Developing advanced shape-morphing, biomolecule-delivering and cell-iaden tissue engineering scaffolds through 3D/4D printing

Abstract

Additive manufacturing (AM), alias 3D printing, has been applied in the tissue engineering field to produce tissue engineering scaffolds with controlled architectures and functionalities by accurately positioning biomaterials, biomolecules, and/or living cells in fabricated structures. However, commonly 3D printed scaffolds are static and the properties remain the same during the whole product lifetime, which cannot meet some specific requirements in regenerative medicine. To solve these problems, in recent years, AM has advanced from 3D printing to 4D printing where time is used as the fourth dimension besides 3D space. 4D printing empowers us to create dynamic scaffolds which can change their shape, properties, and/or functionalities under suitable stimulus/stimuli after in vivo deployment. Developing advanced tissue engineering scaffolds similar to the native tissues or organs through 3D/4D printing is therefore highly appealing in regenerative medicine. This project aims to investigate 3D/4D printing for fabricating advanced scaffolds that integrate biomolecule delivery, live cell incorporation and shape-morphing ability, with the ultimate goal of improving human body tissue regeneration. Firstly, 3D printing was applied to fabricate biomolecule-delivering or/and cell-laden scaffolds using different hydrogel inks/bioinks, including poly(lactide-co-glycolide) (PLGA) nanofiber reinforced alginate hydrogel, Laponite XLG nanoclay reinforced hydrogels, pre-crosslinked alginate hydrogel containing living cells, gelatin and gelatin methacryloyl (GelMA) blend hydrogel bioinks. These inks/bioinks exhibited good rheological properties and printability and hence were suitable for micro extrusion-based 3D printing. Biomolecules, i.e., growth factors, were incorporated into 3D printed scaffolds to promote tissue regeneration. Live rat bone marrow-derived mesenchymal stem cells (rBMSCs) were encapsulated into/onto 3D printed scaffolds via cell printing or cell-seeding strategy for achieving better tissue regeneration outcomes. Culturing tumor cells (i.e., Hela cells) with chemotherapy drug-loaded scaffolds was performed to investigate the chemotherapeutic effect of released drug for cancer treatment. Secondly, 4D printing was applied to fabricate shape-morphing scaffolds which could transform from 2D shapes to 3D shapes according to the preprogrammed design by responding to suitable external stimuli. Three biomaterials for 4D printing were investigated: one shape memory polymer [poly(D, L-lactide-co-trimethylene carbonate) (PDLLA-co-TMC)] and two shape-morphing hydrogels [GelMA and alginate/methylcellulose (Alg/MC)]. PDLLA-co-TMC, a thermal responsive synthetic polymer, was 4D printed into porous scaffolds which could automatically fold into tubes with controlled diameters at the physiological temperature. The two shape-morphing hydrogels were 4D printed into different 2D patterns which could transform into various simple and complex 3D morphologies by responding to water environment. Their shape-morphing ability was attributed to the uneven internal force resulting from the crosslinking degree gradient across the thickness or/and plane of printed samples. Thirdly, shape-morphing, biomolecule-delivering and rBMSC-laden scaffolds were created via 4D bioprinting. Such scaffolds were designed as a bilayer structure: the first layer was composed of PDLLA-co-TMC polymer that provided self-folding ability, while the second layer was composed of GelMA hydrogel containing biomolecules and gelatin/GelMA hydrogel containing rBMSCs that gave bio-functionalities. The bilayer scaffolds could fold automatically into tubular structures in physiological condition. Furthermore, released biomolecules could induce the differentiation of rBMSCs towards smooth muscle cells. Such advanced scaffolds are highly attractive for regenerating tubular body tissues.