Analytical methods

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LYOPOR 3D collagen scaffold - analytical methods

Scaffold characterization

Prepared fibers were visualized using scanning electron microscopy (SEM), and fiber diameter was determined. Samples were coated with thin layer of platinum (3 cycles) and visualized using SEM (Tescan Vega 3, Czech Republic). The acceleration voltage for all samples was 10 kV.

The diameters of the 3D printed microfibers and electrospun nanofibers were measured in the Image J program. The porosity was calculated from the dimensions shown in Figure.

Cell viability analysis

The metabolic activity of cells was measured using an MTS assay (CellTiter 96® AQueous One Solution Cell Proliferation Assay; Promega). Twenty microliters of MTS solution were added to 100 μL of the sample medium and incubated for 2 h at 37°C. Subsequently, 100 μL of the cultivated solution was transferred to a new clean well. The results were examined using spectrophotometry at 490 nm (reference wavelength, 690 nm). 

Cell proliferation analysis

The proliferation of chondrocytes on the scaffold was measured from the amount of DNA (Quant-iT™ dsDNA Assay Kit; Life Technologies). The proliferation of chondrocytes on scaffolds was tested on days 1, 3, 7, 14, 21, and 28. The scaffolds were put into a vial with 500 μL of cell lysis solution (0.2% v/v Triton X-100, 10 mM Tris (pH 7.0), and 1 mM EDTA) and processed through 3 freeze/thaw cycles; the scaffold sample was first frozen at -70°C and thawed at room temperature. Between each freeze/thaw cycle, the scaffolds were roughly vortexed. Sample (10 µl) was mixed with 200 μL of reagent solution measured on a multiplate fluorescence reader (Synergy HT, λex= 485 nm, λem= 525 nm). Results were evaluated from the calibration curve using the standards in the kit. 

Visualization of cell adhesion and proliferation on scaffolds

DiOC6(3) staining was used to detect cell adhesion on the scaffolds on days 1, 7, 14, 21, and 28. Samples were fixed with frozen methylalcohol (-20 °C) for 10 min. Subsequently, the fluorescent probe 3,3′-diethyloxacarbocyanine iodide (DiOC6(3), D273, Invitrogen, Molecular Probes 1 μg/mL in phosphate buffered saline (PBS); pH 7.4) was added and incubated with the samples for 45 min at room temperature. The samples were rinsed with PBS, and propidium iodide (PI; 5 μg/mL in PBS) was added for 10 min, followed by rinsing with PBS, and visualized using a ZEISS LSM 5 DUO confocal microscope (PI: λexc= 561 nm, λem= 630–700 nm; DiOC6(3): λexc= 488 nm, λem = 505–550 nm).

RNA isolation and qRT-PCR analysis

The total RNA was isolated using the Qiagen RNA mini-kit (Qiagen, Hilden, Germany) according to the protocol provided by the manufacturer. Reverse transcription (RT) was performed using the RevertAid H Minus First Strand cDNA Synthesis Kit (Thermo Scientific, Waltham, MA, USA) with 80 ng of total RNA. Transcript levels were evaluated using quantitative real-time RT-PCR. QRT-PCR was performed using the Light Cycler 480 II real-time PCR system (Roche, Basel, Switzerland) with LC 480 TaqMan Master and TaqMan probes (Universal Probe Library, Roche) following the protocols from the manufacturer. The genes whose expression was analyzed by qRT-PCR was listed in Table. All samples were run in triplicates. The thermo cycling parameters were 95 °C for 10 min; 95 °C for 10 s, 60 °C for 10 s (45 cycles); and 40 °C for 1 min. All samples were scaled relative to the median of the GAPDH expression level, which was used as an endogenous control gene.

Detection of chondrogenic marker using indirect immunofluorescence staining

The presence of type II collagen, a typical marker of chondrogenic differentiation, was confirmed using indirect immunofluorescence staining on days 7, 14, 21, and 28. Samples were fixed with 4 % formaldehyde for 10 min, washed with PBS and incubated in 3% FBS in PBS/0.1% Triton for 30 min at room temperature. A primary monoclonal antibody against type II procollagen, clone II-II6B3 was obtained from the Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at the Department of Biology, University of Iowa, Iowa City, IA 52242. (dilution 1:20) and incubated overnight at 2-8°C. After three washes with PBS/0.05% Tween, the samples were incubated with the secondary antibody, Alexa Fluor 488 conjugated anti-mouse antibody, for 45 min. Subsequently, a solution of PI was added for 10 min (5 μg/mL in PBS) for the visualization of the cells’ nuclei. The samples were washed three-times washed in PBS/0.05% Tween again and were then examined with a ZEISS LSM 5 DUO confocal microscope (Alexa Fluor 488: λexc= 488 nm, λem = 505–550 nm PI: λexc= 561 nm, λem= 630–700 nm)

Quantification of selected growth factors released from platelets

To observe distribution of growth factors released from platelets, the sandwich enzyme-linked immunosorbent assay (ELISA) was used. Platelets in concentration 900×109/L were lysate using 3 freeze-melt cycles (-80°C and 37°C) and subsequently centrifuged 4120 rpm for 10 min to remove cell membranes. SDF-1α and bFGF were determined according to the guidelines (Peprotech). P-Selectin, EGF, HGF, KGF, IGF-1, TGF-β, Thrombospondin and VEGF were quantified according to the manufacturer’s instructions (DuoSet ®, R&D Systems, MN, USA). Briefly, the protein-captured antibodies were precoated onto 96-well plates overnight and then blocked by 100 µl blocked buffer and washed 3× with wash solution. Subsequently, 100 μl of sample or standard were added to each of the wells in triplicate, the plates were incubated for 2 h, and washed twice with wash solution. Next, 100 μl of biotinylated primary antibodies was added and the plates were incubated for 2 h. The plates were washed twice with wash solution, each well was incubated with 100 μl streptavidin-conjugated horseradish peroxidase (20 min, 4°C), and again washed third with wash solution. The antigen-antibody complex was detected by colorimetric reactions initiated by the addition of 100 μl of tetramethylbenzidine substrate solution (R&D Systems) to each well, and the reaction was stopped after 20 min with 50 μl of 1N H2SO4. The absorbance of the samples was measured at 450 nm with the Biotek Synergy H1 ELISA reader, and the concentration of proteins was determined by an eight-point calibration curve.