ABSTRACT
To select the optimum method for disinfecting scaffolds before recellularization, the effects of octenisept and chlorhexidine at different concentrations on lung biological matrices before and after decellularization were studied by using morphological methods (studies of biomechanical strength of extracellular matrix fibers) and by analyzing chemiluminescence in rats. Chlorhexidine diluted 1 : 10 had the least damage on the matrix properties and to the greatest extent contributed to disinfection of scaffolds for their further storage and experimental studies.
Subject(s)
Anti-Infective Agents, Local/chemistry , Chlorhexidine/chemistry , Disinfection/methods , Extracellular Matrix/chemistry , Lung/chemistry , Animals , RatsABSTRACT
It was found that the chemiluminescence intensity in native and recellularized tissues of rat muscular organs as well as in their decellularized scaffolds can serve as an express criterion that, along with ultrastructural analysis, makes it possible to perform quantitative assessment of the viability of cellular structures in biological samples of the diaphragm.
Subject(s)
Diaphragm/chemistry , Luminescent Measurements , Tissue Engineering , Tissue Scaffolds/chemistry , Animals , RatsABSTRACT
Based on the data of morphological analysis, we performed histological evaluation of rat tissue reaction to subcutaneous implantation of decellularized matrices of intrathoracic organs and tissues. Cell composition of the inflammatory infiltrate was analyzed, and the dynamics of macrophage and T and B lymphocyte content was assessed on days 7 and 14 of the experiment. It was found that the reaction to implantation depended not only on the quality of decellularization and efficiency of removal of antigen molecules, but also on the original histological structure and quality of preimplantation processing of the transplant.
Subject(s)
Diaphragm/ultrastructure , Extracellular Matrix/ultrastructure , Lung/ultrastructure , Tissue Engineering/methods , Tissue Scaffolds , Animals , Antigens, CD/immunology , Antigens, CD/metabolism , B-Lymphocytes/cytology , B-Lymphocytes/immunology , Biomarkers/metabolism , Deoxycholic Acid/chemistry , Deoxycholic Acid/pharmacology , Deoxyribonucleases/chemistry , Deoxyribonucleases/pharmacology , Diaphragm/cytology , Diaphragm/drug effects , Diaphragm/transplantation , Extracellular Matrix/chemistry , Heart/drug effects , Lectins, C-Type/immunology , Lectins, C-Type/metabolism , Lung/cytology , Lung/drug effects , Macrophages/cytology , Macrophages/immunology , Male , Mannose Receptor , Mannose-Binding Lectins/immunology , Mannose-Binding Lectins/metabolism , Rats , Rats, Wistar , Receptors, Cell Surface/immunology , Receptors, Cell Surface/metabolism , Skin , T-Lymphocytes/cytology , T-Lymphocytes/immunology , Transplantation, HomologousABSTRACT
Biological compatibility of a tissue engineered construct of the trachea (synthetic scaffold) and allogenic mesenchymal stem cells was studied on laboratory Papio hamadryas primates. Subcutaneous implantation and orthotopic transplantations of tissue engineered constructs were carried out. Histological studies of the construct showed chaotically located filaments and mononuclear cells fixed to them. Development of a fine connective tissue capsule was found at the site of subcutaneous implantation of the tissue engineered construct. The intact structure of the scaffold populated by various cell types in orthotopic specimens was confirmed by expression of specific proteins. The results indicated biological compatibility of the tissue engineered construct with the mesenchymal stem cells; no tissue rejection reactions were recorded; simulation of respiratory disease therapy on Papio hamadryas proved to be an adequate model.
Subject(s)
Foreign Bodies/surgery , Mesenchymal Stem Cell Transplantation , Polyethylene Terephthalates/pharmacology , Tissue Engineering/methods , Tissue Scaffolds , Trachea/transplantation , Animals , Biomarkers/metabolism , Cell Adhesion/drug effects , Gene Expression , Keratins/genetics , Keratins/metabolism , Lectins, C-Type/genetics , Lectins, C-Type/metabolism , Leukocytes, Mononuclear/cytology , Leukocytes, Mononuclear/drug effects , Leukocytes, Mononuclear/metabolism , Male , Mannose Receptor , Mannose-Binding Lectins/genetics , Mannose-Binding Lectins/metabolism , Mesenchymal Stem Cells/cytology , Mesenchymal Stem Cells/metabolism , Papio hamadryas , Receptors, Cell Surface/genetics , Receptors, Cell Surface/metabolism , Subcutaneous Tissue/surgery , Transplantation, Autologous , Vimentin/genetics , Vimentin/metabolismABSTRACT
Automated image analysis methods are highly important for biotechnology research. The authors developed and tested a program for the morphometric analysis of photomicrographs of the sections processed using the standard immunohistochemical examination protocols. The color deconvolution method used in the algorithm was proven to be effective in mapping the distribution of DAB chromogen in the sample containing multiple dyes. The experiment demonstrated that the level of extracellular matrix proteins could be comparatively quantified in different groups of samples. The effective methods for the quantitative analysis of the Ki-67 labelling index were also tested using the same algorithms. The developed program was published under free GPL 3.0.
Subject(s)
Antigens, Nuclear/isolation & purification , Extracellular Matrix/ultrastructure , Image Processing, Computer-Assisted/methods , Immunohistochemistry/methods , Algorithms , Antigens, Nuclear/ultrastructure , Coloring Agents/chemistry , Coloring Agents/isolation & purification , Humans , SoftwareABSTRACT
Chronic renal failure (CRF) is one of the most challenging problems of contemporary medicine. Patients with chronic renal failure usually need renal replacement therapy as either hemodialysis, peritoneal dialysis or a kidney transplant. The latter is the most promising option for end-stage kidney disease. However, the shortage of donor organs, the complexity of their delivery, the difficulty in finding an immunologically compatible donor and the need for lifelong immunosuppression triggered advances in modern tissue engineering. In this field, the primary priority is focused on developing bioengineered scaffolds with subsequent recellularization with autologous cells. Using such constructs would allow for solving both ethical and immunological problems of transplantation. The aim of this pilot study was to develop a new method of renal decellularization using small laboratory animals. MATERIALS AND METHODS: The study investigated the morphological structure of the obtained decellularized matrix and quantitatively tested DNA residues in the resulting scaffold. We proposed a new biophysical method for assessing the matrix quality using the EPR spectroscopy and conducted experiments on the matrix recellularization with mesenchymal multipotent stem cells to estimate cytotoxicity, cell viability and metabolic activity. RESULTS: The obtained decellularized renal matrix retained the native tissue architecture after a complete removal of the cell material, had no cytotoxic properties and supported cell adhesion and proliferation. CONCLUSION: All the above suggests that the proposed decellularization protocol is a promising method to produce tissue-engineered kidney constructs with possible clinical application in the foreseeable future.
Subject(s)
Kidney Failure, Chronic/therapy , Kidney/anatomy & histology , Kidneys, Artificial , Tissue Engineering , Tissue Scaffolds , Animals , Cell Separation , Kidney/cytology , RatsABSTRACT
We modified the protocol of obtaining of biological scaffolds of rat lungs based on dynamic recording of specific resistivity of working detergent solution (conductometry) during perfusion decellularization. Termination of sodium deoxycholate exposure after attaining ionic equilibrium plateau did not impair the quality of decellularization and preserved structural matrix components, which was confirmed by morphological analysis and quantitative assay of residual DNA.
Subject(s)
Lung , Tissue Scaffolds , Animals , Conductometry , Deoxycholic Acid/chemistry , Detergents/chemistry , Electric Conductivity , Lung/cytology , Male , Rats, Wistar , Solutions , Tissue Engineering , Tissue FixationABSTRACT
The effect of decellularization on the biomechanical properties of macaque lungs was studied. The quality of the biological scaffold was additionally assessed by morphological methods, and the contents of extracellular matrix (ECM) fibers were determined both qualitatively and quantitatively. Histological analysis revealed no damage of structural integrity of ECM components, but the scaffold elasticity significantly decreased, which was confirmed by the changes in the hysteresis loop without a concomitant decrease in peak loads, with the mechanical strength of the samples being retained. These changes require taking additional measures to prevent a decrease in the effective lung volume.
Subject(s)
Extracellular Matrix/metabolism , Lung/physiology , Tissue Engineering , Animals , Biomechanical Phenomena , Elasticity , Macaca mulatta , Male , Tissue Engineering/methods , Tissue ScaffoldsABSTRACT
Using EPR spectroscopy it was established that the determination of the concentration of paramagnetic centers in lyophilized tissues allows indirect evaluation of the quality of decellularization of intrathoracic organs (diaphragm, heart, and lungs), since the content of paramagnetic particles in them can serve as a criterion of cell viability and points to the necessity to repeat decellularization. Experiments in rats showed that the EPR spectra of the native thoracic organs contained paramagnetic centers with g-factor values ranging from 2.007 to 2.011 at a concentration of 10(-8) to 6.62 × 10(-7) mol/g of lyophilized tissue, whereas in all decellularized tissues of the same organs paramagnetic particles were not detected.
