MaterialsPolyvinyl alcohol (PVA, Mw = 146000–186000, Sigma Aldrich) and potassium tert-butoxide (C4H9OK, ≥ 98%, Shanghai Macklin Biochemical Co., Ltd.) were used as received. Dimethyl sulfoxide (DMSO, ≥ 99.0%), glutaraldehyde (25 vol%), hydrochloric acid (36.5 wt%), sodium hydroxide, sodium ethoxide, sodium methoxide, sodium trifluoroethanol, N-Methylpyrrolidone (NMP) and N,N-Dimethylacetamide (DMAc) were purchased from Sinopharm Chemical Reagent Co., Ltd.Fabrication of PVA-OH hydrogelPVA (3 g) was dissolved in DMSO (12 g), forming a solution with mass concentration of 20%. A certain amount of C4H9OK (0.075, 0.15, 0.3, 0.45 g) was added into the PVA solution and stirred at 90 °C for 2 h. Then, the solutions were cooled down at a certain temperature (75, 50, 25, 4 °C) for 12 h to become organagels. The organogels were immersed in ice-water mixture for 24 h to thoroughly remove DMSO and C4H9OK, forming PVA-OH hydrogels. PVA-OH hydrogel for strengthening and toughening mechanism was prepared by using 0.45 g C4H9OK and cooling down at 4 °C.Fabrication of PVA-EX hydrogelPVA (3 g) was dissolved in DMSO (12 g), forming a solution with mass concentration of 20%. Afterwards, PVA solution that does not contain C4H9OK was immersed directly in ice-water mixture for 24 h, forming a solvent-exchanged hydrogel as control sample, denoted as PVA-EX.Water content measurementFirst, PVA-OH hydrogels were weighed in the air, and their weights were recorded as mwet. Then, the hydrogels were dried thoroughly at 55 °C for 48 h. The obtained solids were weighed in the air, and their weights were recorded as mdry. Finally, the water contents (W) of PVA-OH hydrogels were calculated from the equation:$$W=\frac{{m}_{{{{\rm{wet}}}}}-{m}_{{{{\rm{dry}}}}}}{{m}_{{{{\rm{wet}}}}}}\times 100\%$$
(1)
Crystallinity measurementPVA-OH hydrogels were soaked in a solution containing glutaraldehyde (8 ml), hydrochloric acid (0.4 ml) and water (92 ml). After 2 h, the hydrogels were washed with deionized water to remove excessive glutaraldehyde and hydrochloric acid, followed by drying for crystallinity measurement. In this way, amorphous chains within the hydrogels were fixed by glutaraldehyde to minimize the additional formation of the crystalline domain during the drying process.Differential scanning calorimetry (DSC) was carried out by TA Q2000 to measure the crystallinity16. We first weighed the total mass of the dried sample (containing a little residual water), recorded as m. Then, DSC test was performed under a nitrogen atmosphere with a flow rate of 30 mL min−1, and the temperature was increased from 50 to 250 °C with a heating rate of 10 °C min−1. The heat flow-time curves presented two peaks. One was a broad peak ranging from 60 °C to 180 °C, corresponding to the evaporation of residual water. The other was a narrow peak ranging from 200 °C to 250 °C, corresponding to the melting of crystalline domains. The enthalpy for the evaporation of residual water per unit mass of the dried sample Hresidual was obtained from the integration of the broad peak. The enthalpy for the melting of crystalline domains per unit mass of the dried sample Hcrystalline was obtained from the integration of the narrow peak. The mass of the residual water mresidual was calculated as:$${m}_{{{{\rm{residual}}}}}=m\times \frac{{H}_{{{{\rm{residual}}}}}}{{H}_{{{{\rm{water}}}}}^{0}}$$
(2)
where H0water is the evaporation heat (2260 J g-1) of pure water39. The mass of the crystalline domains mcrystalline was calculated as:$${m}_{{{{\rm{crystalline}}}}}=m\times \frac{{H}_{{{{\rm{crystalline}}}}}}{{H}_{{{{\rm{crystalline}}}}}^{0}}$$
(3)
where H0crystalline is the melting enthalpy (138.6 J g-1) of 100 wt% crystalline PVA40. The crystallinity in the ideally dry sample Xdry was calculated as:$${X}_{{{{\rm{dry}}}}}=\frac{{m}_{{{{\rm{crystalline}}}}}}{m-{m}_{{{{\rm{residual}}}}}}$$
(4)
The crystallinity in PVA-OH hydrogels Xwet was calculated as:$${X}_{{{{\rm{wet}}}}}={X}_{{{{\rm{dry}}}}}\times (1-W)$$
(5)
SAXS measurementSAXS was conducted on a Xenocs Xeuss 2.0 System by using an incident Cu-Kα X-ray beam that was perpendicular to the sample plane. X-ray wavelength, spot size and distance between specimen and detector were 0.154 nm, 172 × 172 μm2 and 1185 mm, respectively. Samples were square slices with a side length of 10 mm. Scattering patterns were collected by a Pilatus 300 k detector. The distance distribution function, P(r), was analyzed by SASfit software.(1) The inter-crystallite distance (Rd) was obtained from the Bragg expression41:$${R}_{{{{\rm{d}}}}}=\frac{2\pi }{{q}_{\max }}$$
(6)
Where qmax is the scattering vector at which Iq2 shows a maximum value.(2) The radius of gyration (Rg) could be calculated at low q according to Guinier equation35:$$I(q)={I}_{0}\exp \left(-\frac{{{R}_{g}}^{2}{q}^{2}}{3}\right)$$
(7)
Where I(q) was the scattering intensity and I0 was the zero angle scattering intensity. Rg was used to represent the average size of crystallites.(3) The pair-distribution function P(r) could be calculated through a Fourier transform of the scattering curve35:$$P(r)=\frac{r}{2{\pi }^{2}}{\int }_{0}^{\infty }I(q)q\,\sin (qr){{{\rm{d}}}}q$$
(8)
(4) The orientation order parameter (f) was calculated to describe the orientation degree of crystal domains. Its value ranges from 0 to 1, where the former corresponds to an isotropic structure and the latter corresponds to a perfect orientation structure along the director. A Maier-Saupe distribution function was used to fit the azimuthal angle profile42:$$I={I}_{0}+A\exp [\omega {\cos }^{2}\,({\varphi }-{{\varphi }}_{0})]$$
(9)
where I0 is the free baseline intensity, φ is the azimuthal angle, φ0 is the azimuth at the position of maximal intensity, and ω is a parameter that determines the width of the distribution. After the fitting, parameter ω was obtained, and the orientation factor f can be determined using the following formula:$$f=\frac{{\int }_{-1}^{1}{P}_{2}(\cos {\varphi })\exp (\omega {\cos }^{2}\,{\varphi })d(\cos {\varphi })}{{\int }_{-1}^{1}\exp (\omega {\cos }^{2}{\varphi })d(\cos {\varphi })}$$
(10)
where the function P2(cosφ) is the second-order Legendre polynomial of cosφ and often referred to as the Hermans orientation function:$${P}_{2}(\cos {\varphi })=\frac{1}{2}(3{\cos }^{2}\,{\varphi }-1)$$
(11)
Tensile testMechanical properties were measured on a Shimadzu AGS-X Tester with a gauge length of 10 mm and a load speed of 20 mm min-1.(1) Young’s modulus (E) was calculated from the stress-strain (σ-ε) curve within the initial linear region. The toughness, a parameter that characterizes the energy required to fracture the sample per unit volume, was calculated by the integral area from ε = 0 to the fracture point (εf) under the stress-strain (σ-ε) curve. Specifically, the toughness (U) was calculated as follows:$$U={\int }_{0}^{{\varepsilon }_{f}}{\sigma }_{{{{\rm{load}}}}}{{{\rm{d}}}}\varepsilon$$
(12)
(2) The cyclic loading-unloading tensile test was performed to investigate the energy dissipated mechanism. The dissipated energy (W) was calculated from the hysteresis area:$$W={\int }_{0}^{\varepsilon }{\sigma }_{{{{\rm{load}}}}}-{\sigma }_{{{{\rm{unload}}}}}{{{\rm{d}}}}\varepsilon$$
(13)
(3) The dissipated ratio was the ratio of the dissipated energy (W) to toughness (U) at a specific strain, i.e., dissipated ratio = W/U.Fracture toughness testThe fracture toughness was determined by a single-notch test43. Two identical hydrogels were fabricated with the same length 20 mm, width 5 mm and thickness about 1 mm for tensile test at a rate of 10 mm min-1 on the Shimadzu AGS-X material testing machine with a load cell of 500 N. A notch 1 mm was introduced across the width of one sample. A critical extension λc was defined when the notch turned into a running crack. Another sample without notch was uniformly stretched to measure the relationship between nominal stress s and strain λ. The work done to reach λc for an unnotched sample was defined as U(λc), and the fracture toughness Γ was calculated as:$${\varGamma }=\frac{6\cdot U({\lambda }_{c})\cdot c}{\sqrt{{\lambda }_{c}}}$$
(14)
where c was the crack length.Fatigue threshold of the hydrogel samplesFatigue threshold of the hydrogel samples was quantified through the single-notch method, following previous protocol16,38. All fatigue tests in this study were performed on fully swollen hydrogel samples within a water bath using a mechanical stretcher (Cellscale), in order to avoid dehydration-induced crack propagation. All cyclic tensile tests were conducted on notched and unnotched samples with identical dog-bone shapes. A notched sample with pre-cut crack (length less than 1/5 of the overall width) was exposed to a cyclic tensile test at a strain of λmax, and we recorded the crack length at the undeformed state cover cycles using a digital microscope (AM4815ZT, 20 mm pixel−1). Meanwhile, the same stretch λmax was also applied on a unnotched sample, and the strain energy density of under the Nth cycle estimated following this formula:$$W({\lambda }_{\max },\,N)={\int }_{1}^{{\lambda }_{\max }}S{{{\rm{d}}}}\lambda$$
(15)
The applied energy release rate G in the notched sample under the Nth cycle with a maximal applied stretch of λmax can be calculated following this formula:$$G({\lambda }_{\max },\,N)=2k({\lambda }_{\max })\cdot c(N)\cdot W({\lambda }_{\max },\,N)$$
(16)
where k is a slowly varying function of the applied stretch as k = 3·λmax−1/2. By varying the applied stretch of λmax, we acquired the curve of crack extension per cycle dc/dN versus the applied energy release rate G. The fatigue threshold can be obtained by linearly extrapolating the curve of dc/dN versus G to the intercept with the abscissa. To validate that the energy release rate value obtained from the linear extrapolation is equal to the real fatigue threshold of the hydrogel sample, a notched hydrogel sample was exposed to a single-notch tensile at this energy release rate for 30000 cycles, and crack propagation was further monitored with the camera.Lap shear adhesion testThe adhesion strength was evaluated by lap shear adhesion test. PVA-O- solution was evenly coated on the surface of one substrate with an overlap area of 1 cm2. After that, it was quickly covered with the other substrate, followed by complexation for adhesion and immersed in DI water for removing extra reagent. Lap shear adhesion tests were performed on a Shimadzu AGS-X Tester with a 500 N load cell at a loading speed of 10 mm min-1. The adhesion strength was calculated as the failure force divided by the overlap area.Rabbit Chondrocytes Isolation and CultureAll animal experiments in this research were approved by the Animal Care and Ethics Committee of the College of Biology, Hunan University. The isolation and culture of rabbit chondrocytes from articular cartilage of 1 week-old New Zealand white rabbits were following previous method44. Concretely, rabbit legs were collected and immersed in PBS in the biosafety cabinet. The surrounding tissues around the cartilage were removed, and the cartilage was washed twice with PBS containing 1% antibiotics. The washed articular cartilage was cut into small pieces ( < 1 mm3) and transferred into centrifugal tubes. Adequate amounts of the DMEM medium (HyClone, USA) containing 15% (w/v) type II collagenase (300 u/mg, Worthington, USA) was added to the centrifuge tube to digest the ECM. Chondrocytes were collected every 4 h, and the collection was repeated once. The collected digestive fluids were filtrated and centrifuged at 3000 rpm for 5 min, and then, the collected chondrocytes were resuspended in the DMEM medium containing 10% fetal bovine serum (Gibco, USA) and cultured in cell culture dishes. The cell culture medium was changed every 2 days. After two passage culture, chondrocytes were used for this study.In vitro cell viability studyThe live/dead cell staining experiment was applied following the manufacturer’s protocol. To obtain the hydrogel extracts, 1 g PVA-OH was added to 10 mL DMEM and incubated in incubator for 24 h at 37 °C. Then the supernatant was collected and filtered through a 0.22 μm filter (Millipore, USA) to obtain hydrogel extracts for the following cell experiments. Rabbit chondrocytes inoculated with 20,000 cells per well in a 12-well plate (Corning, USA). After the cells were attached to the plate, extracts were used instead of media. The cells with AM/PI staining at day 1, 3 and 5 were observed and photographed by fluorescence microscope (BX53; Olympus, Tokyo, Japan). In cell proliferation experiments, chondrocytes were inoculated into 96-well plates (Corning, USA) with a density of 5000 cells per well and cultured with PVA-OH hydrogel extracts or DMEM (control). Cells were counted using the Cell Counting Kit-8 (CCK-8; Beyotime, China) after 1day, 3 days and 5 days of culture. Optical density at 450 nm (OD450) was measured with a microplate reader (BioTek, ELX808, USA). The experiment was repeated at least three times.Material characterizationsFTIR spectra were collected using a Nicolet IS50 spectrometer (Thermo Fisher Scientific) with an attenuated total reflectance mode in the wavenumber range from 4000 cm-1 to 400 cm-1. WAXD was carried out on a MiniFlex X-ray diffractometer (Rigaku) in a scanning range from 10° to 60° under a scanning rate of 10° min-1. A rheological test was carried out on a MARS60 rheometer (HAAKE). Unless otherwise stated, the temperature for test is 90 °C for solution and 25 °C for gels. 13C-NMR spectra were recorded by a 400 MHz nuclear magnetic resonance spectrometer (Bruker) at 25 °C. DMSO-d6 was used as a solvent. The concentration of samples was set as 30 mg ml-1. WAXS was conducted on a Xenocs Xeuss 2.0 System by using an incident Cu-Kα X-ray (λ = 0.154 nm) beam that was perpendicular to the sample plane. The distance between specimen and detector was 100 mm.Molecular dynamics simulationMolecular simulation was used to explore atomic-level details of PVA and PVA-O- solution. Models were constructed using Material Studio, mainly including MS Visualizer, Amorphous Cell and Forcite modules. Molecular models were constructed by MS Visualizer. The polymerization degree of PVA molecules was set to be 20, larger than its value of Characteristic ratio, C∞, which was calculated to be 6.945. PVA-O- molecular model was constructed by deleting four H atoms of hydroxyl groups that occupy 5%.Solution models were constructed by Amorphous Cells. Molecules constructed by MS Visualizer were filled into the empty cell. Two PVA or PVA-O- molecules were put into the empty cell, and the filling density of DMSO was set to 1.1 g cm−3 (the experimental value of DMSO density). Filling with different numbers of cations (K+ ions) to balance the charge of the system, the modeling process is illustrated in the figure below. The box was a cube with a side length of about 40 Å. The volume ratio of solvent DMSO to PVA in the model was about 20:1, in which the amount of solvent was sufficient to reflect the solvent environment around PVA molecules.For molecular dynamics simulation, the ensemble was set to NVT, the initial velocity was adopted as Maxwell-Boltzmann distribution, the simulated temperature was 298 K (25 °C), the Nose-Hoover thermostat was adopted, the time step was 1 fs, and the total simulation time was 5000 ps.Reporting summaryFurther information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
Control nucleation for strong and tough crystalline hydrogels with high water content
