The development of cell-based approaches to the treatment of various cornea pathologies, including limbal stem cell deficiency (LSCD), is an area of current interest in regenerative biomedicine. In this context, the shortage of donor material is urgent, and limbal mesenchymal stem cells (L-MSCs) may become a promising cell source for the development of these novel approaches, being established mainly within the rabbit model. In this study, we obtained and characterized rabbit L-MSCs and modified them with lentiviral transduction to express the green fluorescent protein EGFP (L-MSCs-EGFP). L-MSCs and L-MSCs-EGFP express not only stem cell markers specific for mesenchymal stem cells but also ABCG2, ABCB5, ALDH3A1, PAX6, and p63a specific for limbal epithelial stem cells (LESCs), as well as various cytokeratins (3/12, 15, 19). L-MSCs-EGFP have been proven to differentiate into adipogenic, osteogenic, and chondrogenic directions, as well as to transdifferentiate into epithelial cells. The possibility of using L-MSCs-EGFP to study the biocompatibility of various scaffolds developed to treat corneal pathologies was demonstrated. L-MSCs-EGFP may become a useful tool for studying regenerative processes occurring during the treatment of various corneal pathologies, including LSCD, with the use of cell-based technologies.

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.

This study examines the biocompatibility of 11 modern wound dressings (WDs)―Syspur-derm^®, Parapran^®, Lomatuell^®H, Voskopran^®, Metalline^®, Granuflex^®, Chitopran^®, HydroTac^®transparent, Branolind^®N, Aquacel^TM adhesive foam, Aquacel^TMAg⁺―developed for the treatment of acute and chronic wounds, and their potential use as secondary WD for the hydrogel-based bioengineered skin equivalent (BSE) “Equivalent Dermal, ED”. The study was conducted to better understand the properties of these WDs that influence the healing process. The biocompatibility of WDs was evaluated in vitro based on their effects on the viability of human dermal fibroblasts (DFs). The MTT assay, lifetime analysis of DFs’ morphological state, and analysis of their actin cytoskeletal organization using a WDs’ extracts showed that effects of WD on DFs varied among WDs. It has been revealed that WDs Parapran^®, Lomatuell^®H, Voskopran^®, Metalline^® and Chitopran^® have high biocompatibility and can be effectively used for wound treatment, whereas Granuflex^®, Syspyr-derm^®, HydroTac^® transparent, Branolind^®N, Aquacel^TM adhesive foam and Aquacel^TMAg⁺ have lower biocompatibility, so they could be used for wound therapy with caution. Only Parapran^® with chlorhexidine showed high biocompatibility with the BSE “The Dermal Equivalent, ED” and can be safely used in combination with it as a secondary WD.

The umbilical cord is a material that enhances regeneration and is devoid of age-related changes in the extracellular matrix (ECM). The aim of this work was to develop a biodegradable scaffold from a decellularized human umbilical cord (UC-scaffold) to heal full-thickness wounds. Decellularization was performed with 0.05% sodium dodecyl sulfate solution. The UC-scaffold was studied using morphological analysis methods. The composition of the UC-scaffold was studied using immunoblotting and Fourier transform infrared spectroscopy. The adhesion and proliferation of mesenchymal stromal cells were investigated using the LIVE/DEAD assay. The local reaction was determined by subcutaneous implantation in mice (n = 60). A model of a full-thickness skin wound in mice (n = 64) was used to assess the biological activity of the UC-scaffold. The proposed decellularization method showed its effectiveness in the umbilical cord, as it removed cells and retained a porous structure, type I and type IV collagen, TGF-β3, VEGF, and fibronectin in the ECM. The biodegradation of the UC-scaffold in the presence of collagenase, its stability during incubation in hyaluronidase solution, and its ability to swell by 1617 ± 120% were demonstrated. Subcutaneous scaffold implantation in mice showed gradual resorption of the product in vivo without the formation of a dense connective tissue capsule. Epithelialization of the wound occurred completely in contrast to the controls. All of these data suggest a potential for the use of the UC-scaffold.