Calciprotein particles (CPPs) are essential circulating scavengers of excessive Ca²⁺ and PO₄³⁻ ions, representing a vehicle that removes them from the human body and precludes extraskeletal calcification. Having been internalised by endothelial cells (ECs), CPPs induce their dysfunction, which is accompanied by a remarkable molecular reconfiguration, although little is known about this process’s extracellular signatures. Here, we applied ultra-high performance liquid chromatography-tandem mass spectrometry to perform a secretome-wide profiling of the cell culture supernatant from primary human coronary artery ECs (HCAECs) and internal thoracic artery ECs (HITAECs) treated with primary CPPs (CPP-P), secondary CPPs (CPP-S), magnesiprotein particles (MPPs), or Ca²⁺/Mg²⁺-free Dulbecco’s phosphate-buffered saline (DPBS) for 24 h. Incubation with CPP-P/CPP-S significantly altered the profiles of secreted proteins, delineating physiological and pathological endothelial secretomes. Neither pathway enrichment analysis nor the interrogation of protein–protein interactions detected extracellular matrix- and basement membrane-related molecular terms in the protein datasets from CPP-P/CPP-S-treated ECs. Both proteomic profiling and enzyme-linked immunosorbent assay identified an increased level of protectin (CD59) and reduced levels of osteonectin (SPARC), perlecan (HSPG2), and fibronectin (FN1) in the cell culture supernatant upon CPP-P/CPP-S treatment. Elevated soluble CD59 and decreased release of basement membrane components might be considered as potential signs of dysfunctional endothelium.

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.

Osteogenic differentiation is crucial in normal bone formation and pathological calcification, such as calcific aortic valve disease (CAVD). Understanding the proteomic and transcriptomic landscapes underlying this differentiation can unveil potential therapeutic targets for CAVD. In this study, we employed RNA sequencing transcriptomics and proteomics on a timsTOF Pro platform to explore the multiomics profiles of valve interstitial cells (VICs) and osteoblasts during osteogenic differentiation. For proteomics, we utilized 3 data acquisition/analysis techniques: data-dependent acquisition (DDA)–parallel accumulation serial fragmentation (PASEF) and data-independent acquisition (DIA)–PASEF with a classic library-based (DIA) and machine learning–based library-free search (DIA-ML). Using RNA sequencing data as a biological reference, we compared these 3 analytical techniques in the context of actual biological experiments. We use this comprehensive dataset to reveal distinct proteomic and transcriptomic profiles between VICs and osteoblasts, highlighting specific biological processes in their osteogenic differentiation pathways. The study identified potential therapeutic targets specific for VICs osteogenic differentiation in CAVD, including the MAOA and ERK1/2 pathway. From a technical perspective, we found that DIA-based methods demonstrate even higher superiority against DDA for more sophisticated human primary cell cultures than it was shown before on HeLa samples. While the classic library-based DIA approach has proved to be a gold standard for shotgun proteomics research, the DIA-ML offers significant advantages with a relatively minor compromise in data reliability, making it the method of choice for routine proteomics.

Ocular surface reconstruction is essential for treating corneal epithelial defects and vision recovery. Stem cell-based therapy demonstrates promising results but requires further research to elucidate stem cell survival, growth, and differentiation after transplantation in vivo. This study examined the corneal reconstruction promoted by EGFP-labeled limbal mesenchymal stem cells (L-MSCs-EGFP) and their fate after transplantation. EGFP labeling allowed us to evaluate the migration and survival rates of the transferred cells. L-MSCs-EGFP seeded onto decellularized human amniotic membrane (dHAM) were transplanted into rabbits with a modeled limbal stem cell deficiency. The localization and viability of the transplanted cells in animal tissue were analyzed using histology, immunohistochemistry, and confocal microscopy up to 3 months after transplantation. EGFP-labeled cells remained viable for the first 14 days after transplantation. By the 90th day, epithelialization of the rabbit corneas reached 90%, but the presence of viable labeled cells was not observed within the newly formed epithelium. Although labeled cells demonstrated low survivability in host tissue, the squamous corneal-like epithelium was partially restored by the 30th day after transplantation of the tissue-engineered graft. Overall, this study paves the way for further optimization of transplantation conditions and studying the mechanisms of corneal tissue restoration.

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.

The aim of this meta-analysis — to compare clinical outcomes and complications of the reconstruction of extensive long-bone defects of the limbs of various etiologies in patients older than 18 years using the Masquelet technique, the Ilizarov bone transport, and reconstructive procedures with vascularized bone flaps. Methods. Publications were searched in the eLIBRARY, PubMed, Embase, Cochrane Central Register of Controlled Trials, and Google Scholar databases for the period 2015–2025. Randomized controlled trials and cohort studies addressing the reconstruction of extensive bone defects of the limbs using different techniques (the Masquelet technique (MT), bone transport (BT), and free vascularized flaps (FVF), particulary the fibular graft) were included in the analysis. Results. Nine publications involving a total of 375 patients were included in the meta-analysis to assess the effectiveness and safety of the techniques. The majority of cases involved long bones of the lower limbs (n = 332; 87%), most commonly the tibia (n = 284; 74%), followed by the femur (n = 48; 13%). Among upper limb injuries, extensive bone defects of the forearm were reported in 44 cases (12%) and of the humerus in 4 cases (1%). The overall duration of fixation did not differ significantly between MT and FVF groups, nor between MT and BT groups (p = 0.76 and p = 0.40, respectively). The proportion of patients achieving complete bone union without additional surgical interventions was significantly higher with BT compared to MT (86% vs. 61%, p = 0.02). The analysis of surgical site infection (SSI) rates revealed the following patterns: the proportion of patients with superficial SSI was significantly lower with MT compared to BT (11% vs. 23%, p = 0.0004). Conversely, the incidence of deep SSI was significantly higher with MT compared to BT (15% vs. 8%, p = 0.04). No statistically significant differences were found regarding nonunion rates or the conversion of surgical treatment to amputation. The proportion of patients with excellent or good outcomes according to the ASAMI score was 85% for MT and 71% for BT; however, the difference was not statistically significant (p = 0.48). Conclusion. The systematic literature review confirmed the effectiveness of all three methods: the Masquelet technique, the Ilizarov bone transport, and free vascularized flaps. The meta-analysis did not reveal a significant advantage of any single method in terms of fixation time or time to union; however, distinct safety profiles were identified and should be considered when selecting the surgical strategy. When choosing a technique for the reconstruction of large segmental bone defects, it is essential to take into account the defect location and length, soft tissue status, the presence and activity of infection, as well as individual patient characteristics, particularly treatment adherence and expectations regarding the quality of life.