The effects of TGF-β1 and IGF-I on the biomechanics and cytoskeleton of single chondrocytes

SummaryObjectiveAscertaining how mechanical forces and growth factors mediate normal and pathologic processes in single chondrocytes can aid in developing strategies for the repair and replacement of articular cartilage destroyed by injury or disease. This study examined effects of transforming growth factor-β1 (TGF-β1) and insulin-like growth factor-I (IGF-I) on the biomechanics and cytoskeleton of single zonal chondrocytes.MethodSuperficial and middle/deep bovine articular chondrocytes were seeded on tissue culture treated plastic for 3 and 18h and treated with TGF-β1 (5ng/mL), IGF-I (100ng/mL), or a combination of TGF-β1 (5ng/mL)+IGF-I (100ng/mL). Single chondrocytes from all treatments were individually studied using viscoelastic creep testing and stained with rhodamine phalloidin for the F-actin cytoskeleton. Lastly, real-time RT-PCR was performed for β-actin.ResultsCreep testing demonstrated that all growth factor treatments stiffened cells. Image analysis of rhodamine phalloidin stained chondrocytes showed that cells from all growth factor groups had significantly higher fluorescence than controls, mirroring creep testing results. Growth factors altered cell morphology, since chondrocytes exposed to growth factors remained more rounded, exhibited greater cell heights, and were less spread. Finally, real-time RT-PCR revealed no significant effect of growth factor exposure on β-actin mRNA abundance. However, β-actin expression varied zonally, suggesting that this gene would be unsuitable as a PCR housekeeping gene.ConclusionsThese results indicate that TGF-β1 and IGF-I increase F-actin levels in single chondrocytes leading to stiffening of cells; however, there does not appear to be direct transcriptional regulation of unpolymerized β-actin. This suggests that the observed response is most likely due to signaling cross-talk between growth factor receptors and integrin/focal adhesion complexes

Multiscale Strategies for Cartilage Repair

This thesis uses a multiscale approach to identify and manipulate physiologic and in vitro developmental milieus towards the functional repair of articular cartilage. The overarching goals of this work are to improve knowledge of cartilage physiology and to enhance functional engineering of biologic cartilage replacements. Towards this end, assessment and modulation of cartilage phenotype were undertaken at multiple levels of complexity: gene transcription, cytoskeletal architecture, ion channels, single cells, extracellular matrix, intact tissue, and the whole joint. The first part of this thesis focused on probing cartilage phenotype at the single cell level. A quantitative single cell gene expression assay was developed and used to quantify cell-to-cell variability and the chondrocyte response to growth factors. Next, the viscoelastic compressive properties of single chondrocytes were measured and compared to cytoskeleton organization before and after growth factor exposure. It was found that growth factors increased matrix gene expression and induced cell stiffening in a time- and cartilage zone-dependent manner. The second part of this thesis investigated the modulation of the chondrocyte microenvironment for enhanced cartilage tissue engineering. Tissue constructs were grown in vitro using a chondrocyte self-assembly process. In one study, it was found that TRPV4 ion channel activation significantly increased cartilage matrix production and improved tensile properties in self-assembled constructs. In a second study, constructs were exposed to static or dynamic application of hypo-osmotic and hyper-osmotic stress. Static application of hyper-osmotic stress was found to improve construct compressive and tensile properties, and their corresponding biochemical mediators, significantly. A third study showed that treatment of constructs with ribose, an agent used for non-enzymatic glycation, produced enhanced tissue mechanics and biochemistry in a time-dependent manner. The third part of this thesis describes efforts to improve the potential clinical translatability of in vitro cartilage repair strategies. A technique was developed to decellularize xenogenic self-assembled constructs. Decellularization resulted in histologic and biochemical cell depletion with maintenance of tissue mechanical properties. Additionally, a comprehensive characterization of the major tissues of the immature knee joint revealed and reinforced important structure-function relationships that will inform future cartilage repair strategies. The total body of work contained in this thesis contributes significantly both to a basic understanding of cartilage physiology as well as to evolving strategies for cartilage repair. This thesis advances the field of cartilage tissue engineering by examining chondrocyte phenotype, the cell and tissue microenvironment, and avenues for clinical translation

Tensile Properties, Collagen Content, and Crosslinks in Connective Tissues of the Immature Knee Joint

The major connective tissues of the knee joint act in concert during locomotion to provide joint stability, smooth articulation, shock absorption, and distribution of mechanical stresses. These functions are largely conferred by the intrinsic material properties of the tissues, which are in turn determined by biochemical composition. A thorough understanding of the structure-function relationships of the connective tissues of the knee joint is needed to provide design parameters for efforts in tissue engineering.The objective of this study was to perform a comprehensive characterization of the tensile properties, collagen content, and pyridinoline crosslink abundance of condylar cartilage, patellar cartilage, medial and lateral menisci, cranial and caudal cruciate ligaments (analogous to anterior and posterior cruciate ligaments in humans, respectively), medial and lateral collateral ligaments, and patellar ligament from immature bovine calves. Tensile stiffness and strength were greatest in the menisci and patellar ligament, and lowest in the hyaline cartilages and cruciate ligaments; these tensile results reflected trends in collagen content. Pyridinoline crosslinks were found in every tissue despite the relative immaturity of the joints, and significant differences were observed among tissues. Notably, for the cruciate ligaments and patellar ligament, crosslink density appeared more important in determining tensile stiffness than collagen content.To our knowledge, this study is the first to examine tensile properties, collagen content, and pyridinoline crosslink abundance in a direct head-to-head comparison among all of the major connective tissues of the knee. This is also the first study to report results for pyridinoline crosslink density that suggest its preferential role over collagen in determining tensile stiffness for certain tissues

Impact of the COVID-19 Pandemic on Patient Preferences and Decision Making for Symptomatic Urolithiasis

Background: Pandemic restrictions have changed how patients approach symptomatic kidney stones. We used a mixed-methods digital ethnographic approach to evaluate social media discussions about patient concerns and preferences for urolithiasis care during the COVID-19 pandemic. Materials and Methods: We retrospectively analyzed kidney stone-related discussions on a large social media platform using qualitative analysis and natural language processing-based sentiment analysis. Posts were mined for demographic details, treatments pursued, and health care encounters. Pre-COVID-19 (January 1, 2020-February 29, 2020) and COVID-19 (March 1, 2020-June 1, 2020) posts were extracted from the popular online Reddit discussion board, r/KidneyStones, which is dedicated to discussions related to urolithiasis. Results: We extracted n = 649 posts (250 pre-COVID-19, 399 COVID-19); 150 from each cohort underwent thematic analysis and data extraction. Quantitative sentiment analysis was performed on 418 posts (179 pre-COVID-19, 239 COVID-19) that described stone-related decision making before intervention. Notable discussion themes during COVID-19 focused on barriers to care and concerns about stone management. Discussants exhibited more negative and anxious tones during COVID-19, based on sentiment analysis (p \u3c 0.01). Patient preferences shifted away from in-person visits and procedures (p \u3c 0.001). Mean reported stone size among those visiting emergency room (ER) increased from 5.1 to 10.5 mm (p \u3c 0.001). The proportion of discussants preferring conservative management with stones ≥10 mm increased (12.5% pre-COVID-19 vs 26% during COVID-19, p = 0.002). Opioid mentions increased from 9% to 27% of posts (p \u3c 0.001) and were most associated with conservative management discussions. Conclusions: Online discussion forums provide contemporaneous insight into patients\u27 experiences during a time when traditional patient-centered research methodologies are limited due to social distancing. During the pandemic, patients with symptomatic kidney stones expressed anxiety regarding outpatient encounters and reluctance toward procedural intervention. Patients opted instead for at-home conservative treatment beyond clinical guidelines and reserved ER visits for larger stones, potentially causing self-harm. Opioid discussions proliferated, an alarming consequence of the pandemic

Location Advantages, Governance Quality, Stock Market Development and Firm Characteristics as Antecedents of African M&As

This study explores firm- and country-specific antecedents of African M&As. We use one of the largest datasets to-date consisting of 1,490 unique African firms (11,183 firm-year observations) from 1996 to 2012. Our results suggest that improvements in time-varying country-level factors, including location advantages (market size, human capital and efficiency opportunities), national governance quality, and stock market development are associated with an increase in the volume of M&A activity. Consistent with the resource-curse paradox, high resource endowments are not associated with increased levels of M&A. In support of the management inefficiency but contrary to the traditional firm size hypotheses, African targets are generally characterised by declining stock returns and accounting profitability but are more likely to be larger firms; suggesting the presence of information asymmetry concerns in their selection. Notwithstanding, we find evidence of heterogeneity across countries with inconsistent support for established target prediction hypotheses. A model which combines firm- and country- specific factors better explains observed variations in African M&A activity

Sarcopenia Is an Independent Risk Factor for Proximal Junctional Disease Following Adult Spinal Deformity Surgery

Study Design: Retrospective cohort study. Objectives: Sarcopenia is a risk factor for medical complications following spine surgery. However, the role of sarcopenia as a risk factor for proximal junctional disease (PJD) remains undefined. This study evaluates whether sarcopenia is an independent predictor of proximal junctional kyphosis (PJK) and proximal junctional failure (PJF) following adult spinal deformity (ASD) surgery. Methods: ASD patients who underwent thoracic spine to pelvis fusion with 2-year clinical and radiographic follow-up were reviewed for development of PJK and PJD. Average psoas cross-sectional area on preoperative axial computed tomography or magnetic resonance imaging at L4 was recorded. Previously described PJD risk factors were assessed for each patient, and multivariate linear regression was performed to identify independent risk factors for PJK and PJF. Disease-specific thresholds were calculated for sarcopenia based on psoas cross-sectional area. Results: Of 32 patients, PJK and PJF occurred in 20 (62.5%) and 12 (37.5%), respectively. Multivariate analysis demonstrated psoas cross-sectional area to be the most powerful independent predictor of PJK (P = .02) and PJF (P = .009). Setting ASD disease–specific psoas cross-sectional area thresholds of <12 cm2 in men and <8 cm2 in women resulted in a PJF rate of 69.2% for patients below these thresholds, relative to 15.8% for those above the thresholds. Conclusions: Sarcopenia is an independent, modifiable predictor of PJK and PJF, and is easily assessed on standard preoperative computed tomography or magnetic resonance imaging. Surgeons should include sarcopenia in preoperative risk assessment and consider added measures to avoid PJF in sarcopenic patients

Exploiting stochastic dominance to generate abnormal stock returns

We construct zero cost portfolios based on second and third degree stochastic dominance and show that they produce systematic, statistically significant, abnormal returns. These returns are robust with respect to the single index CAPM, the Fama-French 3-factor model, the Carhart 4-factor model and the liquidity 5-factor model. They are also robust with respect to momentum portfolios, transactions costs, varying time periods and when broken down by a range of risk factors, such as firm size, leverage, age, return volatility, cash flow volatility and trading volume

IGF-1 does not moderate the time-dependent transcriptional patterns of key homeostatic genes induced by sustained compression of bovine cartilage

Objective To determine changes in chondrocyte transcription of a range of anabolic, catabolic and signaling genes following simultaneous treatment of cartilage with Insulin-like growth factor-1 (IGF-1) and ramp-and-hold mechanical compression, and compare with effects on biosynthesis. Methods Explant disks of bovine calf cartilage were slowly compressed (unconfined) over 3-min to their 1 mm cut-thickness (0%-compression) or to 50%-compression with or without 300 ng/ml IGF-1. Expression of 24 genes involved in cartilage homeostasis was measured using qPCR at 2, 8, 24, 32, 48 h after compression ±IGF-1. Clustering analysis was used to identify groups of co-expressed genes to further elucidate mechanistic pathways. Results IGF-1 alone stimulated gene expression of aggrecan and collagen II, but simultaneous 24h compression suppressed this effect. Compression alone up-regulated expression of matrix metalloproteinase (MMP)-3, MMP-13, a disintegrin and metalloproteinase with thrombospondin motif (ADAMTS)-5 and transforming growth factor (TGF)-β, an effect not reversed by simultaneous IGF-1 treatment. Temporal changes in expression following IGF-1 treatment were generally slower than that following compression. Clustering analysis revealed five distinct groups within which the pairings, tissue inhibitor of metalloproteinase (TIMP)-3 and ADAMTS-5, MMP-1 and IGF-2, and IGF-1 and Collagen II, were all robustly co-expressed, suggesting inherent regulation and feedback in chondrocyte gene expression. While aggrecan synthesis was transcriptionally regulated by IGF-1, inhibition of aggrecan synthesis by sustained compression appeared post-transcriptionally regulated. Conclusion Sustained compression markedly altered the effects of IGF-1 on expression of genes involved in cartilage homeostasis, while IGF-1 was largely unable to moderate the transcriptional effects of compression alone. The demonstrated co-expressed gene pairings suggest a balance of anabolic and catabolic activity following simultaneous mechanical and growth factor stimuli.National Institutes of Health (U.S.) (grant R01-AR33236)National Institutes of Health (U.S.) (grant R01-HG003352)National Institutes of Health (U.S.) (grant P42-ES04699)National Institutes of Health (U.S.) (grant T32-EB006348

Markov Chain Models to Estimate the Premium for Extended Hedge Fund Lockups

A lockup period for investment in a hedge fund is a time period after making the investment during which the investor cannot freely redeem his investment. It is routine to have a one-year lockup period, but recently the requested lockup periods have grown longer. We estimate the premium for such extended lockup, taking the point of view of a manager of a fund of funds, who has to choose between two investments in similar funds in the same strategy category, the first having a one-year lockup and the second having an n-year lockup. Assuming that the manager will rebalance his portfolio of hedge funds on a yearly basis, if permitted, we define the annual lockup premium as the difference between the rates of return from these investments. We develop a Markov chain model to estimate this lockup premium. By solving systems of equations, we fit the Markov chain transition probabilities to three directly observable hedge fund performance measures: the persistence of return, the variance of return and the hedge-fund death rate. The model quantifies the way the lockup premium depends on these parameters. Data from the TASS database are used to estimate the persistence, which is found to be statistically significant