Preparation of PDA NP-incorporated membranes
First, 2 mg/mL of a dopamine solution (Sigma, USA) was fabricated by dissolving it in 10 mM of a Tris-HCl solution (pH = 8.5) with stirring for 1 day at 60 °C. Then, the reaction was stopped by centrifugation at 5000 rpm, followed by drying at 60 °C to obtain PDA NP powders.
PCL granules (Mw ≈ 80,000 g/mol, Sigma-Aldrich) with different amounts of PDA NPs (mass ratio of PCL:PDA NPs = 100:1, 100:2, 100:5, and 100:10) were dissolved in hexafluoroisopropanol (HFIP) through ultrasonic agitation overnight to achieve a concentration of 8% (w/v). After that, the prepolymer solution was transferred to a syringe with a 23 G blunt needle. The electrospinning of fibrous membranes was conducted by a conventional electrospinning setup (SS-2535, YongKang Technology, China) according to our previous work23. The resulting membranes were collected on Al films and then vacuum-dried to remove the residual organic solvent. According to the different concentrations of PDA NPs (1, 2, 5, and 10 wt%), these membranes were denoted as 1% PDA/PCL, 2% PDA/PCL, 5% PDA/PCL, and 10% PDA/PCL.
Microstructural analysis of fibrous membranes was investigated with a scanning electron microscope (SEM, JSM-6701F, JEOL, Japan). The chemical analysis of the engineered fibrous membranes was identified through Raman spectroscopy and X-ray photoelectron spectroscopy (XPS, Kratos Analytical, UK). The hydrophilicity of the membranes was tested through contact angle goniometry (SL200B, Kino, USA).
Formation of bone-like apatite
The 1× synthetic body fluid (SBF) (Table S1) was fabricated by dissolving NaCl, NaHCO3, KCl, K2HPO4•3H2O, MgCl2•6H2O, CaCl2, and Na2SO4 in order in deionized water and buffering to pH 7.4 using (CH2OH)3CNH2 and 1 mol/L HCl at 37 °C. The engineered fibrous membranes were soaked in SBF at 37 °C to examine the in vitro bioactivity. After 7 and 14 days, samples were taken away from SBF and gently washed. The chemical constitution and microstructure of nodules were determined by XPS and SEM.
hMSC culture and seeding
Circular samples (1.9 cm2) were cut from the fibrous membranes and placed into 24-well plates. Afterwards, the samples were disinfected using 75% ethanol for 40 min followed by UV light for 30 min and were then washed using sterile PBS before cell culture. The hMSCs were purchased from Sciencell Research Laboratories (USA) and were extracted from healthy human bone marrow. They were expanded in standard growth medium consisting of high-glucose DMEM (HyClone, USA), complemented with 10% fetal bovine serum (HyClone, China), and 1% penicillin–streptomycin (P-S) and were then incubated under a 5% CO2 atmosphere. Cells before passage five were used, and the trypsinized cells were seeded on the microfibers at 2 × 104 cells/cm2. After 5 days of incubation, the medium was changed to an osteoinductive solution composed of low-glucose DMEM containing 10% FBS, 1% P-S, 50 μg/mL ascorbic acid, 10 mM sodium β-glycerophosphate, and 100 nM dexamethasone (Sigma). The medium was refreshed every 2–3 days.
Cytocompatibility of membranes
Early attachment assay
The early cellular attachment on the engineered fibrous membranes was conducted using SEM, F-actin fluorescence staining, and the Cell Counting Kit-8 assay (CCK-8, Dojindo, Japan) after incubating for 6 and 12 h. The morphologies of hMSCs on the fibrous membranes were observed using SEM. The substrates with cells were fixed in 2.5% glutaraldehyde for 30 min and then dehydrated with graded concentrations of ethanol. Finally, the dehydrated samples were dried in a vacuum dryer and visualized under SEM. The rinsed hMSCs were fixed with 4% paraformaldehyde for 0.5 h and permeabilized with 0.1% Triton X-100. Then, 5 µg/mL of FITC-phalloidin (Sigma) was added to stain the hMSCs for 0.5 h. After rinsing with PBS, the samples were reacted for 8 min with 15 µg/mL DAPI (Sigma). Finally, the fluorescence signal was captured under a confocal laser scanning microscope (CLSM). At the desired time, CCK-8 was introduced into each well at a 10% proportion for 3 h in the dark. The optical density (O.D.) of the supernatant was measured by a microplate reader (Model 680, Bio-Rad, USA) according to the manufacturer’s instructions.
Cell proliferation assay
The CCK-8 kit was also used to measure cell proliferation for 6 days, and the cellular morphologies on the fibrous surfaces at 3 days were observed using SEM.
Osteogenic potential evaluation
Alkaline phosphatase activity and mineralization
The intracellular alkaline phosphatase activity of the hMSCs was evaluated with the alkaline phosphatase (ALP) assay kit (Nanjing Jiancheng Bioengineering Institute, China) following the manufacturer’s specification. The results were normalized to the total protein amount obtained from the MicroBCA protein assay kit (Thermo, USA). Furthermore, the ALP distribution on fibrous membranes was viewed using a BCIP/NBT ALP color development kit (Beijing ComWin Biotech, China).
Mineralized matrix formation was examined after 14 and 21 days of osteoinductive culture by staining with Alizarin Red S (ARS, Sigma). The cell layers were fixed and soaked in the ARS solution (2 w/v%, pH = 4.2) for 0.5 h. After rinsing using deionized water, the images were captured by a scanner. To further quantify mineralization, the ARS-stained substrates were immersed in a hexadecylpyridinium chloride solution (100 mM, Sigma) for 12 h to dissolve the calcified matrix. The O.D. of the solution was measured at 570 nm using a microplate reader.
At 2 and 3 weeks of osteoinduction, the total mRNA was isolated from the cells by TRIzol (Invitrogen, USA) treatment and converted into cDNA using the RevertAid First Strand cDNA Synthesis Kit (Thermo, USA) according to the manufacturer’s instructions. RT-PCR analysis was performed on an ABI 7500 real-time PCR machine (Applied Biosystems, USA) using SYBR Green (Roche, USA). Each specimen was run in triplicate, and glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was assigned as an endogenous control. The primers (5′-3′) used in this study are listed in Table S2.
After fixation, permeabilization, and treatment with a 1% BSA/PBST solution, the hMSCs were then exposed to primary antibodies against ALP (1:400, CST), RunX-2 (1:400, CST), OCN (1:400, CST), and Col1a1 (1:400, CST) at 4 °C for 24 h, rinsed with PBS three times, and incubated with FITC-488 goat anti-rabbit (1:100, ZSGB-BIO, Beijing, China) or TRITC-543 goat anti-mouse (1:100, ZSGB-BIO) secondary antibodies for 60 min. The cells were counterstained with DAPI (10 µg/mL) for nucleus staining and imaged by CLSM.
In vivo bone formation tests
Mouse skull defect model
The animal experiment was approved by the Institutional Animal Care and Use Committee at Peking University (approval no.: AAIS-WeiSC-3). The effect of fibrous membranes on in vivo bone regeneration was investigated using 6- to 8-week-old CD-1 mice. Before the operation, the mice were anesthetized by pentobarbital sodium (3%, 40 mg/kg) through intraperitoneal injection. Two critical-sized defects (4 mm diameter) were created on both sides of the skull using a dental trephine24. The right-side cavity was covered with microfibers, while the left one remained without any fiber implantation and served as the negative control. The animals were divided into three groups based on the implanted membranes: Defect only (n = 6), pure PCL microfibers (n = 6), and 2% PDA/PCL fibrous membranes (n = 6). To label the mineralization process, the mice were injected intraperitoneally with calcein (50 mg/kg, Sigma-Aldrich) and ARS (50 mg/kg) at 1 and 2 weeks, respectively. These mice were euthanized at a scheduled time. After sacrifice, the calvaria was harvested and fixed in 10% formalin solution for further evaluation.
The samples were measured by micro-CT scanning (18 μm voxel, Scanco Medical vivaCT40, Switzerland) to reconstruct 3D images. Afterwards, the trabecular bone volume/tissue volume (BT/TV), trabecular number (Tb. N), trabecular thickness (Tb. Th), and trabecular spacing (Tb. Sp) were calculated using the built-in program.
To obtain decalcified paraffin sections, the fixed samples were exposed to a 15% EDTA solution for 3 weeks. After dehydration, the samples were embedded in paraffin and cut into 10 μm longitudinal sections for H&E, Masson trichrome, toluidine blue and immunohistochemical OCN staining via an optical microscope (TE200-E, Nikon Eclipse, Japan). The fluorochrome-labeled samples were dehydrated using different concentrations of ethanol and embedded in methyl methacrylate. Moreover, transverse sections (10 μm) were cut to observe the fluorochrome labels under CLSM.
For quantitative experiments, six samples of each group were tested to provide the mean and standard deviation. One-way ANOVA and Tukey’s post hoc tests were applied to determine the significant differences among the groups.