Cleistanthin A causes peripheral vasoconstriction and myocardial depression in isolated tissue preparations.

BACKGROUND: Cleistanthus collinus is a poisonous shrub commonly used for deliberate self-harm in rural south India. Boiled decoction or a paste made from its leaves is used for suicide. Cleistanthoside A and Cleistanthin A are the major toxins identified from this plant. In this study, we disclose the mechanism of Cleistanthin A toxicity and concentrations of the two toxins in different extracts of Cleistanthus collinus. METHODS: The effect of Cleistanthin A was studied on isolated goat leg arteries using two different preparations namely transverse cylinder and longitudinal strip. The influence of Cleistanthin A on peripheral vascular resistance and myocardial contractility was evaluated by rat hind limb and isolated rat heart experiments, respectively. For the quantification of toxins, five different extracts of C. collinus leaves were prepared. The extracts were subjected to analytical HPLC to quantify Cleistanthoside A and Cleistanthin A. RESULTS AND CONCLUSION: Cleistanthin A increased vascular tension in transverse cylinder preparation and increased peak, trough and mean aortic pressures in the rat hind limb preparations. In isolated rat heart experiments, there was an increase in diastolic and mean ventricular pressure with a significant decrease in ventricular pulse pressure. These observations suggest that the hypotension in C. collinus poisoning patients may be due to cardiotoxicity and not due to vasodilation as is currently believed. Quantification of different extracts showed that boiled extracts had higher quantities of Cleistanthoside A whereas crushed leaf extracts yielded significantly higher amounts of Cleistanthin A.

An in vitro analysis of the effect of hyperosmolarity on the chondrogenic potential of human articular cartilage derived chondroprogenitors.

PURPOSE: Chondroprogenitors display promise for articular cartilage regeneration. It is imperative to standardize culture conditions, to further enhance chondrogenicity and reduce tendency for hypertrophy. Cartilage matrix provides a unique hyperosmolar microenvironment that enables native cells to resist compressive stress. However, commonly used culture media have osmolarities relatively hypoosmotic when compared to in-vivo conditions. Previous reports involving chondrocytes demonstrated enhanced chondrogenic potential secondary to utilization of hyperosmolar culture conditions. The study aimed to assess the effect of hyperosmolarity (either mimicking normal joint conditions or short-term hyperosmotic stress) on chondroprogenitor phenotype. MATERIALS AND METHODS: Fibronectin adhesion assay derived human articular chondroprogenitors (n = 3) were divided into 3 groups: a) Control: cells grown in standard culture conditions (320 mOsm/L), b) Test A: cells grown in hyperosmolar media mimicking joint conditions (409 mOsm/L) and c) Test B: cells exposed to short-term hyperosmotic stress (504 mOsm/L) for 24 h, prior to assessment. Evaluation parameters included population doubling, cell size, surface marker expression, mRNA expression (markers of chondrogenesis, dedifferentiation and hypertrophy) and multilineage potential. RESULTS: Subjecting these cells to increased osmolarity in culture did not demonstrably favor chondrogenesis (control vs Test A: comparable COL2A1) while hyperosmotic stress further increased the tendency for hypertrophy and terminal differentiation (high COL1A1 and low COL2A1, P = 0.006). Additionally, growth kinetics, surface marker expression and multilineage potential were comparable across groups. CONCLUSION: Chondroprogenitors displayed sensitivity to increase in osmolarity as chondrogenic phenotype did not improve, while hypertrophic propensity was heightened, although further analysis of culture and phenotypic parameters will aid in optimizing chondroprogenitor use in cartilage regeneration.

Comparative analysis of human bone marrow mesenchymal stem cells, articular cartilage derived chondroprogenitors and chondrocytes to determine cell superiority for cartilage regeneration.

INTRODUCTION: Chondroprogenitors, a promising therapeutic modality in cell-based therapy, are routinely isolated from articular cartilage by fibronectin differential adhesion assay. However, there is paucity of information regarding their biological profile and the lack of a marker that can reliably distinguish them from cultured chondrocytes due to possible dedifferentiation. Since chondroprogenitors have been classified as mesenchymal stem cells(MSCs), the aim of our study was to compare bone marrow-MSCs, chondroprogenitors and chondrocytes, and assess superiority for cartilage repair. An additional objective was to also compare CD49b as a differentiating marker for isolating chondroprogenitors as a recent report demonstrated significantly high expression in the surfaceome of migratory articular chondroprogenitors. METHODS: Bone marrow aspirate and articular cartilage was obtained from three osteoarthritic knee joints. Study arms included a) bone marrow-MSCs, b) chondroprogenitors, c) cultured chondrocytes, d) chondrocytes cultured with additional growth factors and e) CD49b + sorted chondroprogenitors. Assessment parameters included population doubling, surface expression for positive, negative MSC markers and potential markers of chondrogenesis (CD29, CD49e, CD49b, CD166 and CD146), RT-PCR for markers of chondrogenesis and hypertrophy and trilineage differentiation. RESULTS AND CONCLUSION: Chondroprogenitors exhibited efficient chondrogenesis (SOX-9 and COL2A1) and significantly lower tendency for hypertrophy (RUNX2), which was also reflected in trilineage differentiation where progenitors displayed minimal calcified matrix, efficient glycosaminoglycan deposition and high collagen type II uptake. CD49b did not serve as a marker for isolation as sorted chondroprogenitors performed significantly poorer when compared to fibronectin assay derived cells. Emphasis on preclinical studies utilizing progenitors of higher purity is the future direction.

Pondering the Potential of Hyaline Cartilage-Derived Chondroprogenitors for Tissue Regeneration: A Systematic Review.

OBJECTIVE: Chondroprogenitors have recently gained prominence due to promising results seen in in vitro and animal studies as a potential contender in cell-based therapy for cartilage repair. Lack of consensus regarding nomenclature, isolation techniques, and expansion protocols create substantial limitations for translational research, especially given the absence of distinct markers of identification. The objective of this systematic review was to identify and collate information pertaining to hyaline cartilage-derived chondroprogenitors, with regard to their isolation, culture, and outcome measures. DESIGN: As per Preferred Reporting Items for Systematic Reviews and Meta-Analyses guidelines, a web-based search of Scopus and PubMed databases was performed from January 2000 to May 2020, which yielded 509 studies. A total of 65 studies were identified that met the standardized inclusion criteria which comprised of, but was not limited to, progenitors derived from fibronectin adhesion, migrated subpopulation from explant cultures, and single-cell sorting. RESULT: Literature search revealed that progenitors demonstrated inherent chondrogenesis and minimal tendency for hypertrophy. Multiple sources also demonstrated significantly better outcomes that bone marrow-derived mesenchymal stem cells and comparable results to chondrocytes. With regard to progenitor subgroups, collated evidence points to better and consistent outcomes with the use of migratory progenitors when compared to fibronectin adhesion assay-derived progenitors, although a direct comparison between the two cell populations is warranted. CONCLUSION: Since chondroprogenitors exhibit favorable properties for cartilage repair, efficient characterization of progenitors is imperative, to complete their phenotypic profile, so as to optimize their use in translational research for neocartilage formation.

Comparison of the efficiency of laminin versus fibronectin as a differential adhesion assay for isolation of human articular cartilage derived chondroprogenitors.

Purpose: Cartilage repair following trauma or degeneration is poor, making cell-based therapy an important avenue of treatment. Chondrocytes and mesenchymal stem cells have been extensively studied as potential candidates, although tendency toward hypertrophy and formation of mixed hyaline-fibrocartilage necessitates further optimization. Chondroprogenitors, isolated using fibronectin adhesion assay are reported to show reduced hypertrophy and enhanced chondrogenesis. Laminin, an essential component of extracellular matrix, has been shown to positively modulate chondrocyte proliferation, migration, and survival. The aim of our study was to evaluate the effect of laminin as a differential adhesion assay and obtain an enriched population of chondroprogenitors and assess its efficiency when compared to progenitors obtained via fibronectin.Materials and methods: Chondrocytes were isolated from three osteoarthritic knee joints and subjected to fibronectin and laminin adhesion to obtain chondroprogenitors. After expansion in culture, they were assessed for differences in their biological characteristics based on growth kinetics, surface marker expression, gene expression for assessing markers of chondrogenesis and hypertrophy, and potential for tri-lineage differentiation.Results: Our results showed that cells isolated by laminin and fibronectin both displayed comparable characteristics except in terms of proliferative potential (higher in laminin), gene expression of COL2A1 (lower in laminin) and trilineage potential where the laminin group showed higher osteogenic and adipogenic differentiation.Conclusion: This was the first attempt to successfully isolate human articular cartilage derived chondroprogenitor clones using laminin, which retained stem cell like characteristics. Further evaluation to optimize this method will help enhance chondroprogenitor characteristics, for use in cartilage repair.