Myoblast differentiation on growth factor-immobilized polycaprolactone microparticles: a potential bioactive bulking agent for fecal incontinence

Materials

PCL (Mw 43 000∼50 000 Da; Polysciences, Warrington, PA, USA) and Pluronic F127 (EG₉₉ PG₆₅ EG₉₉, Mw 12 500 Da; Sigma, St. Louis, MO, USA) were used to fabricate microparticles. Heparin and growth factors (NGF and bFGF) were purchased from R & D Systems (Minneapolis, MN, USA) and Celsus Laboratories (Cincinnati, OH, USA), respectively. All other chemicals were of analytical grade and were used as received. Water was purified using a Milli-Q purification system (Millipore Co., Billerica, MA, USA). For in vitro cell culture, the microparticles were sterilized by ethylene oxide (EO).

Fabrication of PCL microparticles

PCL microparticles were prepared by an isolated particle-melting method, described elsewhere [28]. Briefly, PCL pellets were cryo-pulverized into random-shape microparticles using a freezer mill (SPEX 6750, Metuchen, NJ, USA). The crushed particles with a size range of 100∼200 μm were separated by micro-sieving using standard testing sieves (Chunggye Industrial Co., Korea), and then the microparticles were evenly dispersed in a cold Pluronic F127 aqueous solution (20 wt %; sol-gel transition temperature, ∼20 °C) and the gelation of Pluronic F127 solution induced by treatment at ∼25 °C for 1 h [PCL microparticles/Pluronic F127 solution, 1/50 (w/v)]. The PCL microparticles dispersed in the gel matrix were then located in a water bath at 65 °C for 30 min. With this procedure, the random-shape PCL microparticles melted (melting point of PCL, ∼60 °C) and transformed into spherical shapes. After this procedure, the PCL microparticles/Pluronic F127 gel mixture was cooled to ∼4 °C and then centrifuged. After removing the supernatant, the Pluronic F127-entrapped PCL microparticles were freeze-dried. The morphology of the prepared microparticles was observed by a scanning electron microscope (SEM; Model S-3000N, Hitachi, Japan).

Growth factor immobilization and release test

Two different growth factors (NGF and bFGF) were incorporated on the surfaces of the Pluronic F127-entrapped PCL microparticles via heparin binding. Schematic diagrams show the binding mechanism of heparin and subsequent growth factors onto the surface of the Pluronic F127-entrapped PCL microparticles (Fig. 1). To introduce heparin onto the surface of the microparticles, the microparticles were immersed in heparin solution [1 mg/mL (in 2 wt % NaCl solution)] at 4 °C for 3 h. The heparin-immobilized microparticles were rinsed successively with 2 wt % NaCl solution and water, and then freeze-dried. The amount of immobilized heparin was determined using a Toluidine blue assay [29]. To investigate the Pluronic F127 effect on heparin binding on the surface of the microparticles, the heparin was also immobilized on the PCL microparticles without Pluronic F127 and the heparin immobilization amount compared with that of the Pluronic F127-entrapped PCL microparticles.

Fig. 1

Schematic diagrams showing the binding of heparin and growth factors onto the surface of the Pluronic F127-entrapped PCL microparticles.

To incorporate growth factors onto the heparin-bound PCL microparticles, the microparticles were immersed in growth factor solutions of either NGF or bFGF (each 200 ng/mL)] at room temperature for 5 h. The growth factor-immobilized microparticles were washed three times with phosphate buffered saline (PBS; pH ∼7.4) and the amount of immobilized growth factors was determined by a direct ELISA technique [30]. The growth factor-immobilized microparticles were blocked in Reagent diluent [1 % bovine serum albumin (BSA) in PBS] in a 96-well polystyrene (PS) plate (Corning) for 1 h, and then was washed with 0.05 % Tween 20 in PBS three times. The microparticles were then immersed in a detection antibody in Reagent diluent for 2 h, and it was washed repeatedly. Then the microparticles were immersed in Strept Avidin-HRP for 20 min, after which it was washed repeatedly. The microparticles were positioned at the bottom of the wells in a 96-well plate. Finally, 1:1 mixture of Color reagent A (H₂ O₂) and Color reagent B (tetramethylbenzidine) substrate solution was added to each well and the color development was monitored concurrently with a series of soluble NGF and bFGF standards. After 20 min, Stop solution (2 N H₂ SO₄) was added to each well, and the absorbance was measured at 450 nm ultraviolet (UV) plate reader along with standards. To examine the effect of heparin bound on the surface of the microparticles on growth factor immobilization and release behavior, growth factor (NGF or bFGF)-adsorbed PCL microparticles without heparin binding were also prepared using the same procedure as above. The growth factor (NGF and/or bFGF)-immobilized microparticles (10 mg) were incubated in 1 mL PBS supplemented with 1 % bovine serum albumin (BSA; Sigma) at 37 °C for up to 35 days under mild shaking (∼50 rpm) to investigate the release behavior of the growth factors. At preset time intervals, the whole incubation medium was collected and replaced with fresh PBS. The amount of released growth factors from the microparticles in the collected medium was determined by the ELISA kit.

In vitro culture of myoblasts on growth factor-immobilized microparticles

In order to investigate the differentiation potential of myoblasts into myotubes, myoblasts were cultured onto the growth factor (NGF and/or bFGF)-immobilized PCL/Pluronic F127 microparticles. To isolate myoblasts, a muscle biopsy (∼1 cm³) was taken from the hind limb of dog. All biopsies were minced mechanically with scissors into small pieces of <1 mm³. The myoblasts were collected as previously described [31, 32] and were grown in tissue culture polystyrene (PS) dishes (Corning, Lowell, MA, USA) with skeletal muscle growth medium (SkGM-2; Lonza, Switzerland). For seeding of the myoblasts (after three passages) onto the PCL microparticles, the microparticle groups (w/o growth factors; single NGF or bFGF loading, each 10 mg; dual NGF/bFGF loading, 5 mg/5 mg) were placed in a 500 μL cell suspension (cell density, 1.0×10⁷ cells/mL) in the above growth medium and shaken mildly under 50 rpm for 2 h and then 100 rpm for 24 h for even cell adhesion onto the microparticles. Next, the microparticles were carefully transferred to non-treated 24-well PS dishes (Corning), and the growth medium was added into each well (500 μL) and cultured in an incubator at 37 °C in a 5 % CO₂ atmosphere for 28 days with mild shaking (∼50 rpm). The cell culture on the non-treated PS dishes with mild shaking may minimize migration of cells attached on the microparticles to the dish surfaces. The culture medium was exchanged with fresh medium every 3 days. Cell proliferation on the microparticles during the cell culture periods was estimated by measurement of the DNA content. For this, the cells of each microparticle group at predetermined time intervals (0, 7, 14, 21, and 28 days) were digested overnight in a Papain buffer at 60 °C. The DNA content was determined using a DNA-binding fluorochrome, Hoechst 33258 (Sigma-Aldrich), and purified calf thymus DNA as the standard.

Quantitative real time polymerase chain reaction (real-time PCR) analysis

At initial cell seeding (0 day) and after 28 days of cell culture in each microparticle group, RNA was extracted using an RNAspin mini kit (GE Healthcare, Piscataway, NJ, USA) according to the manufacturer’s instructions. The extracted RNA specimens were converted into cDNA using a reverse transcriptase (Superscript III^®; Invitrogen, Carlsbad, CA, USA) with oligo (dT) primers. Polymerase chain reactions were performed using an ABI Prism Sequence Detection System 7500 (Applied Biosystems, Foster City, CA, USA) with a Taqman^® gene expression assay (Applied Biosystems). RT-PCR analysis was conducted in a 25 μL reaction mixture containing 1.25 μL of 20X FAM TM dye-labeled Taqman^® MGB probe and two PCR primers, 1 μL of template. Expression of the following genes was examined: myogenic factor 5 (MyF 5) and Pax 7 (early markers in myoblast phase [33, 34]), myogenin (MyoG) and myosin heavy chain (MHC) (late (myotube-specific) markers in myoblast differentiation [35]), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH, a housekeeping gene). The data were analyzed using Sequence Detection Software. To quantify the gene expression level, the cyclic threshold method (user bulletin #2, Applied Biosystems) of relative quantification was adapted to estimate the number of copies. The data was normalized with GAPDH values. RT-PCR was performed with the following primers: MyF 5 (F: 5′-CTGTCTGGTCCCGAAAGAAC-3′; R: 5′-GAGAGGGAAGCTGTGTCCTG-3′); Pax 7 (F: 5′-GAGTTCGATTAGCCGAGTGC-3′; R: 5′-CGGGTTCTGATTCCACATCT-3′); MyoG (F: 5′-CAGTGAATGCAACTCCCACA-3′; R: 5′-GGCGTCTGTAGGGTCAGC-3′); MHC (F: 5′-AGAGAAACCACATTCGAGTCGTG-3′; R: 5′-TTGATCCTGATGGCGTCATTC-3′); GAPDH (F: 5′-TGTGTCCGTCGTGGATCTGA-3′; R: 5′-TGTGTCCGTCGTGGATCTGACCTGCTTCACCACCTTCTTGA-3′).

Western blot

At initial cell seeding and after 28 days of cell culture in each microparticle group, the total protein was extracted from each group using a cell lysis buffer (50 mM Tris-HCl, 150 mM NaCl, 0.1 % NP-40, 5 mM EDTA, 10 % glycerol) (Cell signaling, Beverly, MA, USA). Lystates were resolved by 12 % SDS-polyacrylamide gel electrophoresis 50 mmol/L Tris (pH 6.8), 10 mmol/L dithiothreitol (freshly added), 2 % SDS, 0.1 % bromophenol blue, and 20 % glycerol (SDS-PAGE) and transferred onto nitrocellulose membranes (Amersham, UK). The membranes were blocked with 5 % skim milk (in 20 mM Tris-HCl, 150 mM NaCl, and 0.1 % Tween 20). Then, the membranes were probed with antibodies against anti-Pax 7, anti-myosin heavy chain (anti-MHC), and anti-GAPDH (anti-glyceraldehyde-3-phosphate dehydrogenase; Sigma), reacted with the bound antibody, and then visualized with horseradish peroxidase-conjugated secondary antibody (GenDEPOT, Barker, TX, USA). Immunoreactivities were detected using enhanced chemiluminescence (ECL). Images were obtained using an X-ray developer (Daesung, Korea).

Immunohistochemical analysis

At initial cell seeding and after 28 days of cell culture in each group, immunohistochemical staining was conducted to visualize Pax 7 and MHC. For this, the cell-adhered microparticles embedded in an optimal cutting temperature (OCT) compound (Triangle Biomedical Sciences, Durham, NC, USA) were frozen (–20 °C) and cut into 10 μm transverse sections. The sections were mounted on positively charged slides and then were fixed in 4 % paraformaldehyde in PBS for 10 min and permeabilized with PBS containing 0.1 % Triton X-100 (0.1 % PBST). Next, they were incubated with Pax 7 antibody (diluted to 1:200) and MHC antibody (diluted to 1:1000) for 12 h (4 °C) and then with Texas Red-conjugated anti-mouse IgG (Pax 7) and fluorescein-conjugated anti-rabbit IgG (MHC) second antibodies (Invitrogen) for 1 h at room temperature. The nuclei were stained with 4′6-diamidino-2-phenylindole (DAPI; Vector Laboratories, Burlingame, CA, USA). Images were visualized with confocal microscopy (LSM510 META, Carl Zeiss, Germany).

Statistical analysis

The data obtained from each microparticle group were averaged and expressed as mean ± standard deviation. The Student’s t-test was used to determine the significance of the differences between the microparticle groups. The differences were considered statistically significant at p< 0.05.

Characterization of growth factor-immobilized PCL microparticles

The PCL microparticles were fabricated by the isolated particle-melting method. During the procedure, the random-shape (cryo-pulverized) microparticles were transformed into spherical-shape microparticles with similar sizes (Fig. 2). From the amount of heparin immobilized on the surfaces of PCL microparticles with and without Pluronic F127 entrapment as determined by Toluidine blue assay (Fig. 3), it was observed that the Pluronic F127-entrapped PCL microparticles (+ F127) provide much larger heparin binding on the surfaces of the microparticles (0.31 ± 0.08 μg/mg) compared with the microparticles without Pluronic F127 (- F127; 0.08 ± 0.01 μg/mg), suggesting efficient interaction between Pluronic F127 and heparin. Consequently, the Pluronic F127-entrapped PCL microparticles were used for the heparin and subsequent growth factor immobilizations.

Fig. 2

SEM photographs of random-shape (cryo-pulverized) and spherical-shape PCL microparticles.

Fig. 3

Amount of heparin immobilized on the PCL microparticles with and without Pluronic F127 entrapment (Toluidine blue assay; n = 3, *p < 0.05).

The amount of growth factors immobilized on the heparin-bound PCL microparticles was determined by the direct ELISA technique [30]. The growth factor immobilizations were significantly higher on the surfaces of the heparin-bound PCL microparticles [NGF (+ Hep), 3.85 ± 0.63 ng/mg; bFGF (+ Hep), 4.50 ± 0.80 ng/mg] than on those of the microparticles without heparin [NGF (- Hep), 1.18 ± 0.18 ng/mg; bFGF (- Hep), 1.48 ± 0.15 ng/mg] (Fig. 4a). The release behavior of the growth factors from the heparin-bound PCL microparticles (Fig. 4b) showed sustained release of growth factors from the microparticles, regardless of growth factor type, over 4 weeks (more than 90 % release of loading amount), probably because of ion exchange in the medium. On the contrary, the microparticles without heparin binding showed an initial burst release of growth factors for 7 days, and then the amount of both growth factor releases was not significant.

Fig. 4

(a) Loading amount and (b) cumulative released amount of NGF and bFGF from the PCL microparticles with and without heparin immobilization (n = 3; *p < 0.05).

In vitro differentiation of myoblasts on growth factor-immobilized microparticles

To investigate the in vitro cell proliferation and differentiation potential of myoblasts by continuously releasing growth factors from the PCL microparticles, the myoblasts were seeded onto the microparticle groups (w/o G. F., single NGF, single bFGF, and dual NGF/bFGF loading). The cell proliferation on the microparticles was estimated by the DNA contents at the predetermined time intervals (0, 7, 14, 21, and 28 days) after seeding. The cells are shown to have proliferated for up to 7 days, and then the cell growth for all groups was not significantly different in the culture (Fig. 5). Higher cell growth patterns were observed in the presence of growth factors, probable because of stimulation for cell growth and protection of cell apoptosis by the growth factors [36]; however, differences of cell proliferation among the growth factor (NGF, bFGF, and dual bFGF/NGF)-immobilized microparticle groups were not significant.

Fig. 5

Total DNA content with time after in vitro myoblast culture on the PCL microparticles with and without growth factor immobilization (n = 3; *p < 0.05).

To investigate the differentiation potential of myoblasts into myotubes on the growth factor-immobilized PCL microparticles, a quantitative RT-PCR analysis for several genes was conducted at 0 and 28 days after seeding. Differentiation of myoblasts was monitored using four specific markers, MyF 5, Pax 7, MyoG, and MHC [33–35]. The RT-PCR results showed decreased MyF 5 and Pax 7 (early markers in myoblast differentiation) expression and increased MyoG and MHC (myotube-specific markers in myoblast differentiation) expression on the growth factor-immobilized PCL microparticles over time, indicating the differentiation of myoblasts into the more mature cell type (myotubes) to regenerate muscles (Fig. 6a). The single bFGF- and dual NGF/bFGF-immobilized microparticle groups produced significantly higher myotube-specific genes (MyoG and MHC) than the other groups (w/o G.F. and single NGF) at 28 days. In particular, the dual NGF/bFGF group showed notably greater MHC gene expression compared with the single bFGF group. This suggests that the dual NGF/bFGF-immobilized microparticles can provide a better environment for the differentiation of myoblasts into myotubes than the other groups.

Fig. 6

(a) RT-PCR and (b) Western blot analyses after in vitro myoblast culture on the PCL microparticles with and without growth factor immobilization at 0 and 28 days (n = 3; *p < 0.05).

In the Western blot analysis, it was also observed that Pax 7 expression on the growth factor-immobilized PCL microparticles decreased dramatically between the initial stage (0 day) and after 28 days, while MHC expression showed the opposite trend (Fig. 6b). The increase in MHC expression at 28 days was in the sequence as follows: Dual NGF/bFGF group > single bFGF group > single NGF group > w/o G. F. group.

From immunohistochemical observations of myoblasts cultured on the PCL microparticles with and without growth factor immobilization (Fig. 7), it was observed that the cells (blue color, cell nucleus) were uniformly distributed on the microparticles, indicating that the PCL microparticles can be an appropriate cell carrier. Pax 7 expression (red color) was detected at 0 day in all microparticle groups, but the expression almost disappeared at 28 days, suggesting that the cells at 0 day are undifferentiated myogenic cells and those at 28 days are differentiation-committed cells [37]. MHC expression was not detected at 0 day, regardless of the presence of growth factors; however, the growth factor-immobilized microparticle groups at 28 days showed greater levels of MHC expression (green color) than the w/o growth factor group, indicating the greater differentiation of myoblasts into myotubes. In particular, the dual NGF/bFGF group had the highest expression of MHC. This observation was consistent with the results of RT-PCR and Western blot analyses (refer to Fig. 6).

Fig. 7

Immunohistochemical observations after in vitro myoblast culture on the PCL microparticles with and without growth factor immobilization at 0 and 28 days [x 100; blue, cell nucleus (DAPI); red, Pax 7; green, MHC].