear stress). To test the hypothesis, human dermal fibroblasts were exposed to laminar steady fluid flow for 20 and 40 hours and RNA was collected for microarray analysis. Gene expression data was processed using gene network analysis, pathway analysis, and gene functional analysis with comparison to data from publicly available data sets. Additional treatment with PI3K/mTOR pathway inhibitor, PI-103, was performed to evaluate pathway involvement in flow modulation of gene expression. Results from overall transcription analysis demonstrated that fluid flow modulated many genes in fibroblasts including those related to differentiation, development and TGF-β pathway regulation.
- ItemOpen AccessSynovial Cellular Composition in Osteoarthritic Knee Joints(2015-06-10) O'Brien, Kate; Krawetz, RomanAbstract Osteoarthritis is a degenerative disorder affecting every tissue within the joint. OA is characterized by the degeneration of articular cartilage leading to inflammation of the underlying bone and synovium, resulting in loss of mobility and pain. Inflammation has been shown to play a key role in the development and progression of OA, but since OA is a multi-factorial disease, its exact pathogenesis has yet to be fully elucidated. However, it has been established that a major influence in the development of OA is the presence of pro-inflammatory cytokines in the synovium; specifically, IL-1-beta has been implicated in cartilage destruction and the TNF pathway in the OA inflammatory cascade. The ability to detect these cytokines is limited until progression of the disease is relatively advanced. To date, few studies have examined the cellular composition (including inflammatory cells) in the synovium of patients with early OA. Furthermore, in studies which do examine cells within the synovium, few if any compare these results to stringently characterized normal joints. The current study examined human synovium taken from OA patients at the time of arthroscopic knee surgery, while normal tissue was harvested from cadaveric donations. Synovium was taken from one to four sites within the joint (osteoarthritic and non-osteoarthritic (normal) human knees) and examined using histological techniques to determine the cellular composition of the synovium. Within joint and between joint comparisons were made. There were no significant trends found either within joint or between joint with regards to cellularity. Early OA synovium was defined as subclinical OA synovium; this being the case, the lack of significant difference could be due to the fact that early OA synovium has few differences from “normal synovium”.