Journal: International Journal of Nanomedicine

Article Title: Positive impact of IGF-1-coupled nanoparticles on the differentiation potential of human chondrocytes cultured on collagen scaffolds

doi: 10.2147/IJN.S72872

Figure Lengend Snippet: Chemical synthesis of IGF-1 nanoparticles and application for in vitro cultivation of dedifferentiated chondrocytes. Notes: ( A ) Conjugation of the thiolated IGF-1 to PEG24-maleimide functionalized red fluorescent silica nanoparticles (sicastar ® -redF). ( B ) In passage three, chondrocytes were seeded either on plastic (2-D; upper panel) or on a collagen scaffold (3-D; lower panel) with a concentration of 1×10 5 cells per 1 cm 2 . Cells were incubated with (a) platelet growth factor lyophilisate (771 pg/μL IGF-1, 517 pg/mL TGF-β1, 2.46 pg/mL VEGF, 2.20 pg/mL basic FGF); (b) 50 ng/mL rhIGF-1; (c) red-fluorescent rhIGF-1-coupled silica nanoparticles (12.5 μg/mL particle suspension =50 ng/mL rhIGF-1); or (d) red-fluorescent control silica nanoparticles (12.5 μg/mL particle suspension =50 ng/mL NH 2 ) for 48 and 96 hours, as well as for 3, 7, and 14 days. The supplements (lyophilisate, rhIGF-1, nanoparticles) were only added in the beginning, when the cells were seeded. During cultivation over 14 days, medium (without supplements) was changed every 2–3 days. Abbreviations: IGF-1, insulin-like growth factor 1; TGF-β1, transforming growth factor beta 1; VEGF, vascular endothelial growth factor; FGF, fibroblast growth factor; rhIGF-1, recombinant human IGF-1.

Article Snippet: Red fluorescent silica nanoparticles (sicastar ® -redF, 40-01-103; micromod Partikeltechnologie GmbH, Rostock, Ger-many) with a diameter of 1 μm and amino groups on the surface ( ) were applied for the conjugation of IGF-1.

Techniques: In Vitro, Conjugation Assay, Concentration Assay, Incubation, Suspension, Control, Recombinant

Journal: International Journal of Nanomedicine

Article Title: Positive impact of IGF-1-coupled nanoparticles on the differentiation potential of human chondrocytes cultured on collagen scaffolds

doi: 10.2147/IJN.S72872

Figure Lengend Snippet: Binding of IGF-1-coupled nanoparticles by chondrocytes grown in a monolayer for 2 and 4 days. Notes: Chondrocytes were isolated from the hyaline cartilage of patients (n=3) undergoing primary total knee joint replacement and cultured in a monolayer (1×10 5 cells per well in a 24-well plate). Cells were suspended with medium supplemented with red-fluorescent rhIGF-1-coupled nanoparticles (12.5 μg/mL particle suspension =50 ng/mL rhIGF-1). During cultivation, no medium change was conducted. After 2 and 4 days, cells were stained with calcein-AM to visualize vital cells (green, upper left and lower left) using a fluorescence microscope. The wavelength of 569–585 nm was used to visualize IGF-1-coupled nanoparticles (red, upper middle and lower middle). The pictures of cells and particles were combined (green, upper right and lower right; overlays). Red darts indicate particles bound by chondrocytes. White bar =100 μm. Abbreviation: calcein-AM, calcein-acetoxymethyl; IGF-1, insulin-like growth factor 1; rhIGF-1, recombinant human IGF-1.

Article Snippet: Red fluorescent silica nanoparticles (sicastar ® -redF, 40-01-103; micromod Partikeltechnologie GmbH, Rostock, Ger-many) with a diameter of 1 μm and amino groups on the surface ( ) were applied for the conjugation of IGF-1.

Techniques: Binding Assay, Isolation, Cell Culture, Suspension, Staining, Fluorescence, Microscopy, Recombinant

Journal: International Journal of Nanomedicine

Article Title: Positive impact of IGF-1-coupled nanoparticles on the differentiation potential of human chondrocytes cultured on collagen scaffolds

doi: 10.2147/IJN.S72872

Figure Lengend Snippet: Effect of IGF-1-coupled nanoparticles on chondrocytes grown in a monolayer for 2 and 4 days. Notes: Chondrocytes were isolated from the hyaline cartilage of patients (n=3) undergoing primary total knee joint replacement and were cultured in a monolayer (1×10 5 cells per well in a 24-well plate). Cells were suspended in serum-free medium supplemented with rhIGF-1-coupled nanoparticles (sNP–IGF-1; 12.5 μg/mL particle suspension =50 ng/mL rhIGF-1), control particles (sNP-Co; 12.5 μg/mL particle suspension =50 ng/mL NH 2 ), and rhIGF-1 (50 ng/mL). No medium change was conducted. After 2 and 4 days, CICP (osteogenic marker) as well as CPII (chondrogenic marker) were quantified in the cell supernatant by means of enzyme-linked immunosorbent assay. Data were normalized by division with WST-1 absorption values. Boxes denote interquartile ranges, horizontal lines within the boxes denote medians, and whiskers denote minimum and maximum values. Analysis of variance post hoc-LSD was conducted (there were no significant differences between 2 and 4 days of incubation nor between the 3 different treatments). Abbreviations: CICP, procollagen type I; CPII, procollagen type II; LSD, least significant difference; sNP, silica nanoparticles; sNP-Co, control nanoparticles; WST-1, water-soluble-tetrazolium salt; IGF-1, insulin-like growth factor 1; rhIGF-1, recombinant human IGF-1.

Article Snippet: Red fluorescent silica nanoparticles (sicastar ® -redF, 40-01-103; micromod Partikeltechnologie GmbH, Rostock, Ger-many) with a diameter of 1 μm and amino groups on the surface ( ) were applied for the conjugation of IGF-1.

Techniques: Isolation, Cell Culture, Suspension, Control, Marker, Enzyme-linked Immunosorbent Assay, Incubation, Recombinant

Journal: International Journal of Nanomedicine

Article Title: Positive impact of IGF-1-coupled nanoparticles on the differentiation potential of human chondrocytes cultured on collagen scaffolds

doi: 10.2147/IJN.S72872

Figure Lengend Snippet: Collagen scaffolds enriched with IGF-1-coupled nanoparticles and growth factor lyophilisate have a positive impact on early chondrogenic redifferentiation. Notes: Chondrocytes were isolated from hyaline cartilage of patients (n≥3) undergoing primary total knee joint replacement and were either cultured on cell culture plastic ( A – C ) or on a collagen scaffold ( D – F ) at a concentration of 1×10 5 cells/1 cm 2 each. Initially, culture medium was supplemented with rhIGF-1-coupled nanoparticles (sNP– IGF-1; 12.5 μg/mL particle suspension =50 ng/mL rhIGF-1), control particles (sNP-Co; 12.5 μg/mL particle suspension =50 ng/mL NH 2 ), rhIGF-1 (50 ng/mL), and growth factor Lyoph. Medium change (without supplements) was conducted every 2–3 days. After 3 and 7 days of cultivation, metabolic cell activity was determined with WST-1 assay, and CICP (osteogenic marker) as well as CPII (chondrogenic marker) were quantified in the cell supernatant by means of enzyme-linked immunosorbent assay. Means and standard deviations are demonstrated. For statistically significance analyses, the post hoc-LSD ANOVA was applied (7 days compared to 3 days: * P <0.05, ** P <0.01, *** P <0.001; groups compared at 3 days: ## P <0.01, ### P <0.001; groups compared at 7 days: + P <0.05, ++ P <0.01, +++ P <0.001). ( G ) Calculation of the ratio (7 days:3 days) of procollagen type II production by cells grown on scaffolds or on plastic with sNP–IGF-1, sNP-Co, rhIGF-1, and Lyoph. The post hoc-LSD ANOVA was applied (comparison of groups: # P <0.05, ## P <0.01; scaffold compared to monolayer: *** P <0.001). ( H ) Cells grown on the collagen scaffold enriched with sNP–IGF-1 were stained with calcein-AM after 7 days (viable cells: fluoresce green; nanoparticles fluoresce: red). The left diagram shows a z-stack and the right picture shows a representative microscopic top view. Abbreviations: ANOVA, analysis of variance; calcein-AM, calcein-acetoxymethyl; CICP, procollagen type I; CPII, procollagen type II; Lyoph, lyophilisate; LSD, least significant difference; OD, optical density; sNP, silica nanoparticles; sNP-Co, control nanoparticles; WST-1, water-soluble-tetrazolium salt; IGF-1, insulin-like growth factor 1; rhIGF-1, recombinant human IGF-1.

Article Snippet: Red fluorescent silica nanoparticles (sicastar ® -redF, 40-01-103; micromod Partikeltechnologie GmbH, Rostock, Ger-many) with a diameter of 1 μm and amino groups on the surface ( ) were applied for the conjugation of IGF-1.

Techniques: Isolation, Cell Culture, Concentration Assay, Suspension, Control, Activity Assay, WST-1 Assay, Marker, Enzyme-linked Immunosorbent Assay, Comparison, Staining, Recombinant