Microenvironment Design for Stem Cell Fate Determination - Tissue Engineering: Cell-Surface Interactions for Tissue Culture - page 251

osteoblasts was limited in the osteogenic layer, there was significantly enhanced
chondrogenic differentiation of MSCs in the chondrogenic layer. This effect was
further

enhanced when MSCs pre-differentiated into osteoblastic cells were

encapsulated in the osteogenic layer [ 192 ].
In addition to the suggested layered composite approach, other studies utilized
delivery of controlled factor gradients via MPs in polymer scaffolds for the
induction of stem cells into a complex tissue [ 193 , 194 ]. For example, BMP2- and
TGFb1-loaded PLGA MPs were utilized with a gradient scaffold fabrication
technology to produce MP-based scaffolds containing opposing gradients of these
signals. The results indicated that hMSC-seeded gradient scaffolds produced
regionalized ECM, similar to that of the osteochondral tissue. Overall, these
studies demonstrate the fabrication of layered hydrogel composites that mimic the
structure and function of osteochondral tissue, along with the application of these
composites as cell and growth factor carriers [ 193 ].

4 Bioreactors for Dynamic Stem Cell Microenvironment

The microenvironment consists of additional cues, such as local blood perfusion
and hypoxia in the bone marrow niche, known to be key players in the determi-
nation of HSC fate. Recently, Winkler et al. [ 195 ] established a positional hier-
archy between HSCs and lineage-restricted hematopoietic progenitor cells (HPCs)
relative to the blood flow rate. The most potent HSCs were exposed to negligible
blood flow, whereas lineage-restricted HPCs as well as stromal cells such as
endothelial cells, MSCs and osteoblast cells were located in vascular niches,
perfused by rapid blood flow. This study along with many others suggests that
stem cell microenvironment design should consider the dynamic nature of the
native niche, i.e., including the parameters of fluid flow kinetics and mass transport
(especially oxygen diffusion) in the 3D cell cultures, as well as other time-
constant-related factors and metabolite diffusion. Ex vivo, in static 3D cell cultures
with the lack of vasculature, the transport of nutrients and dissolved oxygen from
the bulk medium to the surface of the cell constructs is limited, as is the internal
mass transfer rate from the surface of the construct into its core.
In an attempt to overcome mass transport limitations and to enable controlled
biophysical and mechanical stimuli, bioreactors have been implemented for cre-
ating the 3D stem cell microenvironment. The bioreactor systems, from simple
conventional spinner flasks, the rotary wall vessels, up to the latest perfusion
vessels, were found to improve cell viability, proliferation and differentiation. For
example, 3D cultivation in spinner flasks improved, to some extent, tissue
homogeneity and viability of hMSC [ 196 ]. In another study, rotary cell culture
systems (RCCS), developed by NASA, were shown to enable 3D cell cultivation
under medium mixing with a minimal shear stress on the cultivated cells. It was
shown that cultivation of hESCs within a rotating bioreactor increased the cell
proliferation rate and maintained cell viability in the culture [ 197 ].