Agarose gel

Synovial MSCs seeded on PLA films were washed with PBS, fixed in 4% paraformaldehyde (PFA, Fujifilm Wako Pure Chemical Corporation) at 4°C for 1 h, and embedded in 2% agarose gel (Fujifilm Wako Pure Chemical Corporation). The framework of the PLA film was then removed, and the cylindrical agarose gel holding the PLA film was embedded in paraffin, sliced, stained with safranin-o/fast green (Fujifilm Wako Pure Chemical Corporation), and observed with a microscope (BZ-X810).
The cartilage thickness was measured by drawing a single line along the long axis of the cartilage, determining the midpoint of both ends of the cartilage on that line, and then drawing seven perpendicular lines 500 μm on both sides of that midpoint. The midpoints of both ends of the cartilage were determined on each vertical line, and the minimum width through these points was determined. Finally, an average thickness of the cartilage and the coefficient of variance for cartilage thickness at 7 points were calculated using ImageJ.

Total RNA was taken from frozen samples of gastrocnemius skeletal muscles using TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions. ReverTra Ace (Toyobo, Osaka, Japan) synthesized cDNA from 1 µg of the RNA. Primers for human and mouse β-actin were purchased from Taqman Gene Express ion Assays (Applied Biosystems, Foster City, CA, USA). Polymerase chain reaction (PCR) products were visualized by agarose gel (Wako, Osaka, Japan) electrophoresis with ethidium bromide staining.

RNA was extracted from cultured DPSCs and the frozen samples of hindlimb skeletal muscles using TRIzol Reagent (Invitrogen) according to the manufacturer’s instructions. Starting from 1 μg RNA, cDNA was synthesized using ReverTra Ace (Toyobo, Osaka, Japan) according to the manufacturer’s descriptions. Primers and probes for basic fibroblast growth factor (bFGF), vascular endothelial growth factor (VEGF), nerve growth factor (NGF), neurotrophin-3 (NT-3) and 18S rRNA and β2 microglobulin for the endogenous control were purchased from Taqman Gene Expression Assays (Applied Biosystems, Foster City, CA, USA). Polymerase chain reaction (PCR) products were visualized by agarose gel (Wako, Osaka, Japan)/ethidium bromide (Sigma). Real-time quantitative PCR (qRT-PCR) was performed and monitored by the ABI PRISM 7000 Sequence Detction System (Applied Biosystems). The relative quantity was calculated by the by the ΔΔCt method.