Ethics approval for the animal experiments was obtained from the Italian Ministry of Health (protocol #163/2011-B; released on 16 September 2011) and all experiments were conducted in accordance with the rules of good animal experimentation (IACUC, number 432, dated 12 March 2006).
Preparation of mesoangioblasts and culture conditions
Mesoangioblasts were cultured at 37°C (5% CO2) in petri dishes with DMEM (Dulbecco’s modified Eagle’s medium with GlutaMAX; Gibco-BRL,Gaithersburg, MD, USA), supplemented with heat-inactivated 10% FCS (EuroClone), 100 IU/ml penicillin and 100 mg/ml streptomycin . Mesoangioblasts were transduced with third-generation lentiviral vectors encoding the nuclear β-Galactosidase. and mesoangioblasts expressing nuclear lacZ (nlacZ-mesoangioblasts) were cultured and used for in vitro differentiation or intra-muscular injection .
PEG-fibrinogen was produced and polymerized as described previously . Briefly, PEG-fibrinogen was prepared at a desired concentration and diluted with sterile PBS as required. A photoinitiator (Igracure™ 2959; Ciba Specialty Chemicals, USA) was added to the PEG-fibrinogen mixture at a final concentration of 0.1% w/v. Cells were added at the desired concentration and the solution was immediately exposed to UV light (365 nm, 4–5 mW/cm2) for 5 minutes for the in vitro experiments. In vivo experiments were exposed to UV light (365 nm, 200 mW/cm2) using a hand-held light gun (LED-200; Electro-lite Corp., Bethel, CT USA) for 1 minute.
Animals and treatments
Rag2 γ-chain null mice (4 months old) and α-sarcoglycan knockout/severe combined immunodeficiency beige (α-SGKO/SCIDbg) mice  (12 months old) were used for intra-muscular injection. Briefly, mice were anesthetized with an intra-muscular injection of physiologic saline 10 ml/kg containing ketamine 5 mg/ml and xylazine 1 mg/ml. For the liquid nitrogen (N2) muscle-crush injury, a small skin incision was made over the tibialis anterior (TA) muscle of anesthetized mice. A liquid-nitrogen-cooled needle (0.20 mm diameter) was inserted along the craniocaudal axis of the TA twice, 30 seconds for each insertion. For intra-muscular cell delivery, approximately 3 × 105 nlacZ-mesoangioblasts were injected into the TA via a 0.20 mm diameter needle inserted along the craniocaudal axis of the muscle. For PF-embedded nlacZ-mesoangioblast injections, a limited incision was made on the medial side of the leg to separate the TA from the skin and to allow in vivo PF photopolymerization. A subgroup of animals was injected intraperitoneally with 5-bromo-2-deoxyuridine (BrdU) 100 mg/kg (RPN 20; GE Healthcare, Princeton, NJ, USA) to label proliferating cells 2 hours after mesoangioblast transplantation. The BrdU-labeled mice were killed 48 hours after cell injection.
The presence of apoptotic cells was examined using terminal deoxynucleotidyl transferase dUTP nick-end labeling (TUNEL) staining (Roche Diagnostics, Basel, Switzerland) in 10 μm cryosections. Positive control sections were treated with DNaseI (Roche Diagnostics, Basel, Switzerland) for 20 minutes at 37°C. Sections were incubated with the TUNEL reagent at 37°C for 30 minutes before being counterstained with 4,6-diamidino-2-phenylindole (DAPI).
The tissue samples were fixed in 4% paraformaldehyde for 30 minutes at 4°C and washed in PBS, embedded in optimal cutting temperature compound, and flash-frozen in liquid-nitrogen-cooled isopentane. Sections were cut at a thickness of 8 μm on a cryostat (Leica, Heerbrugg, Switzerland) and washed with buffer (PBS containing 0.2% Triton X-100). The sections were then incubated with primary antibody (rabbit anti-laminin; Sigma-Aldrich, St Louis, MO, USA) diluted to a final concentration of 1:100 with blocking buffer (PBS containing 0.2% Triton X-100 and 20% heat-inactivated goat serum) for 20 minutes at room temperature. Sections were washed with washing solution (PBS containing 0.2% Triton X-100 and 1% BSA), and then incubated with the secondary antibody (horseradish peroxidase-conjugated goat anti-rabbit; Chemicon International Inc., Temecula, CA, USA), diluted 1:500 in 20% goat serum. The immune reaction was developed using 3-amino-9 ethylcarbazole substrate (AEC; Sigma-Aldrich).
Afterwards, the sections were stained with X-Gal to reveal β-galactosidase-positive cells as described previously . Briefly, the sections were washed twice with PBS for 5 minutes each and incubated for 24 hours at 37°C with an X-Gal working solution. This solution is composed of the X-Gal stock solution (X-Gal 40 mg/ml in N,N-dimethyl formamide, which was stored at −20°C and protected from light) diluted 1 in 40 in X-Gal dilution buffer (crystalline potassium ferricyanide 5 mmol/l, potassium ferricyanide trihydrate 5 mmol/l, and magnesium chloride 2 mmol/l in PBS, which was protected from light, and stored at 4°C). Sections were washed twice with PBS for 5–10 minutes each, and then covered directly with aqueous mounting medium (Aqua Poly/Mount; Polysciences Inc., Warrington, PA, USA) The lacZ-positive nuclei were counted in five randomly selected fields of three different non-adjacent transverse sections from the largest TA portion taken from three mice per experimental group.
Immunofluorescence procedures were performed essentially as described previously . Briefly, the specimens were prepared as described above, and then incubated with primary antibodies diluted with blocking buffer for 20 minutes at room temperature. The primary antibodies used were: mouse anti-α-SG (Ad1/20A6; Vector Laboratories Inc., Burlingame, CA, USA) 1:100 dilution, rabbit anti-laminin (#9393; Sigma-Aldrich) at 1:500, rabbit anti-lacZ (Cappel Laboratories, Durham, NC, USA) 1:100, mouse anti-Pax7 and anti-Myosin Heavy Chain (MF20) (Developmental Studies Hybridoma Bank, Iowa City, IA, USA) 1:100. After several washes with buffer, sections were incubated with secondary antibodies diluted with blocking buffer for 1 hour at room temperature. The secondary antibodies (all used at 1:500) were anti-mouse FITC (Chemicon International Inc.), anti-rabbit Alexa488, and anti-rat Alexa488 (both Molecular Probes, Eugene, OR, USA). Sections were counterstained with DAPI to detect nuclei, washed several times with wash buffer, and mounted (Vectorshield; Vector Laboratories Inc.). To visualize BrdU, a commercial kit was used, and sections were treated with nuclease/anti-BrdU solution provided in the kit (RPN20, GE Healthcare, Princeton, NJ, USA) for 1 hour at room temperature in accordance with the manufacturer’s instructions. Sections were washed three times in PBS, and incubated for 30 minutes at room temperature with Alexa Fluor 488 secondary antibody against mouse (Molecular Probes). Sections were counterstained with 4′,6-diamidino-2-phenylindole (DAPI), washed in PBS, and mounted as described above.
Tissue samples (n = 3 for each time point per group) of TA treated with PF-embedded mesoangioblasts from α-SG null mice were homogenized in liquid nitrogen, mixed with lysis buffer (50 mmol/l Tris/HCl, pH 7.4, 1 mmol/l EDTA, 1 mmol/l EGTA, 1% Triton X-100, 1 mmol/l), and protease inhibitor cocktail (Sigma-Aldrich), and separated by centrifugation at 12,000 g for 10 minutes at 4°C to remove the nuclei and cellular debris. Protein concentrations were determined by bicinchoninic acid (BCA) protein assay (Pierce Biotechnology Inc., Rockford, IL, USA) using BSA as a standard. Total homogenates were separated by SDS-PAGE. For western blotting analysis, proteins were transferred to membranes (Immobilon; Amersham Biosciences Inc., Piscataway, NJ, USA), saturated with blocking solution (1% BSA and 0.1% Tween-20 (Sigma-Aldrich) in PBS) and hybridized with cleaved caspase-3 rabbit monoclonal antibody (#9669; Cell Signaling Technology, Danvers, MA, USA), α-SG mouse monoclonal antibody (Ad1/20A6; Vector Laboratories) or lacZ polyclonal antibody (#55976; Cappel Laboratories) at 1:1,000 dilution, or with GAPDH monoclonal antibody (GAPDH-71.1; Sigma-Aldrich) at 1:10,000 dilution for 1 hour at room temperature. The blots were washed three times (15 minutes each at room temperature) with blocking solution, and then reacted with anti-mouse or anti-rabbit secondary antibody conjugated with HRP (Bio-Rad Laboratories, Inc., Hercules, CA, USA) at 1:3,000 dilution for 1 hour at room temperature. The blots were then washed three times, and finally visualized with an enhanced chemiluminescent immunoblotting detection system (Pierce Biotechnology Inc).
Statistical significance of the differences between means was assessed by one-way analysis of variance (ANOVA) followed by the Student-Newman-Keuls test to determine which groups were significantly different from the others. When only two groups had to be compared, the unpaired Student’s t-test was used. P<0.05 was considered significant. Values are expressed as means ± standard deviation (SD).