RESUMO
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.
Assuntos
Anti-Infecciosos Locais/química , Clorexidina/química , Desinfecção/métodos , Matriz Extracelular/química , Pulmão/química , Animais , RatosRESUMO
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.
Assuntos
Diafragma/ultraestrutura , Matriz Extracelular/ultraestrutura , Pulmão/ultraestrutura , Engenharia Tecidual/métodos , Alicerces Teciduais , Animais , Antígenos CD/imunologia , Antígenos CD/metabolismo , Linfócitos B/citologia , Linfócitos B/imunologia , Biomarcadores/metabolismo , Ácido Desoxicólico/química , Ácido Desoxicólico/farmacologia , Desoxirribonucleases/química , Desoxirribonucleases/farmacologia , Diafragma/citologia , Diafragma/efeitos dos fármacos , Diafragma/transplante , Matriz Extracelular/química , Coração/efeitos dos fármacos , Lectinas Tipo C/imunologia , Lectinas Tipo C/metabolismo , Pulmão/citologia , Pulmão/efeitos dos fármacos , Macrófagos/citologia , Macrófagos/imunologia , Masculino , Receptor de Manose , Lectinas de Ligação a Manose/imunologia , Lectinas de Ligação a Manose/metabolismo , Ratos , Ratos Wistar , Receptores de Superfície Celular/imunologia , Receptores de Superfície Celular/metabolismo , Pele , Linfócitos T/citologia , Linfócitos T/imunologia , Transplante HomólogoRESUMO
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.
Assuntos
Antígenos Nucleares/isolamento & purificação , Matriz Extracelular/ultraestrutura , Processamento de Imagem Assistida por Computador/métodos , Imuno-Histoquímica/métodos , Algoritmos , Antígenos Nucleares/ultraestrutura , Corantes/química , Corantes/isolamento & purificação , Humanos , SoftwareRESUMO
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.
Assuntos
Falência Renal Crônica/terapia , Rim/anatomia & histologia , Rins Artificiais , Engenharia Tecidual , Alicerces Teciduais , Animais , Separação Celular , Rim/citologia , RatosRESUMO
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.
Assuntos
Pulmão , Alicerces Teciduais , Animais , Condutometria , Ácido Desoxicólico/química , Detergentes/química , Condutividade Elétrica , Pulmão/citologia , Masculino , Ratos Wistar , Soluções , Engenharia Tecidual , Fixação de TecidosRESUMO
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.
Assuntos
Matriz Extracelular/metabolismo , Pulmão/fisiologia , Engenharia Tecidual , Animais , Fenômenos Biomecânicos , Elasticidade , Macaca mulatta , Masculino , Engenharia Tecidual/métodos , Alicerces TeciduaisRESUMO
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.