Bz x700 series microscope

Late osteogenic differentiation of the hMSCs was assessed using Von Kossa staining, using a commercially available kit (Kit KTVKO, American MasterTech). After 5 weeks of culture in growth or osteogenic cell culture medium, the silk rolls were rinsed in PBS, fixed with 4% paraformaldehyde for 30 minutes, rinsed extensively with distilled water, and placed in a 5% silver nitrate solution and exposed to UV light for approximately 1 minute until a dark staining could be observed. The rolls were then rinsed extensively in distilled water, placed for 2–3 minutes in 5% sodium thiosulfate, and counterstained with a nuclear fast red stain for 5 minutes. The silk rolls were once again rinsed extensively with distilled water before being imaged using a BZ-X700 series microscope (Keyence, Itasca, IL).

Cells were seeded at 20,000 cells/well in 12-well plates in a layer of 0.32% agar (SeaKem® LE Agarose (Lonza #50000)/RPMI/FBS/0.64% Antibiotic-Antimycotic (Gibco #15240-096) over a layer of 0.5% agar/RPMI/FBS/0.64% Antibiotic-Antimycotic. Cells were treated with vehicle or INI-43 at 0.5, 1, or 2 μM concentrations in the top agar layer at the time of plating. Cultures were grown for 3 weeks at 37°C in a humidified incubator containing 5% CO₂. Colonies were fixed and stained with 14.3% ethanol and 0.1% crystal violet in H₂0. Plates were imaged using a Keyence BZ-X700 Series microscope (2X magnification, Z-stack, 25.2 μm pitch), and colonies quantified using the BZ-X700 Analysis software, where the Hybrid Cell Count feature was used to define the minimum colony area.

Fluorescein isothiocyanate (FITC)-labeled bovine serum albumin was added to the R5 peptide solution in a molar ratio of 1/1000. The peptide mixture was used to create the ink for printing, followed by the same printing setup and site-specific silicification process as described earlier. FITC-labeled BSA alone served as a control. The FITC-labeled micropatterns on the silk hydrogels were imaged using the BZ-X700 series microscope (Keyence, IL, USA).

Early osteogenic differentiation of the hMSCs was evaluated by measuring the Alkaline Phosphatase (ALP) activity after 21 days of culture, using Alkaline Phosphatase Colorimetric Assay Kit (Abcam, ab83369). Briefly the silk rolls were washed in cold PBS, and the cells were treated with 0.2% Triton X 100 (X110, Sigma) for 10 minutes, followed by sonication. Samples were centrifuged at 4℃ for 15 minutes to remove insoluble materials, and the supernatant was collected and kept on ice. Cell lysates and the assay buffer solution (5 mM p-nitrophenylphosphate) were added to a 96-well plate, incubated for 1 hour, and the absorbance was read at 405 nm using a microplate reader. A standard curve was prepared from standards (0–20 μM) prepared with a pNPP solution. To detect ALP expression, nitro-blue tetrazolium/indolylphosphate (NBT/BCIP) (Thermo Scientific) staining was also performed. Before staining, the cells were washed, NBT/BCIP was added and the samples were incubated at 37°C in a humidified chamber containing 5% CO₂. After 30 mins, the samples were washed with PBS and imaged using a BZ-X700 series microscope (Keyence, Itasca, IL).

In vivo sections were stained with hematoxylin (Sigma-Aldrich, St. Louis, MO) to visualize cell nuclei and Masson’s Trichrome (Sigma-Aldrich, St. Louis, MO) to visualize collagen deposition (blue), muscle fibers and blood vessels (red). Following staining and dehydration, samples were embedded in DPX Mountant (Sigma Aldrich, St. Louis, MO) and imaged using a Keyence® BZ-X700 series microscope. VEGFR-2 (sab4501645, 1/100), CD3 (ab5690, 1/100), CD68 (ab125212, 1/200), CD31 (ab64543, 1/200), alpha smooth muscle actin (sc-32251, 1/200), and C-C chemokine receptor 7 (CCR7, NB100-712, 1/250) antibodies were also used to evaluate cell infiltration in cECM containing hydrogels. Nuclei were stained with DAPI. Secondary visualization of the primary antibodies was completed with Alexa Fluor® 488 goat anti-mouse (Thermofisher, A11001), Alexa Fluor® 555 donkey anti-rabbit (Thermofisher, A31572), and Alexa Fluor® 488 donkey anti-goat (Thermofisher, A11055).