## 2 Materials and Methods
### ADSC Differentiation on Different Surfaces  
Cell differentiation to osteogenic lineages was initiated after 7 days of culture. The cells were exposed to a differentiation media (growth media plus \(10^{-8}\) M dexamethasone, 50 \(\mathrm{\SIUnitSymbolMicro g}\)/mL \(\mathrm{\SIUnitSymbolMicro g}\)-glycerol phosphate, and 6 mM ascorbic acid) to induce osteogenesis. The differentiation media was changed every other day from day 7 to 14. After 14 days of culture, the media was removed from the wells and the surfaces were rinsed twice with PBS. Subsequently, 500 \(\mathrm{\SIUnitSymbolMicro m}\) 0.2% (\(\
u/v\)) Triton X-100 solution in PBS were added to each well, and the surfaces were shaken at 150 rpm for 20 min to remove the proteins from the cells. Osteogenic differentiation was then evaluated using the following assays: total protein content, alkaline phosphatase (ALP) activity, and calcium concentration (Ca):  
* The total protein content was determined using a commercially available microRNA assay. In total, 150 \(\mathrm{\SIUnitSymbolMicro m}\) of working reagent were generated from the assay and 150 \(\mathrm{\SIUnitSymbolMicro m}\) of protein supernatant were added to a 96-well plate and incubated for 2 h at 37 \({}^{\circ}\)C and 5% CO\({}_{2}\). After the incubation period, the absorbance was read at 562 nm. The total protein concentration was determined from a standard absorbance curve versus the known albumin standard provided by the manufacturer.
* ALP activity was determined using a commercially available ALP assay kit from QuantiChromTM. In total, 150 \(\mathrm{\SIUnitSymbolMicro L}\) of working reagent were prepared from the assay kit, and 50 \(\mathrm{\SIUnitSymbolMicro L}\) of protein supernatant were added to a 96-well plate. The absorbance was read at 405 nm and repeated after 4 min. Absorbance was converted to concentration using an ALP standard, and data were normalized using the total protein content.
* Calcium deposition was determined using a commercially available calcium reagent test from Teco Diagnostics (Anhaeim, CA, USA). The protein supernatant was removed, and surfaces were rinsed with DI water. In total, 6 M HCl (Hydrochloric acid) solution were added to the wells, and the surfaces were placed in a shaker for 12 h at 100 rpm to ensure all the calcium dissolved in the solution. After 12 h, 1 mL of working reagent was prepared from the test kit, and 20 \(\mathrm{\SIUnitSymbolMicro L}\) of HCl-calcium solution were added to a 24-well plate. The absorbance was read at 570 nm and was converted to concentration using the calcium standard provided by the manufacturer and the data was normalized using total protein content.  
The differentiation was also characterized using immunofluorescence microscopy for osteoclast. After 14 and 28 days of culture, the media was removed, and the surfaces were rinsed thrice in PBS. Cells were then fixed using a 3.7% (\(\
u/v\)) formaldehyde solution in PBS for 15 min. The surfaces were rinsed three times in PBS before the cells were permeabilized using a 1% Triton X-100 solution in PBS for 3 min and rinsed twice with PBS. The surfaces were immersed in a 10% bovine serum albumin (BSA) solution in PBS for 30 min to block non-specific binding sites in the cells. After BSA was removed, surfaces were washed twice with PBS and an osteoclast (OCN) primary antibody in a 1% BSA solution at a dilution of 1:100 for 60 min. Surfaces were then washed with PBS thrice, followed by the addition of a secondary antibody, FITC, in a 1% BSA solution at a dilution of 1:200 for 45 min. Following washing with PBS, surfaces were stained with rhodamine-phalloidin and DAPI, the process for which was explained in the previous section. The surfaces were then washed twice with PBS and left in PBS until being imaged under a fluorescence microscope.