Fibronectin solution

The growth of HUVECs in the microfluidic channels was compared between the control (without VF) and experimental (with VF) groups. For the control group, media channels were first coated with fibronectin solution (33 µg ml⁻¹, Sigma-Aldrich) in EGM-2 media and incubated at 37°C for 1 h. Channels were then washed twice by adding 70–50 µl (the volume added to the inlet and outlet reservoirs) of EGM-2. The procedures for seeding HUVECs within the chips were identical in both conditions (with/without VF). PDF was created by adding 70–50 µl (inlet-outlet) to the media channel reservoirs. Then, 10 µl of HUVEC suspension at a density of 3 million cells ml⁻¹ was added into the media inlet reservoirs under incubation at 37°C for 15 min. To ensure that the entire surface of the collagen gel was covered by HUVECs, the seeding step was repeated four times. Each time, a 90° rotation of the chip perpendicular to the direction of the flow was performed. Media was changed daily, and HUVECs formed a lumen structure around the VF-formed collagen gel after 2 days of culture.

Planar micropatterned substrates containing alternating 15-μm-wide adhesive and 50-μm-wide non-adhesive stripes were produced using the deep UV light method (Azioune et al. 2010 (link)). Briefly, rectangular and circular glass coverslips were first activated by exposure to air plasma (Harrick Plasma) for 45 s (Fig. 1A). Subsequently, they were incubated for 1 h in 0.1 mg/mL poly-L-lysine-gpoly(ethyleneglycol) (PLL(20)-g[3.5]-PEG(2), SuSoS) in 10 mL HEPES at pH 7.3 for passivation (Fig. 1B). After washing with distilled water, the treated surface was illuminated with deep UV light (UVO-Cleaner, Jelight) through a chromium synthetic quartz photomask (Toppan, TX, USA) for 3 min (Fig. 1C). Unpatterned glass coverslips served as controls. Prior to cell seeding, all patterned and control substrates were incubated for 1 h with 50 $\mathrm{\mu }\text{g}/\text{mL}$ fibronectin solution (Sigma Aldrich Merck KGaA, Darmstadt, Germany) at room temperature (Fig. 1D).Fig. 1

Micropattern fabrication process using the deep UV light method. (1) Glass substrate plasma treatment; (2) PEG coating; (3) UV light treatment; (4) Fibronectin coating. Adapted from Azioune et al. (2010 (link))

MG InHex (MOZO-GRAU®, Valladolid, Spain) implants made from commercially pure grade IV titanium and containing microthreads in the cortical part, self-tapping design in the apical end, and a 45° platform switching shoulder were used in the early healing study (Figure 1). The implants selected were 10 mm long and 3.75 mm in diameter.
After sterilization with UV light overnight, the implant surfaces were treated with glow discharge plasma (GDP) and protein grafting, as described previously [40 (link)]. The implants were cleaned with argon-based GDP (PJ; AST Products Inc., North Billerica, MA, USA) at 85 Watts (W), 13.56 MHz, and 100 millitorr of argon gas at room temperature for 15 minutes (Figure 2). Implants treated with GDP only were used as control and have been defined as “Ar-GDP” in this paper.
Thereafter, the implants were exposed to allylamine gas in the GDP reactor for 30 minutes, immersed in 3% glutaraldehyde (GA) solution (Merck, NJ, USA) for 30 min, and rinsed with 0.1 M phosphate buffered saline (PBS). The implants were then immersed in 5 μg/mL fibronectin solution (Sigma-Aldrich Co., St. Louis, MO, USA). Tris-phosphate buffer (pH 7.4) was used to interrupt the chain reaction of fibronectin links, and these implants were named “GDP-fib.”

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Place the glass bottom dish (WPI's FluoroDish, 35 mm Diameter, 23 mm Well, Petri Dishes) and PDMS chip facing upwards into a plasma cleaner (diener, Germany) and activate the surfaces for 1 min.

Note: If you don't have access to plasma cleaner, you can make a soft PDMS chip by mixing the PDMS (RTV 615 kit, Momentive performance materials, US) and curing agent in a ratio of 30:1. This chip is sticky and it can be attached directly to the glass bottom dish / coverslip.•

Remove the chip and dish from the plasma machine and attach them by applying a gentle press.

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Now the PDMS chip is attached to the glass bottom dish (Fig. 2A).Fig. 2

Stepwise demonstration of generating fibronectin-coated lines on glass bottom dish.

Fig 2:

Fibronectin Coating:•

Load 10 µl of fibronectin solution (25 µg/ml, Sigma) into each hole of the PDMS chip (Fig. 2B).

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Seal the dish with parafilm to avoid drying of fibronectin and incubate for 1 h at room temperature.

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Peel off the PDMS from the glass bottom dish (Fig. 2C).

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Wash the dish 2 times with phosphate-buffered saline (PBS).

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Fibronectin-coated 5 µm lines are generated on the glass bottom dish (Fig. 2E, F).

Cell mechanical stimulation was done as previously described⁷ . Briefly, a 150 μL droplet of a 10 μg/mL fibronectin solution (Sigma) was deposited in the center of the membrane mounted in the ring. After overnight incubation at 4 °C, the fibronectin solution was rinsed, cells were seeded on the fibronectin-coated membranes and allowed to attach during 30–90 min. Then ring-containing membranes were mounted in the stretch system previously described⁷ . Cell images were acquired with a 60x water dipping objective (NIR Apo 60X/WD 2.8, Nikon) and an Orca Flash 4.0 camera (Hamamatsu), in an upright epifluorescence microscope with the Metamorph software. Cells were always imaged in two different channels collecting each fluorophore emission signal, every 3 s. They were imaged for 2 min at rest, 3 min in the 6% stretched state (nominal stretch of the PDMS substrate), and 3 min during the release of the stretch.

Polyacrylamide (PA) gels were prepared on aminosilanized glass cover slips as previously described [14] (link). Briefly, 40% w/v acrylamide and 2% w/v bis-acrylamide stock solutions (Bio-Rad, Hercules, CA) were mixed to prepare a PA solution and then the gel’s stiffness was achieved by varying the final concentration of the PA solution (3% ∼7.5%) and Bis-acrylamide cross-linker (0.06% ∼0.4%). To polymerize the solutions, 2.5 µl of 10% w/v ammonium persulfate (APS; Bio-Rad, Hercules, CA) and 0.25 µl of N,N,N′,N′-Tetramethylethylenediamine (TEMED; Bio-Rad, Hercules, CA) were added to yield a final volume of 500 µl PA solution. To crosslink extracellular matrix molecules onto the gel surface, a photoactivating cross-linker, 0.5 mg/ml of sulfo-SANPAH (sulfosuccinimidyl 6-(4′-azide-2′-nitrophenyl-amino) hexanoate, Pierce, Rockford, IL) solution was used. The powder of sulfo-SANPAH was dissolved in 10 mM HEPES buffer containing 0.5% DMSO and the solution was added on top of the PA gel. Gel dishes were placed at a distance of ∼15 cm from the UV light of the hood for 6 min and rinsed three times with 10 mM HEPES buffer for 10 min. A 200 µl of fibronectin solution (5, 10 or 40 µg/ml) from bovine plasma (Sigma, St. Louis, MO) was incubated on top of the sulfo-SANPAH-coated PA gel at 37°C overnight to deposit fibronectin for subsequent cell seeding.