Effect of low temperatures and ionizing irradiation upon physical-mechanical properties and connective-tissue structures of porcine fibrous pericardium and aortic valve leaflets

Xenogeneic tissue devitalization is one of the creating methods of the tissuereplacing the biocompatible cell-free shells for the regenerative surgery. The work describes the possibility of applying the complex approach based on the continuous usage of cryo and radioactive (electron irradiation exposure) biological tissue damage effects. The pre-implant treatment provides sterilization and a possibility for the low temperature preservation of xenografts. After the transplantation such a cell-free xenoscaffold can be gradually replaced with the autogenic extracellular matrix from the recipient’s cells and forms a stable long-term structure of the biological prosthesis. Fibrous pericardium (FP) and aortic valve leaflets (AVLs) were extracted from the mature pig. The prepared tissues were rinsed with the sterile normal saline solution and frozen down to the liquid-nitrogen temperature. After one time placing on water-bath (37°C) they were exposed to electron irradiation within dosage range of 25-30 kGray and submerged into the liquid nitrogen vapors. After influence of low temperature and ionizing radiation, tissue morphological structure was assessed using the optical microscopy. Deformations, i.e. longitudinal and transverse monoaxial strength were performed to calculate the physical and mechanical properties of FP and AVLs. Such a devitalization method of the FP and AVLs causes significant destructive changes in cell elements, however the spatial arrangement and structural integrity of the connective tissue fiber are preserved. Joint impact of low temperatures and ionizing radiation gives the synergetic effect, increasing the strength and elastic tissue properties. Freezing down to –196 °C and electron irradiation initiate formation of the intra- and intermolecular transverse cross-linking due to the binding activity of fibrous proteins. It leads to a more dense arrangement of the collagen fiber, adds strength to the implant and provides the structural tissue stabilization. The authors believe that during the remodeling in the recipient organism, the biomaterial structure modified in such a manner can successfully prevent physiological tension

Organospecific influence of the extract of cryopreserved piglets’ skin fragments

Researching mechanisms of peptides’ tissue-specific effect is an important task of modern molecular biology, physiology and medicine. In previous studies it was shown that extracts of cryopreserved piglets’ skin fragments and pigs’ spleen fragments accelerate and normalise the healing of skin wound in experiment. Fibroblasts culture is an appropriate model for studying tissue-specific biological activity of peptide complexes on relevant type of cells. This research was aimed to establish the influence of the extract of cryopreserved newborn piglets’ skin fragments (cNPSE) and the extract of cryopreserved pigs’ spleen fragments (cPSE) on proliferative and metabolic activity of skin fibroblasts in culture. Extracts were obtained from cryopreserved skin fragments and cryopreserved spleen fragments. Primary culture of neonatal rat skin fibroblasts was obtained by free cell transfer from skin fragments and subsequent reseeding. Metabolic activity of cells in culture was defined using non-toxic redox indicator AlamarBlue. The number of cells in a well was measured by counting the quantity of cells in wells. On the 7th day the metabolic activity of fibroblasts, cultured with cNPSE (1 μg/ml final peptide concentration), was higher than control by 1.3 times. Adding of cPSE (in the same concentration of peptides) increased cell metabolic activity by 1.2 times. While incubating fibroblasts in the medium with 2% FBS, a decrease of metabolic activity of cells was observed on the 5th day, and by the 7th day it was 51.2% of the control. At 1 and 1.5 μg/ml final peptide concentration of cNPSE, the metabolic activity of fibroblasts remained at the level observed in the control samples with 10% FBS. Adding cPSE to the incubation medium did not affect the metabolic activity of cells. Increased metabolic activity of cells (initially kept for 30 minutes at 4°C) was observed on the 5th and 7th day in cNPSE presence. Thus, it was found that adding cNPSE and cPSE to the culture medium of rats’ skin fibroblasts increase the metabolic activity of cells. A dose-dependent effect is observed. The addition of cNPSE to the medium with 2% FBS maintains the metabolic activity of fibroblasts at the level observed in the control samples incubated with 10% FBS. Adding extract also increases the metabolic activity of fibroblasts after hypothermic impact on the cells

Organospecific influence of the extract of cryopreserved piglets’ skin fragments

Researching mechanisms of peptides’ tissue-specific effect is an important task of modern molecular biology, physiology and medicine. In previous studies it was shown that extracts of cryopreserved piglets’ skin fragments and pigs’ spleen fragments accelerate and normalise the healing of skin wound in experiment. Fibroblasts culture is an appropriate model for studying tissue-specific biological activity of peptide complexes on relevant type of cells. This research was aimed to establish the influence of the extract of cryopreserved newborn piglets’ skin fragments (cNPSE) and the extract of cryopreserved pigs’ spleen fragments (cPSE) on proliferative and metabolic activity of skin fibroblasts in culture. Extracts were obtained from cryopreserved skin fragments and cryopreserved spleen fragments. Primary culture of neonatal rat skin fibroblasts was obtained by free cell transfer from skin fragments and subsequent reseeding. Metabolic activity of cells in culture was defined using non-toxic redox indicator AlamarBlue. The number of cells in a well was measured by counting the quantity of cells in wells. On the 7th day the metabolic activity of fibroblasts, cultured with cNPSE (1 μg/ml final peptide concentration), was higher than control by 1.3 times. Adding of cPSE (in the same concentration of peptides) increased cell metabolic activity by 1.2 times. While incubating fibroblasts in the medium with 2% FBS, a decrease of metabolic activity of cells was observed on the 5th day, and by the 7th day it was 51.2% of the control. At 1 and 1.5 μg/ml final peptide concentration of cNPSE, the metabolic activity of fibroblasts remained at the level observed in the control samples with 10% FBS. Adding cPSE to the incubation medium did not affect the metabolic activity of cells. Increased metabolic activity of cells (initially kept for 30 minutes at 4°C) was observed on the 5th and 7th day in cNPSE presence. Thus, it was found that adding cNPSE and cPSE to the culture medium of rats’ skin fibroblasts increase the metabolic activity of cells. A dose-dependent effect is observed. The addition of cNPSE to the medium with 2% FBS maintains the metabolic activity of fibroblasts at the level observed in the control samples incubated with 10% FBS. Adding extract also increases the metabolic activity of fibroblasts after hypothermic impact on the cells