Abstract
Microcarrier cell scaffolds have potential as injectable cell delivery vehicles or as building blocks for tissue engineering. The use of small cell carriers allows for a ‘bottom up’ approach to tissue assembly when moulding microparticles into larger structures, which can facilitate the introduction of hierarchy by layering different matrices and cell types, while evenly distributing cells through the structure. In this work, silk fibroin (SF), purified from Bombyx mori cocoons, was blended with gelatin (G) to produce materials composed of varying ratios of the two components (SF: G 25:75, 50:50, and 75:25). Cell compatibility to these materials was first confirmed in two-dimensional culture and found to be equivalent to standard tissue culture plastic, and better than SF or G alone. The mechanical properties of the blends were investigated and the blended materials were found to have increased Young's moduli over SF alone. Microcarriers of SF/G blends with defined diameters were generated in a reproducible manner through the use of an axisymmetric flow focussing device, constructed from off-the-shelf parts and fittings. These SF/G microcarriers supported adhesion of rat mesenchymal stem cells with high degrees of efficiency under dynamic culture conditions and, after culturing in osteogenic differentiation medium, cells were shown to have characteristics typical of osteoblasts. This work illustrates that microcarriers composed of SF/G blends are promising building blocks for osteogenic tissue engineering.
Highlights
Scaffold design for tissue engineering has traditionally relied on a top-down approach, generally attempting to seed cells evenly throughout a relatively large pre-formed scaffold
In order to determine the suitability of silk fibroin (SF)/G blends for use as a substrate for mesenchymal stem cell (MSC) proliferation and osteogenic differentiation, cell-material interactions were initially assessed in 2D by seeding Rat mesenchymal stem cell (rMSC) on biomaterial films, which were cast within multiwell plates
Suitable blends were taken forwards to evaluate their ability to form microcarriers which supported MSC adhesion and osteogenesis. 3.1 rMSC adhesion to SF and SF/gelatin blends (SF/G) blends in two-dimensional culture The ability of films of SF, SF/G and gelatin to support rMSC proliferation was assessed over 7 days using the resazurin reduction assay which, unlike a number of other metabolic assays, enables the same population of cells to be examined across multiple time points (Figure 1). rMSCs proliferated poorly on pure gelatin films, which was unexpected as gelatin is well known as a highly cell-adhesive material and is used routinely in tissue engineering scaffolds [36,37,38]
Summary
Scaffold design for tissue engineering has traditionally relied on a top-down approach, generally attempting to seed cells evenly throughout a relatively large pre-formed scaffold. This has distinct disadvantages; it is often difficult to ensure even cell distribution, and central or core scaffold regions can remain underpopulated. Bottom-up approaches have been investigated by a number of groups, whereby smaller scaffold fragments or particles are seeded with cells before being moulded or shaped into the final 3D structure [24] In this way, cells are evenly spread throughout the construct, and complexity can be built in by layering different cell or scaffold types. Particulate growth matrices with diameters of several hundred microns, are ideal candidates for this type of approach, and have wider applications in the scale-up of cell production [5,6,7] and the precise, injectable delivery of therapeutic cells to areas of disease or damage within the body [6, 8, 9]
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