Browsing by Subject "Osteoarthritis"

Background

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Trial registration

Kidney stones disease (KSD) is infamous for the morbidity it renders to an afflicted individual by causing intense pain through abrasions the urinary tract during stone passage and/or by causing increased fluid pressure caused by outflow blockage. Symptomatic kidney stones are typically treated through lithotripsy, in which stones are broken into fragments which are small enough for active retrieval or spontaneous passage. However, many individuals experience incomplete passage and some fragments remain in the kidney indefinitely. These residual stone fragments (RSF) serve as nuclei for further stone growth and cause KSD recurrence. RSFs can grow through two mechanisms: 1) calcium and other ionic stone precursors can directly crystalize on the surface of a fragment, and 2) small urinary crystallites become coated in an adhesive layer of urinary protein and adhere to the RSF. While dietary changes and a variety of medications have been shown to be effective at inhibiting this growth, and ultimately disease recurrence, a lack of patient compliance severely limits the efficacy of these approaches. In this work, we designed an analogue of mucins, biological surface coatings employed by the body as surface protectants and lubricants, to adsorb to the surface of RSFs and inhibit both mechanisms of stone growth. To do so, we performed phage display to identify peptides which bind to kidney stones. We designed genes for these peptides and expressed them as fusion peptides with elastin-like polypeptides to facilitate expression. We also used a calcium depletion assay to probe their ability to inhibit growth of kidney stone. The peptides discovered by phage display were unable to inhibit growth of RSFs through calcium adsorption. Instead, we used oligoanionic binders to synthesize analogue mucins composed of elastin-like polypeptides and synthetic polymers. We characterized these mucin analogues at each step using a combination of NMR, IR, and GPC when appropriate. Stone-targeted mucin analogues successfully inhibited the growth of calcium oxalate monohydrate stone models. Finally, to monitor adsorption of these mucin analogues to model kidney stones, we functionalized sensors with calcium oxalate monohydrate using polyacrylate as an adhesive layer. In sum, this work explores the ability to synthesize mucin analogues to inhibit recurrent kidney stone disease and have potential to shift the paradigm of kidney stone treatment.

Osteoarthritis (OA) is the most common joint disorder causing chronic disability in the world population. By the year 2030, an estimated one fifth of this population will be affected by OA. Although OA is regarded as a multi-factorial disorder with both environmental and genetic components, the exact pathogenesis remains unknown.

In this study, we hypothesize that biomarkers associated with OA can be used as quantitative traits of OA, and provide enough power to identify new genes or replicate known gene associations for OA. We established an extensive family called the CARRIAGE (CARolinas Region Interaction of Aging, Genes and Environment) family. Then, we measured and analyzed seven OA-related biomarkers (HA, COMP, PIIANP, CPII, C2C, hs-CRP and GSP) in this extensive family to evaluate their association with OA clinical phenotypes. These findings suggest that OA biomarkers can reflect hand OA in this large multigenerational family. Therefore, we performed nonparametric variance components analysis to evaluate heritability for quantitative traits for those biomarkers. Finally, based upon OA biomarkers with high heritability, we performed a genome-wide linkage scan. Our results provide the first evidence of genetic susceptibility loci identified by OA-related biomarkers, indicating several genetic loci potentially contributing to the genetic diversity of OA.

Meanwhile, we identified joint hypermobility as a factor which reduces OA risk and has an inverse association with serum COMP levels in this family. The relationship between lower serum COMP and OA have been further validated in another Caucasian GOGO (Genetics of Generalized Osteoarthritis) population. Therefore, we further hypothesize that joint hypermobility, having the characteristic of a decreased OA risk, can serve as a quantitative trait for identifying protective loci for OA. Then, we performed nonparametric variance components analysis to evaluate the heritability of joint hypermobility. The result also shows joint hypermobility has substantial heritable components in this family. Lastly, based on the same genome-wide linkage scan, we identify genetic susceptibility loci for joint hypermobility.

In conclusion, our work provides the first linkage study to identify genetic loci associated with OA using biological markers. Furthermore, we have also shown genetic susceptibility loci for joint hypermobility, possibly implying protective loci for OA.

Osteoarthritis (OA) is a common joint disorder, affecting over 27 million Americans. OA is characterized by the degeneration of cartilage tissue, and presents clinically with joint pain, stiffness, and limited range of motion. As such, it is a leading cause of disability in the United States. Current treatment options for OA focus on relieving pain (either pharmacologically or through surgical joint replacement), but do not treat or reverse cartilage degeneration. A main reason for this is that the diagnosis of OA depends on pain and radiographic findings, which are not present until advanced stages of the disease. Development of therapies focused on treating or reversing OA degeneration would therefore be enhanced if OA pathology was detectable at earlier stages of the disease. Because changes in mechanical properties (i.e. the stiffness and permeability) occur in OA cartilage before pain and radiographic features are visible, measurement of cartilage mechanics may be used for earlier assessment of OA degeneration. As cartilage mechanics are traditionally measured in the ex vivo environment, the goal of this dissertation was to develop a noninvasive methodology for measuring cartilage mechanical properties in vivo.

Specifically, the methodology consists of a combination of noninvasive magnetic resonance imaging (MRI) techniques to quantify in vivo cartilage composition and mechanical response, as well as a statistical model predicting cartilage stiffness based on these MRI measurements. Porcine knee joint cartilage was used to develop the statistical model, where stiffness was quantified in the traditional manner using ex vivo mechanical testing. The statistical model was then applied to in vivo data from a cohort of healthy human volunteers, for whom the noninvasive MRI techniques were used to measure the composition and mechanical response of their tibial cartilage. Thus, human tibial cartilage stiffness in vivo was quantified.

Overall, the in vivo estimates of healthy human tibial cartilage stiffness (ranging from 0.39 ± 0.05 MPa to 1.06 ± 0.24 MPa) compare well with ex vivo measurements of human cadaveric tibial cartilage stiffness (ranging from 0.45 ± 0.28 MPa to 0.65 ± 0.25 MPa). This finding supports the validity of the methodology developed in this dissertation. Future work using this in vivo methodology for measuring cartilage mechanical properties has diverse applications regarding cartilage health. For instance, this technique may be used clinically to provide earlier detection of OA pathology, or it may be used in future biomechanics research to evaluate the efficacy of different therapeutic approaches toward ameliorating OA pathology and restoring healthy cartilage mechanics. Therefore, the methodology for measuring cartilage mechanical properties in vivo developed here represents an important contribution to the fields of biomechanics and OA research.

Osteoarthritis (OA) is a degenerative disease of articular joints characterized by progressive deterioration of the cartilage lining, subchondral bone destruction and thickening of the joint capsule. These tissue changes lead to symptomatic joint pain and joint dysfunction, leading to restrictions on daily life activities. Intra-articular injections of corticosteroids or anti-inflammatory compounds are commonly given to relieve symptoms associated with OA; however, rapid clearance of these compounds from the joint space and into draining synovial lymphatics necessitates the use of drug carriers to increase drug residence and efficacy.

Silk fibroin, a protein polymer from the mulberry silkworm (Bombyx mori) and of slow biodegradation in vivo, has a long history of clinical use. Silk fibroin can be fabricated into nano- and micro-particles capable of entrapping small-molecule drugs to provide for sustained release. For this work, silk microparticles were fabricated entrapping the near-infrared fluorescent dye, Cy7, as a model small-molecule drug. The release kinetics of the Cy7 from the silk microparticles were characterized in vitro and fluorescence in vivo imaging was used to study the clearance of silk microparticles following intra-articular injection in healthy rat knee joints.

Furthermore, a surgically-induced model of OA was used in rat knee joints to study the effect of OA pathology on intra-articular clearance. Fluorescently-labeled dextrans of varying size (10 and 500 kDa molecular weight) were intra-articularly injected in the knee joints of healthy and OA rats, and fluorescence in vivo imaging was employed to detect changes in the intra-articular clearance. Additionally, a new method to characterize the trans-synovial clearance of the fluorescent dextrans was developed using the confocal microscopy of joint tissue sections from healthy and OA joints.

The role of articular cartilage in diarthrodial joints is primarily mechanical as the tissue provides a nearly frictionless, load-bearing surface that supports and distributes forces generated during joint loading. Embedded within the extensive cartilage extracellular matrix (ECM), chondrocytes are surrounded by a narrow, distinct pericellular matrix (PCM) that is thought to regulate the biomechanical microenvironment of the cell and influence chondrocyte metabolism, cartilage homeostasis, and overall joint health. While previous studies of PCM mechanical properties required physical extraction of the cell and PCM from the tissue, atomic force microscopy (AFM) provides a means for high resolution microindentation testing that can be used to measure local mechanical properties in situ. This dissertation develops and applies AFM microindentation techniques to 1) evaluate the microscale elastic properties of the cartilage PCM and ECM in situ and 2) correlate site-specific biochemical composition with biomechanical properties of the PCM and ECM.

An AFM-based stiffness mapping technique was experimentally validated and applied to cartilage sections to evaluate ECM and PCM properties in situ with minimal disruption of native matrix integration. As expected, PCM elastic moduli were significantly less than ECM moduli, uniform with depth, and mechanically isotropic. ECM moduli exhibited distinct depth-dependent anisotropy and unexpectedly, were found to decrease with depth from the articular surface. Both the PCM and ECM exhibited alterations in microscale moduli and their spatial distributions when evaluated in cartilage presenting early degenerative changes associated with osteoarthritis (OA) as compared to healthy tissue.

The ability to correlate site-specific biochemical composition with local biomechanical properties provides a more complete characterization of the chondrocyte microenvironment. To this end, we developed novel immunofluorescence (IF)-guided AFM stiffness mapping and demonstrated that PCM mechanical properties correlate with the presence of type VI collagen. Extending this technique by using dual IF, we presented new evidence for a defining role of perlecan in the PCM, showing that interior regions of the PCM rich in perlecan and type VI collagen exhibit lower elastic moduli than peripheral PCM and ECM regions lacking perlecan. Furthermore, lower moduli at the PCM interior were significantly influenced by the presence of heparan sulfate. IF-guided AFM stiffness mapping was combined with enzymatic digestion to demonstrate that the micromechanical properties of the PCM exhibit high resistance to specific enzymatic digestion of aggrecan and aggrecan-associated glycosaminoglycans but are vulnerable to proteolytic degradation by leukocyte elastase.

Overall, this research generates new insights into the complex structural, compositional, and functional relationships between the cartilage ECM and PCM and provides the tools and framework for further studies to continue to investigate their importance in regulating chondrocyte physiology in health and disease.

Osteoarthritis (OA) is a degenerative disease affecting articular cartilage, leading to loss of its structure and function. Early stage OA is characterized by changes in the extracellular matrix (ECM), including a reduction in proteoglycans (PG) concentration, increased water content within the tissue, and increased synthesis and degradation of matrix molecules with disorganization of collagen network [1, 2]. The ability to noninvasively quantify PG changes in cartilage would therefore be useful for early OA diagnosis, monitoring cartilage response to therapies, and assessing efficacy of cartilage repair procedures [3]. T1rho and T2 weighted magnetic resonance (MR) imaging techniques have been shown to have potential in tracking early biochemical compositional changes within cartilage associated with degeneration [3, 4]. Additionally, this method has the potential to be a powerful tool to better understand how cartilage responds to different loading environments both acutely and over time. The main objective of this work is to validate T1rho and T2 relaxation times as non-invasive measures for the assessment of biochemical and biomechanical properties of cartilage.

The first two studies presented in this work focus on the validation of this T1rho and T2 imaging for non-invasive cartilage assessment. The first study examines both normal and osteoarthritic cartilage containing both OA defect regions and healthy appearing areas. This study aims to comprehensively assess the relationship between OA cartilage composition, biochemical, and biomechanical properties with T1rho and T2 relaxation times in order to validate this technique as an in vivo diagnostic method for early stage OA. The second study utilizes targeted enzymatic depletion of both glycosaminoglycan (GAG) and collagen to determine the specific effect of each ECM component on T1rho and T2 relaxation times. A repeated measures design examines the effect of targeted enzymatic cartilage degradation (to isolate changes in cartilage biochemical composition, mechanical properties, and histology) on the T1rho and T2 relaxation times. These studies utilize confined compression for biomechanical analysis of cartilage, biochemical assays for the determination of S-GAG and collagen content, and histology for visualization of cartilage structure and composition. These measures are compared to the associated T1rho and T2 relaxation times. The results of these studies indicate that increases in T1rho relaxation times are correlated with S-GAG depletion, increased percent extractable collagen, decreases in mechanical strength of cartilage, and areas of OA defects (within which the previously mentioned biomechanical and biochemical conditions exist). Together, the results of these two studies validate T1rho and T2 quantitative imaging techniques for the in vivo diagnosis of early OA and the non-invasive assessment of cartilage biomechanical and biochemical properties.

Altered patterns of mechanical loading can result in morphological and compositional changes to cartilage that lead to cartilage degeneration. Quantitative MR imaging is a unique tool with the potential to provide insight into the relationship between biomechanics and the biophysical environment of cartilage, which is vital to better understanding the development of OA and degeneration of cartilage. The third study presented utilizes T1rho as a method for assessing localized changes to cartilage with dynamic activity. Sagittal MR images were obtained before and immediately after subjects completed a single legged hopping activity to dynamically load cartilage. A system of equally spaced grid points were registered to 3D surface mesh models of the tibial and femoral cartilage surfaces constructed from the MR images. T1rho relaxation times were then determined at each grid point to examine site-specific changes before and after exercise. A significant decrease in relaxation times was found after exercise in both the tibial plateau and the femoral condyle, with a greater decrease observed in the lateral femoral cartilage than in the medial femoral cartilage. No significant correlation between location and exercise was found. At each grid point, T1rho cartilage maps were also divided into superficial and deep regions of cartilage to determine where the greatest changes occurred. Ongoing analysis of the layer specific results will provide insight into where in the cartilage thickness these changes are most localized. The decrease in relaxation times after loading is likely due to the relative increase in PG content that results from the exudation of water from the cartilage ECM due to loading. This study demonstrates how T1rho may be used to non-invasively provide insight into the biophysical environment of cartilage with loading.

T1rho and T2 imaging represent a very powerful tool for the non-invasive assessment of articular cartilage. This work is significant in that it validates this method for the assessment of cartilage biomechanical and biochemical properties. Additionally, these methods can be used in future work to better understand how various risk factors contribute to OA development and to give valuable insight into the connection between biomechanical factors, biochemical composition, and the development of cartilage degeneration.

With growing numbers of increasingly younger patients suffering debilitating arthropathies, the need for simple models that recapitulate the complex interplay between distinct joint tissues, and grafts that emulate these joint structures in their biological properties and their strength have become more urgent. The objective of this study is to engineer constructs of multiple tissue types by controlling the morphogenetic factors that direct stem cell differentiation and tissue formation either exogenously or via transduction of expression vectors. Our hypothesis is that sequential changes in exogenous growth factor delivery and also scaffold-mediated inducible regulation of morphogenetic gene expression and signaling in 3D-constructs of murine iPSCs will lead to the formation of both bone and cartilage tissue types, both as separate tissues, and as osteochondral constructs. In the first study, osteochondral organoids were grown in a scaffold-free system from a single iPSC cell source, creating tissues containing a distinct core with the genetic and extracellular matrix profile of articular cartilage surrounded by a shell with the genetic and extracellular matrix profile of bone. In the second study, chondrogenic, osteogenic, and osteochondral tissue grafts were grown by scaffold-mediated lentiviral delivery of differentiation factors expressed both constitutively and in a temporally inducible manner. These constructs will provide an excellent platform to study diseases of the osteochondral junction, to screen pharmacologic therapies affecting both cartilage and bone tissue, and as a next step toward making an implantable osteochondral graft for the direct treatment of joint defects.

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