Silk sericin/poly (NIPAM/LMSH) nanocomposite hydrogels: Rapid thermo-responsibility and good carrier for cell proliferation

Materials

The monomer N-isopropylacrylamide (NIPAM, Tokyo Chemical Industry Co. Ltd., Tokyo, Japan), silk sericin powder (SS, M_r=4000~20 000, Huzhou Xintiansi Bio-tech Co. Ltd., Huzhou, P.R. China) and Tissue culture polystyrene (TCPS, Sigma-Aldrich Co. LC., USA) plates was purchased. Inorganic nanoclay lithium magnesium silicate(LMSH, (Mg,Li)₃Si₄O₁₀(OH)₂·4H₂O) was a gift of Luancheng Zixin industrial and trading Co., Ltd, Shijiazhuang City, Hebei Province, P.R.China. Dulbecco’s modified eagle medium(DMEM) supplemented with 5 % fetal bovine serum (Minhai biotechnologies), penicillin and streptomycin, Trypsin and Ethylenediaminetetraacetic acid (EDTA) were obtained from Shanghai Sangon Biological Engineering Technology & Services Co., Ltd., Shanghai, P.R. China. L929 were kindly provided by Tianjin Medical University. Redox agent peroxydisulfate (APS, Sinopharm Chemical Reagent Co. Ltd., Shanghai, P.R. China) and N,N,N′,N′-tetra-methylenediamine (TEMED, Sinopharm Chemical Reagent Co. Ltd., Shanghai, P.R. China), and some solvent like ethanol, etc., were of analytic grade and used as received without further purification. All water used in the whole experiment was of Millipore Milli-Q grade (Chongqing Molecular Water System Co. Ltd., Chongqing, P.R. China).

Synthesis of SS/poly(NIPAM/LMSH) nanocomposite hydrogels

According to Table 1, the physical cross-linker LMSH, the monomer NIPAM and SS were fully dissolved in aqueous water at 25 °C. The mixture solution was bubbled with nitrogen for 10 min at 5 °C while stirring, and then APS and TEMED were dropped into above mixture solution to initiate the polymerization. Finally, the pre-reacted solution was injected into the glass mold with 2 mm thickness and reacted at 25 °C for 48 h. As a comparison, the pure PNIPAM nanocomposite hydrogel without any SS was prepared by same method.

The prepared nanocomposite hydrogel samples were cut into disks with 8 mm in diameter and immersed in excess distilled water for 3 days to remove the unreacted monomer or small molecules. The resulting SS/poly(NIPAM/LMSH) nanocomposite hydrogel samples were simply named as HSPX. In the present study, X means the mass ratio of SS/(NIPAm+SS).

Temperature dependence of equilibrium swelling ratio

The temperature-stimulating equilibrium swelling ratios of HSP nanocomposite hydrogels were gravimetrically measured. After swelling for 24 h in distilled water at a required temperature, the HSP discs were taken out for weighting. The value of equilibrium swelling ratio (ESR) at every particular temperature could be calculated according to following equation,

where W_s and W_d are the equilibrium weight and the dry weight of HSP nanocomposite hydrogels, respectively.

Differential scanning calorimeter (DSC) analysis

The thermo-responsibility of HSP hydrogel samples were carried out on a DSC-7 differential scanning calorimeter (Perkin-Elmer Inc., USA) under a nitrogen atmosphere, at a heating rate of 2 °C·min^–1 from 15 to 50 °C. Deionized water was used as the reference in the DSC measurement.

Thermal impulse response behavior

The impulse responsibility of HSP hydrogels with alternating temperature changes was studied. Firstly, equilibrium HSP hydrogel sample at 25 °C was weighted after removing the excess water from the surface. It was subsequently immersed into distilled water for 6 min and weighted. Then, HSP hydrogel sample was quickly transferred to distilled water at 37 °C for another 6 min. Similar process was operated for total four cycles.

Cell adsorption and proliferation on HSP nanocomposite hydrogels

The HSP nanocomposite hydrogels were sterilized by immersed in 75 % ethanol for 1 day, then exposed to UV light in a clean bench for 30 min and finally transferred to plastic cup where they were incubated in DMEM at 37 °C overnight. Fibroblasts (L929) cultured on TCPS dishes were harvested and resuspended in DMEM supplemented with 10 % FBS, 100 units/mL penicillin, and 100 mg/mL streptomycin. L929 cells were seeded onto HSP hydrogels at 0.5 × 10⁴ cells/cm² and cultured at 37 °C under a humidified atmosphere of 5 % CO₂ for up to 7 days.

The L929 cell morphology on the surface of HSP nanocomposite hydrogels was observed by scanning electron microscope (SEM) (model 200, Quanta). Firstly, the specimens of cell-hydrogel complexes were rinsed with 37 °C PBS buffer and fixed in 4 % glutaraldehyde at 37 °C. After fixation for 4 h, the HSP samples were washed in PBS buffer (37 °C, 10 min) three times. Finally, the specimens were dried by freeze drying and sputter-coated with gold.

The seeded substrates were cultured at 37 °C under a humidified atmosphere of 5 % CO₂. After 0.5, 1, 2, and 4 h, the metabolic activity of attached cells was determined by the MTT essay, respectively. Cell adhesion rate was calculated from the metabolic activity of attached L929 cells on the surfaces and the seeding density.

The metabolic activity was qualified by the 3-(4,5)-dimethylthiahiazo (-z-y1)-3,5-di-phenytetrazoliumromide (MTT) essay every 2 days. At the desired time point, the culture medium was drained and resupplied with 500 μL of fresh culture medium. MTT dissolved in HBSS (5 mg/mL) was added to each well. After incubating at 37 °C for 4 h, excess medium was removed. The cells were then dissolved in 200 μL DMSO and the OD value of each well was determined by Auto microplate reader (R960, Metertech Co., USA) at 495 nm.

Detachment of L929 cells

The detachment of L929 cell was performed by decreasing temperature from 37 to 15 °C. L929 cells were firstly plated onto TCPS and HSP nanocomposite hydrogel disc surface and cultured at 37 °C. After 2 days’ cultivation, unattached cells were removed by medium change and each plate was transferred to the incubator equipped with a cooling unit fixed at 15 °C. After 0.5, 1, and 2 h incubation, detached L929 cells were collected and determined by MTT essay.

Cell transshipment

The cells detached by decreasing temperature from 37 to 15 °C was aspirated and transferred to a fresh 96-well TCPS culture plate. The plate was then returned to the 37 °C CO₂ incubator to allow cell attachment and growth for 1 week. The re-growth of cells recovered by enzyme digest was used as control. Proliferation of the transferred cells was also assessed using MTT assay.

The appearance and swelling behavior of HSP nanocomposite hydrogels

The nanocomposite hydrogels using nanoclay like hectorite as physical cross-linker are commonly optical transmittance. Figure 1 shows the digital camera photos of HSP hydrogels. It is very obvious that all swollen HSP hydrogels are transparent because colored curves below the hydrogels can be seen. Furthermore, it is seen that the volume increases with increasing mass ratio of SS/(NIPAm+SS) on account of high hydrophilicity of SS. In fact, as the mass ratio of SS/(NIPAm+SS) is 20 %, the pore shape of HSP nanocomposite hydrogel after freeze-drying turns to layer structure from honey-comb structure, which has been confirmed by previous report [21].

Fig. 1

The appearance of HSP nanocomposite hydrogels.

Thermo-responsibility of HSP nanocomposite hydrogels

Figure 2 shows the curves of equilibrium swelling ratios (ESR) of HSP nanocomposite hydrogels at different temperatures. With increasing temperature from 14 to 50 °C, the ESR values of all HSP hydrogels decrease rapidly at 29.5~33.5 °C, and then keep nearly constant. It means that the volume phase transition temperature (VPTT) value of HSP nanocomposite hydrogels is at 29.5~33.5 °C. Furthermore, with the addition of mass ratio of SS/(NIPAm+SS), the ESR value at each temperature also increases, which is in accordance with the result of Fig. 1. For example, at 14 °C, the ESR values of HSP0, HSP05, HSP10, HSP15 and HSP20 are 44.2, 58.8, 66.3, 70.3 and 104.9 g/g, respectively. A linear equation between mass ratio of SS/(NIPAm+SS) and ESR values can be obtained as following: y=265.7x + 42.3. In the case of change scope of equilibrated swelling ratios, HSP20 nanocomposite hydrogel presents quickest and largest descent at around VPTT. It indicates that the introduction of SS increase the temperature-sensitivity of pure HSP nanocomposite hydrogels. The SS containing high hydrophilic groups like -OH-COOH and -NH₂ and rapid losing water under temperature stimulus are responsible for this.

Fig. 2

Equilibrium swelling ratios of HSP nanocomposite hydrogels as a function of temperature.

To further confirm the phase transition behaviors of HSP nanocomposite hydrogels, the DSC thermograms was performed. As shown in Fig. 3, obvious endothermic peaks appear at all curves of HSP nanocomposite hydrogels, confirming the thermo-responsibility of HSP hydrogels. As temperature exceeds 31~33 °C, the thermo effect happened. It indicates that VPTT values of HSP nanocomposite hydrogels are 31~33 °C, which is in agreement with the result in Fig. 2. It also can be seen that with increasing mass ratio of SS/(NIPAm+SS) from 0 to 20 wt %, the VPTT values increased. The VPTT values of HSP0, HSP10 and HSP20 are 32.12, 32.97, and 33.34 °C, respectively. In other words, the introduction of SS has indeed produced an effect on the VPTT of HSP nanocomposite hydrogels. The reason can be ascribed to that hydrophilic/hydrophobic balance of pure PNIPAM chains was destroyed by the introduction of hydrophilic SS.

Fig. 3

DSC thermograms of HSP nanocomposite hydrogels.

Due to good thermo-responsibility at 31~33 °C, impulse responsibility upon temperature of HSP nanocomposite hydrogels was carried out. As shown in Fig. 4, when temperature was alternatively changed between 37 and 20 °C, the re-swelling ratios of HSP nanocomposite hydrogels are going on a downward trend. It is clear that the shrunken SS/poly(NIPAM/LMSH) molecule chains are difficult to recover within 6 min and to absorb same amount losing water. According to mentioned above, due to high hydrophilicity of SS, SS could increase the temperature-sensitivity of pure HSP nanocomposite hydrogels and present sharply decrease upon equilibrated swelling ratios (ESR) at around VPTT. Therefore, the HSP nanocomposite hydrogels with high mass ratio of SS/(NIPAm+SS) show rapid decrease during shrinking process. Compared to pure HSP0 nanocomposite hydrogel, the incorporation of SS leads to lower swelling ratio under each temperature cycle. However, since plenty of water has been expelled from the matrix, it is not easy to restore original state within short time. For this reason, the shrinking-swelling performance of HSP nanocomposite hydrogel is lack of reversibility. It is seen that after twice temperature cycle, the re-swelling ratios or re-deswelling ratios keep almost the same and below 5 g/g. Therefore, it is much better that only one temperature stimulus was performed when cells on the surface of HSP nanocomposite hydrogels were detached by lowing temperature.

Fig. 4

Impulse response behavior of HSP nanocomposite hydrogels between 25 and 37 °C.

Cell adsorption of HSP nanocomposite hydrogels

Figure 5 shows the adhesion rate of L929 cells attached on the surfaces HSP nanocomposite hydrogels at 37 °C. It can be seen that L929 cells were well attached on the surfaces of HSP nanocomposite hydrogels and TCPS within 4 h. After 4 h, the adsorption rates on the surfaces of HSP nanocomposite hydrogels and TCPS reached about 80 %. Compared to HSP nanocomposite hydrogels, the absorption rate on the surface of TCPS is the fastest. At first 2 h, absorption rates all increase quickly, and then remain in equilibrium. In comparison with HSP0, the attached situation on the surface of HSP20 was much better. However, on culturing 4 h, the HSP05 nanocomposite hydrogel presents highest adsorption rate, which is slightly higher than that of HSP10, HSP15 and HSP20. It indicates that the adsorption rate, which is slightly higher than that of HSP10, HSP15 and HSP20. It indicates that the adsorption rate would decrease after L929 cells were seeded onto the surface of HSP nanocomposite hydrogel. Then, it is reasonable to believe that, at first 2 h, L929 cells could adhere to the surface of HSP nanocomposite hydrogels because of good cell affinity of SS.

Fig. 5

The adhesion rate of L929 cells attached on the surfaces HSP nanocomposite hydrogels at 37 °C.

Cell morphology and cultivation of HSP nanocomposite hydrogels

The L929 cell compatibility of HSP nanocomposite hydrogels was examined by morphology and adhesion assessing. As shown above, HSP20 nanocomposite hydrogel shows the highest adhesion rate within 4 h, then the SEM images of L929 cells on the surface of HSP20 after culturing 1, 3, 5, 7 days were first carried out. As shown in Fig. 6, when culturing time increased from 1 to 7 days, the attached L929 cells on the surface of HSP20 nanocomposite hydrogel disc increases gradually. For pure HSP20, not any L929 cells can be found from surface. But on the first day, seeded L929 cells begin to adhere to the surface of HSP20 nanocomposite hydrogel. On culturing day 5, L929 cells have covered whole surface of HSP20. On culturing day 7, the L929 cell number on the surface of HSP20 hydrogel was nearly saturated, leading to partly dead of L929 cells. The graph from Fig. 6 also indicates that HSP20 nanocomposite hydrogel is very suitable for accelerating proliferation of L929 cells.

Fig. 6

SEM micrographs (300×) of seeded L929 cells attached on the surface of HSP20 nanocomposite hydrogel after culturing 1, 3, 5, and 7 days, respectively.

To further illustrate the effect of HSP nanocomposite hydrogels with various SS content on the L929 cell activity, the SEM images of L929 cells on the surface of HSP0 and HSP10 nanocomposite hydrogels were compared, which can be found from Fig. 7. It can be seen that the surfaces of HSP0 and HSP10 nanocomposite hydrogels fixed by glutaraldehyde are close texture. On culturing 3d, L929 cells present good cell growth on the surfaces of HSP0 and HSP10 nanocomposite hydrogels. After culturing day 7, L929 cells still proliferate on the surface of HSP0, but excessive multiplication or dead cells has occurred on the surface of HSP10. Therefore, it is reasonable to assume that the incorporation of SS could promote the proliferation of L929 cells on the surface of HSP nanocomposite hydrogels.

Fig. 7

SEM micrographs (300×) of L929 cells attached on the surface of HSP0 and HSP10 after culturing 0, 3, and 7 days, respectively.

Figure 8 shows the cell activity of L929 cells on the surfaces of TCPS and HSP nanocomposite hydrogels for culturing 1, 3, 5, and 7 days at 37 °C. It could be seen that, with increasing culturing time from 0 to 7 days at 37 °C, the cell activity increased on the surfaces of both TCPS and HSP nanocomposite hydrogels. But L929 cell activity on the TCPS control surface is always higher than that on HSP nanocomposite hydrogel surface regardless of culturing time. It indicates that L929 cells can adhere and proliferate on the surfaces of HSP nanocomposite hydrogel, similar with TCPS plate. Furthermore, it can be seen that, compared to pure HSP0 nanocomposite hydrogel, the incorporation of SS like HSP05-20 could accelerate cell proliferation to its surface, which is in accordance with the results obtained in Fig. 6 and 7.

Fig. 8

Cell activity on the surface of TCPS and HSP nanocomposite hydrogels using MTT assay. Cells were seeded at 0.5×10⁴ cells/cm² in a medium with 10% FBS. *p<0.05.

Cell detachment via temperature stimulus

As mentioned in Fig. 2, by decreasing the temperature from 37 to 15 °C, the swelling ratios of HSP nanocomposite hydrogels decrease quickly. And then the decrease in volume could accelerate the detachment of L929 cells from the surface of HSP nanocomposite hydrogels. Figure 9 shows the percentage of detached cells from the surfaces of TCPS and HSP nanocomposite hydrogels as a function of treatment time at 15 °C. As shown in Fig. 9, there were almost no L929 cells were detached from TCPS surface within 2 h because TCPS presents nothing thermo-responsive behavior. However, the L929 cells could simultaneously detach from the surface of HSP nanocomposite hydrogels by reducing temperature to below VPTT. Furthermore, in comparison with pure HSP0 hydrogel, more rapid detachment rate of HSP nanocomposite hydrogels with the incorporation of SS could be found. For example, at 0.5 h, the detachment rate of HSP0, HSP05, HSP10, HSP15 and HSP20 are 71.5, 80.4, 83.2, 85.6, and 85.0 %, respectively. In other words, high mass ratios of SS/(NIPAm+SS) could accelerate cell detachment on account of rapid temperature-sensitivity at above and below VPTT of HSP nanocomposite hydrogels.

Fig. 9

The percentage of detached cells from different surfaces as a function of incubation time at 15 °C.

Cell transshipment after lowering temperature and enzymatic digestion

Figure 10 shows the proliferation of L929 cells cultured at 37 °C and detached from the surface of HSP nanocomposite hydrogels after lowering temperature and enzymatic digestion. It is seen that, during the culturing time between day 1 and day 7, the L929 cells via lowering temperature and enzymatic digestion still alive and present good activity, and could re-grow or proliferate, similar with the results of Fig. 8. In addition, the cell activity via lowering temperature from the surface of HSP nanocomposite hydrogel is much higher than that via enzymatic digestion because the damage to L929 cells was avoided.

Fig. 10

Cell transshipment of L929 cells detached from the surface of HSP nanocomposite hydrogels after lowering temperature and enzymatic digestion.