3.1. Synthesis of biodegradable polyurethane
The polymer was synthesized via a two-step polymerization process. PCL-diol (number average molecular weight of 2000, Sigma) in toluene (anhydrous, 99.8%, Sigma) was added to a flask under an argon atmosphere; thereafter hexamethylene diisocyanate (HDI, ≥ 99.0%, Sigma) and tin(II) 2-ethyl hexanoate (SnOct2, 97%, Sigma) were added to the flask. After 2 h of stirring at 90 ℃, (3-Aminopropyl)triethoxysilane (APTES, ≥ 98%, Sigma-Aldrich) was added. The molar feed ratio of the PCL-diol/HDI/APTES was 1:2:2. After stirring for 1 h at 90°C, the flask was maintained at room temperature (RT) overnight. The product was purified via precipitation, in methanol (99.8%), and vacuum-dried overnight. The molecular weights (Mn and Mw) of the polymers were measured using gel permeation chromatography (GPC). The dn/dc of the polymer in CHCl3 was 0.0759. A Bruker Ascend™ 400 proton nuclear magnetic resonance spectrometer (1H NMR) at 400 MHz was used to determine the structure of the polymer.
3.2. Synthesis of inorganic–organic hybrids and sample preparation
The biodegradable polyurethane was dissolved in tetrahydrofuran (THF, ≥ 99.9%, Sigma) and TEOS (98%, Sigma) was hydrolyzed in the following molar ratio: TEOS:water:HCL = 1:4:0.6. The polymer was mixed with the hydrolyzed TEOS at a weight ratio of 70–100 wt. % (with 10 wt. % increments). The mixture was stirred vigorously and 1.9 g of sodium chloride (NaCl, ≥ 99.5%, Biosesang) was gradually added to the 1 ml mixture before gelation. The NaCl particles were collected using a 300–500 µm sieve. The mixture/salt paste was placed in a mold and stored overnight, at 40°C. After air drying at RT for 1 h, the NaCl was leached by soaking in deionized water (DW) for 5 days. For all the tests, except the compression test of the 0–40 wt. % silica hybrids, the samples were cut into cylindrical shapes that were 8.66 mm in diameter and 1 to 2 mm in thickness.
3.3. Hybrid characterization
TGA (TA Instruments Q50) was used to measure the weight percentages of the silica and polymer in the hybrids; the samples were heated at a rate of 20°C/min from 0 to 150°C in N2. The transmittance spectra of the hybrids were measured using a Fourier transform infrared (FTIR) spectrometer (Nicolet iS20 FTIR spectrometer). In the polymer, the ester stretching and aliphatic ether peaks appeared at 1210–1163 cm− 1 and 1150–1085 cm− 1. In the hydrolyzed TEOS, the Si-O-Si bond appeared at 1094 cm− 1 and 798 cm− 1. The gelation time of the hybrids was determined by crossing the storage modulus (G’) and loss modulus (G’) using a rheometer (MSC 102, Anton Paar, DE). All the samples were measured immediately after mixing the hydrolyzed TEOS with the polymer solution. The measurements were conducted at an oscillating frequency of 1 Hz and 2% of the oscillating strain, at 25 ℃.
3.4. Experimental analysis of the annealing effect
3.4.1. Crystalline structure characterizations
Differential scanning calorimetry (DSC) measurements was performed to measure the enthalpy change (ΔΗ) of the hybrids using on a Q10 differential scanning calorimeter. The hybrids were heated at a rate of 10°C/min from 0 to 150°C in N2. The enthalpy change (\(\varDelta {H}_{m})\) was measured for the silica-polymer ratio and the scaffold morphology. The enthalpy of fusion of the 100 % cystalline PCL (\(\varDelta {H}_{m}^{0})\) was 139.5 J/g. The percent crystallinity of the samples was calculated as follows:
%crystallinity =\(\frac{\varDelta {H}_{m}}{\varDelta {H}_{m}^{0}}\times 100\)
3.4.2. Accelerated degradation test
Accelerated degradation was assessed at 5-time points ( 8, 24, 48, 72, and 168 h). The hybrids were immersed in 1 M sodium hydroxide (NaOH, Junsei Chemical Co., Ltd.) at 25°C, rinsed with DW three times, and dried overnight under vacuum. The percentage of mass loss was then measured.
3.4.3. Porous structure evaluation
Cross-sectional images of the hybrids before and after annealing were obtained using SEM after the hybrids were cross-sectioned and sputtered with gold. The pore sizes of the hybrids were measured from the SEM images using ImageJ. The interstitial porosity was measured using an AutoPore IV 9500 Series.
3.4.4. Mechanical properties
The compression tests were performed using a 5966 universal testing machine (5966 UTM). The compression speed was 1 mm/min, and the samples were compressed to 60% of the original sample height, before and after annealing.
BPHS30 was immersed in 1X PBS and incubated at 37°C in an orbital shaker with an rpm of 50, for 2 or 4 weeks. After immersion, the samples were washed three times with DW, to remove the salt from the PBS solution, and dried overnight in a vacuum oven. Compression tests were performed as described above.
3.4.5. Silica ion released from hybrid scaffolds
The silica ions released from BPHS30 were measured using Inductively coupled plasma-optical emission spectroscopy (ICP-OES, iCAP 6500 Duo). A standard curve of the silica ions was prepared at 0.5, 1, 5, and 10 ppm. The hybrid scaffolds (30 mg) were dissolved in 20 ml of PBS (1X, PH 7) and incubated in a shaking incubator for 1, 2, and 4 weeks (at 37°C and 50 rpm). The solution was mixed with 2 M nitric acid in a 1:4 volume ratio. The silica content was measured at a wavelength of 212.412 nm.
3.5. In vitro test
3.5.1. Cell culture and cell seeding
Mouse pre-osteogenic precursor cells (MC3T3-E1, ATCC) were harvested in α-MEM (Gibco) that was supplemented with 10% v/v FBS (HyClone) and 5 ng·mL− 1 of penicillin/streptomycin. The hybrids were sterilized in 70% ethanol for 5 min and washed with α-MEM for 30 min before cell seeding. The MC3T3-E1 cells were seeded at a density of 250,000 cells, on the hybrids, and cultured in a basal media for 7 days and thereafter on an osteogenic differentiation media (α-MEM supplemented with 10 v/v % FBS, 5 ng·mL− 1 penicillin/streptomycin, 5 µg·mL− 1 L-ascorbic acid (Sigma), and 2 mM β-glycerophosphate disodium salt (Sigma)). Ascorbic acid and β-glycerophosphate disodium salt-induced osteogenic differentiation of the MC3T3-E1 cells.
3.5.2. Cytotoxicity test
The MC3T3-E1 cells were used to perform in vitro cytotoxicity tests in accordance with ISO 10993-5 and ISO 10993-12 protocols. The hybrids were dissolved in α-MEM at 37°C for 72 h and thereafter sterilized using a 0.2 µm filter. The negative control for the toxicity test was a high-density polyethylene (PE) film (Hatano Research Institute, FDSC) and the positive control was polyurethane (PU) containing 0.1% zinc diethyldithiocarbamate (ZDEC; Hatano Research Institute, FDSC), which has cytotoxicity. The MC3T3-E1 cells were cultured in 96 well plates, in α-MEM, for 24 h. The medium was changed with dissolution products and incubated for 24 h. The cell viability was measured using a CCK-8 (DOjinDO) cell viability assay kit; 10% CCK-8 of the total volume was added to each well, and the absorbance was measured at 450 nm, using Spectramax 384.
3.5.3. Cell attachment test
An inorganic–organic mixture (sol–state hybrid) was coated onto a glass substrate and dried in an oven, at 40°C, to perform the cell attachment test. The MC3T3-E1 cells were seeded on the hybrid-coated glass substrates at a density of 106 cells/ml and incubated for 3 days in basal α-MEM. The samples were washed in PBS and thereafter 4% of paraformaldehyde (PFA) was added to fix the cells, at 4 ℃, overnight, before the immunohistochemical (IHC) staining. The cells were then treated with 0.5% Triton X-100 in PBS for 20 min, to permeabilize the cell membranes. After permeation, they were blocked with 10 mg/ml BSA in PBS, at 4°C, for 30 min. All the samples were stained with Alexa Fluor™ 594 Phalloidin (1:200 dilution, 10 mg/ml BSA in PBS) and DAPI nuclei staining (0.1 µg/mL in PBS). The labeled F-actin and cell nuclei were obtained using a Zeiss LSM 700 confocal microscope (Carl Zeiss).
3.5.4. DNA quantification and mineralization
The MC3T3-E1 cells were harvested on the hybrids for 1, 7, and 28 days, to measure the DNA concentration, as described in the “Cell culture and cell seeding” section above. The samples were washed in 1X PBS and thereafter left in 0.1% v/v Triton-X; freeze-thaw cycles were repeated for cell lysis. DNA from the cell lysate was quantified using Hoechst staining 33258 (≥ 98%, Sigma), and calf thymus DNA was used as the DNA standard.
Mineralization of the hybrids occurred after 1, 7, and 28 d, and the MC3T3-E1 cells were harvested on the hybrids for 1, 7, and 28 days, as described in the “Cell culture and cell seeding” section above. The scaffolds were then dried in hexamethyldisiloxane (HMDS, Sigma) and cross-sectioned. The cross-sectioned hybrids were sputtered with platinum, and SEM images were obtained.
3.5.5. qRT PCR for mRNA quantification
The hybrid scaffolds were analyzed for the expression of two osteogenic markers, RUNX2 and COL1A1, in the MC3T3-E1 pre-osteoblast cells, using qPCR. All the expression levels were normalized to the housekeeping gene 18S. RNA from the cells on the hybrid scaffold was extracted using an RNeasy Mini Kit (Qiagen) and converted to cDNA using the Maxime RT Premix (iNtRON). The primer sequences are listed in Table 4. Real-time PCT was performed using the SYBR Premix Ex Taq (Applied Biosystems 7500 real-time cyclers).
Table 4
Forward and reverse primer sequences for osteogenic differentiation.
| Forward | Reverse |
---|
18S | 5'-GTAACCCGTTGAACC–3' | 5'-CCATCCAATCGGTAGCG-3' |
ALPL | 5'-GATCGGGACTGGTACTCGGATAA-3' | 5'-CACATCAGTTCTGTTCTTCGGGTAC-3' |
COL1A1 | 5'-TTCTCCTGGTAAAGATGGTGC-3' | 5'-GGACCAGCATCACCTTAACA-3' |
RUNX2 | 5'-CCGCCTCAGTGATTTAGGGC-3' | 5'-GGGTCTGTAATCTGACTCTGTCC-3' |
3.6. In vivo testing
3.6.1. Animal surgical procedure
All the animals were treated according to the protocols of the Institutional Animal Care and Use Committee at the Biomedical Research Institute of the Korea Institute of Science and Technology. The distal femoral defect model was adapted from Tielinen et al. 52. Sprague-Dawley rats (12-week-old, male, 350–400 g; DBL) were used in three different experimental groups (n = 5 animals/group): control (defect without scaffold), BPHS0, and BPHS30. Inflammation and bone regeneration was assessed at two time points (2 and 4 weeks post-surgery). The animals were anesthetized using isoflurane. The lateral epicondyles of the distal femur were shaved and disinfected using povidone-iodine. After incision of the skin and muscles, cylindrical defects were created with a 3 mm diameter and 4 mm depth, in the lateral condyles. The defects were washed with 1X PBS and filled with the samples or left empty. (BPHS0 and BPHS30 were pre-annealed at 60°C for 2 h to prevent the sample missing from shrinkage). The incision was closed using a 3 − 0 black silk suture. At 2 and 4 weeks post-surgery, the animals were sacrificed and the distal femurs were explanted. The femurs were fixed in 10% (v/v) formalin for three days.
3.6.2. Micro-computed tomography for the evaluation of bone mineralization
The explanted specimens were analyzed using micro-CT (Skyscan 1172). The X-ray source was 56 KV, 179 µA, 10 W to set the transmission of samples to 30%. An aluminum filter (0.5 µm) was used to scan the femurs (high-density tissues). The newly formed bone volume (BV) and total bone volume (TV) of the samples were quantified using the CTAn software (Skyscan Ltd). Thereafter, 3D images were generated from the scanned images using the CTvox software.
3.6.3. Histological and immunofluorescent analyses
The specimens were decalcified using a decalcification solution (Sigma, USA) for 36 h, followed by rinsing with tap water. Thereafter, the samples were dehydrated with increasing concentrations of ethanol (70, 80, 90, 95, and 100%) for 1 h each. The samples were embedded in paraffin and sectioned into 7 µm-thick slices for histological and fluorescence staining. The slides for histological analysis were stained with H&E and MT after deparaffinization and rehydration. The collagen density was defined as the ratio of blue intensity to the total area, using the ImageJ software (n = 3 in each group). To evaluate bone regeneration in the defects, immunofluorescence staining for osteogenesis, phagocytic activity, vasculogenesis, and angiogenesis was performed. For osteogenesis evaluation, the cross-sectioned slices were stained with antibodies against type I collagen (ab34710, 1:200; Abcam) and osteocalcin (sc-390877 FITC, Santa Cruz Biotechnology, USA, 1:50). Alexa Fluor 594 (anti-rabbit IgG) was used as the secondary antibody against type I collagen. The nuclei were counterstained with DAPI (Microcular Probes, USA). The positive areas of the stained sections in three random fields were quantified using the ImageJ software (n = 3 in each group). To evaluate phagocytic activity, the samples were stained with antibodies against CD68 (ab955, Abcam, UK, 1:60) and CD206 (sc-34577, Santa Cruz Biotechnology, 1:100). Alexa Fluor 594 (anti-mouse IgG) and Alexa Fluor 488 (anti-goat IgG) were used as the secondary antibodies. The CD68 and CD206 positive areas in the three fields were quantified using the ImageJ software (n = 3 in each group). The CD206+/CD68+ ratio indicates the proportion of M2 macrophages. To verify vasculogenesis, the primary antibodies used were the monoclonal mouse anti-human α-smooth muscle actin antibody (α-SMA, sc-53142 AF594, Santa Cruz Biotechnology, 1:50) for staining the α-smooth muscle cells (SMCs), and the polyclonal rabbit anti-human von Willebrand factor antibody (vWF, sc-365712 FITC, Santa Cruz Biotechnology, 1:50) for staining the endothelial cells. To evaluate vascular cell recruitment in the defect areas, the α-SMA- and vWF-positive areas in the three fields were quantified using the ImageJ software (n = 3 in each group). The vessel maturation index (%) was calculated using the α-SMA-positive/vWF-positive area ratio.
3.7. Statistical analysis
The statistical analysis was performed using a one-way ANOVA and Tukey’s test, for multiple comparisons. The results are shown as the mean ± standard error of the mean (SEM) and were considered statistically significant at *p < 0.05 and **p < 0.01.