Antioxidative therapy in an ex vivo human cartilage trauma-model: attenuation of trauma-induced cell loss and ECM-destructive enzymes by N-acetyl cysteine

Osteoarthritis (OA) is the most common rheumatic disease, characterized by progressive articular cartilage degradation. Raman spectroscopy (RS) has been recently proposed as a label-free tool to detect molecular changes in musculoskeletal tissues. We used cartilage samples derived from human femoral heads to perform an ex vivo study of different Raman signals and ratios, related to major and minor molecular components of articular cartilage, hereby proposed as candidate optical biomarkers for OA. Validation was performed against the radiological Kellgren–Lawrence (K-L) grading system, as a gold standard, and cross-validated against sulfated glycosaminoglycans (sGAGs) and total collagens (Hyp) biochemical contents. Our results showed a significant decrease in sGAGs (SGAGs, A1063 cm−1/A1004 cm−1) and proteoglycans (PGs, A1375 cm−1/A1004 cm−1) and a significant increase in collagen disorganization (ColD/F, A1245 cm−1/A1270 cm−1), with OA severity. These were correlated with sGAGs or Hyp contents, respectively. Moreover, the SGAGs/HA ratio (A1063 cm−1/A960 cm−1), representing a functional matrix, rich in proteoglycans, to a mineralized matrix-hydroxyapatite (HA), was significantly lower in OA cartilage (K-L I vs. III–IV, p < 0.05), whilst the mineralized to collagenous matrix ratio (HA/Col, A960 cm−1/A920 cm−1) increased, being correlated with K-L. OA samples showed signs of tissue mineralization, supported by the presence of calcium crystals-related signals, such as phosphate, carbonate, and calcium pyrophosphate dihydrate (MGP, A960 cm−1/A1004 cm−1, MGC, A1070 cm−1/A1004 cm−1 and A1050 cm−1/A1004 cm−1). Finally, we observed an increase in lipids ratio (IL, A1450 cm−1/A1670 cm−1) with OA severity. As a conclusion, we have described the molecular fingerprint of hip cartilage, validating a panel of optical biomarkers and the potential of RS as a complementary diagnostic tool for OA.

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The Hexosamine Biosynthetic Pathway as a Therapeutic Target after Cartilage Trauma: Modification of Chondrocyte Survival and Metabolism by Glucosamine Derivatives and PUGNAc in an Ex Vivo Model

International Journal of Molecular Sciences ◽

10.3390/ijms22147247 ◽

2021 ◽

Vol 22 (14) ◽

pp. 7247

Author(s):

Jana Riegger ◽

Julia Baumert ◽

Frank Zaucke ◽

Rolf E. Brenner

Keyword(s):

Biosynthetic Pathway ◽

Ex Vivo ◽

Type Ii Collagen ◽

Cartilage Explants ◽

Uridine Diphosphate ◽

Cell Protection ◽

Ex Vivo Model ◽

Human Cartilage ◽

Hexosamine Biosynthetic Pathway ◽

Cellular Processes

The hexosamine biosynthetic pathway (HBP) is essential for the production of uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), the building block of glycosaminoglycans, thus playing a crucial role in cartilage anabolism. Although O-GlcNAcylation represents a protective regulatory mechanism in cellular processes, it has been associated with degenerative diseases, including osteoarthritis (OA). The present study focuses on HBP-related processes as potential therapeutic targets after cartilage trauma. Human cartilage explants were traumatized and treated with GlcNAc or glucosamine sulfate (GS); PUGNAc, an inhibitor of O-GlcNAcase; or azaserine (AZA), an inhibitor of GFAT-1. After 7 days, cell viability and gene expression analysis of anabolic and catabolic markers, as well as HBP-related enzymes, were performed. Moreover, expression of catabolic enzymes and type II collagen (COL2) biosynthesis were determined. Proteoglycan content was assessed after 14 days. Cartilage trauma led to a dysbalanced expression of different HBP-related enzymes, comparable to the situation in highly degenerated tissue. While GlcNAc and PUGNAc resulted in significant cell protection after trauma, only PUGNAc increased COL2 biosynthesis. Moreover, PUGNAc and both glucosamine derivatives had anti-catabolic effects. In contrast, AZA increased catabolic processes. Overall, “fueling” the HBP by means of glucosamine derivatives or inhibition of deglycosylation turned out as cells and chondroprotectives after cartilage trauma.

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Experimental Models to Study COVID-19 Effect in Stem Cells

Cells ◽

10.3390/cells10010091 ◽

2021 ◽

Vol 10 (1) ◽

pp. 91

Author(s):

Rishi Man Chugh ◽

Payel Bhanja ◽

Andrew Norris ◽

Subhrajit Saha

Keyword(s):

Stem Cells ◽

Ex Vivo ◽

Drug Efficacy ◽

Cell Loss ◽

Experimental Models ◽

Model Systems ◽

Virus Infectivity ◽

Ex Vivo Model ◽

Global Pandemic ◽

The Impact

The new strain of coronavirus (severe acute respiratory syndrome coronavirus type 2 (SARS-CoV-2)) emerged in 2019 and hence is often referred to as coronavirus disease 2019 (COVID-19). This disease causes hypoxic respiratory failure and acute respiratory distress syndrome (ARDS), and is considered as the cause of a global pandemic. Very limited reports in addition to ex vivo model systems are available to understand the mechanism of action of this virus, which can be used for testing of any drug efficacy against virus infectivity. COVID-19 induces tissue stem cell loss, resulting inhibition of epithelial repair followed by inflammatory fibrotic consequences. Development of clinically relevant models is important to examine the impact of the COVID-19 virus in tissue stem cells among different organs. In this review, we discuss ex vivo experimental models available to study the effect of COVID-19 on tissue stem cells.

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Characterization of Ex Vivo–Generated Bovine and Human Cartilage by Immunohistochemical, Biochemical, and Magnetic Resonance Imaging Analyses

Tissue Engineering Part A ◽

10.1089/ten.tea.2009.0717 ◽

2010 ◽

Vol 16 (7) ◽

pp. 2183-2196 ◽

Cited By ~ 17

Author(s):

Ashleigh E. Nugent ◽

David A. Reiter ◽

Kenneth W. Fishbein ◽

Denise L. McBurney ◽

Travis Murray ◽

...

Keyword(s):

Magnetic Resonance Imaging ◽

Magnetic Resonance ◽

Ex Vivo ◽

Resonance Imaging ◽

Human Cartilage

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New Therapeutic Strategies for Osteoarthritis by Targeting Sialic Acid Receptors

Biomolecules ◽

10.3390/biom10040637 ◽

2020 ◽

Vol 10 (4) ◽

pp. 637 ◽

Cited By ~ 3

Author(s):

Paula Carpintero-Fernandez ◽

Marta Varela-Eirin ◽

Alessandra Lacetera ◽

Raquel Gago-Fuentes ◽

Eduardo Fonseca ◽

...

Keyword(s):

Articular Cartilage ◽

Ex Vivo ◽

Molecular Model ◽

Cartilage Degradation ◽

Degenerative Joint Disease ◽

Joint Disease ◽

Novel Therapy ◽

Human Cartilage ◽

Sialic Acid Receptors ◽

Cartilage Breakdown

Osteoarthritis (OA) is the most common degenerative joint disease characterized by articular cartilage degradation and joint degeneration. The articular cartilage is mainly formed by chondrocytes and a collagen-proteoglycan extracellular matrix that contains high levels of glycosylated proteins. It was reported that the shift from glycoproteins containing α-2,6-linked sialic acids to those that contain α-2,3 was associated with the onset of common types of arthritis. However, the pathophysiology of α-2,3-sialylation in cartilage has not been yet elucidated. We show that cartilage from osteoarthritic patients expresses high levels of the α-2,3-sialylated transmembrane mucin receptor, known as podoplanin (PDPN). Additionally, the Maackia amurensis seed lectin (MASL), that can be utilized to target PDPN, attenuates the inflammatory response mediated by NF-kB activation in primary chondrocytes and protects human cartilage breakdown ex vivo and in an animal model of arthritis. These findings reveal that specific lectins targeting α-2,3-sialylated receptors on chondrocytes might effectively inhibit cartilage breakdown. We also present a computational 3D molecular model for this interaction. These findings provide mechanistic information on how a specific lectin could be used as a novel therapy to treat degenerative joint diseases such as osteoarthritis.

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EFFECTS OF N-ACETYL-CYSTEINE SUPPLEMENTATION ON EX-VIVO CLONOGENICITY AND OXIDATIVE PROFILE OF LINEAGE-COMMITTED HEMATOPOIETIC STEM/PROGENITOR CELLS

Jurnal Teknologi ◽

10.11113/jt.v80.11419 ◽

2018 ◽

Vol 80 (3) ◽

Cited By ~ 1

Author(s):

Chan Chin Yi ◽

Zariyantey Abd Hamid ◽

Izatus Shima Taib ◽

Tan Hui Yee ◽

Muhd Khairul Akmal Wak Harto ◽

...

Keyword(s):

Progenitor Cells ◽

Ex Vivo ◽

Oxidative Status ◽

Oxygen Species ◽

Hematopoietic Stem ◽

Colony Forming Units ◽

Stem And Progenitor Cells ◽

Acetyl Cysteine ◽

Antioxidant Supplement ◽

B Lymphoid

Hematopoietic stem and progenitor cells (HSPCs) are exposed to oxidative damage acquired during ex vivo expansion which affects their therapeutic potency. Efforts to overcome this limitation includes the use of antioxidants. The effects of N-Acetyl-Cysteine (NAC) supplementation for 48 hours on maintenance of ex vivo HSPCs was investigated by examining the cell viability at concentrations of 0.125 µM, 0.25 µM, 0.5 µM, 1.0 µM and 2.0 µM, followed by clonogenicity and oxidative status assessments of lineage-committed progenitors (myeloid, erythroid and pre-B lymphoid) at selected NAC concentrations (0.25 µM, 0.5 µM, 2.0 µM). NAC supplementation significantly (p< 0.05) enhanced viability of HSPC at 0.25 µM, 0.5 µM, 2.0 µM. The clonogenicity of each progenitor was not affected as no significant changes of Colony Forming Units (CFUs) counts was noted between NAC-supplemented group than control. NAC showed no significant effects on reactive oxygen species (ROS), glutathione (GSH) and superoxide dismutase (SOD) levels of respective progenitors as compared to control. Conclusively, NAC shows potential property as antioxidant supplement for ex vivo maintenance of HSPCs by promoting survivability and maintaining clonogenicity.

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212 EVALUATION OF THE EFFICACY OF IL-1R ANTAGONISTS IN HUMAN CARTILAGE DEGRADATION EX VIVO

Osteoarthritis and Cartilage ◽

10.1016/s1063-4584(11)60239-2 ◽

2011 ◽

Vol 19 ◽

pp. S104-S105

Author(s):

J.T. Hulme ◽

K. Merriam ◽

J.B. Rottman ◽

G.D. Virca ◽

J.E. Sims ◽

...

Keyword(s):

Ex Vivo ◽

Cartilage Degradation ◽

Human Cartilage

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Endothelial cell loss following tissue harvesting by pneumodissection for endothelial keratoplasty: an ex vivo study

British Journal of Ophthalmology ◽

10.1136/bjophthalmol-2014-306560 ◽

2015 ◽

Vol 99 (5) ◽

pp. 710-713 ◽

Cited By ~ 16

Author(s):

Saief L Altaan ◽

Ankur Gupta ◽

Laura E Sidney ◽

Mohamed S Elalfy ◽

Amar Agarwal ◽

...

Keyword(s):

Endothelial Cell ◽

Ex Vivo ◽

Cell Loss ◽

Endothelial Keratoplasty ◽

Endothelial Cell Loss ◽

Ex Vivo Study

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Raman Needle Arthroscopy for In Vivo Molecular Assessment of Cartilage

10.1101/2021.06.15.448529 ◽

2021 ◽

Author(s):

Kimberly Kroupa ◽

Man I Wu ◽

Juncheng Zhang ◽

Magnus Jensen ◽

Wei Wong ◽

...

Keyword(s):

Ex Vivo ◽

Low Cost ◽

Femoral Condyle ◽

Cartilage Explants ◽

Anatomical Site ◽

Superficial Zone ◽

Human Cartilage ◽

Polarized Raman ◽

Bovine Cartilage

The development of treatments for osteoarthritis (OA) is burdened by the lack of standardized biomarkers of cartilage health that can be applied in clinical trials. We present a novel arthroscopic Raman probe that can optically biopsy cartilage and quantify key ECM biomarkers for determining cartilage composition, structure, and material properties in health and disease. Technological and analytical innovations to optimize Raman analysis include: 1) multivariate decomposition of cartilage Raman spectra into ECM-constituent-specific biomarkers (glycosaminoglycan [GAG], collagen [COL], water [H2O] scores), and 2) multiplexed polarized Raman spectroscopy to quantify superficial zone collagen anisotropy via a PLS-DA-derived Raman collagen alignment factor (RCAF). Raman measurements were performed on a series of ex vivo cartilage models: 1) chemically GAG-depleted bovine cartilage explants (n=40), 2) mechanically abraded bovine cartilage explants (n=30), 3) aging human cartilage explants (n=14), and 4) anatomical-site-varied ovine osteochondral explants (n=6). Derived Raman GAG score biomarkers predicted 95%, 66%, and 96% of the variation in GAG content of GAG-depleted bovine explants, human explants, and ovine explants, respectively (p<0.001). RCAF values were significantly different for explants with abrasion-induced superficial zone collagen loss (p<0.001). The multivariate linear regression of Raman-derived ECM biomarkers (GAG and H2O scores) predicted 94% of the variation in elastic modulus of ovine explants (p<0.001). Finally, we demonstrated the first in vivo Raman arthroscopy assessment of an ovine femoral condyle through intraarticular entry into the synovial capsule. This work advances Raman arthroscopy towards a transformative low cost, minimally invasive diagnostic platform for objective monitoring of treatment outcomes from emerging OA therapies.

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Conformational Perturbation of SARS-CoV-2 Spike Protein Using N-Acetyl Cysteine, a Molecular Scissor: A Probable Strategy to Combat COVID-19

10.26434/chemrxiv.12687923.v1 ◽

2020 ◽

Author(s):

Utsab Debnath ◽

Varun Dewaker ◽

Yenamandra S. Prabhakar ◽

Parthasarathi Bhattacharyya ◽

Amit Mandal

Keyword(s):

Disulfide Bond ◽

In Silico ◽

Ex Vivo ◽

Virus Disease ◽

Spike Protein ◽

Early 21St Century ◽

Acetyl Cysteine ◽

Structural Architecture

The infection caused by Severe Acute Respiratory Syndrome–CoronaVirus-2 (SARS-CoV-2) resulted in a pandemic across the globe with a huge death toll. The symptoms from SARS-CoV2 appear somewhat similar to the SARS-CoV-1 infection that appeared in early 21st century but the infectivity is far higher for the SARS-CoV-2. The virus attaches itself to exposed human epithelial cells through the spike protein. Recently discovered crystal structure of the complex of spike protein of SARS-CoV-2 with human angiotensin-converting enzyme 2 (ACE2) receptor indicated that the virus binds with the host cell very strongly. We hypothesized that the perturbation of the functionally active conformation of spike protein through the reduction of a solvent accessible disulfide bond (Cys391-Cys525) that provides its structural architecture, may 2 be a feasible strategy to disintegrate the spike protein from ACE2 receptor and thereby prevent the infection. Using in silico platform we showed that N-acetyl cysteine (NAC), a drug used as antioxidant and mucolytic agent, binds in the close proximity of above disulfide bond. The reduction of the disulfide bond via thiol/disulfide exchange, followed by covalent conjugation of NAC perturbed the stereo specific orientations of interacting key residues of spike protein. This resulted in threefold weakening in the binding affinity of spike protein with ACE2 receptor. This opens avenues for exploring the effect of NAC in vitro, ex vivo and in vivo and on successful observation of the similar effect as in silico, the intervention of NAC may be translated in the pharmacoprevention and treatment of Corona virus disease 2019.

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