Fifty- to sixty-gram Sprague Dawley (SD) rats (purchased from the Animal Center of Shanghai Jiaotong University) were anesthetized with 10% chloral hydrate, and the inguinal subcutaneous fat was excised and soaked three times with 0.25% chloramphenicol. The tissue was then minced and digested with 0.1% type I collagenase (Sigma-Aldrich Co. LLC., MO, USA) for 45 min at 37°C, filtered with a 200-mm nylon strainer (BD Falcon, Corning Inc., NY, USA) and collected by centrifugation (1,500 r/min, 10 min, 37°C). The upper layer of fat and the supernatant were removed and cultured in DMEM (Gibco, Thermo Fisher Scientific Inc.) supplemented with 10% FBS (Gibco, Thermo Fisher Scientific Inc., MA, USA) at 37°C in a 5% CO₂. Forty-eight hours later, the cells were washed with PBS and digested with 0.25% trypsin and passaged with a ratio of 1:3 upon reaching 80%–90% confluence. The culture medium was changed every 2 days. The second passage of ADSCs was identified by flow cytometry analysis of the marker of CD34, CD90, CD29, and CD105.

DsRNAs targeting the MyoD promoter were designed and synthesized by Thermo Fisher Scientific Co., Ltd, China. Then, MyoD saRNA was transfected into ADSCs with Lipofectamine™ RNAiMAX (Thermo Fisher Scientific Co., Ltd, China) following the manufacturer’s instruction. Seventy-two hours after transfection, cells were collected to extract total protein or RNA. The effective cells were analyzed by RT-PCR and western blot and picked with statistical analysis.

The saRNA sequences are as follows: control saRNA forward: 5′-UUCUCCGAACGUGUCACGUTT-3′; control saRNA reverse: 5′-ACGUGACACGUUCGGAGAATT-3′. MyoD saRNA forward:5′-UGCCUGGUAUCCCUACAAAtt-3′; MyoD saRNA reverse: 5′-UUUGUAGGGAUACCAGGCAtt-3′. The above sequence was labeled with green fluorescent FAM.

NPs and saRNA encapsulated NPs were prepared by double emulsion method with a carrier material PEG-PLA and cationic lipid BHEM-Chol. PEG₅₀₀₀-PLA₂₅₀₀₀ and BHEM-Chol are dissolved in 0.5 ml chloroform, 25 μl saRNA solution (including 200 μg saMyoD), and the initial emulsion is formed under the ultrasonic of the probe type ultrasonic breaker (output power 80 W, 30 s). The initial emulsion was added to 1.5 ml of 1% polyvinyl alcohol aqueous solution, and then emulsified again (output power 80 W, 2 min). Then the emulsion was added to 25 ml of 0.3% PVA aqueous solution and the organic solvent was volatilized under reduced pressure, centrifuged (4°C, 30,000g, 1 h). The NPs were collected and suspended twice with ultrapure water, washed by centrifugation, collected. The sample was lyophilized.

ADSCs were seeded in 24-well cell culture plates at a density of 10⁵/well. After overnight culture, the medium was changed to DMEM complete medium (containing 10% FBS) containing FAM-MyoD-saRNA-NPs. The concentration of the particles was 0.366 mg/ml and the FAM-saRNA was 100 nM. The experiments were divided into four groups, ADSCs transfected with PBS group NP), NP(saMyoD) group, NP(scramble) group, and empty NP (Blank) group. The cells were cultured at 37°C for 4 h and then the FAM was detected by Flow cytometry.

ADSCs were incubated with both 100 mg SF(VEGF)/BAMG and 100 mg NP(saMyoD)/BAMG composite scaffold containing DMEM medium, or 200 mg each scaffolds DMEM medium for 3 days (37°C, 5% CO₂₎. The cytotoxicity of 1, 2, and 3 days was detected with an MTT [3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide] based In Vitro Toxicology kit (Sigma-Aldrich, Schnelldorf, Germany) according to the manufacturer’s instructions.

Following the former research (Wang Q. et al., 2017), 200 μl VEGF solution (20 μg/ml) and 8 ml ethanol were added into 20 ml 3% SF aqueous solution. Then, the mixture were stabilized for 5 min under low speed stirring, and frozen at −20°C for 24 h. After ultracentrifugation at 40,000g, the supernatant was removed, and the precipitated silk fibroin particles were lyophilized for further use.

The bladder tissue was cut longitudinally from the rat bladder, Then, carefully peeled off the bladder mucosa, and remove the muscle layer of the pulp by microscopy. After soaking in nuclease-free water for 24 h, the treated tissues were dipped in a decellularized solution (0.1% Triton X-100 and 0.15% ammonia water) for 14 days, the decellularized solution was updated every 3 days. Then, the tissues were frozen at −80°C for 24 h, vacuum dried for 24 h, and stored in 75% alcohol.

SF(VEGF) or NP(saMyoD) were dissolved in PBS to a final concentration of 1% and added to BAMG. Due to ultrasound could improve the kinetic energy of biomolecules and increase the collision frequency with BAMG (Zhang et al., 2018), the BAMG was ultrasonically shaken in SF(VEGF) and NP(saMyoD) solution for 10 min and dried under low temperature pressure. Then the SF(VEGF)/BAMG or NP(saMyoD)/BAMG composite scaffold were well prepared. To get the ADSCs well attached the composite scaffold, 100 µl third generation of ADSCs were incubated with SF(VEGF)/BAMG or NP(saMyoD)/BAMG composite scaffold at 3×10⁶/ml. Two hours later serum-free medium was added, 4 h later, the cells were incubated with medium supplemented with serum. After 24 h, the cells were washed with serum medium and washed twice with PBS to remove unadhered and dead cells.

Scaffold mechanical testing: In brief, five pieces BAMGs were picked randomly and immersed in PBS for 24 h. Then those BAMGs were fixed for tensile test with no shift. Before the test, the length and width within the framework were measured. The frame was stretched in the vertical shift in longitudinally with 10mm/min. We reaped these indexes, the initial elastic modulus (EM), ultimate tensile strength (UTS), and % elongation to failure (ETF) with instron 5542 Tensile Tester (Norwood, MA, USA).

Eight-week-old adult male SD rats were acclimatized and housed in cages with free access to food and water in temperature-controlled, pathogen-free animal room facilities (20°C–22°C, humidity 40%–70%, 12 h day/night cycle) for one week before the experiments. The animals were randomly divided into four groups (12 in each group): the sham operation group, the ADSC-BAMG group, the ADSC-saMyo/BAMG group, the ADSC-saMyoD/SF(VEGF)/BAMG group. All animal procedures were approved and supervised by the Ethical Committee of Shanghai Jiaotong University and were performed as the guidelines of the China Act on Welfare and Management of Animals.

Rat bladder enlargement model was simply described as follows: rats were anesthetized by intraperitoneal injection of pentobarbital (30 mg/kg), and incised under sterile conditions with a 1-cm incision at the lower abdomen skin to expose the bladder. Cut about 40% to 60% bladder tissue in squares (Wang Q. et al., 2017). BAMG with the same size and area was sutured with the 5–0 polypropylene line to repair the defect of the bladder. BAMG was faced to the bladder cavity. Intravesical incision, normal saline was injected into the bladder from the urethra, and the abdominal cavity was closed after no clear leakage.

Total protein extracted from cell were loaded on to sodium dodecyl sulfate‐polyacrylamide gel electrophoresis (SDS‐PAGE) and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, USA). The membranes were incubated first with primary antibody and then with secondary antibody. GAPDH was used as a control to verify the equal loading of proteins. Western blot analysis was carried out by a standard protocol using antibodies for SM22a (ab14106, 1:800); Desmin (#5332, 1:1000); α‐SMA (sc-53142, 1:800) and GAPDH (sc-32233, 1:1000)

Total RNA was extracted from cells using the Trizol reagent (Invitrogen, Thermo Fisher Scientific Inc., NY, USA) as the manufacture’s protocol. The cDNA was acquired by cDNA Reverse Transcription Kit (ABI). Then, the mRNA expression of a-SMA, SM22α and Desmin were conducted by ABI Prism 7900 (Applied Biosystems, Foster City, CA, USA). All reactions were run in triplicate. The cycle threshold (Ct) method was used to calculate values. Target mRNA expression levels were normalized to the housekeeping genes of GAPDH as an internal control.

One hundred milligram of SF(VEGF)/BAMG was suspended in 1 ml of PBS and shakened at 37°C at 100 rpm. A 0.2 ml sample was taken daily from day 1 to day 7 and replenished with an equal volume of fresh PBS. The taken sample concentration of VEGF was measured by ELISA.

Following Rats sacrifice, the bladder tissue was harvested and kept in neutral buffered formalin 10% (v/v) for one day until paraffin embedding for histology. The tissue was sliced in a 5-μm thickness in paraffin block and stained with routine hematoxylin and eosin solutions. Images were captured with a light microscope (Olympus CKX41 microscope, Tokyo, Japan) linked to a computer.

Paraffin-embedded tissue sections were dewaxed with xylene and rehydrated through an ethanol gradient into water. Following blocking of endogenous peroxidase activity with 0.3% hydrogen peroxide for 10 min, the tissue slices were washed with PBS, and incubated with VEGF (sc-80442, 1:200), a-SMA (sc-53142, 1:100) antibody. Next, slices were incubated with biotinylated secondary antibody and then with horseradish peroxidase labeled streptavidin. Diaminobenzidine (DAB) was used as chromogen and antibodies were diluted in the recommended antibody diluting buffer.

All data are expressed as the mean ± standard deviation. According to different data set, using a two-tailed Student’s t-test or one-way analysis of variance (ANOVA) with the Bonferroni post hoc test in GraphPad Prism 8 (GraphPad Software Inc., San Diego, CA, USA). P< 0.05 indicates statistical significance.

In this study, we isolated and cultured rat ADSC cells, and found uniform spindle-like structure in the second generation. Optimistically, the cell began to proliferate rapidly. To identify the isolated cells were functional ADSCs, we detected cell surface specific markers with flow cytometry analysis (Figure 1). The results showed that 85.12% of the detected isolated cells were able to express CD90, 78.87% express CD29 and 81.54% express CD105 (Schäffler and Büchler, 2007; Yamamoto et al., 2007), but few isolated cells express CD34 (4.52%). These results strongly suggested that we had isolated and culture ADSC successfully.

The mean diameter of NP(saMyoD) was 167 ± 7.3nm, with PDI 0.208, indicating homogeneous distribution of the particles. The NP(saMyoD) has a 12.1 mV potential voltage, instructing NP(saMyoD) could bind with negative charged cell membrane epitope. NP(saMyoD) displayed a stable diameter of within 180 nm in fetal bovine serum within 48 h, showing good stability (Figure 2A). In addition, NP(saMyoD) had encapsulate efficiency (92.3%) and drug loading capacity (0.667%), suggesting that siRNA could be encapsulated efficiently in NP. Besides, the release assay showed that NP(saMyoD) release effect could last for 12 days before plateau (Figure 2B).

Myogenic differentiation 1 (MoD) plays a vital role in muscle cell differentiation and muscle regeneration (Weintraub et al., 1991; Clemen et al., 1999; Gaudet et al., 2011). After isolated rat ADSCs in vitro efficiently, we were looking for a method to overexpression Myod in ADSCs more efficiently and keep those ADSCs well-functioned in animal body. Currently, rushing research have shed light on the application of NPs in conjunction with small molecular compounds or nucleotide in the medical area (Moghimi et al., 2001; Fan et al., 2018; Li et al., 2019). In this work, we have developed an saMyod-conjugated-hollow gold NPs NP(saMyoD) and identified its biological function. First, FAM-labeled saMyoD was used to prepare NP(saMyoD), we then transfected the compound into ADSCs. The flow cytometry results showed that NP(saMyoD) was greatly expressed in ADSCs, the same to NP(saScr) (Figure 3A). The quantitative results showed that about 80% of the cells could be transfected with NP(saMyoD) (Figure 3B). Two days after transfection, the expression of MyoD was detected by RT-PCR (Figure 3C) and Western Blot (Figure 3D). These results demonstrated that, after transfection of ADSC with NP(saMyoD), the mRNA and protein expression of MyoD increased dramatically in ADSCs transfected with NP(saMyoD) compared to other groups (Figures 3C, D). Meanwhile, the expression of MyoD in cells transfected with NP was similar to NP (scramble) transfected cells (Figures 3C, D).

As the NP(saMyoD) transfected ADSCs showed high Myod expression level, we further investigate the function of those ADSCs. First, we examined the expression level of smooth muscle cell markers α-SMA, SM22α, and Desmin. As shown in Figure 4A, compared with NP(saScr), the mRNA expression of α-SMA, SM22α, and Desmin were significantly upregulated after 14 days transfection with NP(saMyoD). The protein expression of α-SMA, SM22α, and Desmin were consistent with mRNA results (Figure 4B).

We have evaluated the efficiency and function of NP(saMyoD) on ADSCs, then we’d like to evaluate the SF(VEGF) scaffolds by detecting the sustained release of VEGF on SF-BAGM. The release of VEGF was increased along with the time elapsed and exhibited a burst release in the third day, and then the release ratio of VEGF was slowed down but the release period lasted for more than 7 days (Figure 5A). Then, we have checked other indicators in previous reports, the characterization of SF, BAMG and their composite scaffolds were measured with scanning electron microscopy (Zhu et al., 2016). These results indicated that SF-BAMG was successfully constructed and suitable for drug delivery and release regulation. We performed mechanical test for the scaffolds that we generated, the results showed the biomechanics of ADSC-NP(saMyoD)/SF(VEGF)/BAMG is UTS 0.47 ± 0.12 MPa, EM 1.02 ± 0.35 MPa (Figure 5D). MTT assay was performed to determine the toxic effects ofNP(saMyoD)/BAMG and SF(VEGF)/BAMG scaffolds on ADSCs. Comparing to the blank group, NP(saMyoD)/BAMG slightly inhibited the proliferation of ADSC, which is consistent with our previous study (Wang et al., 2015), but NP(saMyoD)/BAMG is not toxic accroding to the China national standards for medical devices evaluation (GB/T16886.5-2017), because the inhibition ratio is lower than 20%. The cell morphology is normal under the microscope (Figure 5C). SF(VEGF)/BAMG is also not toxic to ADSCs and even promotes the growth of ADSC compared to control group (Figure 5B).

A rat model of bladder enlargement was prepared and different scaffolds were implanted. After 10 weeks, the animals were executed to test the repair function of ADSC-NP(saMyoD)/BAMG and ADSC-NP(saMyoD)/SF(VEGF)/BAMG. First, we did not found obvious deterioration in liver and renal of these groups. After detecting the stone situation of these groups, the data demonstrated the composite group showed the least stones than other groups (Figure 6A). Then, Bladder function was evaluated, the data showed that the bladder capacities of the ADSC-NP(saMyoD)/SF(VEGF)/BAMG groups were significantly higher than sham group (Figure 6B). ADSC-NP(saMyoD)/BAMG also appeared to play a role in bladder repair, but there was no statistical difference compared with sham group (Figure 6B). The gradually increasing bladder capacities indicated that MyoD and VEGF might improve the balance between the degradability and generation of the scaffold. The bladder compliance of three treated groups were gradually increased and the compliance of ADSC-NP(saMyoD)/SF(VEGF)/BAMG group was the max (Figure 6C).

The HE staining examination of bladder tissue slices in the ADSC-NP(saMyoD)/BAMG and ADSC-NP(saMyoD)/SF(VEGF)/BAMG groups illustrated in-growth of connective tissue into both the marginal and central regions of the original implantation sites. In addition, the entire urothelium regenerated well and more densely and regularly arranged smooth muscle fibers were detected in the ADSC-NP(saMyoD)/SF(VEGF)/BAMG groups (Figure 6D). Immunohistochemistry were performed to analyze the regeneration of bladder wall components. The results demonstrated that, comparing with ADSC-NP(saMyoD)/BAMG, there were more α-SMA and VEGF positive signal in ADSC-NP(saMyoD)/SF(VEGF)/BAMG group (Figures 6E, F). Thus, ADSC-NP(saMyoD)/SF(VEGF)/BAMG may better promote the growth of smooth muscle and blood vessels formation. At last, the masson dye results showed the sham group showed more collagen than ADSC-NP(saMyoD)/BAMG group, and the ADSC-NP(saMyoD)/SF(VEGF)/BAMG group just had little fibrosis, indicating that the composite scaffolds have enough ability to antifibrotic and with great safety (Figure 6G).