Sometimes, to move forward, it is necessary to look back. Collagen type I is one of the most commonly used biomaterials in tissue engineering and regenerative medicine. There are a variety of collagen scaffolds and biomedical products based on collagen have been made, and the development of new ones is still ongoing. Materials, where collagen is in the fibrillar form, have some advantages: they have superior mechanical properties, higher degradation time and, what is most important, mimic the structure of the native extracellular matrix. There are some standard protocols for the formation of collagen fibrils in vitro, but if we look more carefully at those methods, we can see some controversies. For example, why is the formation of collagen gel commonly carried out at 37 ​°C, when it was well investigated that the temperature higher than 35 ​°C results in a formation of not well-ordered fibrils? Biomimetic collagen materials can be obtained both using culture medium or neutralizing solution, but it requires a deep understanding of all of the crucial points. One of this point is collagen extraction method, since not every method retains the ability of collagen to reconstitute native banded fibrils. Collagen polymorphism is also often overlooked in spite of the appearance of different polymorphic forms during fibril formation is possible, especially when collagen blends are utilized. In this review, we will not only pay attention to these issues, but we will overview the most prominent works related to the formation of collagen fibrils in vitro starting from the first approaches and moving to the up-to-date recipes.

The functioning of the human cornea heavily relies on the maintenance of its extracellular matrix (ECM) mechanical properties. Within this context, corneal stromal fibroblasts (CSFs) are essential, as they are responsible for remodeling the corneal ECM. In this study, we used a decellularized human amniotic membrane (dHAM) and a custom fibrillar collagen film (FCF) to explore the effects of fibrillar materials on human CSFs. Our findings indicate that substrates like FCF can enhance the early development of focal adhesions (FAs), leading to the activation and propagation of mechanotransduction signals. This is primarily achieved through FAK autophosphorylation and YAP1 nuclear translocation pathways. Remarkably, inhibiting FAK autophosphorylation negated the observed changes. Proteome analysis further confirmed the central role of FAs in mechanotransduction propagation in CSFs cultured on FCF. This analysis also highlighted complex signaling pathways, including chromatin epigenetic modifications, in response to fibrillar substrates. Overall, our research highlights the potential pathways through which CSFs undergo behavioral changes when exposed to fibrillar substrates, identifying FAs as essential mechanotransducers.

A large number of artificial tears are currently available in the pharmaceutical market. Selecting the right drug for the patient remains a challenge for both the doctor and the patient. Comparing the cytotoxicity of artificial tears is one of the criteria for the rational selection of a drug that promotes maximum clinical efficacy and a higher safety profile. It is known that cells grown in vitro retain many metabolic features of the parent host tissues and at the same time lack tissue and organ interrelations and regulatory effects of the nervous and endocrine systems and have very limited compensatory capabilities. These features of cell cultures provide an opportunity to investigate the interaction of chemical agents directly with the cell itself, to identify changes in cellular and subcellular structures that can be masked in whole-organism settings. This study presents the results of assessing the cytotoxicity of tear substitutes, which demonstrate that these drugs can have a cytostatic effect in vitro and differ in their cytotoxic potential. In recent years, the problem of drug therapy of patients with dry eye syndrome has been attracting increasing attention of ophthalmologists, so screening the cytotoxicity of a wide range of tear substitutes using cell culture-based test systems can promote the rational selection of these drugs.

The effect of the concentration of glutaraldehyde, which has the ability to crosslink collagen type I molecules, on its stability, mechanical strength, and cytotoxicity was studied. It is shown that under physiological conditions, collagen containing 10 and 18% glutaraldehyde possesses the greatest stability. The results of the rheological analysis showed that with a minimum concentration of glutaraldehyde (1%), the mechanical characteristics of collagen hydrogels are significantly improved. However, a concentration of glutaraldehyde above 10% reduces cell viability by 50%.