Methacrylated Gelatin as a Scaffold for Mechanically Isolated Stromal Vascular Fraction for Cutaneous Wound Repair.

Mechanically processed stromal vascular fraction (mSVF) is a highly interesting cell source for regenerative purposes, including wound healing, and a practical alternative to enzymatically isolated SVF. In the clinical context, SVF benefits from scaffolds that facilitate viability and other cellular properties. In the present work, the feasibility of methacrylated gelatin (GelMA), a stiffness-tunable, light-inducible hydrogel with high biocompatibility is investigated as a scaffold for SVF in an in vitro setting. Lipoaspirates from elective surgical procedures were collected and processed to mSVF and mixed with GelMA precursor solutions. Non-encapsulated mSVF served as a control. Viability was measured over 21 days. Secreted basic fibroblast growth factor (bFGF) levels were measured on days 1, 7 and 21 by ELISA. IHC was performed to detect VEGF-A, perilipin-2, and CD73 expression on days 7 and 21. The impact of GelMA-mSVF on human dermal fibroblasts was measured in a co-culture assay by the same viability assay. The viability of cultured GelMA-mSVF was significantly higher after 21 days (p < 0.01) when compared to mSVF alone. Also, GelMA-mSVF secreted stable levels of bFGF over 21 days. While VEGF-A was primarily expressed on day 21, perilipin-2 and CD73-positive cells were observed on days 7 and 21. Finally, GelMA-mSVF significantly improved fibroblast viability as compared with GelMA alone (p < 0.01). GelMA may be a promising scaffold for mSVF as it maintains cell viability and proliferation with the release of growth factors while facilitating adipogenic differentiation, stromal cell marker expression and fibroblast proliferation.

Supramolecular Reinforcement of Polymer-Nanoparticle Hydrogels for Modular Materials Design.

Moldable hydrogels are increasingly used as injectable or extrudable materials in biomedical and industrial applications owing to their ability to flow under applied stress (shear-thin) and reform a stable network (self-heal). Nanoscale components can be added to dynamic polymer networks to modify their mechanical properties and broaden the scope of applications. Viscoelastic polymer-nanoparticle (PNP) hydrogels comprise a versatile and tunable class of dynamic nanocomposite materials that form via reversible interactions between polymer chains and nanoparticles. However, PNP hydrogel formation is restricted to specific interactions between select polymers and nanoparticles, resulting in a limited range of mechanical properties and constraining their utility. Here, a facile strategy to reinforce PNP hydrogels through the simple addition of α-cyclodextrin (αCD) to the formulation is introduced. The formation of polypseudorotoxanes between αCD and the hydrogel components resulted in a drastic enhancement of the mechanical properties. Furthermore, supramolecular reinforcement of CD-PNP hydrogels enabled decoupling of the mechanical properties and material functionality. This allows for modular exchange of structural components from a library of functional polymers and nanoparticles. αCD supramolecular binding motifs are leveraged to form CD-PNP hydrogels with biopolymers for high-fidelity 3D (bio)printing and drug delivery as well as with inorganic NPs to engineer magnetic or conductive materials.

Development of alginate dialdehyde-gelatin based bioink with methylcellulose for improving printability.

This study used methylcellulose (MC) to improve the printability of the alginate dialdehyde-gelatin (ADA-GEL) based bioink. The printability as well as the capability to maintain shape fidelity of ADA-GEL could be enhanced by the addition of 9% (w/v) MC. Moreover, the properties of the ink crosslinked with Ca2+ and Ba2+ were investigated. The samples crosslinked with Ba2+ were more stable and stiffer than the Ca2+ crosslinked samples. However, both Ca2+ and Ba2+ crosslinked samples exhibited a similar trend of MC release during incubation under cell culture conditions. The toxicity test indicated that both samples (crosslinked with Ca2+ and Ba2+) exhibited no toxic potential. The fabrication of cell-laden constructs using the developed bioinks was evaluated. The viability of ST2 cells in Ba2+ crosslinked samples increased while for Ca2+ crosslinked samples, a decreased viability was observed over the incubation time. After 21 days, cell spreading in the hydrogels crosslinked with Ba2+ occurred. However, a certain degree of cell damage was observed after incorporating the cells in the high viscosity bioink.

Hierarchical biomaterials via photopatterning-enhanced direct ink writing.

Recent advances in additive manufacturing (AM) technologies provide tools to fabricate biological structures with complex three-dimensional (3D) organization. Deposition-based approaches have been exploited to manufacture multimaterial constructs. Stimulus-triggered approaches have been used to fabricate scaffolds with high resolution. Both features are useful to produce biomaterials that mimic the hierarchical organization of human tissues. Recently, multitechnology biofabrication approaches have been introduced that integrate benefits from different AM techniques to enable more complex materials design. However, few methods allow for tunable properties at both micro- and macro-scale in materials that are conducive for cell growth. To improve the organization of biofabricated constructs, we integrated direct ink writing (DIW) with digital light processing (DLP) to form multimaterial constructs with improved spatial control over final scaffold mechanics. Polymer-nanoparticle hydrogels were combined with methacryloyl gelatin (GelMA) to engineer dual inks that were compatible with both DIW and DLP. The shear-thinning and self-healing properties of the dual inks enabled extrusion-based 3D printing. The inclusion of GelMA provided a handle for spatiotemporal control of cross-linking with DLP. Exploiting this technique, complex multimaterial constructs were printed with defined mechanical reinforcement. In addition, the multitechnology approach was used to print live cells for biofabrication applications. Overall, the combination of DIW and DLP is a simple and efficient strategy to fabricate hierarchical biomaterials with user-defined control over material properties at both micro- and macro-scale.

Cell-laden alginate dialdehyde-gelatin hydrogels formed in 3D printed sacrificial gel.

Alginate dialdehyde-gelatin (ADA-GEL) hydrogels have been reported to be suitable matrices for cell encapsulation. In general, application of ADA-GEL as bioink has been limited to planar structures due to its low viscosity. In this work, ring shaped constructs of ADA-GEL hydrogel were fabricated by casting the hydrogel into sacrificial molds which were 3D printed from 9% methylcellulose and 5% gelatin. Dissolution of the supporting structure was observed during the 1st week of sample incubation. In addition, the effect of different crosslinkers (Ba2+ and Ca2+) on the physicochemical properties of ADA-GEL and on the behavior of encapsulated MG-63 cells was investigated. It was found that Ba2+ crosslinked network had more than twice higher storage modulus, and mass decrease to 70% during incubation compared to 42% in case of hydrogels crosslinked with Ca2+. In addition, faster increase in cell viability during incubation and earlier cell network formation were observed after Ba2+ crosslinking. No negative effects on cell activity due to the use of sacrificial materials were observed. The approach presented here could be further developed for cell-laden ADA-GEL bioink printing into complex 3D structures.