The interactions of astrocytes and fibroblasts with defined pore structures in static and perfusion cultures

Abstract

Open pores to maintain nutrient diffusion and waste removal after cell colonization are crucial for the successful application of constructs based on assembled membranes, in our case tubular scaffolds made of ɛ-polycaprolactone (PCL), for use in tissue engineering. Due to the complex three-dimensional structure and large size of such scaffolds needed for transplantable tissues, it is difficult to investigate the cell–pore interactions in situ. Therefore miniaturized bioreactors inside Petri dishes (30 mm in diameter), containing porous PCL or poly-dimethylsiloxane (PDMS) membranes, were developed to allow the interactions of different cells with defined pores to be investigated in situ during both static and perfusion cultures. Investigation of two different cell types (fibroblasts and cortical astrocytes) and how they interact with a range of pores (100–350 μm in diameter) for up to 50 days indicated that the cells either ‘covered’ or ‘bridged’ the pores. Three distinct behaviors were observed in the way cortical astrocytes interacted with pores, while fibroblasts were able to quickly bridge the pores based on consistent “joint efforts”. Our studies demonstrate that the distinct pore sealing behaviors of both cell types were influenced by pore size, initial cell density and culture period, but not by medium perfusion within the range of shear forces investigated. These findings form important basic data about the usability of pores within scaffolds that could inform the design and fabrication of suitable scaffolds for various applications in tissue engineering.

The Development of a ɛ-Polycaprolactone Scaffold for Central Nervous System Repair

“…Its potential use as a scaffold for cell transplantation has added support by its long degradation time. 23,42 The degradation of radioactive-tagged PCL transplanted subcutaneously into rats and followed over 3 years was very slow. Over the first 2 years it broke down into small molecular weight material by random, spontaneous hydrolysis, eventually the degraded material would be metabolized, although its overall shape still was recognizable (Sun et al 2006).…”

“…Myelin ensheathment observed in these cultures is similar to myelination and sheath formation in vivo. [23][24][25][26] One important feature of the myelinating culture is ensuring that the neurite density is comparable between , markers of increasing oligodendroglial maturity at both 7 DIV and 14 DIV, with the percentage of A2B5 + cells and NG2 + cells calculated from a total cell number as indicated by DAPI (blue) staining. The total coverage of O4 + processes was also calculated and divided by the total DAPI count to give a ratio of O4 + coverage to total cell number.…”

“…To quantify myelination as defined by the percentage of fibers ensheathed by a myelin sheath, the cultures were labeled with SMI-31 (green), anti-PLP myelin (red), and DAPI (blue). Ten images per coverslip were acquired at · 10 magnification as previously described 23 and initially opened using ImageJ with the blue (DAPI) channel removed. The red and green images were then opened with Adobe Photoshop Elements 7.0 and areas of myelin that was a clear sheath were overlaid by hand using a brush tool in blue in a new, lightened layer.…”

“…22 The pores enable mass transfer within the 3D scaffold and previous work has demonstrated that a pore diameter of at least 200 mm prevents occlusion by astrocytes. 22,23 This 3D scaffold will eventually be coated with glial cells, as part of a 1 combinatorial approach to treat SCI, to promote the ingrowth of endogenous CNS cells in an aligned manner to aid crossing the graft. Before the implantation of this complicated 3D Swiss roll, we have adopted a systematic approach to investigate the different features of the scaffold design to fully assess their potential effects on CNS cells using dissociated rat spinal cord cells.…”

Engineering Mechanical, Biochemical, and Topographical Niche Cues by Photocrosslinkable, Microribbon-Like Hydrogels

“…Prefabricated scaffolds with cell-sized microstructures, on the other hand, are widely used to control cell behaviors via ECM-mimicking microstructures, such as macropores [15][16][17][18][19], microchannels [20,21], and microfibrous textures [22]. These microarchitectures can promote desired cell fates by controlling cell morphology, supporting tissue deposition, and facilitating nutrient diffusion [16,18,19,23]. …”