Osteogenic supplement

Hydrogel constructs were cultured in glutamine, cystine, and methionine-free high glucose DMEM (Life Technologies) supplemented with 10% FBS, 100 μg/mL sodium pyruvate, 0.201 mM cystine, 75 μM azidohomoalanine, 25 μM methionine, 1% penicillin/streptomycin, and osteogenic supplement (R&D System). Blocking of nascent fibronectin adhesion was performed using a monoclonal antibody against human fibronectin (2.5–10 μg/mL HFN7.1, Developmental Studies Hybridoma Bank)^{19 (link),53 (link)}.
For nascent ECM labeling, hydrogels were washed twice in PBS with 2% BSA, followed by 30 min incubation in 30 μM DBCO-488 (Click Chemistry Tools) at 37 °C/5% CO₂. Following three washes (3 min each) with PBS/2% BSA, hydrogels were fixed in 10% formalin for 30 min at room temperature and washed with PBS three times. For imaging, z-stack images at 20 × 0.75 NA and 100 × 1.4 NA were acquired using a Nikon A1R Confocal Microscope. Local ECM thickness was determined by creating binary masks of z-stack images in ImageJ with Otsu’s thresholding, and the ImageJ plugin “BoneJ” was used to calculate the average local ECM thickness per slice^{19 (link),54} . (Nascent ECM thickness: BoneJ Plugin ImageJ (×180, 0.13 μm/pixel), 3–6 slices measured and averaged for each cell)

For cell preparation, mononuclear cells (MNC) were isolated from BM aspirates using density gradient centrifugation with LymphoPrep™ (PROGEN Biotechnik, cat. #1,114,547, Heidelberg, Germany).
To expand the mesenchymal stromal cell fraction, isolated MNCs were cultured in the Mesencult medium [Mesencult™ Proliferation Kit (human), STEMCELL Technologies Inc, cat. #05411, Vancouver, BC, Canada], with an addition of 10% AB human serum and 1% penicillin–streptomycin solution. BM stromal cells were generated up to passage (P) 3. To enrich and expand the MSC fraction, the isolated MNCs were cultured in the starvation medium RPMI (Biological Industries, cat. #1640, Beit-Haemek, Israel), with an addition of 10% FBS/human serum and 1% penicillin–streptomycin solution.
To induce MSC differentiation into adipocytes/osteocytes, BM-MSCs were stimulated using adipogenic or osteogenic differentiation inducing factors. In brief, P0 MSCs were cultured for 21 days at 37⁰C in either adipogenic or osteogenic medium (basal medium: MEM-α Biological Industries, cat. #01–042-1A; adipogenic supplement: R&D Systems, cat. #CCM011, Minneapolis, MN, USA; osteogenic supplement: R&D Systems, cat. #CCM008). The obtained cells are further referred to as “induced” MSCs, adipocytes or osteocytes. The MSCs that have not undergone such stimulation are further termed “non-induced”.

GBM CD105⁺ cells at low passage (P3-P5) were used for mesenchymal stem cell (MSC) differentiation assays using a human MSC identification kit (R&D) according to the protocol provided by the manufacturer. Briefly, GBM CD105⁺ cells were cultured in human/mouse StemXVivo Osteogenic/ Adipogenic Base Media (R&D) with Adipogenic Supplement(R&D) and Osteogenic Supplement(R&D), respectively, for up to 14 days for adipogenic di­fferentiation or osteogenic differentiation. For chondrogenic diff­erentiation, cells were culture in human StemXVivo Chondrogenic Media (R&D) supplemented with ITS Supplement (R&D) and pelleted in 5 ml tube for up to 20 days. Cells were fixed and detected by anti-hFABP4, anti-hOsteocalcin and anti-hAggrecan immunohistochemistry.