Scientific Papers

Time-sequential fibroblast-to-myofibroblast transition in elastin-variable 3D hydrogel environments by collagen networks | Biomaterials Research


Materials

Tris–HCl buffer (pH 8.0), phosphate buffered saline (PBS), sodium chloride (NaCl), formic acid (FA), ammonium bicarbonate (AmBic), dithiothreitol (DTT), iodoacetamide (IAA), L-cysteine, acetic acid (Sigma, #45754-100ml), sodium bicarbonate (Sigma, #144-55-8), 4% formaldehyde (Sigma, #HT5011), triton X-100 (Sigma, #X100), normal horse serum (Sigma, #H1138), DAPI (Sigma, #9542), and collagenase (Sigma, #C0130) were purchased from Sigma-Aldrich (St. Louis, MO, USA). Protease inhibitor cocktail was purchased from Roche Diagnostic GmbH (Mannheim, Germany). The HLB cartridges were purchased from Waters (MA, USA). Acetonitrile (ACN) (with 0.1% FA), water (with 0.1% FA), n-dodecyl beta-D-maltoside (DDM), trypsin, and bicinchoninic acid (BCA) protein assay reagent were purchased from Thermo Fisher Scientific (Rockford, IL, USA). Rat tail collagen type I solution (Biomatrix, #5010) was purchased from Advanced Biomatrix (Carlsbad, CA, USA). BrdU primary antibody (Cell signaling, #5292) was purchased from Cell Signaling Technology (MA, USA), and mouse anti-S100A4 primary antibody was purchased from Invitrogen (Invitrogen, #MA5-31332). Anti-β-tubulin primary antibody (Abcam, #ab15568), anti-PDGFR-β primary antibody (Abcam, #ab32570), and anti-α-SMA primary antibody (Abcam, #7817) were purchased from Abcam (Cambridge, UK). Alexa Fluor 549 (Thermo Fisher #A21203, #A21207) and Alexa Fluor 488 (Thermo Fisher, #A11029) were purchased from Thermo Fisher Scientific (MA, USA). A Sircol™ inSoluble Collagen Assay kit (Biocolor, #S6000) and Fastin – Elastin assay kit (Biocolor, #F4000) were purchased from Biocolor (Co Antrim, UK). Finally, recombinant human TGF-β protein (Biotechne, #240-B-010) was purchased from Bio-Techne (MN, USA).

Fabrication of fibroblast-laden collagen hydrogels with four different gradients of elastin

Hydrogels were fabricated according to previously reported methods after cell-friendly optimization [21]. First, insoluble elastin from bovine neck ligament (Sigma, #1625) was suspended in 0.1 M acetic acid (Sigma, #45754 100 ml) at 25 mg/ml as the elastin stock solution (ES). Rat tail collagen type I solution (Biomatrix, #5010) at a concentration of 6.2 mg/ml was used as the collagen stock solution (CS). The four types of elastin-variable hydrogels (100C, 4C1E, 1C1E, 1C4E) were fabricated by mixing appropriate volumes of CS, ES, completed cell culture medium, and 7.5% sodium bicarbonate (Sigma, #144-55-8). The pH of the mixture was adjusted to 7.4, the final concentration of collagen was maintained at 2 mg/ml in each hydrogel, and the final concentration of elastin was varied (0–8 mg/ml) based on the collagen/elastin ratios of 1:0, 4:1, 1:1, and 1:4, respectively. Then the mixture was added into a 96-well plate at a volume of 35 µl/well and incubated in an incubator (37 °C, 5% CO2) for 2 h to allow gelation. The hydrogels, without cells, were then fixed with 4% formaldehyde (Sigma, #HT5011) for 30 min and washed with 1X PBS several times before storage at 4 °C until further analysis. After the hydrogel components were mixed with a neutralization solution, human dermal fibroblasts (HDFs) at passage 7 were added at a density of 1 × 105 cells/ml. Then 35 μl of hydrogel mixture, including cells, was gently added into a 96-well plate to avoid bubbles. Finally, gelation of the hydrogel mixture was induced in an incubator (37 °C, 5% CO2) for 2 h. Completed cell culture media with or without TGF-β or BAPTA-AM was added to each well for a fibroblast culture within hydrogels.

Morphological characterization by scanning electron microscopy imaging

The hydrogels were dehydrated by washing with ethanol and freeze-dried for 6 h (FD8512 Floor, ilShinBioBase, Rep. of Korea). Then the dried hydrogels were coated with an ultrathin layer of gold using an ion sputter coater (SPT-20, Coxem, Rep. of Korea) set at 4 mA for 120 s and visualized with a Hitachi S-4800 scanning electron microscope operating at 10 kV (FESEM, S-4800, Hitachi, Japan).

Assessment of cell proliferation in 3D gels with 5-Bromo-2-deoxyuridine (BrdU) assay

To measure fibroblast proliferation, we used a BrdU assay as described in a previous report [24]. First, 1 × 105 cells/ml of fibroblasts were seeded into hydrogel mixtures in a 96-well plate. After gelation, completed cell culture medium containing 10 μM BrdU was added into each well. Cells were continuously cultured up to 72 h. At a certain period of time, hydrogels with or without BrdU-treated cells were fixed using 4% formaldehyde (Sigma, #HT5011). After being washed with PBS 1X three times (30 min each time), the hydrogels were washed with 0.1% Triton X-100 (Sigma, #X100) and incubated for 30 min in 0.1% Triton for permeabilization. Then the cells inside the hydrogels were blocked with 5% normal horse serum (Sigma, #H1138) for 1 h. Hydrogels were incubated with mouse anti-BrdU primary antibody (1:200, Cell Signaling, #5292) overnight at 4 °C. Next, hydrogels were washed with 0.1% Triton X-100 three times (30 min each time). After washing, the hydrogels were incubated with Alexa Fluor 549 (1:200, Abcam #A21203) for 1 h. Then, the remaining dye in hydrogels was washed with 0.1% Triton X-100 and hydrogels were subsequently stained with DAPI (Sigma, #9542) for 15 min. Cell imaging was performed using a laser scanning confocal microscope (LSCM, Olympus FV3000) and Olympus FluoView FV10-ASW software. To observe the fluorescence signals from the cells, 5.0 μm thick images in a z-stack picture were analyzed. All samples were imaged using identical exposure times and laser power settings. Qualification imaging analysis was done using IMARIS 7.6 software.

Microscopic characterization of collagen and elastin in elastin-variable hydrogels with nonlinear optics imaging

A previously developed method was used for imaging collagen fibers employing second harmonic generation (SHG) signals [25]. Collagen fibers in the hydrogels were observed via SHG signals via setting the wavelength of a fs pulsed laser to 810 nm. Briefly, HDFs cultured inside fixed hydrogels were permeabilized using 0.1% Triton X-100 and then blocked with 5% normal horse serum in 1X PBS containing 0.1% Triton X-100 for 90 min. Rabbit anti-β-tubulin primary antibody (1: 200, Abcam, #15568) was incubated overnight with the hydrogels at 4 °C. The hydrogels were then were washed 3 times (30 min each time) with 0.1% Triton X-100 in PBS 1X. After incubating the hydrogels with a secondary antibody conjugated with Alexa Fluor 594 (1:200, Thermo Fisher, #A21207) for 60 min at room temperature, the hydrogels were washed with PBS 1X twice (30 min each time) and the collagen fibers were observed using multimodal nonlinear optical (MNLO) microscopy and SHG signals setting the wavelength of the fs pulsed laser to 810 nm. Z-depth images were set at a thickness of 5.0 μm. All images were obtained using FluoView software (FV10-ASW; Olympus Corp., Tokyo, Japan).

Measurement of collagen fiber length

Measurements of collagen fiber length were carried out following a previously reported method using IMARIS 7.4 software [26]. All SHG images were converted to black and white images and set at the same threshold level, which allowed for the separation of collagen fibers. After that, the freehand tool was utilized to measure the length of the collagen fiber. Approximately 25 fibers were randomly selected for each measurement.

Measurement of collagen by colorimetric assay

Collagenase (Sigma, #C0130) was used to digest the hydrogels. After hydrogel gelation, 100 µl of 1 mg/ml collagenases was added to the hydrogels in culture medium followed by incubation at 37 °C until all hydrogels were digested. Then the collagen level was measured using the Sircol™ inSoluble Collagen Assay kit based on Sircol dye reagent, which binds the basic groups of soluble collagen molecules. Following manufacturer’s instructions, collagen in the digested hydrogel mixture was isolated using cold isolation and concentration reagent (200 μl/sample). After vortexing to mix the contents for a few minutes every 15 min, the mixture was left at 4 °C for 30 min. The mixture was centrifuged at 12,000 rpm for 10 min. The collagen pellet was retained and 1000 μl of cold diluted acid-salt wash reagent was added to each sample to wash the collagen pellet. The collagen after collection using a centrifuge (12,000 rpm, 10 min) was labeled by adding Sircol dye reagent (1000 μl each sample). Samples were gently shaken for 30 min, and the collagen pellet was collected after centrifugation (12,000 rpm, 10 min). Then any unbound dye from the surface of the collagen pellet was washed off by adding 750 μl of ice-cold acid-salt wash reagent into each sample. After centrifugation (12,000 rpm, 10 min), an alkali reagent was added to each sample to solubilize the collagen (250 μl). The absorbance was observed at 550 nm using a BioTek Synergy H1 microplate reader (BioTek Instruments, Inc., USA).

Measurement of elastin by colorimetric assay

Hydrogels were digested using 1 mg/ml collagenase at 37 °C for around 1 h. Then an elastin measurement assay was performed following the manual of the manufacturer using a Fastin – Elastin assay kit (Biocolor, #F4000). Briefly, insoluble elastin in the mixture was extracted using 0.25 M oxalic acid while heating at 100 °C for 60 min. After cooling down, the mixture was centrifuged at 12,000 rpm for 10 min. The supernatant after centrifugation was saved and mixed with an equal volume of elastin-precipitating reagent. After 10 min, the mixture was centrifuged at 12,000 rpm for 10 min. The supernatant was carefully poured out and the pellet was retained. Dye reagent was added to the pellet at a volume of 1 ml per sample. Then the mixture was vortex mixed and incubated for 90 min at room temperature. After drying, the mixture was centrifuged at 12,000 rpm for 10 min to remove any remaining dye reagent. Finally, dye dissociation reagent was added into each tube. After mixing well, the mixture was transferred to a 96-well plate and read with a BioTek Synergy H1 Microplate Reader at a wavelength of 513 nm.

Nanoflow LC–ESI–MS/MS analysis

To extract the protein from HDFs, HDF cell pellets of each condition were mixed with 100 μl of a 0.2% DDM solution containing 150 mM NaCl, 50 mM Tris–HCl, and one tablet of protease. The cell mixtures were mixed vigorously and dissolved at 95 °C for 10 min. Then the solution was centrifuged at 12,000 rpm for 15 min to remove cell debris and take the supernatants. The extracted proteins in the supernatant were subjected to a BCA assay to measure total protein concentration.

Twenty micrograms of protein were reduced using a 50 mM AmBic solution containing 10 mM DDT. To prevent re-folding of the disulfide bonds, 40 nM IAA was added and reacted at room temperature for 30 min. Thereafter, trypsin (protein/enzyme = 20:1) was added to the solution and digested at 37 °C for 18 h to form peptides. Utilizing an HLB cartridge for purification, the peptide solution was then dried under vacuum. The dried sample was stored at –20 °C before nanoflow liquid chromatography–electrospray ionization–tandem mass spectrometry (nLC-ESI–MS/MS).

A solution of H2O/ACN (98:2, v/v) containing 0.1% FA was used to reconstitute the dried samples, and 250 ng of the resulting peptide mixture was introduced into a NanoElute LC system connected to a hybrid trapped ion mobility spectrometry–quadrupole time-of-flight mass spectrometer (timsTOF Pro, Bruker Daltonics, Bremen, Germany) equipped with a modified nano-electrospray ion source (CaptiveSpray, Bruker Daltonics). In a handmade column (75 μm inner diameter, 250 mm length) packed with C18 resins (1.9 μm, 120 Å, Dr. Maisch, Germany), the peptide mixtures were separated at 50 °C with a constant flow of 400 nl/min and then eluted using the following binary gradient of mobile phases A (0.1% FA in H2O) and B (0.1% FA in ACN): 2% to 17% B for 45.0 min, 17% to 25% for 22.5 min, 25% to 37% for 7.5 min, 37% to 80% for 5.0 min, and then kept at this level for 10 min to rinse the analytical column. The timsTOF Pro device was operated in parallel accumulation serial fragmentation (PASEF) acquisition mode using Bruker Compass HyStar 5.0.37.1. The settings for MS and MS/MS scans were as follows: mass range of 100 to 1700 m/z, 1/K0 start at 0.6 Vs/cm2 and end at 1.6 Vs/cm2, capillary voltage of 1500 V, dry gas flow rate of 3 l/min, and dry temperature of 180 °C; PASEF mode: 10 MS/MS scans (total cycle time 1.16 s), charge range of 0 to 5, active exclusion for 0.4 min, scheduling target intensity of 20,000, and intensity threshold of 2500, depending on precursor mass and charge.

Proteomic data analysis

The obtained raw data were submitted to PEAKS Studio 10.5 (Bioinformatics Solutions, Waterloo, Canada) for protein identification and label-free quantification (LFQ) searches against the SwissProt database of Homo sapiens (human, UP00000564, downloaded 22/11/2019, 20,379 entries) from Uniprot (www.uniprot.org/) with a false discovery rate (FDR) of 0.01. The search parameters for identification were as follows: (a) trypsin as the specific enzyme, two missed cleavages allowed; (b) fixed modification: carbamidomethylation of cysteine, and variable modification: oxidation of methionine and acetylation of protein N-term, allowing for three variable post-translational modifications per peptide; (c) precursor mass error tolerance of 20.0 ppm; (d) fragment mass error tolerance of 0.05 Da. Following the completion of protein identification, LFQ was performed using the analyzed PEAKS dataset. The analysis of variance (ANOVA) method was used to conduct the LFQ analysis, and the significance thresholds were set at two unique peptides, a data filter in at least two samples per group, a significance of 20 (p value = 0.01), and a 1.5-fold change. Total ion chromatography (TIC) was used to perform data normalization. GO term enrichment of HDFs was carried out using the ShinyGO program (version 0.77) after the data were exported to Microsoft Excel.

Immunofluorescence-based cell staining

The proteins S100A4, PDGFR-β, and α-smooth muscle actin (α-SMA) were identified via immunofluorescence staining. After being washed and permeabilized with PBS 1X and 0.1% Triton X-100 respectively, the 100C and 4C1E hydrogels were incubated overnight with the following primary antibodies: mouse anti-S100A4 primary antibody (1:200, Invitrogen, #MA5-31332), mouse anti-α-SMA primary antibody (1: 200, Abcam, #ab7817), and rabbit anti-β-tubulin primary antibody (1: 200, Abcam, #ab15568). In addition, the hydrogels were incubated for 48 h with rabbit anti-PDGFR-β primary antibody (1:200, Abcam, #ab32570). The hydrogels were then labeled with the following secondary antibodies: goat anti-mouse Alexa Fluor 488 (1:200, Thermo Fisher, #A11029), and donkey anti-rabbit Alexa Fluor 549 (1:200, Thermo Fisher, #21207). DAPI (1 µg/ml, Sigma, #9542) was used to stain the nuclei. To observe the fluorescence signals from the cells, laser scanning confocal microscopy (LSCM, Olympus FV3000) and Olympus FluoView FV31S-SW software were applied. Images 5.0 μm thick in a z-stack were analyzed. All samples were imaged using identical exposure times and laser power settings. Qualification imaging analysis was performed using IMARIS 7.6 software.



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