Biomaterials with the potential to recognize specific molecules have received considerable attention in the fields of bioscience and bioengineering. The biomolecules including protein antibodies and enzymes are often used as signal-capturing moieties to develop biological substitutes for the therapeutic replacement and tremendous need for organs and tissues ^[1]. However,these biomolecules are not very stable,they cost a lot and their affinity reduces significantly under artificial conditions ^{[2, 3]}. Molecular imprinting is a technique involving artificial recognition sites that can memorize the shape and chemical properties of the target molecules. Compared to the affinity matrices prepared using antibodies or enzymes,the molecularly imprinted polymers (MIP) possess unique properties,such as high stability,low cost,and easy preparation ^{[3, 4]}. The MIPs have been used in various applications including separations ^[5],solid-phase extraction,catalysis ^[6], biomimetic sensor ^[7],and drug delivery ^[8]. Pan et al. ^[9] employed a molecular imprinting methodology to introduce the cell-adhesive peptide RGDS onto a thermo-responsive cell culture substrate,which was innovatively used as a highly efficient novel system for harvesting cell sheets. Fukazawa et al. ^[10] proposed a new molecular imprinting procedure for the purpose of cell capture using the cell-adhesive protein fibronectin (FN) as the template. The binding of FN from the cell culture medium with the fetal calf serum was achieved on the FN imprinting substrate,and induced the cell adhesion.

The imprinting of low-molecular weight compounds has been well established. However,several challenges remain in the imprinting of proteins due to the large size,the structural complexity and the flexible conformation of the bio-macromolecular ^[11]. Polyacrylamide (PAM) hydrogels have been regarded as suitable imprinting matrices for proteins ^[12]. But the poor mechanical property and poor regeneration of the soft texture limited its application. Bovine serum albumin (BSA) imprinted polypropylene (PP) fiber-grafted polyacrylamide hydrogel was prepared using non-woven PP fiber as matrix,BSA as template molecule,and acrylamide as functional monomer ^[13]. In order to improve the strength of the PAMhydrogel,BSA imprinted calcium alginate/polyacrylamide hydrogel film was prepared by using BSA as template,acrylamide and sodium alginate as functional monomers. Acetic acid solution containing sodium dodecyl sulfate (SDS) was used to remove BSA. However,it is different to completely remove SDS and the residual SDS can lead to the death of cells ^[14]. In this paper,PP non-woven supported FN imprinted calcium alginate/polyacrylamide hydrogel membrane (PP-s-CA/PAMMIP) was prepared using FN as template molecule. Amoderate elution solution was used to remove the template FN, and the resulted PP-s-CA/PAM MIP was used for the culture of mouse fibroblast cells (L929).

Non-woven polypropylene (PP) fiber (22 g/m²) was obtained from Xianghehuaxin Non-woven Company,LTD (Langfang,China). Sodium alginate (SA) was purchased from Tianjin Yuanhang chemical company. Acrylamide (AM) and N,N0-methylenebisacrylamide (MBA) was analytical reagent and purchased from Chemistry Reagent Factory of Tianjin (Tianjin,China). CaCl₂ was supplied by Yingda Chemicals (Tianjin,China). Trihydroxymethyl aminomethane (Tris) and ammonium persulfate (APS) was obtained from the Institute of Tianjin Guangfu Fine Chemicals (Tianjin,China). Fibronectin (FN,440KD) was purchased from Shanghai Qianchen Biological Technology Company (Shanghai, China). Mouse fibroblast cells (L929) were obtained from the Cellular Biology Institute of the Chinese Academy of Sciences (Shanghai,China).

Fig. 1 shows the schematic representation of the fabrication procedure of the FN-imprinted PP-s-CA/PAM. Non-woven PP fiber (200 mg) was immersed into 10 mL aqueous solution consisting of FN (2 mg),AM (1 g),APS (10 mg),MBA (0.8 mg),and SA (50 mg). The mixture was incubated for 1 h at room temperature for the pre-assembly between the template molecules and functional monomers. Then the non-woven PP fiber immersed in the mixture was transferred into the quartz glass sheet,purged with nitrogen for 8 min to remove oxygen,and sealed. The polymerization was conducted according to the literature ^[15]. The hydrogel supported on non-woven PP was cross-linked with 1% CaCl₂ for 3 h,and washed repeatedly with deionized water to remove unreacted monomer and cross-linker. The protein in the hydrogel was eluted with Tris-HCl buffer solution (pH 8.3). Then FN imprinted PP nonwoven supported calcium alginate/polyacrylamide hydrogel film (PP-s-CA/PAM MIP) was prepared after extensively washed with deionized water to remove remnant Tris-HCl buffer solution.

Fig. 1

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Fig. 1.Schematic representation of the fabrication procedure of imprinted PP-s-CA/PAM and cell adhesion.

The non-imprinted polymer was synthesized by using the same procedure in the absence of template molecule and was noted as PP-s-CA/PAM NIP.

The morphologies of PP-s-CA/PAM MIP were observed using a scanning electron microscope (FESEM; S-4800,HITACHI,Japan).

About 0.2 g of wet PP-s-CA/PAM MIP or NIP (using filter to absorb the surface water) was placed in a glass bottle containing 5 mL 0.2 mg/mL FN solution. At specific time intervals,samples were withdrawn from the supernatant and determined with a UV spectrophotometer. The adsorption capacity Q (mg/g) of the protein adsorbed onto PP-s-CA/PAM MIP or NIP was calculated according to the literature ^[14].

The PP-s-CA/PAM MIP and NIP were sterilized by rinsing with ethanol and then inserted into a 24-well tissue culture plate. L929 cells were seeded onto PP-s-CA/PAM MIP and NIP at a density of 2×10⁴ cells/well and cultured at 37 ℃ under a humidified atmosphere of air containing 5% CO₂. At the desired time point,the viability of cells cultured on the MIP and NIP was observed by microscopy (OLYMPUS IX71,Japan). The adhesion behavior of L929 cells was researched according to the literature ^[9].

Fig. 2 shows the digital (in 24-well tissue culture plate containing deionized water) and SEM images of PP-s-CA/PAM MIP. The digital photo of PP non-woven was also shown on the left side of Fig. 2(a). The CA/PAM hydrogel was hydrophilic and transparent,so the PP-s-CA/PAM MIP was translucent. PP nonwoven was hydrophobic and some air bubbles were trapped in the PP fiber when it was immersed in the water,so the PP non-woven was opaque. Significant lamellar macroporous structure was found on the surface of PP-s-CA/PAM MIP (Fig. 2(b)). The macroporous structure on the surface of MIP facilitated the diffusion of the protein template. But the imprinting pores were not found because they were too small and the water evaporation in vacuum resulted in the shrinkage of pores.

Fig. 2

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Fig. 2.Digital image of PP (left) and wet PP-s-CA/PAM MIP (right) in 24 well cell culture plate (a) and surface SEM of PP-s-CA/PAM MIP (b).

The adsorption dynamic curves of PP-s-CA/PAM MIP and NIP are shown in Fig. 3. It is found that an abrupt increase in the adsorption capacity of PP-s-CA/PAM MIP and NIP was observed during the first 4 h,and the adsorption capacity almost reached equilibrium after 6 h. The PP-s-CA/PAM MIP adsorbed more FN than NIP,and the adsorption capacity of MIP was 2.6 times of that of NIP. The higher adsorption capacity of MIP for FN was attributed to the generation of FN high-affinity adsorption sites and the complementary cavities in the hydrogel during polymerization. There were no high-affinity adsorption sites and the complementary cavities in the NIP,which was prepared using the same procedure in the absence of FN,so the adsorption capacity of NIP for FN was much lower.

Fig. 3

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Fig. 3.Curves of FN adsorption capacity (a) and L929 cell adhesion (b) on PP-s-CA/PAM MIP and NIP.

L929 cells were cultured on the PP-s-CA/PAM MIP and NIP hydrogel films. The cell adhesion rate on the hydrogels is shown in Fig. 3(b). Compared with cells on NIP,the number of living cells adhered to MIP was much more,indicating that the MIP was more suitable for cell culture. Because the PP-s-CA/PAM MIP can absorb more FN,which is helpful for the adhesion of cells.

As shown in Fig. 4,the cell morphology by phase-contrast microscopy revealed a remarkable difference of adherent L929 cells on the PP-s-CA/PAM MIP and NIP. Compared with cells on NIP, the number of cells adhered to MIP was much more. In the case of PP-s-CA/PAM MIP substrate,cells adhered and spread significantly from the culture medium after 1 h incubation,while the cells on NIP film did not adhere and spread rapidly. After culturing for 4 h, cells were distributed evenly on the surface of PP-s-CA/PAM MIP. The cell morphology of the PP-s-CA/PAM MIP and NIP within 4 h culture was comparable.

Fig. 4

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Fig. 4.Photographs of adhesive L929 cells on PP-s-CA/PAM MIP and NIP after cultured for 1 h and 4 h.

Fibronectin (FN) imprinted polypropylene (PP) non-woven supported calcium alginate/polyacrylamide hydrogel film (PP-s- CA/PAM MIP) was prepared using non-woven PP fiber as matrix,FN as template molecule,sodium alginate (SA) and acrylamide (AM) as functional monomers,via UV radiation-reduced polymerization. The PP-s-CA/PAM MIP exhibited an obvious improvement in terms of adsorption capacity for FN compared with non-imprinted polymer (NIP). The PP-s-CA/PAM MIP was successfully used for the culture of mouse fibroblast cells (L929),which exhibited better cell adherence performance than the NIP did. PP-s-CA/PAM MIP will display great potential in cell culture and tissue engineering.

The research is supported by the National Natural Science Foundation of China (Nos. 51103102,51103180,81100100, 31200674) and Tianjin Municipal Natural Science Foundation (No. 15JCZDJC38300).