Background. Chronic wounds pose a serious clinical and economic burden, and their effective treatment requires the development of new biomaterials that promote tissue regeneration. Acellular dermal matrices (ADMs) represent a promising regenerative strategy; however, their biological properties largely depend on the manufacturing protocol. Objective. To compare the biocompatibility and cytotoxicity of two porcine acellular dermal matrix variants, dry (d-ADM) and wet (w-ADM), and to evaluate the therapeutic efficacy of the biocompatible matrix in an experimental porcine model of chronic full-thickness wounds. Methods. Biocompatibility was assessed in vitro using human umbilical cord-derived mesenchymal stem cells (hU-MSCs) by evaluating cell morphology, viability, proliferation, and lactate dehydrogenase (LDH) release. The therapeutic efficacy of the selected ADM was investigated in a porcine chronic full-thickness excisional wound model and compared with non-adhesive dressings and Promogran Prisma. Wound healing was evaluated after 28 days using histological, immunohistochemical (TNF-α, TGF-β, Ki-67), and morphometric analyses. Results. The wet ADM preserved hU-MSC morphology, viability, and proliferative capacity, whereas the dry ADM induced marked cytotoxicity, increased LDH release, and extensive cell death. In vivo, treatment with w-ADM significantly accelerated wound healing compared with both control dressings, resulting in complete and stable re-epithelialization, minimal inflammatory infiltration, organized collagen remodeling, and improved tissue architecture. Immunohistochemical analysis demonstrated significantly lower expression of TNF-α, TGF-β, and Ki-67, indicating resolution of inflammation and transition toward tissue remodeling and regenerative stabilization. Conclusions. Wet acellular dermal matrix demonstrated excellent biocompatibility and superior regenerative performance, whereas the dry matrix exhibited unacceptable cytotoxicity. Wet ADM represents a promising scaffold for regenerative treatment of chronic wounds and warrants further investigation as a clinically applicable biomaterial, including in combination with mesenchymal stem cell-based therapies. Keywords: acellular dermal matrix, biocompatibility, chronic wounds, cytotoxicity, human umbilical mesenchymal stem cells, lyophilization, morphology
Background. Chronic kidney disease (CKD) is a progressive disorder that affects 10–13% of the worldwide population. The conventional biomarkers, like serum creatinine and estimated glomerular filtration rate (eGFR), have poor sensitivity in detecting early stage CKD, especially as it relates to Type 2 Diabetes Mellitus (T2DM). Recent studies suggest that circulating microRNAs (miRNAs) are sensitive molecular markers of renal pathology. Objectives. This study quantified the serum levels of miR-21, miR-155 and miR-192 in early-stage CKD patients with or without co-existing T2DM and determined the diagnostic performance of these microRNAs compared to cystatin C and eGFR. Methods. A case-control study was conducted with a total of 100 participants classified into three groups; healthy controls (n = 30), CKD patients with T2DM (n = 35) and CKD patients without T2DM (n = 35). The expression of serum miRNA was measured with real-time quantitative PCR using the 2⁻ΔΔCt method, U6 snRNA as internal reference. Cystatin C was determined by particle-enhanced immunonephelometry; eGFR was calculated using the CKD-EPI equation. Statistical analyses were performed using Kruskal-Wallis test, Spearman partial correlations, receiver operating characteristic (ROC) analysis, and multivariate logistic regression. Results. Significant upregulation of all three miRNAs in both CKD groups (P < 0.001) with a differing magnitude of fold-change compared with controls and the highest amplification for miR-192 was detected. ROC analysis identified miR-192 to be the best performing single biomarker (AUC = 0.937; 95% CI: 0.880–0.978). Spearman partial correlation networks showed strong positive relations of miRNAs with cystatin C (ρ = 0.74–0.80) and stronger negative associations with eGFR (ρ = −0.69 to −0.78). Conclusions. Circulating miR-21, miR-155 and miR-192 are promising minimally invasive biomarkers better than conventional markers for early CKD detection. Cystatin C was co-measured with it, yielding a highly accurate diagnostic panel especially in the diabetic nephropathy sub-phenotype. Clinical applicability requires validation in large multicentre cohorts. Keywords: biochemical markers, chronic kidney disease, cystatin C, eGFR, microRNA, ROC analysis
Background. Plasma membrane Na+,K+-ATPase plays an important role in maintaining ionic homeostasis and regulating myometrial contractility. Its inhibition may alter uterine smooth muscle responses to uterotonic stimulation. Objective. This study aimed to determine the mechanism by which calix[4]arene C-1130 inhibits Na+,K+-ATPase activity and to evaluate its effects on oxytocin-induced myometrial contractions. Methods. Na+,K+-ATPase activity was studied in suspensions of perforated plasma membranes isolated from myometrial cells. The kinetic parameters of ATP hydrolysis were determined at different ATP and Mg2+ concentrations. The effects of C-1130 on oxytocin-induced isometric and isotonic contractions were examined in myometrial preparations with preserved endometrium. Results. Calix[4]arene C-1130 decreased the maximum rate of Na+,K+-ATPase-mediated ATP hydrolysis without significantly affecting the apparent affinity or cooperativity parameters for ATP and Mg2+. At a concentration of 10 μM, C-1130 enhanced oxytocin-induced isometric myometrial contraction and increased the normalized maximal rates of both contraction and relaxation phases. Under isotonic conditions, C-1130 increased the velocity and enhanced the power characteristics of muscle contraction. Conclusions. Calix[4]arene C-1130 inhibits myometrial Na+,K+-ATPase through a noncompetitive mechanism. The enhancement of oxytocin-induced contractile muscle activity is likely associated with disruption of ionic homeostasis in myocytes caused by inhibition of the plasma membrane Na+,K+-ATPase. Keywords: ATPase kinetic properties, calix[4]arenes, K+-ATPase, myometrium, Na+, plasma membrane, smooth muscle cells
Background. Calixarenes are supramolecular compounds with a unique three-dimensional structure, the biological activity of which is determined by the chemical groups on the upper or lower rim. It was found that thiacalix[4]arene C-1087 selectively (compared to other ATPases) inhibits plasma membrane Ca2+,Mg2+-ATPase activity that playіs a crucial role in maintaining Ca2+ homeostasis in myometrial cell. To expand our understanding of the thiacalix[4]arene C-1087 effects on Ca2+ exchange in myometrial cells, it is necessary to analyze its possible effects on Ca2+ exchange in intracellular pools. Objective. Therefore, the aim of this study was to investigate whether thiacalix[4]arene C-1087 affects Ca ion exchange in myometrial mitochondria. Methods. Mitochondria from myometrium of non-pregnant rats were isolated. Ionized calcium concentration in the mitochondria matrix was determined using the fluorescent probe Fluo-4 AM. Baseline Ca2+ concentration in the absence of exogenous (added) Ca2+ and Ca2+ accumulation in its presence were determined. Ca2+ concentration in the incubation medium was determined using fluorescent probe 1 µM Fluo5F, Pentapotassium Salt. Relative value of mitochondria membrane potential (Δψ) was assayed using a fluorescent probe 100 nM TMRM. The Microsoft Office Excel software package was used for data processing. Results. It was proven that mitochondria incubation in Mg2+– and Mg2+,АТР-medium with 10 μM thiacalix[4]arene C-1087 was accompanied by an increase of both the baseline Са2+ concentration and accumulation of this cation in the matrix and did not affect Са2+ efflux from mitochondria. Mitochondria incubation with 10 μM thiacalix[4]arene C-1087 resulted in reducing of TMRM-loaded mitochondria fluorescence intensity, indicatings the depolarizing effect of this compound. Conclusions. The results obtained indicate the possibility of thiacalix[4]arene C-1087 modulating effect on the Са2+ exchange in mitochondria and polarization of its membranes. Keywords: membrane polarization, mitochondria, thiacalix[4]arene C-1087, Са2+ accumulation and efflux, Са2+ concentration
Background. Thrombin is the key enzyme of blood coagulation that converts fibrinogen into fibrin and activates multiple coagulation factors. However, the temporal relationship between thrombin-mediated fibrin formation and the availability of thrombin for other physiological substrates during the initiation of coagulation remains insufficiently understood. Objective. To investigate the role of fibrinogen and fibrin in regulating thrombin activity during the initiation phase of thrombin generation in plasma. Methods. Thrombin generation was initiated in human plasma using activated partial thromboplastin time (APTT) reagent or Thromborel. Fibrin formation was monitored turbidimetrically at 405 nm. Thrombin amidolytic activity toward the chromogenic substrate S2238 was measured spectrophotometrically at 405 nm. Fibrin polymerization was inhibited with the synthetic peptide GPRP. The effects of plasma dilution and inhibition of fibrin polymerization on the temporal profiles of fibrin formation and thrombin activity were analyzed. Results. During initiation of thrombin generation with both APTT-reagent and Thromborel, the beginning of cleavage of S2238 by thrombin coincided with the end of fibrin clot formation, marked by turbidimetric curve. At 1:3 plasma dilution, fibrin formation lag time was 210 s, amidase activity lag phase was 410 s. At 1:10 dilution, the lag time for fibrin formation was 73 s, amidase activity was 200 s, indicating that dilution (and lower fibrinogen) accelerates thrombin generation. With 1 mM GPRP, fibrin and amidase activity lag times increased by an order of magnitude. Conclusions. Thrombin generated during initiation of thrombin generation acts primarily on fibrinogen, remaining bound to the molecule throughout its conversion into fibrin and clot formation. Its amidase activity emerges only after protofibrils undergo lateral association into fibrin clot. Once the binding sites in the fibrin clot are saturated, thrombin becomes available for other substrates (FV, FVIII, FXI) and physiological functions. Keywords: fibrin, fibrinogen, thrombin, thrombin generation assay
