(84g) 3-Dimensional Osteochondral Tissue Engineering

Osteoarthritis affects
millions of individuals and is characterized by joint pain caused by articular
cartilage wear. Cartilage, due to its avascular nature, has poor self-repair
capabilities, which result in mechanically inferior repair tissue. One of the
more successful current clinical techniques involves replacing the damaged
cartilage and part of the underlying subchondral bone with osteochondral
grafts. However, the availability of such grafts is limited and if taken from
the patient, results in another defect site. Therefore, tissue engineering has
been investigated as a promising solution for cartilage defects. In this study,
we developed a novel gradient generating bioreactor that was used to
investigate tissue engineering of osteochondral tissues by using molecular
gradients to control human mesenchymal cell (MSC) differentiation. Within the
bioreactor, a flow channel with two inlets allows for the flow
of molecular gradients across 3-dimensional tissue constructs (Fig. 1 and
Fig. 2). Using computational fluid dynamics simulations, we designed the
reactor and showed that these flow established gradients can be created and
maintained indefinitely (Fig. 2). Further, through fluorescent tracer
experiments, we demonstrated the generation and maintenance of the gradients of
bioactive molecules (Fig. 3). To test the effect of molecular gradients
on chondrogenesis, we exposed human MSCs within 3-dimensional collagen
scaffolds to gradients of transforming growth factor beta 1 (TGF-β1).
Chondrogenesis was visualized via toluidine blue staining for
glycosaminoglycans and was only observed within the construct regions exposed
to higher TGF- β1 concentrations (Fig. 4). To develop osteochondral
constructs, the tissue constructs were exposed to opposing gradients of
chondrogenic and osteogenic media. The bioreactor designed here allows for the
production of stable molecular gradients that can be controlled by adjusting
various parameters including the inlet flow rates, inlet molecular
concentrations, and tissue construct position within the reactor (distance from
inlets). The reactor can also serve as a platform to study the effect of
molecular gradients on other composite tissue constructs.

Figure 1:
Bioreactor Design. Two inlets combine into a single flow
channel to form a continuous molecular gradient across the width of the
channel. Three dimensional tissue constructs are placed inside circular wells
below the flow channel.

Figure 2:
Bioreactor and Gradient Simulation.  A. Assembled bioreactor containing MSC-collagen sponge
constructs within the first 4 wells.   B. Gradient simulation across each of the wells of the bioreactor.

Figure 3:
Fluorescent Gradient Characterization. 
Fluorescent intensity (normalized grey value) was measured across the width of
the flow channel at each well containing collagen sponges. Wells are numbered
starting from the well closest to the inlets (well 1). A. Wells 1-4.  B. Wells 5-8.

Figure 4:
Chondrogenesis of MSC-Collagen Sponge Constructs.
Toluidine blue staining for MSC-collagen sponge constructs exposed to A. higher TGF-β1 concentrations  and   B. lower TGF-β1 concentrations along the gradient.