Poly(ethylene glycol) diacrylate (PEGDA), hydroxyapatite (HAp),LiBr,APS, TEMED, FITC, and DAPI were purchased from Sigma-Aldrich (USA). All other chemicals were purchased from Sigma-Aldrich unless otherwise indicated.RUNX2, OCN, OPG, and BSP antibodies were purchasedfrom SantaCruz (USA). The raw silks were obtained from SimatechCo. (JiangSu, China).
2.2 Scaffold fabrication
Cocoons from the mulberry silkworm Bombyxmori were boiled for 30 min in 0.02 M Na2CO3 solution and then rinsed thoroughlywith double-distilled water to extract the glue-like sericin proteins. Next, the extractedsilk proteins were dried briefly at 37°Cfor 1h. The resulting silk fibroin (SF) was thendissolved in 9.3 M LiBr solution at 60°C for 4 h. The SF solution was dialyzed against distilledwater with a dialysis membrane (12 kDa) for 3 dayswith a continuousrenewalof thedistilledwater. The dialysate was centrifuged (5,000 rpm; 10 minat 4°C) to removethe impurities and aggregates formed duringdialysis22. The final concentration of theSF aqueous solutionwas 7–8% (wt/vol), which was determined by weighingdried films at 60°C.
Next, 60 mg PEGDA (molecular weight, 700kDa) was dissolved in 500 μLof distilled water to form a solution with a predetermined concentration (12% wt/vol) at room temperature. The solution was cooled to 0°C, followed by the addition ofSF (38.6 μg), four different amounts of HAp in separate tubes(25, 50, 75, or 100 mg), ammonium persulfate (APS; 20% aqueous solution, 15 μL), and tetramethylethylenediamine (TEMED; 3 μL). In allfourgroups, the solution waspoured into a polypropylene tube (diameter, 8 mm; height, 20 mm), which was kept close tothe surface offrozen isopropyl alcohol (-80°C)23,24. After completely freezing each solution, the solidified samples were transferred into a freezer to complete the polymerization process at -20°C for 12 h. Then, dry unidirectional cryogelswere prepared using a freeze dryer for 24 h until they reached a constant weight. After this, thecryogelswere immersed in excess distilled water atambient temperature for 48 h. The water wasreplaced every 4 h to remove the unreacted materials and obtain the finalcryogel.
2.3 Scanning electron microscopy (SEM)
SEM (Hitachi S-3000N) was performed to determine the scaffold pore structure and surface topography. In brief, scaffolds were washed in phosphate-buffered saline (PBS), fixed in 2% glutaraldehyde overnight,washed in alcohol (with a strength up to 70%), and then freeze-dried overnight in a lyophilizer. To prepare the samples for SEM, the freeze-dried scaffold samples were coated with gold for 10 min in advance, and then cut in both the parallel and perpendicular directions to the freezing direction.
2.4 Fluorescein isothiocyanate (FITC) staining
To observe the morphology of the scaffold during swelling, FITC (a fluorescent probe) was selected to stain thecryogel samples: FITC was dissolved in NaOH aqueous solution to form a solution (5 mg/L). The resultingcryogel samples were cut into slices with a thickness of approximately 0.5 mm and immersed in the FITC solution. After 30 min, the stained samples were washed with PBS until no dye was detected in the discarded PBS.
2.5 Scaffold characterization
2.5.1 Fourier-transform infrared (FTIR) spectrometry
Fourier-transform infrared (FTIR) spectra ofPEG/SF/HAp with different proportionsof HApwere characterized with a spectrometer (Bruker VERTEX 33, Germany). Peaks at different wavenumbers correspond to particular linkages and functional groups; therefore, spectra from 4,000 to 400 cm-1were recorded. The presence of different polymers in the cryogel blend, possible bonds, and crosslinking were confirmed by FTIR. The FTIR spectra of pure PEG, SF, and HApwere measured as controls.
2.5.2 Thermogravimetric analysis (TGA)
The thermal stability ofthe PEG/SF/HAp scaffoldswas examined usingthermogravimetric analysis (TGA). To determine the weight loss of the PEG/SF/HAp scaffolds at different temperatures, TGA measurements were performed up to 1000°C (at a heating rate was 10 °C/min) in a nitrogen atmosphere using theSTA449F5 instrument (Netzsch, Germany).
2.5.3 X-ray diffraction (XRD)
The crystal structure was determined by X-ray diffraction (XRD)using an Empyreandiffractometer system (PANalytical) and Cu Kα radiation (with a wavelength of 1.5406 A(。)). Data were collected for 2θ values of 20–90°, with a step width of 0.02° and a counting time of 1 s per step.
2.6 Mechanical properties of cryogels
Using a dynamic mechanical analyzer (Bose 3220-AT Series II), Young’s modulus (compressive elastic modulus) of the cryogelswas measured parallel to the freezing direction. Young’s modulus (E, kPa) represents the slope of a straightline fitted forthe stress-strain curves in the range from 5% to 15% strain. The E was calculated based on the following equation: E=Δσ/Δε, whereΔσ is the tensile stress, and Δε is the tensile strain. The fully swollen(in PBS) cryogel columns were cut into cubes with a length of approximately 8mm. Theywere then saturated with 0.1 M PBS (pH 7.4) and subjected to unconfined compression. The saturated samples were compressed to as much as 50% of their original length by placing them between two arms of the dynamic mechanical analyzer (DMA) at room temperature. The speed of compression between the two parallel plates was 1mm/min.
2.7 Swelling ratio (SR) of the cryogels
Dry cryogels, which had been prepared using a freeze dryer for 24 h (until they reached a constant weight),were incubated in distilled water, and the weight of the swollen cryogelswas measured atregular time intervals. The SR of the cryogels was calculated based on the following equation: SR=WS/WD, where WS is the weight of the swollen cryogel,and WD is the original weight of the dry cryogel.
2.8 Rat BMSC isolation and seeding
All animal experiments were approved by the Southern Medical University Animal Ethics Committee, and were performed in compliance with the regulations for the use of rat BMSCs. Fresh bone marrow aspirates were obtained from the upper femurs and tibias of 4-week-old Sprague Dawley (SD) rats, and BMSCs were isolated and characterized as previously described25,26. Briefly, the BMSCs were expanded in growth medium (Dulbecco’s Modified Eagle Medium (DMEM)/F12 (Gibco, USA) supplemented with 10% fetal bovine serum (FBS,Gibco, USA) and 1% penicillin-streptomycin (Gibco, USA). The BMSCs were cultured up to the third passage and then seeded on the scaffolds (with 3×107 cells/mL of scaffold)in growth medium without any osteogenic factors, for 21 days. A 40 μL aliquot of BMSCs suspension was pipetted onto the scaffolds and allowed to percolate through. For 2 h,the scaffolds were flipped 180° every 20 min and 10 μL ofgrowth medium was added every 20 min to prevent the cells from drying out.
2.9 Alkaline phosphatase (ALP) staining
Rat BMSCs (1×104 cells/cm2) were seeded onto the scaffolds and cultured in growth medium. After 7 and 14 days, ALP activity was assayed using a 5-bromo-4-chloro-3-indolyl phosphate (BCIP)/nitro blue tetrazolium (NBT) ALP color development kit (Beyotime Institute of Biotechnology, China). The ALP-positive area was measured usingImageJ softwarefor three randomlyselected fields under an optical microscope (Olympus BX51, Japan).
2.10 Immunofluorescence staining
After being cultured on the scaffolds for 7, 14, and 21 days, the cellswere washed three times with DMEM and fixed in 4% paraformaldehyde for 10min at 37°C. Cells were then washed with PBS three times, and treated with 0.1% (v/v) Triton X-100 in PBS at 4°C for 10min to increase the permeability of the cell membranes. After three additional washes with PBS, the samples were incubated in 2% BSA in PBS at 37°C for 30 min to block non-specific interactions. Thecellswere then incubated overnight at 4°C with a rabbit monoclonal antibody against RUNX2 (1:200, mouse) orOCN (1:200, mouse)(Santa Cruz, USA). After being washed twice in PBS, they were further stained with a fluorescent-labeled secondary antibody (1:200) (Beyotime, China) and 4’,6-diamidino-2-phenylindole (DAPI, 1:500) at room temperature for 0.5 h, followed by three washes with PBS. The cellswere observed using a confocal laser scanning microscope (C2, Nikon, Japan). The images obtained were further analyzed usingImageJ software (National Institutes of Health, USA).
2.11 RNA isolation and real-time quantitative PCR (RT-qPCR)
The mRNA expression levels of important osteogenesis-related genes, including OCN, OPG, BSP, and RUNX2, were determined by RT-qPCRat 7, 14, and 21 days after culturing on the scaffolds. Total RNA was isolated and purified using an RNA extraction kit (Tiangen, China) according to the manufacturer’s instructions. First-strand cDNAs were generated by reverse transcription of 1 mg total RNA with a RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific). RT-qPCR was performed in triplicate with an ABI StepOnePlusSystem (Applied Biosystems, USA) and a fluorescence-labeled SYBR Green/ROX qPCR Master Mix Kit (Thermo Scientific). A melting curve analysis was used to confirm the PCR specificity, and the datawere analyzed using SOS 2.1 software (Applied Biosystems). The relative expression levels were analyzed using the 2-ΔΔCt method andnormalizedto those of the housekeeping gene glyceraldehyde 3-phosphate dehydrogenase (GAPDH).Calibration was performedusingBMSC growth on the four PEG/SF/HAp scaffolds at 7, 14, and 21days. Theforward and reverse primer sequences for RT-qPCR of each gene were as follows:
2.12Application of BMSC-laden PEG/SF/HAp scaffolds for bone regeneration in vivo
The BMSCs-laden PEG/SF/HAp scaffolds were investigated in a rat calvarial defect model.Two groups were designed as follows: namely unladen (Group I) and laden (Group II) with BMSCs of PEG/SF/HAp scaffolds implanted into rats skull defects. Autograft bone was implanted into the calvarial defect as the positive control (n = 6), the calvarial defects were created without any treatment as the negative control (n = 6). PEG/SF/HAp scaffolds with different magnitudes of stiffness (laden with or without BMSCs) were implanted into parietal bone defects (n = 6) as the experimental group.Seventy-two SD rats (weight range 180–220 g) were anesthetized (0.2mL/100g b.w.chloral hydrate). Two 5 mm diameter circular acute calvarial bone defects were created in each specimen, scalp skin was incised, and the periosteum was removed to visualize the skull. A 5 mm diameter cylindrical defect was created on each side of the parietal bone (without any damage to the dura mater) using a dental bur, under irrigation with sterile saline solution (Fig. 5A-a). The circular bone plug was removed gently, every two different PEG/SF/HAp scaffolds (n=3) laden with or without BMSCs was placed on each side of the rat skull, each rat being its own control (Fig. 5A-b). Calvarial defects created in the skulls were left empty as negative controls and autograftswerepositive controls (n=3). Wound closure was achieved by a two-layer suturing (periosteum, skin) using absorbable sutures. Immediate post-operative care included intraperitoneal injection of penicillin to prevent infection and buprenorphine (0.02mg/kg b.w.) for analgesia. The animals were fed with a standard diet and housed individually under standard conditions. Wound healing progressed without any sign of infection, material exposure, or other complication.
2.13 Micro-CT examination of samples
For bone regeneration analysis, calvarial specimens were obtained from euthanized rats and scanned using a micro-CT to observethe morphological features of the defect site. The micro-CT device was set at 85kV and 135mA, and a tomographic rotation of 180°. The results were reconstructed with Mimics 20.0 software.
2.14 Biomechanical analysis of regenerated tissue
At 8and 12 weekspost-operation,samples fromthe HAp(25, 50, 75, and 100 mg) groups, blank control group, and autologous bone graft group were collected. An ElectroForce 3510 Bose instrument was used to conduct a biomechanical test on the defect site, and astress-strain curve was obtained. The loading speed of the biomechanical instrument was set at 3 mm/min, and the maximum load on theregeneration tissue wasdefined according to the stress-strain curve of the vertices.
2.15 Histological analysis
Regenerated tissue samples obtained from the defect were washed with PBS and then fixed in 4% phosphate-buffered formalin (pH=7.4) for 48 hours. Decalcification was performed in 20% EDTA for 4 weeks, with the EDTA solution changed every 3 days. The tissue samples were dehydrated through a graded series of ethanol treatments and embedded in paraffin. Finally, bone samples weresectioned into5μm thick sections. To analyze bone regeneration, hematoxylin and eosin (H&E) and Masson trichrome staining were performed.
2.16 Statistical analysis
At least three independent experiments were carried out. Results are reported as mean ± standard deviation (SD), and the means were compared between groups using Student’s t-tests. The level of statistical significance was set at P < 0.05.