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.

There is limited data assessing the cytotoxic effects of organosolv lignin with cells commonly used in tissue engineering. Structural and physico-chemical characterization of fractionated organosolv lignin showed that a decrease of the molecular weight (MW) is accompanied by a less branched conformation of the phenolic biopolymer (higher S/G ratio) and an increased number of aliphatic hydroxyl functionalities. Enabling stronger polymer−solvent interactions, as proven by the Hansen solubility parameter analysis, low MW organosolv lignin (2543 g/mol) is considered to be compatible with common biomaterials. Using low MW lignin, high cell viability (70–100%) was achieved after 2 h, 24 h and 7 days using the following lignin concentrations: MSCs and osteoblasts (0.02 mg/mL), gingival fibroblasts and keratinocytes (0.02 to 0.04 mg/mL), periodontal ligament fibroblasts and chondrocytes (0.02 to 0.08 mg/mL). Cell viability was reduced at higher concentrations, indicating that high concentrations are cytotoxic. Higher cell viability was attained using 30/70 (w/v) NaOH vs. 40/60 (w/v) EtOH as the initial lignin solvent. Hydrogels containing low MW lignin (0.02 to 0.3 mg/mL) in agarose dose-dependently increased chondrocyte attachment (cell viability 84–100%) and hydrogel viscosity and stiffness to 3–11 kPa, similar to the pericellular matrix of chondrocytes. This suggests that low MW organosolv lignin may be used in many tissue engineering fields.

Despite lignin’s global abundance and its use in biomedical studies, our understanding of how lignin regulates disease through modulation of cell morphology and associated phenotype of human cells is unknown. We combined an automated high-throughput image cell segmentation technique for quantitatively measuring a panel of cell shape descriptors, droplet digital Polymerase Chain Reaction for absolute quantification of gene expression and multivariate data analyses to determine whether lignin could therapeutically modulate the cell morphology and phenotype of inflamed, degenerating diseased human cells (osteoarthritic (OA) chondrocytes) towards a healthier cell morphology and phenotype. Lignin dose-dependently modified all aspects of cell morphology and ameliorated the diseased shape of OA chondrocytes by inducing a less fibroblastic healthier cell shape, which correlated with the downregulation of collagen 1A2 (COL1A2, a major fibrosis-inducing gene), upregulation of collagen 2A1 (COL2A1, a healthy extracellular matrix-inducing gene) and downregulation of interleukin-6 (IL-6, a chronic inflammatory cytokine). This is the first study to show that lignin can therapeutically target cell morphology and change a diseased cells’ function towards a healthier cell shape and phenotype. This opens up novel opportunities for exploiting lignin in modulation of disease, tissue degeneration, fibrosis, inflammation and regenerative medical implants for therapeutically targeting cell function and outcome.