Glycosaminoglycan Content Research Articles

Proteomic, mechanical, and biochemical characterization of cartilage development

The objective of this work is to examine the development of porcine cartilage by analyzing its mechanical properties, biochemical content, and proteomics at different developmental stages. Cartilage from the knees of fetal, neonatal, juvenile, and mature pigs was analyzed using histology, mechanical testing, biochemical assays, fluorophore-assisted carbohydrate electrophoresis, and bottom-up proteomics. Mature cartilage has 2.2-times the collagen per dry weight of fetal cartilage, and fetal cartilage has 2.1-times and 17.9-times the glycosaminoglycan and DNA per dry weight of mature cartilage, respectively. Tensile and compressive properties peak in the juvenile stage, with a tensile modulus 4.7-times that of neonatal. Proteomics analysis reveals increases in collagen types II and III, while collagen types IX, XI, and XIV, and aggrecan decrease with age. For example, collagen types IX and XI decrease 9.4-times and 5.1-times, respectively from fetal to mature. Mechanical and biochemical measurements have their greatest developmental changes between the neonatal and juvenile stages, where mechanotransduction plays a major role. Bottom-up proteomics serves as a powerful tool for tissue characterization, showing results beyond those of routine biochemical analysis. For example, proteomic analysis shows significant drops in collagen types IX, XI, and XIV throughout development, which shows insight into the permanence of cartilage's matrix. Changes in overall glycosaminoglycan content compared to aggrecan and link protein indicate non-enzymatic degradation of aggrecan structures or hyaluronan in mature cartilage. In addition to tissue characterization, bottom-up proteomics techniques are critical in tissue engineering efforts toward repair or regeneration of cartilage in animal models. Statement of significanceIn this study, the development of porcine articular cartilage is interrogated through biomechanical, biochemical, and proteomic techniques, to determine how mechanics and extracellular matrix composition change from fetal to mature cartilage. For the first time, a bottom-up proteomics approach is used to reveal a wide variety of protein changes through aging; for example, the collagen subtype composition of the cartilage increases in collagen types II and III, and decreases in collagen types IX, XI, and XIV. This analysis shows that bottom-up proteomics is a critical tool in tissue characterization, especially toward developing a deeper understanding of matrix composition and development in tissue engineering studies.

The degenerative impact of hyperglycemia on the structure and mechanics of developing murine intervertebral discs.

IntroductionDiabetes has long been implicated as a major risk factor for intervertebral disc (IVD) degeneration, interfering with molecular signaling and matrix biochemistry, which ultimately aggravates the progression of the disease. Glucose content has been previously shown to influence structural and compositional changes in engineered discs in vitro, impeding fiber formation and mechanical stability.MethodsIn this study, we investigated the impact of diabetic hyperglycemia on young IVDs by assessing biochemical composition, collagen fiber architecture, and mechanical behavior of discs harvested from 3‐ to 4‐month‐old db/db mouse caudal spines.ResultsWe found that discs taken from diabetic mice with elevated blood glucose levels demonstrated an increase in total glycosaminoglycan and collagen content, but comparable advanced glycation end products (AGE) levels to wild‐type discs. Diabetic discs also contained ill‐defined boundaries between the nucleus pulposus and annulus fibrosus, with the latter showing a disorganized and unaligned collagen fiber network at this same boundary.ConclusionsThese compositional and structural changes had a detrimental effect on function, as the diabetic discs were twice as stiff as their wild‐type counterparts and demonstrated a significant resistance to deformation. These results indicate that diabetes may predispose the young disc to DDD later in life by altering patterns of extracellular matrix deposition, fiber formation, and motion segment mechanics independently of AGE accumulation.

Inhibition of BMP signaling with LDN 193189 can influence bone marrow stromal cell fate but does not prevent hypertrophy during chondrogenesis.

SummaryBone morphogenetic protein (BMP) cascades are upregulated during bone marrow-derived stromal cell (BMSC) chondrogenesis, contributing to hypertrophy and preventing effective BMSC-mediated cartilage repair. Previous work demonstrated that a proprietary BMP inhibitor prevented BMSC hypertrophy, yielding stable cartilage tissue. Because of the significant therapeutic potential of a molecule capable of hypertrophy blockade, we evaluated the capacity of a commercially available BMP type I receptor inhibitor with similar properties, LDN 193189, to prevent BMSC hypertrophy. Using 14-day microtissue chondrogenic induction cultures we found that LDN 193189 permitted BMSC chondrogenesis but did not prevent hypertrophy. LDN 193189 was sufficiently potent to counter mineralization and adipogenesis in response to exogenous BMP-2 in osteogenic induction cultures. LDN 193189 did not modify BMSC behavior in adipogenic induction cultures. Although LDN 193189 is effective in countering BMP signaling in a manner that influences BMSC fate, this blockade is insufficient to prevent hypertrophy.

Brain Cancer Cell-Derived Matrices and Effects on Astrocyte Migration

Cell-derived matrices are useful tools for studying the extracellular matrix (ECM) of different cell types and testing the effects on cell migration or wound repair. These matrices typically are generated using extended culture with ascorbic acid to boost ECM production. Applying this technique to cancer cell cultures could advance the study of cancer ECM and its effects on recruitment and training of the tumor microenvironment, but ascorbic acid is potently cytotoxic to cancer cells. Macromolecular crowding (MMC) agents can also be added to increase matrix deposition based on the excluded volume principle. We report the use of MMC alone as an effective strategy to generate brain cancer cell-derived matrices for downstream analyses and cell migration studies. We cultured the mouse glioblastoma cell line GL261 for 1 week in the presence of three previously reported MMC agents (carrageenan, Ficoll 70/400, and hyaluronic acid). We measured the resulting deposition of collagens and sulfated glycosaminoglycans using quantitative assays, as well as other matrix components by immunostaining. Both carrageenan and Ficoll promoted significantly more accumulation of total collagen content, sulfated glycosaminoglycan content, and fibronectin staining. Only Ficoll, however, also demonstrated a significant increase in collagen I staining. The results were more variable in 3D spheroid culture. We focused on Ficoll MMC matrices, which were isolated using the small molecule Raptinal to induce cancer cell apoptosis and matrix decellularization. The cancer cell-derived matrix promoted significantly faster migration of human astrocytes in a scratch wound assay, which may be explained by focal adhesion morphology and an increase in cellular metabolic activity. Ultimately, these data show MMC culture is a useful technique to generate cancer cell-derived matrices and study the effects on stromal cell migration related to wound repair.

Influence of chloditan on the content of glycosaminoglycans in the blood and liver of rats with alloxan diabetes

Hypoglucocorticoidemia caused by systemic injection of chloditane {[2(chlorphenil)-2-(4- chlorphenil)-l]-l dichlorethane} promotes normalizing the content of glycosaminoglykanes in blood of rats with alloxan diabetes. The increase of concentration of the total glycosaminoglykanes in liver tissue of these rats on the 30th day occurs as a result of the accumulation. The determination of the content W sulphatized glycosaminoglykanes can be used to reveal the metabolic disorders in the connective matrix in alloxan diabetes.

Autologous platelet-rich plasma effects on Staphylococcus aureus–induced chondrocyte death in an in vitro bovine septic arthritis model

To investigate the chondroprotective effects of autologous platelet-rich plasma (PRP), ampicillin-sulbactam (AmpS), or PRP combined with AmpS (PRP+AmpS) in an in vitro chondrocyte explant model of bovine Staphylococcus aureus-induced septic arthritis. Autologous PRP and cartilage explants obtained from 6 healthy, adult, nonlactating Jersey-crossbred cows. Autologous PRP was prepared prior to euthanasia using an optimized double centrifugation protocol. Cartilage explants collected from grossly normal stifle joints were incubated in synovial fluid (SF) alone, S aureus-inoculated SF (SA), or SA supplemented with PRP (25% culture medium volume), AmpS (2 mg/mL), or both PRP (25% culture medium volume) and AmpS (2 mg/mL; PRP+AmpS) for 24 hours. The metabolic activity, percentage of dead cells, and glycosaminoglycan content of cartilage explants were measured with a resazurin-based assay, live-dead cell staining, and dimethylmethylene blue assay, respectively. Treatment effects were assessed relative to the findings for cartilage explants incubated in SF alone. Application of PRP, AmpS, and PRP+AmpS treatments significantly reduced S aureus-induced chondrocyte death (ie, increased metabolic activity and cell viability staining) in cartilage explants, compared with untreated controls. There were no significant differences in chondrocyte death among explants treated with PRP, AmpS, or PRP+AmpS. In this in vitro explant model of S aureus-induced septic arthritis, PRP, AmpS, and PRP+AmpS treatments mitigated chondrocyte death. Additional work to confirm the efficacy of PRP with bacteria commonly associated with clinical septic arthritis in cattle as well as in vivo evaluation is warranted.

A novel nonsense MPS VI mutation in a south Moroccan patient

A 12-years-old, Moroccan child with a history of skeletal deformities, gait disorder, Short stature, valvulopathy, facial dysmorphy, corneal opacity, umbilical hernia, and hepatomegaly. He has onset from birth, delayed first milestones (setting, gait, and language) and severe dysostosis multiplex. Elevated urinary glycosaminoglycans concentration was found, enzymatic assays show a low level of ARSB, genetic testing confirmed a diagnosis of Mucopolysaccharidosis type VI with a novel mutation in ARSB gene. There is a homozygous frameshift nonsense mutation by two thymine deletion in 770 and 771 codons (c. [770_771del TT]; [770_771del TT]). This is the first case and mutation of Mucopolysaccharidosis type VI detected in south Morocco. Keywords: : mucopolysaccharidosis VI; ARSB gene; nonsense mutation; ERT

The Effect of Neonatal, Juvenile, and Adult Donors on Rejuvenated Neocartilage Functional Properties.

Cartilage does not naturally heal, and cartilage lesions from trauma and wear-and-tear can lead to eventual osteoarthritis. To address long-term repair, tissue engineering of functional biologic implants to treat cartilage lesions is desirable, but the development of such implants is hindered by several limitations, including (1) donor tissue scarcity due to the presence of diseased tissues in joints, (2) dedifferentiation of chondrocytes during expansion, and (3) differences in functional output of cells dependent on donor age. Toward overcoming these challenges, (1) costal cartilage has been explored as a donor tissue, and (2) methods have been developed to rejuvenate the chondrogenic phenotype of passaged chondrocytes for generating self-assembled neocartilage. However, it remains unclear how the rejuvenation processes are influenced by donor age and, thus, how to develop strategies that specifically target age-related differences. Using histological, biochemical, proteomic, and mechanical assays, this study sought to determine the differences among neocartilage generated from neonatal, juvenile, and adult donors using the Yucatan minipig, a clinically relevant large animal model. Based on the literature, a relatively young adult population of animals was chosen due to a reduction in functional output of human articular chondrocytes after 40 years of age. After isolation, costal chondrocytes were expanded, rejuvenated, and self-assembled, and the neocartilages were assessed. The aggregate modulus values of neonatal constructs were at least 1.65-fold of those from the juvenile or adult constructs. Poisson's ratio also significantly differed among all groups, with neonatal constructs exhibiting values 49% higher than adult constructs. Surprisingly, other functional properties such as tensile modulus and glycosaminoglycan content did not significantly differ among groups. Total collagen content was slightly elevated in the adult constructs compared to neonatal and juvenile constructs. A more nuanced view using bottom-up mass spectrometry showed that Col2a1 protein was not significantly different among groups, but protein content of several other collagen subtypes (i.e., Col1a1, Col9a1, Col11a2, and Col12a1) was modulated by donor age. For example, Col12a1 protein content in adult constructs was found to be 102.9% higher than neonatal-derived constructs. Despite these differences, this study shows that different aged donors can be used to generate neocartilages of similar functional properties. Impact statement Tissue-engineered neocartilage can be generated with functional properties that mimic native cartilage tissue. However, cell sourcing challenges hinder clinical translation of tissue-engineered cartilage. Chondrocytes can be expanded and rejuvenated for the generation of functional self-assembled cartilage, making an allogeneic approach feasible. However, it is currently unclear if donor age impacts functional properties. In this study, using the Yucatan minipig as a clinically relevant large animal model, we demonstrate that functional properties of self-assembled neocartilage are relatively consistent regardless of donor age, suggesting that a wider range of donor ages may be used for cartilage tissue engineering than previously expected.

Integration of a miniaturized DMMB assay with high-throughput screening for identifying regulators of proteoglycan metabolism

Defective biosynthesis or function of proteoglycans causes pathological conditions in a variety of tissue systems. Osteoarthritis (OA) is a prevalent degenerative joint disorder characterized by progressive cartilage destruction caused by imbalanced proteoglycan synthesis and degradation. Identifying agents that regulate proteoglycan metabolism may benefit the development of OA-modifying therapeutics. High-throughput screening (HTS) of chemical libraries has paved the way for achieving this goal. However, the implementation and adaptation of HTS assays based on proteoglycan measurement remain underexploited. Using primary porcine chondrocytes as a model, we report a miniaturized dimethyl-methylene blue (DMMB) assay, which is commonly used to quantitatively evaluate sulfated glycosaminoglycan (GAG) content, with an optimized detection range and reproducibility and its integration with HTS. Treatment with TGF-β1 and IL1-α, known as positive and negative proteoglycan regulators, respectively, supported the assay specificity. A pre-test of chemical screening of 960 compounds identified both stimulators (4.48%) and inhibitors (6.04%) of GAG production. Fluorophore-assisted carbohydrate electrophoresis validated the activity of selected hits on chondroitin sulfate expression in an alginate culture system. Our findings support the implementation of this simple colorimetric assay in HTS to discover modifiers of OA or other diseases related to dysregulated proteoglycan metabolism.

Engineered Cell-Secreted Extracellular Matrix Modulates Cell Spheroid Mechanosensing and Amplifies Their Response to Inductive Cues for the Formation of Mineralized Tissues.

The clinical translation of mesenchymal stromal cell (MSC)-based therapies remains challenging due to rapid cell death and poor control over cell behavior. Compared to monodisperse cells, the aggregation of MSCs into spheroids increases their tissue-forming potential by promoting cell-cell interactions. However, MSCs initially lack engagement with an endogenous extracellular matrix (ECM) when formed into spheroids. Previously the instructive nature of an engineered, cell-secreted ECM is demonstrated to promote survival and differentiation of adherent MSCs. Herein, it is hypothesized that the incorporation of this cell-secreted ECM during spheroid aggregation would enhance MSC osteogenic potential by promoting cell-matrix and cell-cell interactions. ECM-loaded spheroids contained higher collagen and glycosaminoglycan content, and MSCs exhibited increased mechanosensitivity to ECM through Yes-associated protein (YAP) activation via integrin α2β1 binding. ECM-loaded spheroids sustained greater MSC viability and proliferation and are more responsive to soluble cues for lineage-specific differentiation than spheroids without ECM or loaded with collagen. The encapsulation of ECM-loaded spheroids in instructive alginate gels resulted in spheroid fusion and enhanced osteogenic differentiation. These results highlight the clinical potential of ECM-loaded spheroids as building blocks for the repair of musculoskeletal tissues.

A review on 3D printing in tissue engineering applications

Abstract In tissue engineering, 3D printing is an important tool that uses biocompatible materials, cells, and supporting components to fabricate complex 3D printed constructs. This review focuses on the cytocompatibility characteristics of 3D printed constructs, made from different synthetic and natural materials. From the overview of this article, inkjet and extrusion-based 3D printing are widely used methods for fabricating 3D printed scaffolds for tissue engineering. This review highlights that scaffold prepared by both inkjet and extrusion-based 3D printing techniques showed significant impact on cell adherence, proliferation, and differentiation as evidenced by in vitro and in vivo studies. 3D printed constructs with growth factors (FGF-2, TGF-β1, or FGF-2/TGF-β1) enhance extracellular matrix (ECM), collagen I content, and high glycosaminoglycan (GAG) content for cell growth and bone formation. Similarly, the utilization of 3D printing in other tissue engineering applications cannot be belittled. In conclusion, it would be interesting to combine different 3D printing techniques to fabricate future 3D printed constructs for several tissue engineering applications.

Analysis of normal levels of free glycosaminoglycans in urine and plasma in adults

Plasma and urine glycosaminoglycans (GAGs) are long, linear sulfated polysaccharides that have been proposed as potential noninvasive biomarkers for several diseases. However, owing to the analytical complexity associated with the measurement of GAG concentration and disaccharide composition (the so-called GAGome), a reference study of the normal healthy GAGome is currently missing. Here, we prospectively enrolled 308 healthy adults and analyzed their free GAGomes in urine and plasma using a standardized ultra-high-performance liquid chromatography coupled with triple-quadrupole tandem mass spectrometry method together with comprehensive demographic and blood chemistry biomarker data. Of 25 blood chemistry biomarkers, we mainly observed weak correlations between the free GAGome and creatinine in urine and hemoglobin or erythrocyte counts in plasma. We found a higher free GAGome concentration – but not a more diverse composition - in males. Partitioned by gender, we also established reference intervals for all detectable free GAGome features in urine and plasma. Finally, we carried out a transference analysis in healthy individuals from two distinct geographical sites, including data from the Lifelines Cohort Study, which validated the reference intervals in urine. Our study is the first large-scale determination of normal free GAGomes reference intervals in plasma and urine and represents a critical resource for future physiology and biomarker research.

Intervertebral disc degeneration in warmblood horses: Histological and biochemical characterization.

Gross morphology of healthy and degenerated intervertebral discs (IVDs) is largely similar in horses as in dogs and humans. For further comparison, the biochemical composition and the histological and biochemical changes with age and degeneration were analyzed in 41 warmblood horses. From 33 horses, 139 discs and 2 fetal vertebral columns were evaluated and scored histologically. From 13 horses, 73 IVDs were assessed for hydration, DNA, glycosaminoglycans, total collagen, hydroxyl-lysyl-pyridinoline, hydroxylysine, and advanced glycation end-product (AGE) content. From 7 horses, 20 discs were assessed for aggrecan, fibronectin, and collagen type 1 and 2 content. Histologically, tearing of the nucleus pulposus (NP) and cervical annulus fibrosus (AF), and total histological score (tearing and vascular proliferation of the AF, and chondroid metaplasia, chondrocyte-like cell proliferation, presence of notochordal cells, matrix staining, and tearing of the NP) correlated with gross degeneration. Notochordal cells were not seen in IVDs of horses. Age and gross degeneration were positively correlated with AGEs and a fibrotic phenotype, explaining gross degenerative changes. In contrast to dogs and humans, there was no consistent difference in glycosaminoglycan content and hydration between AF and NP, nor decrease of these variables with age or degeneration. Hydroxylysine decrease and collagen 1 and AGEs increase were most prominent in the NP, suggesting degeneration started in the AP. In caudal cervical NPs, AGE deposition was significantly increased in grossly normal IVDs and total collagen significantly increased with age, suggesting increased biomechanical stress and likelihood for spinal disease in this part of the vertebral column.

Serum collected from rats with myocardial infarction increases extracellular matrix accumulation by myofibroblasts isolated from myocardial infarction scar

Abstract The effect on extracellular matrix content is believed to be an average of several serum derived compounds acting in opposition. The aim of the study is to determine whether whole serum of rats with myocardial infarction may modify the accumulation of extracellular matrix in cultures of myofibroblasts isolated from the myocardial infarction scar. A second aim is to determine whether the tested serum can also degranulate the mast cells. Serum was collected from rats with sham myocardial infarction, rats with myocardial infarction induced by coronary artery ligation and control animals. The experiments were carried out on myocardial infarction scar myofibroblasts or mast cells from the peritoneal cavity. The cultures were divided into three groups containing eight cultures each: one treated with serum from control rats, from animals after sham operation or from those after myocardial infarction. In all groups, the serum was used at concentrations of 10%, 20% or 30%. The total collagen content (Woesner method) glycosaminoglycan level (Farandale method), cell proliferation (BrdU), histamine secretion from mast cells (spectrofluorymetry), β1 integrin and α-smooth muscle actin expression (flow cytometry) were evaluated. Isolated cells were α-smooth muscle actin positive and identified as myofibroblasts. Serum derived from rats with myocardial infarction increased collagen and glycosaminoglycan content in the cultures and modified myofibroblast proliferation in a concentration-dependent manner. The serum also results in an imbalance between collagen and glycosaminoglycan levels. The content of β1 integrin was not influenced by myocardial infarction serum. The serum of rats with myocardial infarction is involved in regulation of collagen and glycosaminoglycan content in myofibroblast cultures, as well as the modification of their proliferation. These changes were not accompanied with integrin β1 density variations. The serum of the myocardial infarction rats did not influence the mast cell degranulation.

Novel insights into the pathogenesis of tendon injury: mechanotransduction and neuroplasticity.

Novel insights into the pathogenesis of tendon injury: mechanotransduction and neuroplasticity.

Evaluating Initial Integration of Cell-Based Chondrogenic Constructs in Human Osteochondral Explants.

Integration of an implant with the surrounding tissue is a major challenge in cartilage regeneration. It is usually assessed with in vivo animal studies at the end-stage of implant development. To reduce animal experimentation and at the same time increase screening throughput and speed up implant development, this study examined whether integration of allogeneic cell-based implants with the surrounding native cartilage could be demonstrated in an ex vivo human osteochondral culture model. Chondrocytes were isolated from smooth cartilage tissue of fresh human tibial plateaus and condyles. They were expanded for 12 days either in three-dimensional spinner flask cultures to generate organoids, or in two-dimensional culture flasks for standard cell expansion. Three implant groups were created (fibrin+organoids, fibrin+cells, and fibrin only) and used to fill a Ø 6 mm full-depth chondral defect created in human osteochondral explants (Ø 10 mm, bone length cut to 4 mm) harvested from a second set of fresh human tibial plateaus. Explants were cultured for 1 or 28 days in a double-chamber culture platform. Histology showed that after 28 days the organoids on the interface of the defect remodeled and merged, and cells migrated through the fibrin glue bridging the space between the organoids and between the organoids and the native cartilage. For both conditions, newly formed tissue rich in proteoglycans and collagen type II was present mainly on the edges and in the corners of the defect. In these matrix-rich areas, cells resided in lacunae and the newly formed tissue integrated with the surrounding native cartilage. Biochemical analysis revealed a statistically significant effect of culture time on glycosaminoglycan (GAG) content, and showed a higher hydroxyproline (HYP) content for organoid-filled implants compared with cell-filled implants at both timepoints. This ex vivo human osteochondral culture system provides possibilities for exploration and identification of promising implant strategies based on evaluation of integration and matrix production under more controlled experimental conditions than possible in vivo.

DGEMRIC T1 IS REDUCED IN HIP CARTILAGE OVERLYING BONE MARROW LESIONS

Bone marrow lesions are associated with pain and rapidly worsening osteoarthritis. Previous quantitative MRI research of knees with BMLs has found that the cartilage immediately overlying BMLs is less healthy than the adjacent non-overlying cartilage in patients with knee OA. It is not clear how BMLs affect overlying cartilage in the hip or in early OA. Determine whether dGEMRIC T1 (T1Gd) is reduced in hip cartilage overlying BMLs in a cross-sectional sample of adults aged 20-49. dGEMRIC and PD-weighted fat-suppressed (PDw-FS) images were acquired for 128 adults aged 20-49 recruited from a larger population-based study using a 3T MRI. Acetabular and femoral cartilage was segmented manually. BMLs were identified and segmented semi-automatically from the PDw-FS images and were identified as containing cysts or not. Cysts were defined as bright areas on the PDw-FS images within the BML with well-defined borders. Cartilage T1Gd maps and segmented BMLs were aligned using landmark-based registration. Age and sex-matched control subjects (without BMLs) were identified for each BML subject and were aligned using landmark-based registration. The overlying cartilage was defined as the projection of the BML shape onto the adjacent cartilage surface (ace/fem). The surrounding cartilage was defined as the cartilage not included in the overlying region. We compared characteristics between the BML and control group using a two-sample t-test or Wilcoxon sum rank test for continuous variables, and a χ 2 or Fisher's exact test for categorical variables. We applied a linear mixed-effects model to compare the change score (mean T1Gd of overlying minus surrounding cartilage) between the BML and control group. The model was adjusted for BMI, physical activity (MET), FAI, hip pain, and location (ace/fem). Change scores were compared between cystic and non-cystic BML subjects. BML (n=32) and control (n=32) participants were similar on: mean BMI (27.1 vs 27.0), mean MET (10473 vs 10121), and presence of hip pain (53.1% vs 56.3%). The BML group had significantly higher rates of FAI (65.6% vs 37.5%, p=0.02). The change score was significantly larger for the BML subjects compared to controls: -80ms (95% CI: -158, -2), p=0.04. There was no significant difference between the change scores for cystic and non-cystic BML subjects: -32ms (95% CI: -154, 91), p=0.60. Change scores for each individual group are listed in Table 1. Hip cartilage overlying BMLs has reduced glycosaminoglycan (GAG) concentration, consistent with findings in knee OA. A strength of our approach is that we controlled for natural variations in T1Gd in the hip. This work demonstrates that BMLs are associated with osteoarthritis progression in the hip, even at early stages, and that progression is most pronounced overlying BMLs. Canadian Institutes of Health Research (PAF-107513). Arthritis Society (TGP-18-0229). BBF has an equity position in a private imaging clinic which includes MRI in Van BC. The IMPAKT-HiP participants and study team members. CORRESPONDENCE ADDRESS: carly.jones@hiphealth.ca .

Endogenous Glycosaminoglycans in Various Pathologic Plasma Samples as Measured by a Fluorescent Quenching Method.

Endogenous glycosaminoglycans (GAGs) with a similar structure to heparin are widely distributed in various tissues. A fluorescence probe, namely Heparin Red, can detect polyanionic GAGs in plasma samples. The purpose of this study is to measure endogenous GAGs in various plasma samples obtained from different pathologic states in comparison to healthy controls utilizing this method. Plasma samples were obtained from patient groups including atrial fibrillation (AF), end-stage-renal-disease (ESRD), diabetes mellitus (DM), sepsis, cancer, liver disease (LD), and pulmonary embolism (PE). Normal human plasma (NHP) was used as healthy controls. The Heparin Red kit from Red Probes (Münster, Germany) was used for the quantification of endogenous GAGs in each sample before and after heparinase I degradation. All results were compiled as group means ± SD for comparison. NHP was found to have relatively low levels of endogenous GAGs with a mean concentration of 0.06 μg/mL. The AF, ESRD, DM, and sepsis patient samples had a mean endogenous GAG concentration of 0.55, 0.72, 0.92, and 0.94 μg/mL, respectively. The levels of endogenous GAGs were highest in cancer, LD, and PE patient plasma samples with a mean concentration of 1.95, 2.78, and 2.83 μg/mL, respectively. Heparinase I degradation resulted in a decline in GAG levels in plasma samples. These results clearly show that detectable Heparin Red sensitive endogenous GAGs are present in circulating plasma at varying levels in various patient groups. Additional studies are necessary to understand this complex pathophysiology.

Effect of FLU-ACIL RHINO nasal spray on the inflammatory process and recovery of patients after FESS

Topicality: The main method of treatment of chronic rhinosinusitis is functional endoscopic rhinosurgery (FESS). However, the regeneration of tissues damaged during surgery remains important. It has three stages – alteration (primary and secondary), exudation and proliferation – and takes place with the formation of reactive oxygen species. Therefore, determination of total NO synthase activity (NOS), inducible synthase activity (iNOS) and constitutive (cNOS) isoforms are biochemical markers of the inflammatory response. The activity of the alteration process and the degree of postoperative damage is characterized by the concentration of glycosaminoglycans and L-oxyproline. Materials and methods: This study determined the multimodal (multi-vector) effect of Flu-Acyl rhino spray (6% N-acetylcysteine in 3% hypertonic pH-controlled saline) on the inflammatory response of the nasal mucosa in the postoperative period. The study was conducted at the Department of Otolaryngology of NMU named after O.O. Bogomolets on the basis of "Alexander Clinical Hospital of Kyiv". Clinical and laboratory examinations of 52 patients aged 18 to 60 years who underwent FESS were performed. Patients were randomized into two groups. 24 patients received standard treatment (control group), 26 patients – with the addition of nasal spray Flu-Acyl rhino (main group). Results: It was found that the indicators of total activity of NO synthase (NOS) and its isoforms, as well as the concentration of glycosaminoglycans and L-oxyproline were lower in patients of the main group on days 7 and 14 of the postoperative period compared with patients in the control group (р<0,05). These results can be explained by the anti-inflammatory effect of Flu-Acyl rhino spray due to the presence of N-acetylcysteine, which is able to inhibit the activation of the transcription factor NF-kB, which leads to decreased iNOS activity on the 7th and 14th day of treatment. N-acetylicysteine also reduces the degradation of amorphous connective tissue, shifts the balance in the proteolysis / antiproteolysis system towards the predominance of the antiproteolytic component, participates in the normalization of physiological production and utilization of sulfide anion. Conclusions: The use of Flu-Acyl rhino nasal spray in the postoperative period due to multimodal action promotes physiological changes from alteration and exudation to proliferation processes, normalizes mucociliary clearance in the postoperative period, improves healing and reduces recovery and disability.

Investigation of the Effect of Agarose Gel Concentration and Culture Period on Bio and Mechanical Properties of Chondrocyte Tissue Engineering

As a gel scaffold for chondrocyte tissue engineering, agarose concentration plays a significant role in the relationship between porosity and nutrition. In this work, the effect of concentration and period cultured on Glycosaminoglycan (GAG) and mechanical properties have been studied. A bovine chondrocytes have been isolated and seeded in different agarose gel scoffed concentrations, about 4% and 6%, for different period cultured, 0 and 7 days. The MTS machine and Spectrophotometric with calibration curve method were used to measure mechanical properties, and GAG concentration of the prepared samples, respectively. The results of mechanical tests and GAG contents shown that there are a wide range of dispersion in the most of the samples, which attribute to different factors. For mechanical properties, these factors could be attributed to anisotropic of the produced chondrocyte with agarose scaffolds, insufficient cells' dispersion within the gel scaffold during seeding and cultured time, and some test procedure condition, such as EBSS hydration. While for GAG results, those factors could be the differences of the cell growth environment between in-vitro and in vivo media.