Figure 1.
Phenylboronate-cis diol chemistry.
In aqueous solutions, phenylboronate (PBA) exists in equilibrium between uncharged trigonal and charged tetrahedral forms. Anionic PBA forms reversible covalent interactions with 1,2- or 1,3-cis diols at alkaline pH (>8.8). A tertiary amine protects boronate moiety from the nucleophilic attack by water molecule and promotes PBA-cis diol interactions at neutral pH.
Figure 2.
Terpolymer-poly(vinylalcohol) (PVA) complexation and fructose induced gel-sol phase transition at physiological pH.
Phenylboronate containing copolymer (PBCC) forms complex gels with polyols like PVA via intermolecular multipoint monodiols under alkaline conditions. A tertiary amine introduced into PBCC structure promotes multipoint monodiols at neutral pH. Monosaccharides such as fructose, by virtue of their higher affinity towards phenylboronate, competitively displace PVA from monodiol interactions and disintegrate the gels.
Figure 3.
Culture and Recovery of NIH3T3 fibroblasts on terpolymer-poly(vinylalcohol) (PVA) hydrogel films.
Fig. 3A shows fructose induced gel-sol transition of AVDT-PVA hydrogel. Figs. 3B-E shows culture of NIH3T3 fibroblasts on DVDT-PVA hydrogel films at day 2, 6, 8 and 12, respectively. Initial aggregation of fibroblasts at specific positions on the gel can be seen in Fig. 3B. After day 2, the fibroblasts have begun to spread out on to the surrounding gel (Figs. 3C, D). On DVDT-PVA hydrogel, the growth has reached to confluence by day 12 (Fig. 3E). Figs. 3F, G show the fibroblast growth after 12 days in culture on AVDT-PVA and NVDT-PVA hydrogels, respectively. Figs. 3H, I show detached fibroblast monolayer from AVDT-PVA gel at the edges (arrows) on fructose (200 mM) treatment with stirring (60 rpm) for 20 min. Continued fructose treatment has resulted in the recovery of individual and small clumps of cells. The gel remnants are indicated by arrows (Figs 3J-L).
Figure 4.
Conceptual depiction of phenylboronate containing copolymer (PBCC)-PVA cryogel synthesis.
Freezing initiates the phase separation of the initial monomer-PVA solution into unfrozen liquid microphase (UFLMP) and frozen solvent crystals (FSCs). As a result UFLMP gets concentrated with monomers and PVA. Within UFLMP, APS and TEMED initiates the free radical polymerization of monomers into PBCC chains that get crosslinked via PVA upon attaining sufficient length. This results in solid cryogel walls separated by FSCs. Freeze drying sublimes FSCs leaving behind interconnected voids/pores.
Figure 5.
Phenylboronate containing copolymer (PBCC)-poly(vinylalcohol) (PVA) cryogels.
Figs. 5A-C shows sheets and monoliths of NVP cryogels. SEM micrographs of NVP cryogels (Figs. 5D, E), AVP cryogels (Figs. 5F, G) and AADP cryogels (Figs. 5H, I) reveal their open macroporous structure.
Figure 6.
Fructose induced disintegration of phenylboronate containing copolymer (PBCC)-poly(vinylalcohol) (PVA) cryogels.
Fig.6 plots the percentage reduction in dry weights of different PBCC-PVA cryogels after 15 days in response to three different fructose concentrations (100 mM, 500 mM and 1000 mM). The cryogels treated with fructose have exhibited significant amounts of degradation compared to controls i.e., cryogels treated with phosphate buffer devoid of monosaccharides. AADP cryogel has showed superior degradability even at low fructose concentrations (100 mM) followed by AVP and NVP cryogels, respectively.
Figure 7.
FDA/DAPI double staining of cryogel sections after 12 days in culture.
Fig. 7A shows the bright field image of AVP cryogel with FDA/DAPI double stained NIH3T3 fibroblasts at day 12. Fig. 7B shows FDA stained viable cells (greenish fluorescence) and Fig. 7C shows DAPI stained viable and non-viable cells (bluish fluorescence) in AVP cryogel. Fig. 7D is a merged RGB image of Figs. 7A-C, respectively where cyan color indicates cell viability while non-viable cells show blue fluorescence alone. Figs. 7E, F are merged RGB images of double stained AADP and NVP cryogels, respectively (day 12). The merged images (Figs. 7D-F) of all the three cryogels are rich in cyan colored portions indicating their ability to support cell adhesion and viability.
Figure 8.
MTT assay of NIH3T3 fibroblasts cultured on AADP cryogels.
Fibroblasts cultured on tissue culture polystyrene (TCPS) plates have served as controls. The fibroblasts in cryogel sections have recorded relatively low levels of metabolic activity during the first few days as compared to the controls. From day 6 onwards, cryogels have displayed good levels of cell metabolic activity whereas controls have recorded a significant decline in cell activity. This indicates the ability of cryogels to offer high surface area for the growing cells as compared to 2-D controls and further demonstrates their support for cell growth and viability.