Hydrogel-supported neural cell cultures are more in vivo-relevant compared to monolayers formed on glass or plastic substrates. However, there is a lack of synthetic microenvironment available for obtaining standardized and easily reproducible cultures characterized by tissue-mimicking cell composition, cell–cell interactions, and functional networks. Synthetic peptides representing the biological properties of the extracellular matrix (ECM) proteins have been reported to promote the adhesion-driven differentiation and functional maturation of neural cells. Thus, such peptides can serve as building blocks for engineering a standardized, all-synthetic environment. In this study, we have compared the effect of two chemically crosslinked hydrogel compositions on primary cerebellar cells: collagen-like peptide (CLP), and CLP with an integrin-binding motif arginine-glycine-aspartate (CLP-RGD), both conjugated to polyethylene glycol molecular templates (PEG-CLP and PEG-CLP-RGD, respectively) and fabricated as self-supporting membranes. Both compositions promoted a spontaneous organization of primary cerebellar cells into tissue-like clusters with fast-rising Ca²⁺ signals in soma, reflecting action potential generation. Notably, neurons on PEG-CLP-RGD had more neurites and better synaptic efficiency compared to PEG-CLP. For comparison, poly-L-lysine-coated glass and plastic surfaces did not induce formation of such spontaneously active networks. Additionally, contrary to the hydrogel membranes, glass substrates functionalized with PEG-CLP and PEG-CLP-RGD did not sufficiently support cell attachment and, subsequently, did not promote functional cluster formation. These results indicate that not only chemical composition but also the hydrogel structure and viscoelasticity are essential for bioactive signaling. The synthetic strategy based on ECM-mimicking, multifunctional blocks in registry with chemical crosslinking for obtaining tissue-like mechanical properties is promising for the development of fast and well standardized functional in vitro neural models and new regenerative therapies.

Purpose: To determine feasibility of plant-derived recombinant human collagen type I (RHCI) for use in corneal regenerative implants Methods: RHCI was crosslinked with 1-ethyl-3-(3-dimethyl aminopropyl) carbodiimide (EDC) and N-hydroxysuccinimide (NHS) to form hydrogels. Application of shear force to liquid crystalline RHCI aligned the collagen fibrils. Both aligned and random hydrogels were evaluated for mechanical and optical properties, as well as in vitro biocompatibility. Further evaluation was performed in vivo by subcutaneous implantation in rats and corneal implantation in Göttingen minipigs. Results: Spontaneous crosslinking of randomly aligned RHCI (rRHCI) formed robust, transparent hydrogels that were sufficient for implantation. Aligning the RHCI (aRHCI) resulted in thicker collagen fibrils forming an opaque hydrogel with insufficient transverse mechanical strength for surgical manipulation. rRHCI showed minimal inflammation when implanted subcutaneously in rats. The corneal implants in minipigs showed that rRHCI hydrogels promoted regeneration of corneal epithelium, stroma, and nerves; some myofibroblasts were seen in the regenerated neo-corneas. Conclusion: Plant-derived RHCI was used to fabricate a hydrogel that is transparent, mechanically stable, and biocompatible when grafted as corneal implants in minipigs. Plant-derived collagen is determined to be a safe alternative to allografts, animal collagens, or yeast-derived recombinant human collagen for tissue engineering applications. The main advantage is that unlike donor corneas or yeast-produced collagen, the RHCI supply is potentially unlimited due to the high yields of this production method. Lay Summary: A severe shortage of human-donor corneas for transplantation has led scientists to develop synthetic alternatives. Here, recombinant human collagen type I made of tobacco plants through genetic engineering was tested for use in making corneal implants. We made strong, transparent hydrogels that were tested by implanting subcutaneously in rats and in the corneas of minipigs. We showed that the plant collagen was biocompatible and was able to stably regenerate the corneas of minipigs comparable to yeast-produced recombinant collagen that we previously tested in clinical trials. The advantage of the plant collagen is that the supply is potentially limitless.

A non-raster scanning technique for atomic force microscopy (AFM) imaging which combines rotational and translational motion is presented. The use of rotational motion for the fast scan axis allows us to significantly increase the scanning speed while imaging a large area (diameter > 30 μ m). An image reconstruction algorithm and the factors influencing the resolution of the technique are discussed. The experimental results show the potential of the rotational scanning technique for high-throughput large area AFM investigation.

Increasing the imaging rate of atomic force microscopy (AFM) without impairing of the imaging quality is a challenging task, since the increase in the scanning speed leads to a number of artifacts related to the limited mechanical bandwidth of the AFM components. One of these artifacts is the loss of contact between the probe tip and the sample. We propose to apply an additional nonlinear force on the upper surface of a cantilever, which will help to keep the tip and surface in contact. In practice, this force can be produced by the precisely regulated airflow. Such an improvement affects the AFM system dynamics, which were evaluated using a mathematical model that is presented in this paper. The model defines the relationships between the additional nonlinear force, the pressure of the applied air stream, and the initial air gap between the upper surface of the cantilever and the end of the air duct. It was found that the nonlinear force created by the stream of compressed air (aerodynamic force) prevents the contact loss caused by the high scanning speed or the higher surface roughness, thus maintaining stable contact between the probe and the surface. This improvement allows us to effectively increase the scanning speed by at least 10 times using a soft (spring constant of 0.2 N/m) cantilever by applying the air pressure of 40 Pa. If a stiff cantilever (spring constant of 40 N/m) is used, the potential of vertical deviation improvement is twice is large. This method is suitable for use with different types of AFM sensors and it can be implemented practically without essential changes in AFM sensor design.

Neuronal-glial cell cultures are usually grown attached to or encapsulated in an adhesive environment as evenly distributed networks lacking tissue-like cell density, organization and morphology. In such cultures, microglia have activated amoeboid morphology and do not display extended and intensively branched processes characteristic of the ramified tissue microglia. We have recently described self-assembling functional cerebellar organoids promoted by hydrogels containing collagen-like peptides (CLPs) conjugated to a polyethylene glycol (PEG) core. Spontaneous neuronal activity was accompanied by changes in the microglial morphology and behavior, suggesting the cells might play an essential role in forming the functional neuronal networks in response to the peptide signalling. The present study examines microglial cell morphology and function in cerebellar cell organoid cultures on CLP-PEG hydrogels and compares them to the cultures on crosslinked collagen hydrogels of similar elastomechanical properties. Material characterization suggested more expressed fibril orientation and denser packaging in crosslinked collagen than CLP-PEG. However, CLP-PEG promoted a significantly higher microglial motility (determined by time-lapse imaging) accompanied by highly diverse morphology including the ramified (brightfield and confocal microscopy), more active Ca²⁺ signalling (intracellular Ca²⁺ fluorescence recordings), and moderate inflammatory cytokine level (ELISA). On the contrary, on the collagen hydrogels, microglial cells were significantly less active and mostly round-shaped. In addition, the latter hydrogels did not support the neuron synaptic activity. Our findings indicate that the synthetic CLP-PEG hydrogels ensure more tissue-like microglial morphology, motility, and function than the crosslinked collagen substrates.