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Collagen, the primary structural protein of the extracellular matrix (ECM), is widely used in biomaterials for tissue engineering and repair. However, its limited mechanical properties constrain its use in load-bearing applications. Here, we present a strategy to fabricate dense collagen–silica hybrid hydrogels (16 wt.% collagen) that have high mechanical strength, flexibility, printability, and biofunctionality. Using borate-mediated templating, we synchronize the formation of nanostructured silica networks with collagen fibrillogenesis, and subsequently covalently bind the two phases. This approach yields a compressive modulus ∼1.5 MPa for the hybrid hydrogels with a water content as high as 85%. Characterization confirms that borates act transiently during processing, allowing network formation without being retained in the final material. The hybrid hydrogels support high cell viability, elongation, and alignment in 3D cultures, while in vivo, initially cell-free scaffolds, implanted subcutaneously display minimal inflammation, vascularized tissue integration, and controlled, cell-mediated degradation. Taken together, this work establishes a robust framework for creating printable, ECM-mimetic collagen–silica hybrids with nanoscale reinforcement, offering new opportunities in regenerative medicine and scaffold fabrication.

Extracellular vesicles (EVs) derived from corneal epithelial cells have shown great promise in promoting corneal wound healing and stromal regeneration, but they face challenges with rapid clearance from the eye. This study addresses these challenges by developing a biocompatible collagen-hydrogel sustained delivery system. We successfully isolated, purified, and characterized corneal epithelial EVs (CE-EVs), assessed their efficacy in corneal epithelial healing in vitro, and demonstrated their sustained delivery over 10 days followed by an on-demand release through enzymatic degradation of the hydrogel, which mimics the in vivo scenario. To develop a microscale understanding of the EV diffusion inside the hydrogel matrix, we probed the hydrogel network with several model compounds and nanoparticles by using advanced confocal microscopy analyses, followed by fitting our results to established diffusion models. Our findings suggest this innovative approach offers a safe and effective strategy to promote corneal wound healing. This technology has the potential to revolutionize corneal injury treatment and improve patient outcomes. Moreover, the possibility to tailor EV-release kinetics broadens the scope of EV research in clinical practices, as varying short- and long-term release profiles will be required for diverse medical applications.

Background: Technological constraints limit 3D printing of collagen structures with complex trabecular shapes. However, the Freeform Reversible Embedding of Suspended Hydrogels (FRESH) method may allow for precise 3D printing of porous collagen scaffolds that carry the potential for repairing critical size bone defects.

Methods: Collagen type I scaffolds mimicking trabecular bone were fabricated through FRESH 3D printing and compared either with 2D collagen coatings or with 3D-printed polyethylene glycol diacrylate (PEGDA) scaffolds. The porosity of the printed scaffolds was visualized by confocal microscopy, the surface geometry of the scaffolds was investigated by scanning electron microscopy (SEM), and their mechanical properties were assessed with a rheometer. The osteoconductive properties of the different scaffolds were evaluated for up to four weeks by seeding and propagation of primary human osteoblasts (hOBs) or SaOS-2 cells. Intracellular alkaline phosphatase (ALP) and lactate dehydrogenase (LDH) activities were measured, and cells colonizing scaffolds were stained for osteocalcin (OCN).

Results: The FRESH technique enables printing of constructs at the millimetre scale using highly concentrated collagen, and the creation of stable trabecular structures that can support the growth osteogenic cells. FRESH-printed collagen scaffolds displayed an intricate and fibrous 3D network, as visualized by SEM, whereas the PEGDA scaffolds had a smooth surface. Amplitude sweep analyses revealed that the collagen scaffolds exhibited predominantly elastic behaviour, as indicated by higher storage modulus values relative to loss modulus values, while the degradation rate of collagen scaffolds was greater than PEGDA. The osteoconductive properties of collagen scaffolds were similar to those of PEGDA scaffolds but superior to 2D collagen, as verified by cell culture followed by analysis of ALP/LDH activity and OCN immunostaining.

Conclusions: Our findings suggest that FRESH-printed collagen scaffolds exhibit favourable mechanical, degradation and osteoconductive properties, potentially outperforming synthetic polymers such as PEGDA in bone tissue engineering applications.

Phosphoserine (pSER) is a phosphorylated amino acid commonly found in non-collagenous bone proteins and is implicated in the regulation of calcium phosphate (CaP) mineral formation and interfacial interactions in native bone. Here, we soaked 3D printed collagen scaffolds, with or without prior mineralisation, in pSER and investigated the early cellular response by assessing pre-osteoblast viability through ATP content measurements at day 3. The scaffolds were further evaluated in a rat uni-cortical femoral defect model and analysed by micro-computed tomography (µCT) and histology after 6 weeks. In parallel, standard 2D cultures of MC3T3-E1 cells and rat mesenchymal stromal cells (rMSCs) were exposed to pSER, nanohydroxyapatite (nHA), or a combination of both to assess dose-dependent effects in the absence of a scaffold environment. Using a dose-dependent screening approach, we identified 0.05% pSER on mineralised collagen scaffolds as the concentration that resulted in the highest cell viability, whereas higher concentrations were cytotoxic. This concentration was further evaluated in a time-dependent setup over 7 days and again showed increased cell viability compared to untreated collagen scaffolds. Similar trends were observed in vivo where CaP-containing groups demonstrated a higher bone volume fraction than CaP-free collagen groups, and pSER-associated effects were detectable but less pronounced. Overall, the results indicate that collagen scaffolds combining pSER and CaP lead to a dose-dependent enhanced pre-osteoblast viability in vitro. Future work will explore how these pSER-modified collagen bioinks influence bone cell differentiation and aim to elucidate the mechanisms through which pSER supports bone regeneration in vivo.