Marko Mihajlovic

Electrowriting of rheologically optimized silk fibroin solutions allows controlled fabrication of microfiber scaffolds. A salt treatment induces scaffold gelation, imparting low stiffness and high mechanical stability to guide renal cell growth.

Despite extensive research, current methods for creating three-dimensional (3D) silk fibroin (SF) scaffolds lack control over molecular rearrangement, particularly in the formation of β-sheet nanocrystals, as well as hierarchical fiber organization at both micro- and macroscale. In this study, we introduce a fabrication process based on electrowriting of aqueous SF-based solutions followed by post-processing using an aqueous solution of sodium dihydrogen phosphate (NaH ₂ PO ₄ ). This approach enables hierarchical assembly of SF chains via β-sheet and α-helix formation. Moreover, this process allows for precise control over micro- and macro-architectures in microfiber scaffolds, enabling the creation of 3D flat and tubular macrogeometries with square-based and crosshatch microarchitectures, featuring inter-fiber distances of 400 µm and approximately 97% open porosity. Remarkably, the printed structures demonstrated restored β-sheet and α-helix structures, which imparted an elastic response of up to 20% deformation and the ability to support cyclic loading without plastic deformation. Furthermore, the printed constructs supported in vitro adherence and growth of human conditionally immortalized proximal tubular epithelial cells and glomerular endothelial cells, with cell viability above 95%. These cells formed uniform, aligned monolayers that deposited their own extracellular matrix. These findings represent a significant development in fabricating organized SF scaffolds with unique fiber structures, mechanical and biological properties, making them highly promising for regenerative medicine applications.

For three-dimensional (3D) bioprinting to fulfill its promise and enable the automated fabrication of complex tissue-mimicking constructs, there is a need for developing bioinks that are not only printable and biocompatible but also have integrated cell-instructive properties. Toward this goal, we here present a scalable technique for generating nanofiber 3D printing inks with unique tissue-guiding capabilities. Our core methodology relies on tailoring the size and dispersibility of cellulose fibrils through a solvent-controlled partial carboxymethylation. This way, we generate partially negatively charged cellulose nanofibers with diameters of ∼250 nm and lengths spanning tens to hundreds of microns. In this range, the fibers structurally match the size and dimensions of natural collagen fibers making them sufficiently large to orient cells. Yet, they are simultaneously sufficiently thin to be optically transparent. By adjusting fiber concentration, 3D printing inks with excellent shear-thinning properties can be established. In addition, as the fibers are readily dispersible, composite inks with both carbohydrates and extracellular matrix (ECM)-derived proteins can easily be generated. We apply such composite inks for 3D printing cell-laden and cross-linkable structures, as well as tissue-guiding gel substrates. Interestingly, we find that the spatial organization of engineered tissues can be defined by the shear-induced alignment of fibers during the printing procedure. Specifically, we show how myotubes derived from human and murine skeletal myoblasts can be programmed into linear and complex nonlinear architectures on soft printed substrates with intermediate fiber contents. Our nanofibrillated cellulose inks can thus serve as a simple and scalable tool for engineering anisotropic human muscle tissues that mimic native structure and function.

The polarization of neurons into axons and dendrites depends on extracellular cues, intracellular signaling, cytoskeletal rearrangements, and polarized transport, but the interplay between these processes during polarization remains unresolved. Here, we show that axon specification is determined by differences in microtubule network mobility between neurites, regulated by Rho guanosine triphosphatases (GTPases) and extracellular cues. In developing neurons, retrograde microtubule flow prevents the entry of the axon-selective motor protein Kinesin-1 into most neurites. Using inducible assays to control microtubule network flow, we demonstrate that local inhibition of microtubule mobility is sufficient to guide Kinesin-1 into a specific neurite, whereas long-term global inhibition induces the formation of multiple axons. We furthermore show that extracellular mechanical cues and intracellular Rho GTPase signaling control the local differences in microtubule network flow. These results reveal a novel cytoskeletal mechanism for neuronal polarization.

The load-bearing function of articular cartilage tissue contrasts with the poor load-bearing capacity of most soft hydrogels used for its regeneration. The present study explores whether a hydrogel based on the methacrylated natural polymers chondroitin sulfate (CSMA) and hyaluronic acid (HAMA), injected into warp-knitted spacer fabrics, could be used to create a biomimetic construct with cartilage-like mechanical properties. The swelling ratio of the combined CSMA/HAMA hydrogels in the first 20 days was higher for hydrogels with a higher CSMA concentration, and these hydrogels also degraded quicker, whereas those with a 1.33 wt% of HAMA were stable for more than 120 days. When confined by a polyamide 6 (PA6) spacer fabric, the volumetric swelling of the combined CSMA/HAMA gels (10 wt%, 6.5 × CSMA:HAMA ratio) was reduced by ~53%. Both the apparent peak and the equilibrium modulus significantly increased in the PA6-restricted constructs compared to the free-swelling hydrogels after 28 days of swelling, and no significant differences in the moduli and time constant compared to native bovine cartilage were observed. Moreover, the cell viability in the CSMA/HAMA PA6 constructs was comparable to that in gelatin–methacrylamide (GelMA) PA6 constructs at one day after polymerization. These results suggest that using a HydroSpacer construct with an extracellular matrix (ECM)-like biopolymer-based hydrogel is a promising approach for mimicking the load-bearing properties of native cartilage.

Retinal diseases are the leading cause of visual impairment worldwide. The effectiveness of antibodies for the treatment of retinal diseases has been demonstrated. Despite the clinical success, achieving sufficiently high concentrations of these protein therapeutics at the target tissue for an extended period is challenging. Patients suffering from macular degeneration often receive injections once per month. Therefore, there is a growing need for suitable systems that can help reduce the number of injections and adverse effects while improving patient complacency. This study systematically characterized degradable "in situ" forming hydrogels that can be easily injected into the vitreous cavity using a small needle (29G). After intravitreal injection, the formulation is designed to undergo a sol-gel phase transition at the administration site to obtain an intraocular depot system for long-term sustained release of bioactives. A Diels-Alder reaction was exploited to crosslink hyaluronic acid-bearing furan groups (HAFU) with 4 arm-PEG10K-maleimide (4APM), yielding stable hydrogels. Here, a systematic investigation of the effects of polymer composition and the ratio between functional groups on the physicochemical properties of hydrogels was performed to select the most suitable formulation for protein delivery. Rheological analysis showed rapid hydrogel formation, with the fastest gel formation within 5 min after mixing the hydrogel precursors. In this study, the mechanical properties of an ex vivo intravitreally formed hydrogel were investigated and compared to the in vitro fabricated samples. Swelling and degradation studies showed that the hydrogels are biodegradable by the retro-Diels-Alder reaction under physiological conditions. The 4APM-HAFU (ratio 1:5) hydrogel formulation showed sustained release of bevacizumab > 400 days by a combination of diffusion, swelling, and degradation. A bioassay showed that the released bevacizumab remained bioactive. The hydrogel platform described in this study offers high potential for the sustained release of therapeutic antibodies to treat ocular diseases.

We present a novel neural implicit representation for articulated human bodies. Compared to explicit template meshes, neural implicit body representations provide an efficient mechanism for modeling interactions with the environment, which is essential for human motion reconstruction and synthesis in 3D scenes. However, existing neural implicit bodies suffer from either poor generalization on highly articulated poses or slow inference time. In this work, we observe that prior knowledge about the human body's shape and kinematic structure can be leveraged to improve generalization and efficiency. We decompose the full-body geometry into local body parts and employ a part-aware encoder-decoder architecture to learn neural articulated occupancy that models complex deformations locally. Our local shape encoder represents the body deformation of not only the corresponding body part but also the neighboring body parts. The decoder incorporates the geometric constraints of local body shape which significantly improves pose generalization. We demonstrate that our model is suitable for resolving self-intersections and collisions with 3D environments. Quantitative and qualitative experiments show that our method largely outperforms existing solutions in terms of both efficiency and accuracy. The code and models are available at https://neuralbodies.github.io/COAP/index.html

Soft materials, such as hydrogels, are used as scaffolds in tissue engineering and regenerative medicine to help tissues regenerate and heal. Recently, supramolecular hydrogels, based on non-covalent interactions, have grown in popularity, especially in the development of materials for biomedical use. Their potential lies in the dynamic, reversible and temporary nature of their crosslinks, which can make them responsive to stimuli, injectable and suitable for 3D printing. Such versatility and processability is important when developing new biomaterials for drug delivery or as implantable scaffolds. The behavior and properties of such hydrogels are different compared to those of chemically crosslinked hydrogels. In this review, we give an overview on supramolecular hydrogels which contain hyaluronic acid (HA) as one of the building blocks. HA is particularly interesting, due to its hydrophilicity, biofunctionality and ease of chemical modification. Specifically, we focus on HA-based hydrogels that make use of hydrogen bonding, hydrophobic interactions, electrostatic interactions, metal–ion coordination and guest–host interactions, and are intended for applications in the biomedical field, with potential for clinical translation.