Noninvasive ultrasound imaging for assessment of intact microstructure of extracellular matrix in tissue engineering.

Development of artificial tissues or organs is one of the actual tasks in regenerative medicine that requires observation and evaluation of intact volume microstructure of tissue engineering products at all stages of their formation, from native donor tissues and decellularized scaffolds to recipient cell migration in the matrix. Unfortunately in practice, methods of vital noninvasive imaging of volume microstructure in matrixes are absent. In this work, we propose a new approach based on high-frequency acoustic microscopy for noninvasive evaluation and visualization of volume microstructure in tissue engineering products. The results present the ultrasound characterization of native rat diaphragms and lungs and their decellularized scaffolds. Verification of the method for visualization of tissue formation in the matrix volume was described in the model samples of diaphragm scaffolds with stepwise collagenization. Results demonstrate acoustic microscopic sensitivity to cell content concentration, variation in local density, and orientation of protein fibers in the volume, micron air inclusions, and other inhomogeneities of matrixes.

Biophysical methods for quality evaluation of decellularized and recellularized tissue-engineered constructs of organs and tissues.

Tissue engineering is rapidly growing now and can become a promising alternative to transplantation of organs and tissues, as it is devoid of major shortcomings of transplantology, such as acute shortage, complexity of selection, delivery and storage of donor material, lifelong immunosuppressive therapy. One of the most widely known methods of obtaining biological scaffolds for the subsequent creation of tissue-engineered constructs of organs and tissues is decellularization. The evaluation of the quality of the obtained scaffolds, based on the study of the viability of cell structures in decellularized and recellularized matrices, is one of the priorities of modern regenerative medicine worldwide. In this investigation, the biophysical criteria of decellularization and recellularization of tissue-engineered constructs based on the evaluation of the generation of free radicals in native, decellularized and recellularized tissues by EPR spectroscopy and chemoluminescence in a complex assessment of the quality of biological matrixes obtained are considered using intrathoracic organs and tissues of rats. It has been established that the intensity indices of free radical generation in native and recellularized tissues of animal organs, as well as in decellularized matrices, can serve as one of the express criteria for quantitative assessment of cell structures viability.

Orthotopic transplantation of a tissue engineered diaphragm in rats.

The currently available surgical options to repair the diaphragm are associated with significant risks of defect recurrence, lack of growth potential and restored functionality. A tissue engineered diaphragm has the potential to improve surgical outcomes for patients with congenital or acquired disorders. Here we show that decellularized diaphragmatic tissue reseeded with bone marrow mesenchymal stromal cells (BM-MSCs) facilitates in situ regeneration of functional tissue. A novel bioreactor, using simultaneous perfusion and agitation, was used to rapidly decellularize rat diaphragms. The scaffolds retained architecture and mechanical properties and supported cell adhesion, proliferation and differentiation. Biocompatibility was further confirmed in vitro and in vivo. We replaced 80% of the left hemidiaphragm with reseeded diaphragmatic scaffolds. After three weeks, transplanted animals gained 32% weight, showed myography, spirometry parameters, and histological evaluations similar to native rats. In conclusion, our study suggested that reseeded decellularized diaphragmatic tissue appears to be a promising option for patients in need of diaphragmatic reconstruction.

Dynamic decellularization and cross-linking of rat tracheal matrix.

Decellularized tissues and organs represent a suitable option for tissue engineering when specific scaffolds are needed. However, the optimal conditions to completely remove all the cellular components and minimally affect the biochemical and structural properties of the extracellular matrix are still to be found. For this aim, bioreactors could be an alternative means to dynamically treat the biological samples, automatically controlling all the variables involved in the process and speeding up the entire procedure in order to deal with a suitable scaffold within a limited time period. This paper presents the characterization of rat tracheae decellularized in dynamic conditions, implementing a detergent-enzymatic method, previously considered. Only 6 cycles were enough to generate a tracheal matrix that was histologically and structurally similar to the native one. The network of collagen, reticular and elastic fibers was well preserved, such as the epithelial cilia, the luminal basement membrane and the main matrix components. The elastin content decreased, even if not significantly, after the decellularization protocol. Mechanical properties of the treated tissues were slightly affected by the procedure, and were partially recovered after crosslinking with genipin, a naturally-derived agent. The use of bioreactors could enhance the decellularization procedure of tissues/organs, but a careful selection of the processing parameters is needed in order to prevent large modifications compared to the native condition.

Electrospun gelatin scaffolds incorporating rat decellularized brain extracellular matrix for neural tissue engineering.

The fabrication of an instructive bioabsorbable scaffold is one of the main goals for tissue engineering applications. In this regard, genipin cross-linked gelatin scaffolds, produced by electrospinning, were tested as a platform to include decellularized rat brain extracellular matrix as an active agent to provide fundamental biochemical cues to the seeded cells. This approach is expected to furnish a suitable natural-based polymeric scaffold with sufficient temporal stability to support cell attachment and spreading, also providing tissue-specific signals that can contribute to the expression of the requested cellular phenotype. We first demonstrated the effectiveness of the proposed decellularization protocol and the cytocompatibility of the resulting brain matrix. Then, the in vitro biological assays of the conditioned electrospun scaffolds, using rat allogeneic mesenchymal stromal cells, confirmed their biocompatibility and showed a differentiative potential in presence of just 1% w/w decellularized rat brain extracellular matrix.