Cell morphology as a biological fingerprint of chondrocyte phenotype in control and inflammatory conditions

Abstract

IntroductionLittle is known how inflammatory processes quantitatively affect chondrocyte morphology and how single cell morphometric data could be used as a biological fingerprint of phenotype.MethodsWe investigated whether trainable high-throughput quantitative single cell morphology profiling combined with population-based gene expression analysis can be used to identify biological fingerprints that are discriminatory of control vs. inflammatory phenotypes. The shape of a large number of chondrocytes isolated from bovine healthy and human osteoarthritic (OA) cartilages was quantified under control and inflammatory (IL-1β) conditions using a trainable image analysis technique measuring a panel of cell shape descriptors (area, length, width, circularity, aspect ratio, roundness, solidity). The expression profiles of phenotypically relevant markers were quantified by ddPCR. Statistical analysis, multivariate data exploration, and projection-based modelling were used for identifying specific morphological fingerprints indicative of phenotype.ResultsCell morphology was sensitive to both cell density and IL-1β. In both cell types, all shape descriptors correlated with expression of extracellular matrix (ECM)- and inflammatory-regulating genes. A hierarchical clustered image map revealed that individual samples sometimes responded differently in control or IL-1β conditions than the overall population. Despite these variances, discriminative projection-based modeling revealed distinct morphological fingerprints that discriminated between control and inflammatory chondrocyte phenotypes: the most essential morphological characteristics attributable to non-treated control cells was a higher cell aspect ratio in healthy bovine chondrocytes and roundness in OA human chondrocytes. In contrast, a higher circularity and width in healthy bovine chondrocytes and length and area in OA human chondrocytes indicated an inflammatory (IL-1β) phenotype. When comparing the two species/health conditions, bovine healthy and human OA chondrocytes exhibited comparable IL-1β-induced morphologies in roundness, a widely recognized marker of chondrocyte phenotype, and aspect ratio.DiscussionOverall, cell morphology can be used as a biological fingerprint for describing chondrocyte phenotype. Quantitative single cell morphometry in conjunction with advanced methods for multivariate data analysis allows identifying morphological fingerprints that can discriminate between control and inflammatory chondrocyte phenotypes. This approach could be used to assess how culture conditions, inflammatory mediators, and therapeutic modulators regulate cell phenotype and function.

Prediction of six macrophage phenotypes and their IL-10 content based on single-cell morphology using artificial intelligence

“…To determine if there were quantitative significant differences in shape descriptors (area, length, width, circularity, aspect ratio, roundness, and solidity), single-cell macrophage analysis was performed using a Fiji-based analysis algorithm ( 13 , 29 ). When comparing different groups of macrophages, the violin box plots ( Figure 5 ) revealed that the GM-CSF/TNFα/IFNγ-M1 (largest) and GM-CSF-M1 macrophages were larger in cell area than the other groups.…”

“…The panel of morphological descriptors that we used here was successfully used by our group to phenotype differentiated human mesenchymal stromal cells (MSCs) ( 30 – 33 ) and healthy vs. inflamed and degenerating diseased human chondrocytes ( 13 ). Moreover, using this panel as a phenotypic marker, combined with multivariate data analysis, we showed that the cell morphology and phenotype, i.e., the “biological fingerprint” of those inflamed and degenerated diseased human cells could be reverted to a healthier cell shape via therapeutic modulation and their healthier cell shape correlated with positive changes in major fibrosis- and inflammatory-regulating genes ( 29 ).…”

“…Single macrophage analysis was performed using a Fiji-based ( 38 ) single-cell shape analysis algorithm that we previously used to phenotype differentiated mesenchymal stromal cells (MSCs) ( 6 , 30 – 33 ) and healthy vs. inflamed and degenerating chondrocytes ( 13 , 29 – 33 ). The fluorescent staining with DAPI and phalloidin visualized the cell’s nucleus and body (F-actin).…”

Colloidal Phenol-Amine Coating on Implants for Improved Anti-Inflammation and Osteogenesis

“…The quantification results of CCK-8 revealed that the proliferative activity of cells on CT-C was significantly higher than that of the Control (Figure B). Since the cell shape is strongly coupled with cell growth, the cell area, the aspect ratio, and roundness were also determined. , As shown in Figure A-ii, the cell area of CT-C was significantly higher than that of Control on the fifth day (Figure C), suggesting that the CT-C could promote cell spreading with the extension of culturing time. The cell aspect ratio (Figure D) in the CT-C group was significantly higher, while the roundness (Figure E), which is related to cell proliferation, was significantly lower.…”

Injectable Thermosensitive Hyaluronic Acid Hydrogels for Chondrocyte Delivery in Cartilage Tissue Engineering

“…The SEM image in Figure 9 indicates chondrocytes cultured in HA–CPN for 28 days were embedded within the secreted ECM, and the cells can retain the correct phenotypes. Instead of an elongated morphology on TCPS due to the dedifferentiation of the chondrocytes, chondrocytes in HPN maintain a spherical morphology, which is one of the characteristics of differentiated chondrocytes [ 47 ]. The dedifferentiation of chondrocytes is common after an in vitro cell culture on TCPS, where chondrocytes tend to lose the original morphology and show a diffused actin network with pronounced stress fiber formation [ 47 ].…”