Choloroform

Real-time PCR was performed using the protocol reported by Washington et al (Washington and others 2013 (link)). In preparation for RT-PCR, samples of muscle from the defect site of the right and left TA (n=4 animals/experimental group) were homogenized with Trizol (Ambion, Carlsbad, CA)/choloroform (Sigma Aldrich, St. Louis, MO). Samples were treated with DNase (Invitrogen, Carlsbad, CA) and then RNA was extracted using an RNAeasy kit (Invitrogen, Carlsbad, CA). After quantification of RNA with a plate reader (BioTek, Winooski, VT), RNA was converted to cDNA with a kit (Invitrogen, Carlsbad, CA). TAQMAN primers (Invitrogen, Carlsbad, CA) for MyoD, Collagen I, Collagen III, ratios of Collagen I to MyoD and Collaen I to Collagen III, and 18S rRNA housekeeping were used to quantify the expression of desired genes. Experimental sample group expressions were normalized to 18S rRNA and then referenced to the contralateral normal limb. Gene expression levels are reported as fold change using the 2^−(ΔΔCt) method.

Hexamethylene diisocyanate (HMDI), dibutyltin dilaurate (DBTDL), butylated hydroxytoluene (BHT), 2,2dimethoxy-2-phenylacetophenone (DMPA), trimethylamine (TEA), dichloromethane (DCM), ethyl ether, choloroform (≥99.5%) were obtained from Sigma-Aldrich. m-Xylene diisocyanate (XDI), 1,3-bis(isocyanatomethyl)cyclohexane (BIC), 2-isocyanatoethyl methacrylate (IEM), triethylene glycol dimethacrylate (TEGDMA) were purchased from TCI America. Polycaprolactone tetra(3mercaptopropionate) (PCL4MP, M_n = 1350), ethoxylated-trimethylolpropan tri(3-mercaptopropionate) (ETTMP, M_n= 700) were donated by Evans Chemetics. All materials were used as received.