Influence of the perfusion bioreactor on Stratified and Distributed approaches for multilayered tissue engineering on biodegradable scaffolds.

Despite wide use and approval of poly lactic-coglycolic acid (PLGA) for surgical applications, there have been very few studies on tissue constructions that mimic physiological multilayer structures by combining PLGA scaffolds with tissue engineering. In our study, we developed a bioreactor system to maintain, and to train two types of three-layered vascular-like structures. Then we examined how the perfusion conditions and different tissue engineering approaches affected the formation of the layered structure and degradation of the PLGA scaffolds. In the proposed Distributed Method, the cells were seeded layer by layer on a single scaffold, using spheroids bigger than scaffold fiber gaps and we achieved the higher cell density compared with the Stratified Method where we stacked three PLGA sheets seeded with individual vascular cell types. At the histological level, scaffold degradation was more prominent in the bioreactor compared to the same time interval in vivo. In addition, the faster flow accelerated the decomposition of PLGA fibers. Moreover, bioreactor perfusion culture at lower flow rates could balance cell adhesion and survival, improve the cell density and promote self-organization of multilayer structure with desirable rate of PLGA scaffolds degradation.

Dynamic flow priming programs allow tuning up the cell layers properties for engineered vascular graft.

Tissue engineered vascular grafts (TEVG) are potentially clear from ethical and epidemiological concerns sources for reconstructive surgery for small diameter blood vessels replacement. Here, we proposed a novel method to create three-layered TEVG on biocompatible glass fiber scaffolds starting from flat sheet state into tubular shape and to train the resulting tissue by our developed bioreactor system. Constructed tubular tissues were matured and trained under 3 types of individual flow programs, and their mechanical and biological properties were analyzed. Training in the bioreactor significantly increased the tissue burst pressure resistance (up to 18 kPa) comparing to untrained tissue. Fluorescent imaging and histological examination of trained vascular tissue revealed that each cell layer has its own individual response to training flow rates. Histological analysis suggested reverse relationship between tissue thickness and shear stress, and the thickness variation profiles were individual between all three types of cell layers. Concluding: a three-layered tissue structure similar to physiological can be assembled by seeding different cell types in succession; the following training of the formed tissue with increasing flow in a bioreactor is effective for promoting cell survival, improving pressure resistance, and cell layer formation of desired properties.

Long-term safety of the carbon fiber as an implant scaffold material.

Permanent therapeutically placed implants often used in situations when regeneration or transplantation are not practical or possible. They include metallic grafts for osteosynthesis, bulk metallic glasses, ceramics, and non-resorbable polymers providing mechanical support. Repair of the tissues on micro scale can also benefit from the biocompatible permanent implants. Vascular graft engineering and repairs of the spinal cord and peripheral nerves are among the most demanding application. Carbon fibers (CF) have superior mechanical and chemical properties, however, their long-time safety was never systematically estimated. The biggest concern comes from residual polymers used for pyrolysis and epoxy laminating resins. Here we attempted to investigate survival of the cells cultured on carbon fibers and to evaluate the tissue responses towards the long-term implanted material. Immortalized rat Schwann cells displayed efficient sporadic attachment to the carbon fibers with survival rate over 90%. Carbon fiber implants in adipose and on connective tissues were tolerable by animals during about 40% of their lifespan with no signs of inflammation on physiological, morphological or gene expression level.

Development of biomimetic system for scale up of cell spheroids - building blocks for cell transplantation.

Artificial assembly of mature tissues in vitro is challenging from many viewpoints. Therefore, production of intermediate building blocks - cell spheroids expected to be a viable alternative. The purpose of this research is to develop a biomimetic system for scale up maintenance of spheroids in vitro, and to confirm basic performance of the device. The system consists of a 3D culture unit and a medium perfusion unit. The 3D culture unit is dedicated for spheroid culture without using scaffolds, eliminating concerns about biocompatibility of artificial materials. our culture vessel allows easy disassembly and tissue extraction, as well as the resulting tissue can be formed into an any desirable shape. The spheroids are cultured in a sealed environment and their life are sustained by hollow fiber perfusion fluidics. We confirmed by visual and by microscopic examination that no contamination did occur before and after spheroid inoculation. Moreover, we confirmed growth and fusion between cells when C2C12 spheroids were cultured in this system.