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Tomographic volumetric bioprinting (VBP) enables fast photofabrication of cell‐laden hydrogel constructs in one step, addressing the limitations of conventional layer‐by‐layer additive manufacturing. However, existing biomaterials that fulfill the physicochemical requirements of VBP are limited to gelatin‐based photoresins of high polymer concentrations. The printed microenvironments are predominantly static and stiff, lacking sufficient capacity to support 3D cell growth. Here a dynamic resin based on thiol–ene photo‐clickable polyvinyl alcohol (PVA) and thermo‐sensitive sacrificial gelatin for fast VBP of functional ultrasoft cell‐laden hydrogel constructs within 7–15 s is reported. Using gelatin allows VBP of permissive hydrogels with low PVA contents of 1.5%, providing a stress‐relaxing environment for fast cell spreading, 3D osteogenic differentiation of embedded human mesenchymal stem cells and matrix mineralization. Additionally, site‐specific immobilization of molecules‐of‐interest inside a PVA hydrogel is achieved by 3D tomographic thiol–ene photopatterning. This technique may enable spatiotemporal control of cell‐material interactions and guides in vitro tissue formation using programmed cell‐friendly light. Altogether, this study introduces a synthetic dynamic photoresin enabling fast VBP of functional ultrasoft hydrogel constructs with well‐defined physicochemical properties and high efficiency.

Mechanical characterization of living cells undergoing substantial external strain promises insights into material properties and functional principles of mechanically active tissues. However, due to the high strains that occur in physiological situations (up to 50%) and the complex structure of living cells, suitable experimental techniques are rare. In this study, we introduce a new system composed of an atomic force microscope (AFM), a cell stretching system based on elastomeric substrates, and light microscopy. With this system, we investigated the influence of mechanical stretch on monolayers of keratinocytes. In repeated indentations at the same location on one particular cell, we found significant stiffening at 25% and 50% strain amplitude. To study the contribution of intermediate filaments, we used a mutant keratinocyte cell line devoid of all keratins. For those cells, we found a softening in comparison to the wild type, which was even more pronounced at higher strain amplitudes.

Abstract3D bioprinting has emerged as a powerful tool for custom fabrication of biomimetic hydrogel constructs that support the differentiation of stem cells into functional bone tissues. Existing stem cell-derived in vitro bone models, however, often lack terminally differentiated bone cells named osteocytes which are crucial for bone homeostasis. Here, we report ultrafast volumetric tomographic photofabrication of centimeter-scale heterocellular bone models that enabled successful 3D osteocytic differentiation of human mesenchymal stem cells (hMSCs) within hydrogels after 42 days co-culture with human umbilical vein endothelial cell (HUVECs). It is hypothesized that after 3D bioprinting the paracrine signaling between hMSCs and HUVECs will promote their differentiation into osteocytes while recreating the complex heterocellular bone microenvironment. To this, we formulated a series of bioinks with varying concentrations of gelatin methacryloyl (GelMA) and lithium Phenyl(2,4,6-trimethylbenzoyl)phosphinate (LAP). A bioink comprising 5% GelMA and 0.05% LAP was identified as an optimal material with high cell viability (>90%) and excellent structural fidelity. Increasing LAP concentration led to much lower degree of cell spreading, presumably due to phototoxicity effects. Biochemical assays evidenced significantly increased expression of both osteoblastic markers (collagen-I, ALP, osteocalcin) and osteocytic markers (Podoplanin, PDPN; dentin matrix acidic phosphoprotein 1, Dmp1) after 3D co-cultures for 42 days. Additionally, we demonstrate volumetric 3D bioprinting of perfusable, pre-vascularized bone models where HUVECs self-organized into an endothelium-lined channel within 2 days. Altogether, this work leverages the benefits of volumetric tomographic bioprinting and 3D co-culture, offering a promising platform for scaled biofabrication of 3D bone-like tissues with unprecedented long-term functionality.

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