Chitosan solution (5 wt%) was prepared by dissolving the chitosan (M_w = 50,000, degree of deacetylation = 97%) in 2 wt% CH₃COOH solution. Typically, electrospinning was performed at 20 kV voltage, 10 cm distance between the needle tip and the collector (Taksan Meditech Co., Ltd., Korea). The flow rate of the solution was controlled by a syringe pump maintained at 10 m/min from the needle outlet. A grounded aluminum foil was used as the collector. The microfibrous chitosan was collected on the surface of aluminum foil. A porosimeter (Auto Pore IV 9500 V 1.03; Instruments Co., USA) was used to examine the porosity and mean pore diameter of the obtained microfibrous chitosan.

The major hind-limb muscles of the rats were removed, trimmed of excess connective tissue and fat, hand minced, and washed twice with PBS. After centrifugation at 2000 rpm for 5 min, the resultant pellets were enzymatically dissociated by adding 0.2% collagenase-type XI (Sigma, Germany) for 1 h at 37 °C, dispase (20 mL, Gibco BRL) for 45 min, and 0.1% trypsin (Sigma, Germany) for 30 min. Cells were separated from muscle fiber fragments and tissue debris by differential centrifugation and were plated on collagen-coated flasks in DMEM containing 5% fetal bovine serum (Gibco BRL), 5% horse serum (Gibco BRL), penicillin (100 U/mL), and streptomycin (100 μg/mL) for 1 h. The muscle cell extract was pre-plated on collagen-coated flasks. Unattached cells floating in the medium after 1 h incubation were then transferred onto fresh collagen-coated flasks. The subsequent pre-plates were performed in the next 2 h, 3 h, 1 day, 2 days and 3 days. Finally, the rMDSCs were seeded into normal tissue culture flasks at 1 × 10⁵ cells/cm². The flasks were rinsed three times with PBS on the second day of expansion. The medium was changed every 2 days throughout the studies. Adherent cells, rMDSCs, were rinsed thoroughly with PBS and detached by 0.05% trypsin-EDTA for experiment use. The final rMDSCs used were taken at passage 5.

The rMDSCs were labeled using the PKH67 Fluorescent Cell Linker Kit (Sigma, USA) according to the manufacturer’s instructions. In brief, the cultured rMDSCs were washed with serum-free media and centrifuged for 5 min at 400 g. The provided diluent C (500 μL) was added to 3 × 10⁵ rMDSCs and immediately mixed with 500 μL of PKH67 stock solution (4 × 10⁻⁶ M) in diluent C. After incubation for 5 min at room temperature, 1 mL of FBS was added and samples were incubated for 1 min to stop the labeling reaction. Finally, the rMDSCs were pelleted for 5 min at 400 g, transferred to a fresh tube and washed three times with complete DMEM.

Electronspun chitosan microfibers were sterilized by EO gas at Hansbiomed Company. For cell culture experiments, the chitosan microfibers were prepared and placed individually into the wells of a 24-well tissue culture plate (Falcon, USA) and then incubated for 1 h in culture media. After suction of the media, the PKH67 labeled rMDSCs (3 × 10⁴ cells/well) were transferred to each well. The culture media was changed every 2 days throughout the studies. For SEM measurements, at 1 and 7 days, the microfibrous scaffolds without or with rMDSCs were fixed with 2.5% glutaraldehyde for 24 h, followed by ethanol dehydration. The fixed chitosan microfibers were coated with a conductive layer of gold using a plasma-sputtering apparatus (Emitech, K575, Kent, UK), and scanning electron microscopy (SEM, S-2250N, Hitachi, Japan) images were obtained. Cytotoxicity was measured using a WST-1 kit (Roche, Germany) after 1, 3, and 7 days. Briefly, 100 μL of WST-1 reagent was added to rMDSCs in 1 mL medium per well, the plates were incubated at 37 °C for 4 h, and the samples were then shaken for 1 min. An aliquot from each well (100 μL) was transferred to a 96-well plate, and absorbance at 450 nm was measured with a microplate reader (EL808 ultra microplate reader; Bio-Tek Instrument, USA).

The rats were housed in sterilized cages with sterile food, water and filtered air, and were handled under a laminar flow hood following aseptic techniques. All animals were treated in accordance with the Institutional Animal Experiment Committee at Ajou University School of Medicine. Twenty-four Fischer rats (140–160 g, 4 weeks), divided into four groups of three rats each, were used in the animal tests. The chitosan microfibers without and with rMDSCs (3 × 10⁴ cells) were implanted subcutaneously under the dorsal skin and then allowed to develop, and biopsied in vivo over 4 weeks. At each of the post-implantation points, the rats were sacrificed and the implants were dissected individually and removed from the subcutaneous dorsum.

SEM was used to examine morphology of in vivo chitosan microfibers. The in vivo chitosan microfibers without or with rMDSCs after 1 and 4 weeks were fixed with 2.5% glutaraldehyde for 24 h, followed by ethanol dehydration. The fixed chitosan microfibers were coated with a conductive layer of gold using a plasma-sputtering apparatus (Emitech, K575, Kent, UK), and scanning electron microscopy (SEM, S-2250N, Hitachi, Japan) images were obtained.

At 3 days, one, two and four weeks after implantation, the rats were sacrificed and the implants were individually dissected and removed from the subcutaneous dorsum. The implants were immediately fixed with 10% formalin and embedded in paraffin. The embedded specimens were sectioned (4 μm) along the longitudinal axis of the implant, and the sections were stained with hematoxylin and eosin (H&E), 6-diamino-2-phenylindoadihydrochloride (DAPI, Sigma-Aldrich, St Louis, MO, USA), and mouse anti-rat CD68 (ED1; 1:1000; Serotec, Oxford, UK). The staining procedures for DAPI and ED1 were as follows. The slides were washed with PBS-T (0.05% Tween 20 in PBS), blocked with 5% bovine serum albumin (BSA; Roche, Mannheim, Germany) and 5% horse serum (HS; GIBCO, Paisley, UK) in PBS for 1 h at 37 °C. The sections were incubated overnight at 4 °C with ED1 antibodies, washed with PBS-T, and then incubated with the secondary antibody (goat anti-mouse Alexa Fluor^®594; Invitrogen, San. Diego, CA, USA) for 3 h at room temperature in the dark. The slides were washed again with PBS-T, counterstained with DAPI, and then mounted with fluorescent mounting solution (DAKO, Calif, USA). Immunofluorescent images were visualized under an Axio Imager A1 (Carl Zeiss Microimaging GmbH, Göttingen, Germany) equipped with Axiovision Rel. 4.8 software (Carl Zeiss Microimaging GmbH, Göttingen, Germany). Before acquisition of immunofluorescent images, the delimitation between implant and host tissue was determined from a differential interference contrast (DIC) optical microscopic image. The sections were also stained with hematoxylin and eosin (H&E) using standard procedures.

Cytotoxicity data were obtained from independent experiments in which three wells per culture plate or chitosan microfibers were examined and ED-1 assays were carried out in independent experiments with n = 16 for each data point, with data given as the mean and standard deviation (SD). Statistical analysis using the one-way ANOVA method was carried out with Bonferroni’s multiple comparisons.