Poly(vinyl alcohol) (PVA) may be used in 3D printing as a sacrificial material in the formation of tissue engineering constructs with complicated architectures. Our research aims to combine 3D printing scaffold fabrication based on PVA as a sacrificial material with enzymatic mineralization and chemical crosslinking of hydrogels, which are done by incorporating specific chemicals into PVA filament. The method to obtain the scaffold involves pouring gelan gum-gelatin (GG-Gel) hydrogel into PVA 3D printed molds. A 3D printed scaffold, being a selectively mineralized and cross-linked hydrogel, can potentially be used in bone and cartilage regeneration. In this study, as a proof of concept, we established a 2D simplified setup of the molding process to assess the ongoing enzymatic mineralization and chemical crosslinking of hydrogels.
PVA (Mowiol, 4-88) was formed into 0.2 mm foils containing calcium glycerophosphate (CaGP) or tannic acid (TA) as a substitute for 3D printed mold material. The hydrogel containing defined amounts of gellan gum (GG), gelatin, CaCl2, and alkaline phosphatase (ALP) was poured on PVA enclosed in a rectangular container. The mineralization of the hydrogels in CaGP or chemical crosslinking with TA were performed over 1 day. Enzymatic mineralization involved the activity of ALP incorporated in the hydrogel cases and the release of inorganic phosphate from CaGP. Chemical crosslinking was obtained by reaction between the amine groups of gelatin and TA. The modified hydrogel strips were washed in water. Hydrogels as well as PVA blends were analysed under light microscopy and FTIR. The percentage of dry mass percentage of hydrogels was measured. Biological properties were tested by culture of MG-63 cells on materials for 3 days. Alamar blue and live-dead (calcein AM, propidium iodide) staining tests were performed.
CaGP-containing PVA foils were translucent and mostly homogenic, although they contained gas bubbles, while TA-PVA was completely homogenic and transparent with an orange tint. GG-Gel hydrogels in contact with PVA foils underwent chemical and physical modifications. In the case of the TA-PVA, the whole mass of material changed color to bronze, and the material was homogeneous with rough, wavy surface. The CaGP-PVA-modified materials were opaque with small crystalline precipitates on the surface. Mineralized was only part of the hydrogel in contact with PVA (0.5 mm depth). FTIR spectra confirmed presence of TA phenol groups as well as PO43–. TA-modified GG-Gel material had the largest cell growth supporting ability compared to the unmodified control. For enzymatically mineralized material, increased cell spreading and attachment was also observed, but this effect was smaller.
Enzymatic mineralization by ALP and CaGP as well as chemical cross-linking by TA can be obtained by incorporation of desired chemicals into potentially 3D-printable PVA. Modifications significantly improved the attachment and viability of MG-63 bone cells. However, chemical cross-linking of GG-Gel resulted in overall better morphology and cell attachment. Unfortunately, the method combining enzymatic mineralization with TA-crosslinking was not possible so far because of the inactivation of ALP by TA.
This study was supported by the National Science Centre Poland (No 2018/29/N/ST8/01544).