Journal: iScience

Article Title: Genetically Engineered Flagella Form Collagen-like Ordered Structures for Inducing Stem Cell Differentiation

doi: 10.1016/j.isci.2019.06.036

Figure Lengend Snippet: Display of a Collagen-like Peptide (GPP) 8 on Flagella, Biomimetic Assembly and Mineralization of the Resultant GPP8 Flagella, and BMSCs' Differentiation on the GPP8 Flagella Film (A) Schematic of a flagellum. It is mainly composed of about 30,000 flagellin (FliC) subunits. It contains 11 subunits per 2 turns. The N and C termini of flagellin (D0 and D1) are highly conserved and face the center of the filament. However, the central D2 and D3 regions are hypervariable and can be genetically engineered to display (GPP) 8 . (B) Schematic of the production and self-assembly of HAP-mineralized flagellar filaments into a matrix, a closed-packed monolayer of mineralized flagella, which can support the adhesion, proliferation, and early osteogenic differentiation of BMSCs. After the peptide is displayed on the flagella by genetically engineering the bacteria (1), the flagella are purified from the surface of bacteria by vortexing (2). Then the flagella are first allowed to interact with Ca 2+ ions and then with an HAP precursor solution to initiate the self-assembly and mineralization of the flagella (3). The resultant mineralized flagella bundles are deposited onto a polylysine-coated glass slide to form a matrix (4) to induce osteogenic differentiation of BMSCs (5 and 6).

Article Snippet: Biomimetic Self-Assembly and Mineralization of GPP8 Flagella under Different Flagella Concentrations in the HAP-Supersaturated Solution after Pretreatment with Calcium Ions (A) Only a few bundles were formed at a low flagella concentration (∼10 μg/mL). (B) At a higher flagella concentration (∼40 μg/mL), more parallel bundles were formed. (C) A close-packed monolayer was observed when the concentration of flagella reached ∼80 μg/mL. (D) SAED pattern at the relatively straight segment of flagella bundles, showing the c -axis preferred orientation of HAP along the flagella (inset). (E) Energy-dispersive X-ray spectroscopic analysis confirming a Ca/P molar ratio of 1.65.

Techniques: Bacteria, Purification

Journal: iScience

Article Title: Genetically Engineered Flagella Form Collagen-like Ordered Structures for Inducing Stem Cell Differentiation

doi: 10.1016/j.isci.2019.06.036

Figure Lengend Snippet: Transmission Electron Microscopic Images of GPP8 Flagella with or without Mineralization (A and C) Transmission electron microscopic (TEM) images (A, low magnification; C, high magnification) of non-mineralized GPP8 flagella (due to the lack of pretreatment), showing that in water, flagella were randomly stacked and aggregated with a characteristic curly morphology. The flagella were stained with uranyl acetate. Owing to the localization of stain on the periphery of the fibers and hollow structure of flagella, tube-like filaments were observed at high magnification. (B and D) Low (B) and high (D) magnification TEM images of flagella after pretreatment and mineralization. SAED clearly showed the characteristic (211), (002), and (004) planes of HAP (inset in B). The HAP nanocrystals located on the surface of flagella and between flagellar filaments when mineralized flagella form bundles are seen in (D). It should be noted that the TEM samples in (B) and (D) were not stained before imaging. Scale bars, 1 μm in (A and B) and 400 nm in (C and D).

Article Snippet: Biomimetic Self-Assembly and Mineralization of GPP8 Flagella under Different Flagella Concentrations in the HAP-Supersaturated Solution after Pretreatment with Calcium Ions (A) Only a few bundles were formed at a low flagella concentration (∼10 μg/mL). (B) At a higher flagella concentration (∼40 μg/mL), more parallel bundles were formed. (C) A close-packed monolayer was observed when the concentration of flagella reached ∼80 μg/mL. (D) SAED pattern at the relatively straight segment of flagella bundles, showing the c -axis preferred orientation of HAP along the flagella (inset). (E) Energy-dispersive X-ray spectroscopic analysis confirming a Ca/P molar ratio of 1.65.

Techniques: Transmission Assay, Staining, Imaging

Journal: iScience

Article Title: Genetically Engineered Flagella Form Collagen-like Ordered Structures for Inducing Stem Cell Differentiation

doi: 10.1016/j.isci.2019.06.036

Figure Lengend Snippet: Biomimetic Self-Assembly and Mineralization of GPP8 Flagella under Different Flagella Concentrations in the HAP-Supersaturated Solution after Pretreatment with Calcium Ions (A) Only a few bundles were formed at a low flagella concentration (∼10 μg/mL). (B) At a higher flagella concentration (∼40 μg/mL), more parallel bundles were formed. (C) A close-packed monolayer was observed when the concentration of flagella reached ∼80 μg/mL. (D) SAED pattern at the relatively straight segment of flagella bundles, showing the c -axis preferred orientation of HAP along the flagella (inset). (E) Energy-dispersive X-ray spectroscopic analysis confirming a Ca/P molar ratio of 1.65. Some residues of Na and Cl came from the HAP precursor solution. (F) An optical density measurement of flagella under different concentrations after mixing with 4 mM HAP-supersaturated solution. The solid line represents the curve-fitting result. (G and H) A series of CD spectra of flagella under different concentrations of CaCl 2 (G, wild-type flagella; H, GPP8 flagella) during pretreatment. (I) Schematic illustration of self-assembly due to interactions with Ca 2+ ions. Ca 2+ ions first interacted with carbonyl groups of GPP8 on flagella by chelation and induced a side-by-side aggregation. With the incorporation of phosphate ions, HAP nuclei were formed, leading to the formation of HAP on flagella. Scale bars, 400 nm in (A–C) and 250 nm in (D).

Article Snippet: Biomimetic Self-Assembly and Mineralization of GPP8 Flagella under Different Flagella Concentrations in the HAP-Supersaturated Solution after Pretreatment with Calcium Ions (A) Only a few bundles were formed at a low flagella concentration (∼10 μg/mL). (B) At a higher flagella concentration (∼40 μg/mL), more parallel bundles were formed. (C) A close-packed monolayer was observed when the concentration of flagella reached ∼80 μg/mL. (D) SAED pattern at the relatively straight segment of flagella bundles, showing the c -axis preferred orientation of HAP along the flagella (inset). (E) Energy-dispersive X-ray spectroscopic analysis confirming a Ca/P molar ratio of 1.65.

Techniques: Concentration Assay, Circular Dichroism

Journal: iScience

Article Title: Genetically Engineered Flagella Form Collagen-like Ordered Structures for Inducing Stem Cell Differentiation

doi: 10.1016/j.isci.2019.06.036

Figure Lengend Snippet: Fluorescence Images, Surface Spreading Area, and Proliferation of BMSCs (A) Morphologies of BMSCs on the different substrates after 24 h. Most BMSCs exhibited a spindle-like morphology on the collagen and flagella-bearing film (WT and GPP8) but presented a well-spread morphology on polylysine (control). Scale bar, 100 μm. Cell nuclei and F-actin were marked by DAPI (blue) and fluorescein isothiocyanate-labeled phalloidin (green), respectively. (B) Surface spreading area analysis of BMSCs. (C) Cell proliferation on the different substrates. Collagen promoted cell proliferation, but cell proliferation on the flagella-bearing films decreased. Compared with control, *p < 0.05, **p < 0.01; compared with collagen, @ p < 0.05, @@ p < 0.01; compared with GPP8 flagella, # p < 0.05). Collagen, collagen-coated surface; WT, wild-type flagella-coated surface; GPP8, mineralized GPP8 flagella bundles; control, polylysine surface.

Article Snippet: Biomimetic Self-Assembly and Mineralization of GPP8 Flagella under Different Flagella Concentrations in the HAP-Supersaturated Solution after Pretreatment with Calcium Ions (A) Only a few bundles were formed at a low flagella concentration (∼10 μg/mL). (B) At a higher flagella concentration (∼40 μg/mL), more parallel bundles were formed. (C) A close-packed monolayer was observed when the concentration of flagella reached ∼80 μg/mL. (D) SAED pattern at the relatively straight segment of flagella bundles, showing the c -axis preferred orientation of HAP along the flagella (inset). (E) Energy-dispersive X-ray spectroscopic analysis confirming a Ca/P molar ratio of 1.65.

Techniques: Fluorescence, Control, Labeling

Journal: iScience

Article Title: Genetically Engineered Flagella Form Collagen-like Ordered Structures for Inducing Stem Cell Differentiation

doi: 10.1016/j.isci.2019.06.036

Figure Lengend Snippet: Differentiation of BMSCs on the Mineralized Flagella Films (A) Immunofluorescence images showed strong expression of OPN and OCN in the cells on the mineralized self-assembled GPP8 flagella film. Collagen and polylysine also promoted the expression of OPN and OCN but at a lower level than mineralized GPP8 flagella. DAPI (blue) marked the cell nuclei, and fluorescein isothiocyanate-labeled phalloidin (green) labeled the F-actin. OPN and OCN were labeled through rhodamine-tagged antibody (red). Scale bar, 25 μm. (B) RT-qPCR analysis showing that the flagella film could enhance mRNA expression of Runx2, OPN, and OCN. Owing to the mineralization on the GPP8 flagella film, OPN expression was significantly increased. However, gene expression between the cells on the collagen and polylysine films did not show significant difference (compared with control, *p < 0.05, **p < 0.01; compared with collagen, @ p < 0.05, @@ p < 0.01; compared with GPP8 flagella # p < 0.05). (C) The calcium-containing minerals (calcified nodules) were stained by alizarin red S after 2 weeks. Scale bar, 400 μm. An increased number of calcified nodules indicated a higher degree of mineralization. The cells were more matured on the mineralized flagella film, indicating accelerated differentiation of BMSCs. (**p < 0.01, versus control; @@ p < 0.01, versus collagen; # p < 0.05, versus GPP8 flagella). Collagen, Col I-coated surface; WT, wild-type flagella-coated surface; GPP8, mineralized GPP8 flagella bundles; control, polylysine surface.

Article Snippet: Biomimetic Self-Assembly and Mineralization of GPP8 Flagella under Different Flagella Concentrations in the HAP-Supersaturated Solution after Pretreatment with Calcium Ions (A) Only a few bundles were formed at a low flagella concentration (∼10 μg/mL). (B) At a higher flagella concentration (∼40 μg/mL), more parallel bundles were formed. (C) A close-packed monolayer was observed when the concentration of flagella reached ∼80 μg/mL. (D) SAED pattern at the relatively straight segment of flagella bundles, showing the c -axis preferred orientation of HAP along the flagella (inset). (E) Energy-dispersive X-ray spectroscopic analysis confirming a Ca/P molar ratio of 1.65.

Techniques: Immunofluorescence, Expressing, Labeling, Quantitative RT-PCR, Gene Expression, Control, Staining