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Fourier transform infrared spectroscopy (FTIRS) can provide rich information on the composition and content of samples, enabling the detection of subtle changes in tissue composition and structure. This study represents the first application of FTIRS to investigate cartilage under microgravity. Simulated microgravity cartilage model was firstly established by tail-suspension (TS) for 7, 14 and 21 days, which would be compared to control samples. A self-developed hollow optical fiber attenuated total reflection (HOF-ATR) probe coupled with a FTIR spectrometer was used for the spectral acquisition of cartilage samples in situ, and one-way analysis of variance (ANOVA) was employed to analyze the changes in the contents of cartilage matrix at different stages. The results indicate that cartilage degenerates in microgravity, the collagen content gradually decreases with the TS time, and the structure of collagen fibers changes. The trends of proteoglycan content and collagen integrity show an initial decrease followed by an increase, ultimately significantly decreasing. The findings provide the basis for the cartilage degeneration in microgravity with TS time, which must be of real significance for space science and health detection., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Background: To determine whether a decrease in serum (1,3)-β-D-glucan (BDG) was associated with reduced mortality and to investigate the performance of BDG downslope in predicting clinical outcome in invasive candidiasis., Methods: Observational cohort study in ICU patients over a ten-year period (2012-2022) in Italy. Proven invasive candidiasis with at least 2 BDG determinations were considered., Results: In the study population of 103 patients (age 47 [35-62] years, SAPS II score 67 [52-77]) 68 bloodstream and 35 intrabdominal infections were recorded. Serial measurements showed that in 54 patients BDG decreased over time (BDG downslope group) while in 49 did not (N-BDG downslope group). Candida albicans was the pathogen most frequently isolated (61%) followed by C. parapsilosis (17%) and C. glabrata (12%), in absence of any inter-group difference. Invasive candidiasis related mortality was lower in BDG downslope than in N-BDG downslope group (17% vs 53%, p < 0.01). The multivariate Cox regression analysis showed the association of septic shock at infection occurrence and chronic liver disease with invasive candidiasis mortality (HR [95% CI] 3.24 [1.25-8.44] p = 0.02 and 7.27 [2.33-22.66] p < 0.01, respectively) while a BDG downslope was the only predictor of survival (HR [95% CI] 0.19 [0.09-0.43] p < 0.01). The area under the receiver operator characteristic curve for the performance of BDG downslope as predictor of good clinical outcome was 0.74 (p = 0.02) and our model showed that a BDG downslope > 70% predicted survival with both specificity and positive predictive value of 100%., Conclusions: A decrease in serum BDG was associated with reduced mortality and a steep downslope predicted survival with high specificity in invasive candidiasis., (© 2024. The Author(s).)

Background: Strategies for articular cartilage repair need to take into account topographical differences in tissue composition and architecture to achieve durable functional outcome. These have not yet been investigated in the equine stifle., Objectives: To analyse the biochemical composition and architecture of three differently loaded areas of the equine stifle. We hypothesise that site differences correlate with the biomechanical characteristics of the cartilage., Study Design: Ex vivo study., Methods: Thirty osteochondral plugs per location were harvested from the lateral trochlear ridge (LTR), the distal intertrochlear groove (DITG) and the medial femoral condyle (MFC). These underwent biochemical, biomechanical and structural analysis. A linear mixed model with location as a fixed factor and horse as a random factor was applied, followed by pair-wise comparisons of estimated means with false discovery rate correction, to test for differences between locations. Correlations between biochemical and biomechanical parameters were tested using Spearman's correlation coefficient., Results: Glycosaminoglycan content was different between all sites (estimated mean [95% confidence interval (CI)] for LTR 75.4 [64.5, 88.2], for intercondylar notch (ICN) 37.3 [31.9, 43.6], for MFC 93.7 [80.1109.6] μg/mg dry weight), as were equilibrium modulus (LTR2.20 [1.96, 2.46], ICN0.48 [0.37, 0.6], MFC1.36 [1.17, 1.56] MPa), dynamic modulus (LTR7.33 [6.54, 8.17], ICN4.38 [3.77, 5.03], MFC5.62 [4.93, 6.36] MPa) and viscosity (LTR7.49 [6.76, 8.26], ICN16.99 [15.88, 18.14], MFC8.7 [7.91,9.5]°). The two weightbearing areas (LTR and MCF) and the non-weightbearing area (ICN) differed in collagen content (LTR 139 [127, 152], ICN176[162, 191], MFC 127[115, 139] μg/mg dry weight), parallelism index and angle of collagen fibres. The strongest correlations were between proteoglycan content and equilibrium modulus (r: 0.642; p: 0.001), dynamic modulus (r: 0.554; p < 0.001) and phase shift (r: -0.675; p < 0.001), and between collagen orientation angle and equilibrium modulus (r: -0.612; p < 0.001), dynamic modulus (r: -0.424; p < 0.001) and phase shift (r: 0.609; p < 0.001)., Main Limitations: Only a single sample per location was analysed., Conclusions: There were significant differences in cartilage biochemical composition, biomechanics and architecture between the three differently loaded sites. The biochemical and structural composition correlated with the mechanical characteristics. These differences need to be acknowledged by designing cartilage repair strategies., (© 2023 The Authors. Equine Veterinary Journal published by John Wiley & Sons Ltd on behalf of EVJ Ltd.)

Introduction: Aberrant gait variability has been observed after anterior cruciate ligament reconstruction (ACLR), yet it remains unknown if gait variability is associated with early changes in cartilage composition linked to osteoarthritis development. Our purpose was to determine the association between femoral articular cartilage T1ρ magnetic resonance imaging relaxation times and gait variability., Methods: T1ρ magnetic resonance imaging and gait kinematics were collected in 22 ACLR participants (13 women; 21 ± 4 yr old; 7.52 ± 1.43 months post-ACLR). Femoral articular cartilage from the ACLR and uninjured limbs were segmented into anterior, central, and posterior regions from the weight-bearing portions of the medial and lateral condyles. Mean T1ρ relaxation times were extracted from each region and interlimb ratios (ILR) were calculated (i.e., ACLR/uninjured limb). Greater T1ρ ILR values were interpreted as less proteoglycan density (worse cartilage composition) in the injured limb compared with the uninjured limb. Knee kinematics were collected at a self-selected comfortable walking speed on a treadmill with an eight-camera three-dimensional motion capture system. Frontal and sagittal plane kinematics were extracted, and sample entropy was used to calculate kinematic variability structure (KV structure ). Pearson's product-moment correlations were conducted to determine the associations between T1ρ and KV structure variables., Results: Lesser frontal plane KV structure was associated with greater mean T1ρ ILR in the anterior lateral ( r = - 0.44, P = 0.04) and anterior medial condyles ( r = - 0.47, P = 0 .03). Lesser sagittal plane KV structure was associated with greater mean T1ρ ILR in the anterior lateral condyle ( r = - 0.47, P = 0.03)., Conclusions: The association between less KV structure and worse femoral articular cartilage proteoglycan density suggests a link between less variable knee kinematics and deleterious changes joint tissue changes. The findings suggest that less knee kinematic variability structure is a mechanism linking aberrant gait to early osteoarthritis development., (Copyright © 2023 by the American College of Sports Medicine.)

Intervertebral disc degeneration (IDD) is closely related to changes in the intervertebral disc (IVD) composition and the resulting viscoelastic properties. IDD is a severe condition because it decreases the disc's ability to resist mechanical loads. Our research aims to understand IDD at the cellular level, specifically the changes in the viscoelastic properties of the nucleus pulposus (NP), which are poorly understood. This study employed a system integrating nanoindentation with Raman spectrometry to correlate biomechanics with subtle changes in the biochemical makeup of the NP. The characterization was, in turn, correlated with the degenerative severity of IVD as assessed using magnetic resonance imaging (MRI) of different patients with spinal stenosis, degenerative spondylolisthesis, and degenerative scoliosis. It is shown that there is an increase in the crosslinking ratio in collagen, a reduction in proteoglycan, and a build-up of minerals upon the rise in the severity level of the disc damage in the NP. Assessment of mechanical characteristics reveals that the increasing disc degeneration makes the NP lose its elasticity, becoming more viscous. This shows that the tissue undergoes abnormalities in weight-bearing ability, which contributes to spinal instability. The correlation of the individual discs shows that grades III and IV have similarities in the changes of Amide I and III toward the storage modulus. In contrast, grades IV and V correlate with mineralization toward the storage modulus. Reduction of proteoglycan has the highest impact on the changes of the storage modulus in all grades of IDD. Connecting compositional alterations to IVD micromechanics at various degrees of degeneration expands our understanding of tissue behavior and provides critical insight into clinical diagnostics, treatment, and tissue engineering., (© 2022 Wiley Periodicals LLC.)

Fibrillar collagen-integrin interactions in the extracellular matrix (ECM) regulate a multitude of cellular processes and cell signalling. Collagen I fibrils serve as the molecular scaffolding for connective tissues throughout the human body and are the most abundant protein building blocks in the ECM. The ECM environment is diverse, made up of several ECM proteins, enzymes, and proteoglycans. In particular, glycosaminoglycans (GAGs), anionic polysaccharides that decorate proteoglycans, become depleted in the ECM with natural aging and their mis-regulation has been linked to cancers and other diseases. The impact of GAG depletion in the ECM environment on collagen I protein interactions and on mechanical properties is not well understood. Here, we integrate ELISA protein binding assays with liquid high-resolution atomic force microscopy (AFM) to assess the effects of GAG depletion on the interaction of collagen I fibrils with the integrin α2I domain using separate rat tails. ELISA binding assays demonstrate that α2I preferentially binds to GAG-depleted collagen I fibrils in comparison to native fibrils. By amplitude modulated AFM in air and in solution, we find that GAG-depleted collagen I fibrils retain structural features of the native fibrils, including their characteristic D-banding pattern, a key structural motif. AFM fast force mapping in solution shows that GAG depletion reduces the stiffness of individual fibrils, lowering the indentation modulus by half compared to native fibrils. Together these results shed new light on how GAGs influence collagen I fibril-integrin interactions and may aid in strategies to treat diseases that result from GAG mis-regulation., (© 2022 The Protein Society.)

Aims: (a) The aim of this study was to investigate both the formation of dense connective tissue within the dental pulp, and its association with pulpal inflammation in teeth with advanced carious lesions; and (b) to investigate in vitro whether inflammation affects the expression of markers related to chondrogenesis/osteogenesis in pulp cells., Materials and Methods: Radiology and Histology: Forty-six teeth with advanced carious lesions were radiographically investigated for intra-pulpal radiodense structures. Specimens were processed for histology and stained with haematoxylin/eosin and proteoglycan-specific stains. The intra-pulpal connective tissue was scored as pulp stones or ectopic connective tissue. Cell culture: pulpal cells from human third molars (n = 5) were cultured in chondrogenic medium +/- TLR2/4 agonists. Expression of the genes IL6, TLR2/4, SOX9, COL1A1, COL2A1, TGFB1, RUNX2 and ALPL was assessed by qPCR. Proteoglycan content within cultures was assessed spectrophotometrically., Results: Radiodense structures were discovered in about half of all pulps. They were associated with ectopic connective tissue (χ 2  = 8.932, p = .004, OR = 6.80, 95% CI: [1.84, 25.19]) and with pulp stones (χ 2  = 12.274, df = 1, p < .001, OR = 22.167, 95% CI: [2.57, 200.00]). The morphology of the ectopic tissue resembled cartilage and was associated with inflammatory infiltration of the pulp (χ 2  = 10.148, p = .002, OR = 17.77, 95% CI: [2.05, 154.21]). After continuous stimulation of cultured cells with TLR2/4 agonists, the expression of two inflammatory markers increased: IL6 at Days 7 (p = .020) and 14 (p = .008); TLR2 at Days 7 (p = .023) and 14 (p = .009). Similarly, expression of chondrogenic markers decreased: SOX9 at Day 14 (p = .035) and TGFB1 at Day 7 (p = .004), and the osteogenic marker COL1A1 at Day 7 (p = .007). Proteoglycan content did not differ between unstimulated and stimulated cells., Conclusions: Ectopic connective tissue resembling cartilage can form in teeth affected by advanced carious lesions. This tissue type is radiographically visible and is associated with inflammatory infiltration of the pulp. Although TLR2/4 agonists led to an inflammatory response in cell culture of pulp cells, the effect on the expression of osteogenic/chondrogenic markers was limited, suggesting that immune cells are needed for connective tissue formation in vivo., (© 2022 The Authors. International Endodontic Journal published by John Wiley & Sons Ltd on behalf of British Endodontic Society.)

Proinflammatory cytokines target vascular endothelial cells during COVID-19 infections. In particular, the endothelial glycocalyx (eGC), a proteoglycan-rich layer on top of endothelial cells, was identified as a vulnerable, vasoprotective structure during infections. Thus, eGC damage can be seen as a hallmark in the development of endothelial dysfunction and inflammatory processes. Using sera derived from patients suffering from COVID-19, we could demonstrate that the eGC became progressively worse in relation to disease severity (mild vs severe course) and in correlation to IL-6 levels. This could be prevented by administering low doses of spironolactone, a well-known and highly specific aldosterone receptor antagonist. Our results confirm that SARS-CoV-2 infections cause eGC damage and endothelial dysfunction and we outline the underlying mechanisms and suggest potential therapeutic options., (© 2022. The Author(s).)

Proteoglycans are heterogeneous macromolecular glycoconjugates that orchestrate many important cellular processes. While much attention has focused on the poly-sulfated glycosaminoglycan chains that decorate proteoglycans, other important elements of their architecture, such as core proteins and membrane localization, have garnered less emphasis. Hence, comprehensive structure-function relationships that consider the replete proteoglycan architecture as glycoconjugates are limited. Here we present an extensive approach to study proteoglycan structure and biology by fabricating defined semisynthetic modular proteoglycans that can be tailored for cell surface display. The expression of proteoglycan core proteins with unnatural amino acids permits bioorthogonal click chemistry with functionalized glycosaminoglycans for methodical dissection of the parameters required for optimal binding and function of various proteoglycan-binding proteins. We demonstrate that these sophisticated materials can recapitulate the functions of native proteoglycan ectodomains in mouse embryonic stem cell differentiation and cancer cell spreading while permitting the analysis of the contributing architectural elements toward function., (© 2022. The Author(s), under exclusive licence to Springer Nature America, Inc.)

Antiplatelet and anticoagulant drugs are classified antithrombotic agents with the purpose to reduce blood clot formation. For a successful treatment of many known complex cardiovascular diseases driven by platelet and/or coagulation activity, the need of more than one antithrombotic agent is inevitable. However, combining drugs with different mechanisms of action enhances risk of bleeding. Dual anticoagulant and antiplatelet (APAC), a novel semisynthetic antithrombotic molecule, provides both anticoagulant and antiplatelet properties in preclinical studies. APAC is entering clinical studies with this new exciting approach to manage cardiovascular diseases. For a better understanding of the biological function of APAC, comprehensive knowledge of its structure is essential. In this study, atomic force microscopy (AFM) was used to characterize APAC according to its structure and to investigate the molecular interaction of APAC with von Willebrand factor (VWF), since specific binding of APAC to VWF could reduce platelet accumulation at vascular injury sites. By the optimization of drop-casting experiments, we were able to determine the volume of an individual APAC molecule at around 600 nm 3 , and confirm that APAC forms multimers, especially dimers and trimers under the experimental conditions. By studying the drop-casting behavior of APAC and VWF individually, we depictured their interaction by using an indirect approach. Moreover, in vitro and in vivo conducted experiments in pigs supported the AFM results further. Finally, the successful adsorption of APAC to a flat gold surface was confirmed by using photothermal-induced resonance, whereby attenuated total reflection-Fourier transform infrared spectroscopy (ATR-FTIR) served as a reference method., (© 2021. The Author(s).)

Objective: Spectroscopic techniques, such as near-infrared (NIR) spectroscopy, are gaining significant research interest for characterizing connective tissues, particularly articular cartilage, because there is still a largely unmet need for rapid, accurate and objective methods for assessing tissue integrity in real-time during arthroscopic surgery. This study aims to identify the NIR spectral range that is optimal for characterizing cartilage integrity by ( a ) identifying the contribution of its major constituents (collagen and proteoglycans) to its overall spectrum using proxy constituent models and ( b ) determining constituent-specific spectral contributions that can be used for assessment of cartilage in its physiological state., Design: The NIR spectra of cartilage matrix constituent models were measured and compared with specific molecular components of organic compounds in the NIR spectral range in order to identify their bands and molecular assignments. To verify the identified bands, spectra of the model compounds were compared with those of native cartilage. Since water obscures some bands in the NIR range, spectral measurements of the native cartilage were conducted under conditions of decreasing water content to amplify features of the solid matrix components. The identified spectral bands were then compared and examined in the resulting spectra of the intact cartilage samples., Results: As water was progressively eliminated from cartilage, the specific contribution of the different matrix components was observed to correspond with those identified from the proxy cartilage component models., Conclusion: Spectral peaks in the regions 5500 to 6250 cm -1 and 8100 to 8600 cm -1 were identified to be effective for characterizing cartilage proteoglycan and collagen contents, respectively.

This study compared the performance of near-infrared spectroscopy (NIRS) models on fresh and freeze-dried beef muscle samples to predict intramuscular connective tissue (IMCT) components and to determine whether the accuracy of the models differed among different muscles from beef cattle. The hypothesis was that the water content of muscle samples would negatively influence the accuracy of the models, which would differ among muscles. Fresh and freeze-dried samples (n = 171) of four muscles were used to develop NIRS models to predict the contents IMCT. For the total collagen content, the standard error of cross validation (SECV) for model using freeze-dried samples (0.75 mg OH-prol/g DM) was lower than that for model using fresh samples (0.84 mg OH-prol/g DM). For cross-links and proteoglycans, the SECV for models using fresh sample spectra was lower than that for models using freeze-dried sample spectra. The accuracy of the prediction of the models also differed among predicted muscle types., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Meniscal tears are a common orthopedic injury, yet their healing is difficult to assess post-operatively. This impedes clinical decisions as the healing status of the meniscus cannot be accurately determined non-invasively. Thus, the objectives of this study were to explore the utility of a goat model and to use quantitative magnetic resonance imaging (MRI) techniques, histology, and biomechanical testing to assess the healing status of surgically induced meniscal tears. Adiabatic T1ρ, T2, and T2* relaxation times were quantified for both operated and control menisci ex vivo. Histology was used to assign healing status, assess compositional elements, and associate healing status with compositional elements. Biomechanical testing determined the failure load of healing lesions. Adiabatic T1ρ, T2, and T2* were able to quantitatively identify different healing states. Histology showed evidence of diminished proteoglycans and increased vascularity in both healed and non-healed menisci with surgically induced tears. Biomechanical results revealed that increased healing (as assessed histologically and on MRI) was associated with greater failure load. Our findings indicate increased healing is associated with greater meniscal strength and decreased signal differences (relative to contralateral controls) on MRI. This indicates that quantitative MRI may be a viable method to assess meniscal tears post-operatively., (© 2021. The Author(s).)

Cutaneous leishmaniasis (CL) is one of the illnesses caused by Leishmania parasite infection, which can be asymptomatic or severe according to the infecting Leishmania strain. CL is commonly diagnosed by directly detecting the parasites or their DNA in tissue samples. New diagnostic methodologies target specific proteins (biomarkers) secreted by the parasite during the infection process. However, specific bioreceptors for the in vivo or in vitro detection of these novel biomarkers are rather limited in terms of sensitivity and specificity. For this reason, we here introduce three novel peptides as bioreceptors for the highly sensitive and selective identification of acid phosphatase (sAP) and proteophosphoglycan (PPG), which have a crucial role in leishmaniasis infection. These high-affinity peptides have been designed from the conservative domains of the lectin family, holding the ability to interact with the biological target and produce the same effect than the original protein. The synthetic peptides have been characterized and the affinity and kinetic constants for their interaction with the targets (sAP and PPG) have been determined by a surface plasmon resonance biosensor. Values obtained for K D are in the nanomolar range, which is comparable to high-affinity antibodies, with the additional advantage of a high biochemical stability and simpler production. Pep2854 exhibited a high affinity for sAP (K D  = 1.48 nM) while Pep2856 had a good affinity for PPG (K D 1.76 nM). This study evidences that these peptidomimetics represent a novel alternative tool to the use of high molecular weight proteins for biorecognition in the diagnostic test and biosensor devices for CL.

Objective: The clinical success of focal metallic resurfacing implants depends largely on the friction between implant and opposing cartilage. Therefore, the present study determines the lubricating ability of the synovial fluid components hyaluronic acid (HA), proteoglycan 4 (PRG4) and a surface-active phospholipid (1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine, POPC), on the articulation between cartilage and a Cobalt Chromium Molybdenum (CoCrMo) implant surface, compared with two cartilage surfaces., Methods: A ring-on-disk geometry was used to perform repeated friction measurements at physiologically relevant velocities (6 and 60 mm/s) using lubricants with an increasing number of components present. Shear measurements were performed in order to evaluate the viscosity. To ensure that it is clinically relevant to explore the effect of these components, the presence of PRG4 in synovial fluid obtained from primary and revision knee and hip implant surgeries was examined., Results: PRG4 in the presence of HA was found to significantly reduce the coefficient of friction for both cartilage-cartilage and cartilage-CoCrMo interface. This is relevant, as it was also demonstrated that PRG4 is still present at the time of revision surgeries. The addition of POPC had no effect for either configurations. HA increased the viscosity of the lubricating fluid by one order of magnitude, while PRG4 and POPC had no effect., Conclusion: The present study demonstrates the importance of selecting the appropriate lubrication solution to evaluate implant materials with biotribology tests. Because PRG4 is a key component for reducing friction between cartilage and an opposing surface, developing coatings which bind PRG4 is recommended for cartilage resurfacing implants., (Copyright © 2021 The Authors. Published by Elsevier Ltd.. All rights reserved.)

Cranial cruciate ligament disease and rupture (CCLD/R) is one of the most common orthopaedic conditions in dogs, eventually leading to osteoarthritis of the stifle joint. Certain dog breeds such as the Staffordshire bull terrier have an increased risk of developing CCLD/R. Previous studies into CCLD/R have found that glycosaminoglycan levels were elevated in cranial cruciate ligament (CCL) tissue from high-risk breeds when compared to the CCL from a low-risk breed to CCLD/R. Our objective was to determine specific proteoglycans/glycosaminoglycans in the CCL and to see whether their content was altered in dog breeds with differing predispositions to CCLD/R. Disease-free CCLs from Staffordshire bull terriers (moderate/high-risk to CCLD/R) and Greyhounds (low-risk to CCLD/R) were collected and key proteoglycan/glycosaminoglycans were determined by semi-quantitative Western blotting, quantitative biochemistry, quantitative reverse transcription polymerase chain reaction, and immunohistochemistry. Gene expression of fibromodulin (P = 0.03), aggrecan (P = 0.0003), and chondroitin-6-sulphate stubs (P = 0.01) were significantly increased, and for fibromodulin this correlated with an increase in protein content in Staffordshire bull terriers compared to Greyhound CCLs (P = 0.02). Decorin (P = 0.03) and ADAMTS-4 (P = 0.04) gene expression were significantly increased in Greyhounds compared to Staffordshire bull terrier CCLs. The increase of specific proteoglycans and glycosaminoglycans within the Staffordshire bull terrier CCLs may indicate a response to higher compressive loads, potentially altering their risk to traumatic injury. The higher decorin content in the Greyhound CCLs is essential for maintaining collagen fibril strength, while the increase of ADAMTS-4 indicates a higher rate of turnover helping to regulate normal CCL homeostasis in Greyhounds., (Copyright © 2021 Elsevier Ltd. All rights reserved.)

Aims: Severe reduction in nephron numbers that are characteristic of renal hypodysplasia (RHD) are one of the cause of childhood chronic kidney disease (CKD). Glomerular hyperfiltration, glomerular hypertrophy, progressive glomerular scarring, and interstitial fibrosis due to reduced nephron number are risk factors for CKD. In recent years, studies on specific markers for early diagnosis of renal failure and mortality have been carried out. The objectives of this study were to identify serum and urinary endocan levels that are expressed in glomerular endothelial cells and tubular epithelial cells in RHD., Materials and Methods: 29 children with RHD were compared to 26 healthy controls in terms of serum and urinary endocan levels., Results: The mean serum endocan level in the RHD group and the control group was 700.72 ± 323.19 and 426.86 ± 233.14 pg/mL, respectively. The mean serum endocan level was significantly higher (p = 0.003) in the RHD group. The mean urinary endocan level in the RHD group was 63.62 ± 92.46 pg/mL, and in the control group it was 80.26 ± 142.49 pg/mL. The mean urinary endocan level did not change between groups (p = 0.95). There was also a significant correlation between serum endocan level and uric acid level in the study group (r = 0.325, p = 0.028)., Conclusion: To our knowledge, this was the first study that evaluated serum and urinary endocan levels in children with RHD. Although serum endocan level was found to be significantly higher in patients with RHD, further studies are needed to validate whether endocan could serve as a marker of poor renal prognosis in RHD.

Background: Environmental conditions strongly influence the healing capacity of connective tissues. Well-vascularized extrasynovial tendons typically undergo a robust wound-healing process following transection and repair. In contrast, avascular intrasynovial tendons do not mount an effective repair response. The current study tests the hypothesis that flexor tendons, as a function of their synovial environment, exhibit unique inflammatory, angiogenic, and metabolic responses to injury and repair., Methods: Flexor tendons present a distinct opportunity to test the study hypothesis, as they have proximal regions that are extrasynovial and distal regions that are intrasynovial. In an internally controlled study design, the second and fifth forepaw flexor tendons were transected and repaired in either the extrasynovial or the intrasynovial anatomical region. Histological, gene expression, and proteomics analyses were performed at 3 and 7 days to define the early biological events that drive synovial environment-dependent healing responses., Results: Uninjured intrasynovial tendons were avascular, contained high levels of proteoglycans, and expressed inflammatory factors, complement proteins, and glycolytic enzymes. In contrast, extrasynovial tendons were well vascularized, contained low levels of proteoglycans, and were enriched in inflammation inhibitors and oxidative phosphorylation enzymes. The response to injury and repair was markedly different between the 2 tendon regions. Extrasynovial tendons displayed a robust and rapid neovascularization response, increased expression levels of complement proteins, and an acute shift in metabolism to glycolysis, whereas intrasynovial tendons showed minimal vascularity and muted inflammatory and metabolic responses., Conclusions: The regional molecular profiles of intact and healing flexor tendons revealed extensive early differences in innate immune response, metabolism, vascularization, and expression of extracellular matrix as a function of the synovial environment. These differences reveal mechanisms through which extrasynovial tendons heal more effectively than do intrasynovial tendons., Clinical Relevance: To improve outcomes after operative repair, future treatment strategies should promote features of extrasynovial healing, such as enhanced vascularization and modulation of the complement system and/or glucose metabolism., Competing Interests: Disclosure: This study was supported by the NIH/NIAMS (R01 AR062947). The WU-PSR is supported in part by the WU Institute of Clinical and Translational Sciences (NIH NCATS UL1 TR000448), the Mass Spectrometry Research Resource (NIH NIGMS P41 GM103422), and the Siteman Comprehensive Cancer Center Support Grant (NIH NCI P30 CA091842). The Washington University Musculoskeletal Research Center is supported by NIH P30 AR074992). The funding source did not play a role in this investigation. The Disclosure of Potential Conflicts of Interest forms are provided with the online version of the article (http://links.lww.com/JBJS/G307)., (Copyright © 2021 by The Journal of Bone and Joint Surgery, Incorporated.)

Protein tyrosine phosphatase receptor σ (PTPσ) is highly expressed by murine and human hematopoietic stem cells (HSCs) and negatively regulates HSC self-renewal and regeneration. Previous studies of the nervous system suggest that heparan sulfate proteoglycans can inactivate PTPσ by clustering PTPσ receptors on neurons, but this finding has yet to be visually verified with adequate resolution. Here, we sought to visualize and quantify how heparan sulfate proteoglycans regulate the organization and activation of PTPσ in hematopoietic stem/progenitor cells (HSPCs). Our study illustrates that syndecan-2 promotes PTPσ clustering, which sustains phospho-tyrosine and phospho-ezrin levels in association with augmentation of hematopoietic colony formation. Strategies that promote clustering of PTPσ on HSPCs may serve to powerfully augment hematopoietic function., Competing Interests: Conflict of interest disclosure The authors declare no competing financial interests., (Copyright © 2021. Published by Elsevier Inc.)

Background: Endothelial dysfunction and injury is a major pathophysiologic feature of sepsis. Sepsis is also the most frequent cause of acute kidney injury (AKI) in critically ill patients. Though most studies of AKI in sepsis have focused on tubular epithelial injury, the role of endothelial dysfunction and injury is less well studied. The goal of this study was first to investigate whether endothelial dysfunction and injury biomarkers were associated with severe AKI in sepsis patients. The second goal was to determine the best performing biomarker for severe AKI and whether this biomarker was associated with severe AKI across different etiologies of sepsis and clinical outcomes., Methods: We studied adults with severe sepsis and acute respiratory failure (ARF) enrolled in the prospective observational Validating Acute Lung Injury markers for Diagnosis (VALID) study. Plasma endothelial dysfunction and injury biomarkers, including angiopoietin-2, soluble vascular endothelial cadherin (sVE-cadherin), endocan and syndecan-1, were measured at study enrollment. Primary analysis focused on the association between endothelial biomarker levels with severe AKI (defined as Kidney Disease: Improving Global Outcomes [KDIGO] AKI stage 2 or 3), other organ dysfunctions (defined by Brussels organ failure scores), and comparison of pulmonary versus non-pulmonary sepsis., Results: Among 228 sepsis patients enrolled, 141 developed severe AKI. Plasma levels of angiopoietin-2, endocan, sVE-cadherin, and syndecan-1 were significantly higher in sepsis patients with severe AKI compared to those without severe AKI. Among four endothelial biomarkers, only angiopoietin-2 was independently associated with severe AKI (odds ratio 6.07 per log increase, 95% CI 2.34-15.78, p < 0.001). Plasma angiopoietin-2 levels by quartile were significantly higher in sepsis patients with hepatic, coagulation, and circulatory failure. Plasma angiopoietin-2 levels were also significantly higher in patients with non-pulmonary sepsis compared to subjects with pulmonary sepsis., Conclusion: Among four biomarkers of endothelial dysfunction and injury, angiopoietin-2 had the most robust independent association with development of severe AKI in patients with severe sepsis and ARF. Plasma angiopoietin-2 levels were also associated with other organ dysfunctions, non-pulmonary sepsis, and death. These findings highlight the importance of early endothelial dysfunction and injury in the pathogenesis of sepsis-induced AKI.

Neonatal infectious diseases are a serious threat to the health of newborns. The aim was to establish a new detection method for the simultaneous measurement of (1,3)-β-d-glucan and procalcitonin in serum for the early screening and efficacy testing of neonatal infectious diseases. We established a sandwich dual-label time-resolved fluorescence immunoassay (TRFIA): anti-(1,3)-β-d-glucan/procalcitonin antibodies immobilized on 96-well plates captured (1,3)-β-d-glucan/procalcitonin antigens and then banded together with the detection antibodies labeled with europium(III) (Eu 3+ )/samarium(III) (Sm 3+ ) chelates. Finally, time-resolved fluorometry was used to measure the fluorescence intensity. The linear correlation coefficient (R 2 ) of the (1,3)-β-d-glucan standard curve was 0.9913, and the R 2 of the procalcitonin standard curve was 0.9911. The detection sensitivity for (1,3)-β-d-glucan was 0.4 pg/mL (dynamic range: 0.6-90 pg/mL), and the average recovery was 101.55%. The detection sensitivity for procalcitonin was 0.02 ng/mL (dynamic range: 0.05-95 ng/mL), and the average recovery was 104.61%. There was a high R 2 between the present TRFIA method and a commercially available assay (R 2  = 0.9829 for (1,3)-β-d-glucan and R 2  = 0.9704 for procalcitonin). Additionally, the cutoff values for (1,3)-β-d-glucan and procalcitonin were 23.95 pg/mL and 0.055 ng/mL, respectively. The present TRFIA method has high sensitivity, accuracy, and specificity and is an effective method for early screening and efficient testing of neonatal invasive fungal infection., (© 2020 International Union of Biochemistry and Molecular Biology, Inc.)

Advances in reagents, methodologies, analytic platforms, and tools have resulted in a dramatic transformation of the research pathology laboratory. These advances have increased our ability to efficiently generate substantial volumes of data on the expression and accumulation of mRNA, proteins, carbohydrates, signaling pathways, cells, and structures in healthy and diseased tissues that are objective, quantitative, reproducible, and suitable for statistical analysis. The goal of this review is to identify and present how to acquire the critical information required to measure changes in tissues. Included is a brief overview of two morphometric techniques, image analysis and stereology, and the use of artificial intelligence to classify cells and identify hidden patterns and relationships in digital images. In addition, we explore the importance of preanalytical factors in generating high-quality data. This review focuses on techniques we have used to measure proteoglycans, glycosaminoglycans, and immune cells in tissues using immunohistochemistry and in situ hybridization to demonstrate the various morphometric techniques. When performed correctly, quantitative digital pathology is a powerful tool that provides unbiased quantitative data that are difficult to obtain with other methods.

Relationships between composition, structure and constituent-specific functional properties of human articular cartilage at different stages of osteoarthritis (OA) are poorly known. We established these relationships by comparison of elastic, viscoelastic and fibril-reinforced poroelastic mechanical properties with microscopic and spectroscopic analysis of structure and composition of healthy and osteoarthritic human tibial cartilage (n = 27). At a low frequency (0.005 Hz), proteoglycan content correlated negatively and collagen content correlated positively with the phase difference (i.e. tissue viscosity). At a high-frequency regime (> 0.05 Hz), proteoglycan content correlated negatively and collagen orientation angle correlated positively with the phase difference. Proteoglycans were lost in the early and advanced OA groups compared to the healthy group, while the superficial collagen orientation angle was greater only in the advanced OA group compared to the healthy group. Simultaneously, the initial fibril network modulus (fibril pretension) was smaller in the early and advanced OA groups compared to the healthy group. These findings suggest different mechanisms contribute to cartilage viscosity in low and high frequencies, and that the loss of superficial collagen pretension during early OA is due to lower tissue swelling (PG loss), while in advanced OA, both collagen disorganization and lower swelling modulate the collagen fibril pretension.

Objective: The aim of the study was to determine whether magnetic resonance imaging (MRI) can be used in assessment of biologic activity of intraluminal thrombus (ILT) and proteolytic processes of the abdominal aortic aneurysm wall., Methods: Using MRI, 50 patients with asymptomatic infrarenal abdominal aortic aneurysm were analyzed at the maximum aneurysm diameter on T1-weighted images in the arterial phase after administration of contrast material. Relative ILT signal intensity (SI) was determined as the ratio between ILT SI and psoas muscle SI. During surgery, the full thickness of the ILT and the adjacent part of the aneurysm wall were harvested at the maximal diameter for biochemical analysis. The concentrations of matrix metalloproteinase 9 and neutrophil elastase (NE/ELA) were analyzed in harvested thrombi, and the concentrations of collagen type III, elastin, and proteoglycans were analyzed in harvested aneurysm walls., Results: A significant positive correlation was found between the NE/ELA concentration of the ILT and the relative SI (ρ = 0.309; P = .029). Furthermore, a negative correlation was observed between the elastin content of the aneurysm wall and the relative SI (ρ = -0.300; P = .034). No correlations were found between relative SI and concentration of matrix metalloproteinase 9, NE/ELA, collagen type III, or proteoglycan 4 in the aneurysm wall., Conclusions: These findings indicate a potential novel use of MRI in prediction of thrombus proteolytic enzyme concentrations and the extracellular matrix content of the aneurysm wall, thus providing additional information for the risk of potential aneurysm rupture., (Copyright © 2019 Society for Vascular Surgery. Published by Elsevier Inc. All rights reserved.)

The extracellular matrix (ECM) spatiotemporally controls cell fate; however, dysregulation of ECM remodeling can lead to tumorigenesis and cancer development by providing favorable conditions for tumor cells. Proteoglycans (PGs) and glycosaminoglycans (GAGs) are the major macromolecules composing ECM. They influence both cell behavior and matrix properties through direct and indirect interactions with various cytokines, growth factors, cell surface receptors, adhesion molecules, enzymes, and glycoproteins within the ECM. The classical features of PGs/GAGs play well-known roles in cancer angiogenesis, proliferation, invasion, and metastasis. Several lines of evidence suggest that PGs/GAGs critically affect broader aspects in cancer initiation and the progression process, including regulation of cell metabolism, serving as a sensor of ECM's mechanical properties, affecting immune supervision, and participating in therapeutic resistance to various forms of treatment. These functions may be implemented through the characteristics of PGs/GAGs as molecular bridges linking ECM and cells in cell-specific and context-specific manners within the tumor microenvironment (TME). In this review, we intend to present a comprehensive illustration of the ways in which PGs/GAGs participate in and regulate several aspects of tumorigenesis; we put forward a perspective regarding their effects as biomarkers or targets for diagnoses and therapeutic interventions.

Chemical exchange saturation transfer of glycosaminoglycans, gagCEST, is a quantitative MR technique that has potential for assessing cartilage proteoglycan content at field strengths of 7 T and higher. However, its utility at 3 T remains unclear. The objective of this work was to implement a rapid volumetric gagCEST sequence with higher gagCEST asymmetry at 3 T to evaluate its sensitivity to osteoarthritic changes in knee articular cartilage and in comparison with T 2 and T 1ρ measures. We hypothesize that gagCEST asymmetry at 3 T decreases with increasing severity of osteoarthritis (OA). Forty-two human volunteers, including 10 healthy subjects and 32 subjects with medial OA, were included in the study. Knee Injury and Osteoarthritis Outcome Scores (KOOS) were assessed for all subjects, and Kellgren-Lawrence grading was performed for OA volunteers. Healthy subjects were scanned consecutively at 3 T to assess the repeatability of the volumetric gagCEST sequence at 3 T. For healthy and OA subjects, gagCEST asymmetry and T 2 and T 1ρ relaxation times were calculated for the femoral articular cartilage to assess sensitivity to OA severity. Volumetric gagCEST imaging had higher gagCEST asymmetry than single-slice acquisitions (p = 0.015). The average scan-rescan coefficient of variation was 6.8%. There were no significant differences in average gagCEST asymmetry between younger and older healthy controls (p = 0.655) or between healthy controls and OA subjects (p = 0.310). T 2 and T 1ρ relaxation times were elevated in OA subjects (p < 0.001 for both) compared with healthy controls and both were moderately correlated with total KOOS scores (rho = -0.181 and rho = -0.332 respectively). The gagCEST technique developed here, with volumetric scan times under 10 min and high gagCEST asymmetry at 3 T, did not vary significantly between healthy subjects and those with mild-moderate OA. This further supports a limited utility for gagCEST imaging at 3 T for assessment of early changes in cartilage composition in OA., (© 2020 John Wiley & Sons, Ltd.)

Diabetic retinopathy is a potentially blinding eye disease that threatens the vision of one-ninth of patients with diabetes. Progression of the disease has long been attributed to an initial dropout of pericytes that enwrap the retinal microvasculature. Revealed through retinal vascular digests, a subsequent increase in basement membrane bridges was also observed. Using cell-specific markers, we demonstrate that pericytes rather than endothelial cells colocalize with these bridges. We show that the density of bridges transiently increases with elevation of Ang-2, PDGF-BB, and blood glucose; is rapidly reversed on a timescale of days; and is often associated with a pericyte cell body located off vessel. Cell-specific knockout of KLF4 in pericytes fully replicates this phenotype. In vivo imaging of limbal vessels demonstrates pericyte migration off vessel, with rapid pericyte filopodial-like process formation between adjacent vessels. Accounting for off-vessel and on-vessel pericytes, we observed no pericyte loss relative to nondiabetic control retina. These findings reveal the possibility that pericyte perturbations in location and process formation may play a role in the development of pathological vascular remodeling in diabetic retinopathy., (© 2020 by the American Diabetes Association.)

Pulmonary artery smooth muscle cells (PASMCs) and pericytes are NG2 + mural cells that provide structural support to pulmonary arteries and capillaries. In pulmonary arterial hypertension (PAH), both mural cell types contribute to PA muscularization, but whether similar mechanisms are responsible for their behavior is unknown. RNA-seq was used to compare the gene profile of pericytes and PASMCs from PAH and healthy lungs. NG2-Cre-ER mice were used to generate NG2-selective reporter mice (NG2tdT) for cell lineage identification and tamoxifen-inducible mice for NG2-selective SDF1 knockout (SDF1 NG2-KO ). Hierarchical clustering of RNA-seq data demonstrated that the genetic profile of PAH pericytes and PASMCs is highly similar. Cellular lineage staining studies on NG2tdT mice in chronic hypoxia showed that, similar to PAH, tdT + cells accumulate in muscularized microvessels and demonstrate significant upregulation of SDF1, a chemokine involved in chemotaxis and angiogenesis. Compared with control mice, SDF1 NG2-KO mice in chronic hypoxia had reduced muscularization and lower abundance of NG2 + cells around microvessels. SDF1 stimulation in healthy pericytes induced greater contractility and impaired their capacity to establish endothelial-pericyte communications. In contrast, SDF1 knockdown reduced PAH pericyte contractility and improved their capacity to associate with vascular tubes in coculture. SDF1 is upregulated in NG2 + mural cells and is associated with PA muscularization. Targeting SDF1 could help prevent and/or reverse muscularization in PAH.

Atopic cataracts develop under the ages of 40 years, after which visual acuity rapidly declines. However, the mechanism underlying the development of atopic cataracts is not yet clear. We focused on the eosinophil granule major basic protein (MBP), which was detected in the aqueous humor of atopic cataracts previously, and which was cytotoxic. Specifically, we investigated its origin in this fluid and its effects on lens epithelial cells (LECs). MBP immunostaining was positive in atopic cataract-derived LECs, but negative in age-related cataract-derived LECs. MBP mRNA was not detected in either type of cataract, but protein was detected in the aqueous humor. Furthermore, the flare values associated with atopic cataracts were higher than those with age-related cataracts. When MBP was purified from eosinophils or recombinant MBP was added to LEC culture medium, cell viability decreased in a concentration-dependent manner, but an MBP antibody neutralized the cytotoxic effect of this protein towards these cells. These results were consistent with the flow of MBP into the aqueous humor from the blood due to a compromised blood-aqueous barrier. Thus, MBP could further penetrate the lens capsule and adhere to LECs, resulting in decreased cell viability and the development of atopic cataracts.

Sprifermin, recombinant human fibroblast growth factor 18 (rhFGF18), induces cartilage regeneration in knees of patients with osteoarthritis (OA). We hypothesized that a temporal multiphasic process of extracellular matrix (ECM) degradation and formation underlie this effect. We aimed to characterize the temporal ECM remodeling of human knee OA articular cartilage in response to sprifermin treatment. Articular cartilage explants from patients with knee OA (n patients  = 14) were cultured for 70 days, with permanent exposure to sprifermin (900, 450, 225 ng/mL), FGF18 (450 ng/mL), insulin-like growth factor-1 (100 ng/mL, positive control) or vehicle (n replicates/treatment/patient  = 2). Metabolic activity (AlamarBlue) and biomarkers of type IIB collagen (PIIBNP) formation (Pro-C2 enzyme-linked immunosorbent assay [ELISA]) and aggrecanase-mediated aggrecan neo-epitope NITEGE (AGNx1 ELISA) were quantified once a week. At end of culture (day 70), gene expression (quantitative reverse transcription polymerase chain reaction) and proteoglycan content (Safranin O/Fast green staining) were quantified. The cartilage had continuously increased metabolic activity, when treated with sprifermin/FGF18 compared to vehicle. During days 7-28 PIIBNP was decreased and NITEGE was increased, and during days 35-70 PIIBNP was increased. At end of culture, the cartilage had sustained proteoglycan content and relative expression of ACAN < COL2A1 < SOX9 < COL1A1, indicating that functional chondrocytes remained in the explants. Sprifermin induces a temporal biphasic cartilage remodeling in human knee OA articular cartilage explants, with early-phase increased aggrecanase activity and late-phase increased type II collagen formation.

In Reinke's space of human vocal fold, type III collagen forms a three dimensional network and this contains numerous minute chambers in between these fibers. These compartments are occupied by glycosaminoglycans and glycoproteins. In laryngeal fold lesions, such as Reinke's edema and vocal fold polyps, proteoglycan (PG)/hyaluronic acid (HA) components of extracellular matrix increased. We investigated the size and quantity of the minute chambers within Reinke's space, filled with PG/HA with the aid of transmission electron microscopy. Eight vocal fold polyps and 10 mucosal biopsies (as control group) were all evaluated by light microscopy and electron microscopy. We detected that PG/HA in extracellular matrix had been increased in vocal fold lesions when compared with control group, by Alcian Blue-pH 2.5 stain. The mean volume of the chambers in Reinke's space of normal larynx was measured as 0.040233 µm 2 whereas the mean volume of these chambers in vocal fold polyps was measured as 6.420221 µm 2 . The difference between the volumes of these chambers in vocal fold polyps and in control group was statistically significant (P = 0.001). Within these chambers PG/HA were found and PG/HA filling these chambers were increased in vocal fold polyps. We think proteoglycan and glycosaminoglycans, especially HA, play an important role in determining biochemical properties of vocal fold lesions., (Copyright © 2018 The Voice Foundation. Published by Elsevier Inc. All rights reserved.)

Current tissue engineering approaches for treatment of injured or diseased articular cartilage use ultraviolet light (UV) for in situ photopolymerization of biomaterials to fill chondral and osteochondral defects as well as resurfacing, stiffening and bonding the extracellular matrix and tissue interfaces. The most commonly used UV light wavelength is UVA 365 nm, the least cytotoxic and deepest penetrating. However, little information is available on the transmission of UVA 365 nm light through the cartilage matrix. In the present study, 365 nm UV light transmission was measured as a function of depth through 100 μm thick slices of healthy articular cartilage removed from mature bovine knees. Transmission properties were measured in normal (Native) cartilage and after swelling equilibration in phosphate-buffered saline (Swollen). Single-factor and multiple linear regression analyses were performed to determine depth-dependencies between the effective attenuation coefficients and proteoglycan, collagen and water contents. For both cartilages, a significant depth-dependency was found for the effective attenuation coefficients, being highest at the articular surface (superficial zone) and decreasing with depth. The effective attenuation coefficients for full-thickness cartilages were approximately a third lower than the total attenuation coefficients calculated from the individual slices. Analysis of absorption and scattering effects due to the ECM and chondrocytes found that UV light scatter coefficients were ∼10 times greater than absorption coefficients. The greater transmittance of UV light through the thicker cartilage was attributed to the collagen within the ECM causing significant backscatter forward reflectance., (© 2019 Wiley Periodicals, Inc.)

The basis of traditional flow cytometry allergy diagnosis is measurement of the expression of basophilic surface activation and/or degranulation markers. Basophils, upon encounter with a specific allergen that cross-links surface FcRI-bound IgE antibodies, not only secrete and release quantifiable bioactive mediators but also upregulate the expression of different markers (e.g., CD63, CD203c) which can be detected by multicolor flow cytometry using specific monoclonal antibodies. Here, we describe a novel technique that relies upon the staining of exteriorized anionic proteoglycans from a basophil granule matrix by cationic fluorescent avidin probes.

Background: We identify and validate accurate diagnostic biomarkers for prostate cancer through a systematic evaluation of DNA methylation alterations., Materials and Methods: We assembled three early prostate cancer cohorts (total patients = 699) from which we collected and processed over 1300 prostatectomy tissue samples for DNA extraction. Using real-time methylation-specific PCR, we measured normalized methylation levels at 15 frequently methylated loci. After partitioning sample sets into independent training and validation cohorts, classifiers were developed using logistic regression, analyzed, and validated., Results: In the training dataset, DNA methylation levels at 7 of 15 genomic loci (glutathione S-transferase Pi 1 [GSTP1], CCDC181, hyaluronan, and proteoglycan link protein 3 [HAPLN3], GSTM2, growth arrest-specific 6 [GAS6], RASSF1, and APC) showed large differences between cancer and benign samples. The best binary classifier was the GAS6/GSTP1/HAPLN3 logistic regression model, with an area under these curves of 0.97, which showed a sensitivity of 94%, and a specificity of 93% after external validation., Conclusion: We created and validated a multigene model for the classification of benign and malignant prostate tissue. With false positive and negative rates below 7%, this three-gene biomarker represents a promising basis for more accurate prostate cancer diagnosis., (© 2019 Wiley Periodicals, Inc.)

Objective: Autologous articular cartilage (AC) harvested for repair procedures of high weight bearing (HWB) regions of the femoral condyles is typically obtained from low weight bearing (LWB) regions, in part due to the lack of non-destructive techniques for cartilage composition assessment. Here, we demonstrate that infrared fiber optic spectroscopy can be used to non-destructively evaluate variations in compositional and mechanical properties of AC across LWB and HWB regions., Design: AC plugs ( N = 72) were harvested from the patellofemoral groove of juvenile bovine stifle joints, a LWB region, and femoral condyles, a HWB region. Near-infrared (NIR) and mid-infrared (MIR) fiber optic spectra were collected from plugs, and indentation tests were performed to determine the short-term and equilibrium moduli, followed by gravimetric water and biochemical analysis., Results: LWB tissues had a significantly greater amount of water determined by NIR and gravimetric assay. The moduli generally increased in tissues from the patellofemoral groove to the condyles, with HWB condyle cartilage having significantly higher moduli. A greater amount of proteoglycan content was also found in HWB tissues, but no differences in collagen content. In addition, NIR-determined water correlated with short-term modulus and proteoglycan content ( R = -0.40 and -0.31, respectively), and a multivariate model with NIR data was able to predict short-term modulus within 15% error., Conclusions: The properties of tissues from LWB regions differ from HWB tissues and can be determined non-destructively by infrared fiber optic spectroscopy. Clinicians may be able to use this modality to assess AC prior to harvesting osteochondral grafts for focal defect repair.

Kidney fibrosis is the common histological end-point of progressive, chronic kidney diseases (CKDs) regardless of the underlying etiology. The hallmark of renal fibrosis, similar to all other organs, is pathological deposition of extracellular matrix (ECM). Renal ECM is a complex network of collagens, elastin, and several glycoproteins and proteoglycans forming basal membranes and interstitial space. Several ECM functions beyond providing a scaffold and organ stability are being increasingly recognized, for example, in inflammation. ECM composition is determined by the function of each of the histological compartments of the kidney, that is, glomeruli, tubulo-interstitium, and vessels. Renal ECM is a dynamic structure undergoing remodeling, particularly during fibrosis. From a clinical perspective, ECM proteins are directly involved in several rare renal diseases and indirectly in CKD progression during renal fibrosis. ECM proteins could serve as specific non-invasive biomarkers of fibrosis and scaffolds in regenerative medicine. The gold standard and currently only specific means to measure renal fibrosis is renal biopsy, but new diagnostic approaches are appearing. Here, we discuss the localization, function, and remodeling of major renal ECM components in healthy and diseased, fibrotic kidneys and the potential use of ECM in diagnostics of renal fibrosis and in tissue engineering.

Purpose: Quadriceps weakness following anterior cruciate ligament reconstruction (ACLR) is linked to decreased patient-reported function, altered lower extremity biomechanics and tibiofemoral joint space narrowing. It remains unknown if quadriceps weakness is associated with early deleterious changes to femoral cartilage composition that are suggestive of posttraumatic osteoarthritis development. The purpose of the cross-sectional study was to determine if quadriceps strength was associated with T1ρ relaxation times, a marker of proteoglycan density, of the articular cartilage in the medial and lateral femoral condyles 6 months following ACLR. It is hypothesized that individuals with weaker quadriceps would demonstrate lesser proteoglycan density., Methods: Twenty-seven individuals (15 females, 12 males) with a patellar tendon autograft ACLR underwent isometric quadriceps strength assessments in 90°of knee flexion during a 6-month follow-up exam. Magnetic resonance images (MRI) were collected bilaterally and voxel by voxel T1ρ relaxation times were calculated using a five-image sequence and a monoexponential equation. Following image registration, the articular cartilage for the weight-bearing surfaces of the medial and lateral femoral condyles (MFC and LFC) were manually segmented and further sub-sectioned into posterior, central and anterior regions of interest (ROI) based on the corresponding meniscal anatomy viewed in the sagittal plane. Univariate linear regression models were used to determine the association between quadriceps strength and T1ρ relaxation times in the entire weight-bearing MFC and LFC, as well as the ROI in each respective limb., Results: Lesser quadriceps strength was significantly associated with greater T1ρ relaxation times in the entire weight-bearing MFC (R 2  = 0.14, P = 0.05) and the anterior-MFC ROI (R 2  = 0.22, P = 0.02) of the ACLR limb. A post hoc analysis found lesser strength and greater T1ρ relaxation times were significantly associated in a subsection of participants (n = 18) without a concomitant medial tibiofemoral compartment meniscal or chondral injury in the entire weight-bearing MFC, as well as anterior-MFC and central-MFC ROI of the ACLR and uninjured limb., Conclusions: The association between weaker quadriceps and greater T1ρ relaxation times in the MFC suggests deficits in lower extremity muscle strength may be related to cartilage composition as early as 6 months following ACLR. Maximizing quadriceps strength in the first 6 months following ACLR may be critical for promoting cartilage health early following ACLR., Level of Evidence: Prognostic level 1.

Conventional arthroscopic evaluation of articular cartilage is subjective and insufficient for assessing early compositional and structural changes during the progression of post-traumatic osteoarthritis. Therefore, in this study, arthroscopic near-infrared (NIR) spectroscopy is introduced, for the first time, for in vivo evaluation of articular cartilage thickness, proteoglycan (PG) content, and collagen orientation angle. NIR spectra were acquired in vivo and in vitro from equine cartilage adjacent to experimental cartilage repair sites. As reference, digital densitometry and polarized light microscopy were used to evaluate superficial and full-thickness PG content and collagen orientation angle. To relate NIR spectra and cartilage properties, ensemble neural networks, each with two different architectures, were trained and evaluated by using Spearman's correlation analysis (ρ). The ensemble networks enabled accurate predictions for full-thickness reference properties (PG content: ρ in vitro, Val = 0.691, ρ in vivo = 0.676; collagen orientation angle: ρ in vitro, Val = 0.626, ρ in vivo = 0.574) from NIR spectral data. In addition, the networks enabled reliable prediction of PG content in superficial (25%) cartilage (ρ in vitro, Val = 0.650, ρ in vivo = 0.613) and cartilage thickness (ρ in vitro, Val = 0.797, ρ in vivo = 0.596). To conclude, NIR spectroscopy could enhance the detection of initial cartilage degeneration and thus enable demarcation of the boundary between healthy and compromised cartilage tissue during arthroscopic surgery.

The function of connective tissues depends on the physical and biochemical properties of their extracellular matrix (ECM), which are in turn dictated by ECM protein composition. With the primary objective of obtaining quantitative estimates for absolute and relative amounts of ECM proteins, we performed a systematic review of papers reporting protein composition of human connective tissues. Articles were included in meta-analysis if they contained absolute or relative quantification of proteins found in the ECM of human bone, adipose tissue, tendon, ligament, cartilage and skeletal muscle. We generated absolute quantitative estimates for collagen in articular cartilage, intervertebral disk (IVD), skeletal muscle, tendon, and adipose tissue. In addition, sulfated glycosaminoglycans were quantified in articular cartilage, tendon and skeletal muscle; total proteoglycans in IVD and articular cartilage, fibronectin in tendon, ligament and articular cartilage, and elastin in tendon and IVD cartilage. We identified significant increases in collagen content in the annulus fibrosus of degenerating IVD and osteoarthritic articular cartilage, and in elastin content in degenerating disc. In contrast, collagen content was decreased in the scoliotic IVD. Finally, we built quantitative whole-tissue component breakdowns. Quantitative estimates improve our understanding of composition of human connective tissues, providing insights into their function in physiology and pathology.

Prostate cancer (PCa) is a common malignancy in males and has a relatively slower progression than other cancers. Our goal was to evaluate the clinical role of SPARC (secreted protein acidic and cysteine rich, osteonectin), cwcv, and kazal-like domains' proteoglycan 1 (SPOCK1) in PCa. SPOCK1 expression was studied through the immunohistochemical staining of specimens from 71 patients with PCa. The correlation between SPOCK1 expression and clinicopathological features was quantitatively analyzed. We used Kaplan-Meier analysis and Cox proportional hazard models to analyze the prognostic value. Of 71 PCa patients, high SPOCK1 expression was more likely to be seen in those with an advanced stage ( p = 0.018) of the disease and an advanced tumor (T) value ( p = 0.014). Patients in Gleason grade groups 3 and 4 had significantly higher SPOCK1 expression ( p = 0.044 and 0.003, respectively) compared to those of Gleason grade group 1. However, this trend was not observed in patients in Gleason grade group 5. For the survival analysis, although it was not statistically significant, patients with a high SPOCK1 expression had a shorter median overall survival (6.2 years) compared to those with low expression (7.8 years). High SPOCK1 expression may be related to advanced clinicopathological features and possibly a poor prognosis. Further analysis with a larger patient base would help clarify this issue.

Objectives: Staining with toluidine blue is a well-established procedure for the histological assessment of cartilaginous- and chondrogenic-differentiated tissues. Being a cationic dye, toluidine blue staining visualizes proteoglycans in a tissue because of its high affinity for the sulfate groups in proteoglycans. It is generally accepted that metachromatic staining with toluidine blue represents cartilaginous matrix and that the degree of positive staining corresponds with the amount of proteoglycans., Design: Articular cartilage and pellets of chondrocytes or bone marrow stromal cells were analyzed with a standardized staining procedure for toluidine blue., Results: In the present study, we illustrate why such an assumption is invalid unless a detailed description of the procedure and/or reference to a detailed published method are provided. This is because the staining specificity and intensity depend, as we have shown, on the pH of the staining solution, the use of dehydration, and on staining time., Conclusions: We can, therefore, suggest a well-controlled standardized protocol for toluidine blue staining, which provides an easy and simple selective staining technique for the assessment of cartilage tissue and proteoglycan development in chondrogenic differentiation. If this procedure is not used, then investigators must provide sufficient technical information concerning the staining protocol to allow an assessment of the validity of the staining results.

Pacinian corpuscles are onion bulb-like multilayered mechanoreceptors that consist of a complicated structure of axon terminals, Schwann related cells (inner core), endoneural related cells (intermediate layer) and perineurial related cells (outer core-capsule). The cells forming those compartments are continuous and share the properties of that covering the nerve fibers. Small leucine-rich proteoglycans are major proteoglycans of the extracellular matrix and regulate collagen fibrillogenesis, cell signalling pathways and extracellular matrix assembly. Here we used immunohistochemistry to investigate the distribution of class I (biglycan, decorin, asporin, ECM2 and ECMX) and class II (fibromodulin, lumican, prolargin, keratocan and osteoadherin) small leucine-rich proteoglycans in human cutaneous Pacinian corpuscles. The distribution of these compounds was: the inner core express decorin, biglycan, lumican, fibromodulin, osteoadherin; the intermediate layer display immunoreactivity for osteoadherin; the outer core biglycan, decorin, lumican, fibromodulin and osteoadherin; and the capsule contains biglycan, decorin, fibromodulin, and lumican. Asporin, prolargin and keratocan were undetectable. These results complement our knowledge about the distribution of small leucine-rich proteoglycans in human Pacinian corpuscles, and help to understand the composition of the extracellular matrix in these sensory formations., (Copyright © 2019 Elsevier GmbH. All rights reserved.)

Background: The present study investigated the therapeutic effect of resveratrol on cardiac injury resulting from blunt chest trauma and the utility of endocan as a biomarker of the inflammation process using rats., Methods: The rats were randomly divided into the following four groups (n=7 in each group): a control group (no treatment or trauma); trauma-induced group (trauma group); resveratrol group (resveratrol 0.3 mg/kg administered via the intraperitoneal [i.p.] route group); and resveratrol + trauma group (resveratrol 0.3 mg/kg administered via the i.p. route 1 hour prior to the induction of trauma)., Results: The immunoreactivity of tumor necrosis factor-α and inducible nitric oxide synthase in the trauma group was increased, whereas the reaction intensity in resveratrol + trauma group was deceased. The mean endocan values of the differed between the groups (p<0.001). The mean endocan value in the resveratrol + trauma group was higher than that of the other groups., Conclusion: Resveratrol exhibited anti-inflammatory and antioxidant effects in lung injury after blunt chest trauma and contributed favorably to the treatment process. We believe that there is a need for further studies on the clinical use of endocan as a biomarker of inflammation in cardiac injury after blunt chest trauma.

Focal defects in articular cartilage are unable to self-repair and, if left untreated, are a leading risk factor for osteoarthritis. This study examined cartilage degeneration surrounding a defect and then assessed whether infilling the defect prevents degeneration. We created a focal chondral defect in porcine osteochondral explants and cultured them ex vivo with and without dynamic compressive loading to decouple the role of loading. When compared to a defect in a porcine knee four weeks post-injury, this model captured loss in sulfated glycosaminoglycans (sGAGs) along the defect's edge that was observed in vivo, but this loss was not load dependent. Loading, however, reduced the indentation modulus of the surrounding cartilage. After infilling with in situ polymerized hydrogels that were soft (100 kPa) or stiff (1 MPa) and which produced swelling pressures of 13 and 310 kPa, respectively, sGAG loss was reduced. This reduction correlated with increased hydrogel stiffness and swelling pressure, but was not affected by loading. This ex vivo model recapitulates sGAG loss surrounding a defect and, when infilled with a mechanically supportive hydrogel, degeneration is minimized., (Copyright © 2019 Elsevier Inc. All rights reserved.)

Background: Mast cells (MCs) in the brain can respond to environmental cues and relay signals to neurons that may directly influence neuronal electrical activity, calcium signaling, and neurotransmission. MCs also express receptors for neurotransmitters and consequently can be activated by them. Here, we developed a coculture model of peritoneal MCs, incubated together with dissociated hippocampal neurons for the study of cellular mechanisms involved in the mast cell-neuron interactions., Methods: Calcium imaging was used to simultaneously record changes in intracellular calcium [Ca 2+ ] i in neurons and MCs. To provide insight into the contribution of MCs on neurotransmitter release in rat hippocampal neurons, we used analysis of FM dye release, evoked by a cocktail of mediators from MCs stimulated by heat., Results: Bidirectional communication is set up between MCs and hippocampal neurons. Neuronal depolarization caused intracellular calcium [Ca 2+ ] i oscillations in MCs that produced a quick response in neurons. Furthermore, activation of MCs with antigen or the secretagogue compound 48/80 also resulted in a neuronal [Ca 2+ ] i response. Moreover, local application onto neurons of the MC mediator cocktail elicited Ca 2+ transients and a synaptic release associated with FM dye destaining. Neuronal response was partially blocked by D-APV, a N-methyl-D-aspartate receptor (NMDAR) antagonist, and was inhibited when the cocktail was pre-digested with chondroitinase ABC, which induces enzymatic removal of proteoglycans of chondroitin sulfate (CS)., Conclusions: MC-hippocampal neuron interaction affects neuronal [Ca 2+ ] i and exocytosis signaling through a NMDAR-dependent mechanism.

Ovine forestomach matrix (OFM) is a decellularized extracellular matrix (dECM) biomaterial that serves as a scaffold for remodeling damaged soft tissue. dECM biomaterials are used in a variety of clinical applications, and their regenerative capacity is encoded not only in their biophysical properties but also in their molecular diversity. In this study, the proteome of OFM was characterized via both targeted and global mass spectrometry (MS) with the use of heavy isotope labeled (SIL) internal standards. Proteins were identified following either chemical digestion or extraction using saline or guanidine hydrochloride, followed by high resolution size exclusion chromatography. Identified proteins were annotated using the matrisome database and molecular function using the gene ontology database. The characterization identified 153 unique matrisome proteins, including 25 collagens, 58 glycoproteins, 12 proteoglycans, 13 ECM affiliated proteins, 20 ECM regulators, and 23 secreted factors. This inventory represents a comprehensive array of matrix proteins that are retained in OFM after processing. The diversity of proteins identified may contribute to OFM's remodeling capacity in clinical applications.

Purpose: Aberrant walking biomechanics after anterior cruciate ligament reconstruction (ACLR) are hypothesized to be associated with deleterious changes in knee cartilage. T1ρ magnetic resonance imaging (MRI) is sensitive to decreased proteoglycan density of cartilage. Our purpose was to determine associations between T1ρ MRI interlimb ratios (ILR) and walking biomechanics 6 months after ACLR., Methods: Walking biomechanics (peak vertical ground reaction force (vGRF), vGRF loading rate, knee extension moment, knee abduction moment) were extracted from the first 50% of stance phase in 29 individuals with unilateral ACLR. T1ρ MRI ILR (ACLR limb/uninjured limb) was calculated for regions of interest in both medial and lateral femoral (LFC) and medial and lateral tibial condyles. Separate, stepwise linear regressions were used to determine associations between biomechanical outcomes and T1ρ MRI ILR after accounting for walking speed and meniscal/chondral injury (P ≤ 0.05)., Results: Lesser peak vGRF in the ACLR limb was associated with greater T1ρ MRI ILR for the LFC (posterior ΔR = 0.14, P = 0.05; central ΔR = 0.15, P = 0.05) and medial femoral condyle (central ΔR = 0.24, P = 0.01). Lesser peak vGRF loading rate in the ACLR limb (ΔR = 0.21, P = 0.02) and the uninjured limb (ΔR = 0.27, P = 0.01) was associated with greater T1ρ MRI ILR for the anterior LFC. Lesser knee abduction moment for the injured limb was associated with greater T1ρ MRI ILR for the anterior LFC (ΔR = 0.16, P = 0.04) as well as the posterior medial tibial condyle (ΔR = 0.13, P = 0.04)., Conclusion: Associations between outcomes related to lesser mechanical loading during walking and greater T1ρ MRI ILR were found 6 months after ACLR. Although preliminary, our results suggest that underloading of the ACLR limb at 6 months after ACLR may be associated with lesser proteoglycan density in the ACLR limb compared with the uninjured limb.