Uni Ghent – Exploring the impact of volumetric additive manufacturing of photo-crosslinkable gelatin on mesenchymal stromal cell behavior and differentiation

Volumetric additive manufacturing (VAM) is an emerging 3D-printing technology that creates an object in one single step, rather than building it layer by layer. A rotating vial filled with liquid resin is illuminated from multiple angles, and wherever enough light overlaps, the resin solidifies. This approach is extremely fast, gentle and well suited for printing soft, bioactive materials. In our recent publication in Additive Manufacturing, our team – Nele Pien, Bryan Bogaert, Marguerite Meeremans, Cezar-Stefan Popovici, Peter Dubruel, Catharina De Schauwer and Sandra Van Vlierberghe – explored how VAM can be combined with cell-friendly gelatin-based hydrogels to guide the behavior and fate of mesenchymal stromal cells (MSCs).

We developed photo-crosslinkable gelatin bioresins based on thiolated gelatin (Gel-SH) and gelatin-norbornene (Gel-NB). By tuning the degree of substitution (DS) and concentration of these components, we were able to adjust stiffness, swelling and degradation of the resulting hydrogels over a wide range, while keeping them highly biocompatible. Using VAM printing, we could rapidly fabricate complex cm³-scale constructs with features down to a few hundred micrometers.

Next, we compared two ways of making MSC-laden scaffolds from the same material system: conventional film-casting versus VAM printing. In both approaches, equine adipose-derived MSCs were mixed homogeneously into the bioresin before crosslinking. This allowed us to directly assess how the fabrication route – and the mechanical properties it gave rise to – influenced cell behavior. Cell viability and metabolic activity remained high for at least 21 days in both film-cast and VAM-printed hydrogels, demonstrating that the printing conditions and light exposure are fully compatible with living cells.

 

Schematic overview of our approach: development of photo-crosslinkable gelatin bioresins, tomographic volumetric 3D (bio)printing, and subsequent differentiation of encapsulated mesenchymal stromal cells into bone, cartilage or adipogenic lineages. Data from Pien et al., Additive Manufacturing 109 (2025) 104850.

We then investigated how the different hydrogels guided MSC differentiation towards bone, cartilage or adipogenic lineages, using lineage-specific osteogenic, chondrogenic and adipogenic media alongside a standard expansion medium. VAM-printed constructs were mechanically stiffer and, in osteogenic medium, showed strongly enhanced osteogenesis with higher alkaline phosphatase activity and calcium deposition. Softer film-cast hydrogels instead favored chondrogenic and adipogenic outcomes, with increased glycosaminoglycans, lipid accumulation and chondrogenic markers. These results illustrate that MSC fate is never dictated by one single parameter, but by the combination of multiple actors, including substrate stiffness and architecture, surface topography, physico-mechanical loading and biomolecular cues such as TGF-β signaling or protein tethering.

The inherent differences in crosslinking density and mechanics between VAM-printed and film-cast scaffolds therefore highlight that material composition alone does not tell the full story. Our gelatin system is highly tunable: softer VAM gels could be obtained by adapting the chemistry (degree of substitution, gelatin concentration), while film casting could be driven towards stiffer constructs by increasing the DS or concentrations, or through extended irradiation or post-curing. Yet, when it comes to fabricating true 3D scaffolds, VAM clearly stands out, uniquely offering complex architectures and excellent design freedom. This opens perspectives for advanced in vitro models, patient-specific bone grafts or hybrid implants that combine stiff, load-bearing regions with softer cartilage- or adipose-like zones. Because VAM is fast and scalable, libraries of scaffold designs and formulations can be printed and screened in parallel, laying the foundation for future studies that further optimize differentiation across multiple lineages and firmly position tomographic VAM at the interface of 3D printing, biomaterials and regenerative medicine.