Cartilage develops specific forms, sizes, and functional properties under the guidance of naturally present biophysical and biochemical stimuli during in vivo growth and maturation. Analogously, controlling the physical and biochemical environment of cartilage during culture may facilitate the in vitro manipulation of tissue properties. This dissertation explores flexural deformation as a physical stimulus capable of inducing changes in the free- swelling shape of articular cartilage explants and specific biochemical agents in modulating the shape plasticity, size, and maturity of cartilage through altered matrix metabolism and remodeling. The bulk of the work described herein was conducted with a model system of immature articular cartilage explants from bovine calves, with consideration of the potential translation of the experimental approaches to other native and engineered chondral tissues. A system for analyzing mechanically- induced reshaping of cartilage was developed through the creation of a novel bioreactor to apply flexure to cartilage explants and a means of sensitively quantifying specimen shape. Using these tools, static flexure was shown to produce significant changes in the free-swelling shape of cartilage in a duration-dependent manner. The flexural stimulus was characterized with micromechanical strain analysis techniques, and the observed asymmetrical strain distributions within the tissue were found to be a result of the pronounced tension-compression nonlinearity of cartilage mechanical properties. Inhibitors of chondrocyte and cartilage extracellular matrix metabolism were used to probe potential mechanisms of cartilage reshaping, and findings indicated that the process of reshaping was largely independent of chondrocyte-mediated matrix synthesis or remodeling. However, a strong temperature-dependent response suggested a possible biophysical mechanism of matrix remodeling. Altering matrix metabolism and remodeling by supplementing cultures with specific biochemical agents or growth factors resulted in changes in tissue composition with respect to the predominant collagen and glycosaminoglycan matrix components. These treatments differentially modulated cartilage volume and functional properties, including shape plasticity and mechanical properties in compression. These studies further elucidate the regulation of the shape, size, and maturity of cartilage by biophysical and biochemical stimuli. The tools and techniques developed here may be translatable to creating chondral grafts with desired properties for joint repair or craniofacial reconstruction

BACKGROUND:Patients can present with symptomatic gallbladder disease after cholecystectomy due to a remnant gallbladder. This is a rare problem and challenging diagnosis with limited prior characterization; thus, we present a large series of patients with a gallbladder remnant. METHODS:A retrospective review was performed of all patients presenting with symptomatic gallbladder remnant at a tertiary care center from 2002 to 2016. Data on presenting symptoms, diagnostic tests, treatments, and follow-up were collected. RESULTS:Thirty-one patients diagnosed and treated for a symptomatic gallbladder remnant were identified. The most common presenting symptoms included right upper quadrant pain (87%) and nausea (55%). The median time from symptom presentation to definitive diagnosis was 60 days. Diagnostic modalities utilized in the evaluation of these patients demonstrated that endoscopic retrograde cholangiopancreatography and magnetic resonance cholangiopancreatography were effective with sensitivities of 85% and 90%, respectively. Twenty-three (76.2%) patients underwent completion cholecystectomy, which proved to be definitive treatment. Additionally, eight (25.8%) patients were non-operative candidates and underwent endoscopic retrograde cholangiopancreatography and sphincterotomy, three of whom developed recurrent symptoms. CONCLUSION:A symptomatic gallbladder remnant after cholecystectomy is infrequently seen; however, the diagnosis should be considered in patients with recurrent biliary symptoms after cholecystectomy. Completion cholecystectomy can be challenging but is highly effective for definitive treatment.

Rat posterolateral lumbar fusion (PLF) models have been used to assess the safety and effectiveness of new bone substitutes and osteoinductive growth factors using palpation, radiography, micro-computed tomography (μCT), and histology as standard methods to evaluate spinal fusion. Despite increased numbers of PLF studies involving alternative bone substitutes and growth factors, the quantitative assessment of treatment efficacy during spinal motion has been limited. The purpose of this study was to evaluate the effect of spinal fusion on lumbar spine segment stability during lateral bending using a μCT-based three-dimensional (3D) kinematic analysis in the rat PLF model. Fourteen athymic male rats underwent PLF surgery at L4/5 and received bone grafts harvested from the ilium and femurs of syngeneic rats (Isograft, n=7) or no graft (Sham, n=7). At 8 weeks after the PLF surgery, spinal fusion was assessed by manual palpation, plain radiography, μCT, and histology. To determine lumbar segmental motions at the operated level during lateral bending, 3D kinematic analysis was performed. The Isograft group, but not the Sham group, showed spinal fusion on manual palpation (6/7), solid fusion mass in radiographs (6/7), as well as bone bridging in μCT and histological images (5/7). Compared to the Sham group, the Isograft group revealed limited 3D lateral bending angular range of motion and lateral translation during lateral bending at the fused segment where disc height narrowing was observed. This μCT-based 3D kinematic analysis can provide a quantitative assessment of spinal fusion in a rat PLF model to complement current gold standard methods used for efficacy assessment of new therapeutic approaches.

Parkinson's disease (PD) is a multi-stage neurodegenerative disorder with largely unknown etiology. Recent findings have identified PD-associated autoimmune features including roles for T cells. To further characterize the role of T cells in PD, we performed RNA sequencing on PBMC and peripheral CD4 and CD8 memory T cell subsets derived from PD patients and age-matched healthy controls. When the groups were stratified by their T cell responsiveness to alpha-synuclein (α-syn) as a proxy for an ongoing inflammatory autoimmune response, the study revealed a broad differential gene expression profile in memory T cell subsets and a specific PD associated gene signature. We identified significant enrichment of transcriptomic signatures previously associated with PD, including for oxidative stress, phosphorylation, autophagy of mitochondria, cholesterol metabolism and inflammation, and the chemokine signaling proteins CX3CR1, CCR5, and CCR1. In addition, we identified genes in these peripheral cells that have previously been shown to be involved in PD pathogenesis and expressed in neurons, such as LRRK2, LAMP3, and aquaporin. Together, these findings suggest that features of circulating T cells with α-syn-specific responses in PD patients provide insights into the interactive processes that occur during PD pathogenesis and suggest potential intervention targets.

Background and objectives

Parkinson's disease (PD) is associated with a heightened inflammatory state, including activated T cells. However, it is unclear whether these PD T cell responses are antigen specific or more indicative of generalized hyperresponsiveness. Our objective was to measure and compare antigen-specific T cell responses directed towards antigens derived from commonly encountered human pathogens/vaccines in patients with PD and age-matched healthy controls (HC).

Methods

Peripheral blood mononuclear cells (PBMCs) from 20 PD patients and 19 age-matched HCs were screened. Antigen specific T cell responses were measured by flow cytometry using a combination of the activation induced marker (AIM) assay and intracellular cytokine staining.

Results

Here we show that both PD patients and HCs show similar T cell activation levels to several antigens derived from commonly encountered human pathogens/vaccines in the general population. Similarly, we also observed no difference between HC and PD in the levels of CD4 and CD8 T cell derived cytokines produced in response to any of the common antigens tested. These antigens encompassed both viral (coronavirus, rhinovirus, respiratory syncytial virus, influenza, cytomegalovirus) and bacterial (pertussis, tetanus) targets.

Conclusions

These results suggest the T cell dysfunction observed in PD may not extend itself to abnormal responses to commonly encountered or vaccine-target antigens. Our study supports the notion that the targets of inflammatory T cell responses in PD may be more directed towards autoantigens like α-synuclein (α-syn) rather than common foreign antigens.