(217bs) Modular Biomaterial Scaffolds for Scalable Tissue Assembly and Rapid Vascularization

Statement
of Purpose: Tissue engineering aims to create
functional biological tissues to treat diseases and damaged organs. A primary
goal is to fabricate 3D constructs that can promote cell-cell interactions and
tissue level organization. Accomplishing these prerequisites with the currently
available conventional scaffolds and fabrication techniques still remains a
challenge. To reproduce the full functionality there is a need to engineer
tissue constructs that mimic the innate architecture and complexity of natural
tissues. The emerging field of modular tissue engineering aims to fabricate
more efficient and complex tissue constructs from the bottom up, with the
desire to recreate the native architecture and to promote extensive
vasculature.

Using
this strategy, we propose to develop methods for fabricating modular tissue
constructs by assembling ECM based microscale modules. These biodegradable
modules possess tunable interior environments, can be seeded internally and
externally, and can be assembled into tissue constructs with parenchymal and
vascular components. The proposed work is based on the hypotheses that the
modular constructs assembled from micro scale modules permeated by a network of
interconnected, endothelial cell-lined channels can facilitate extensive vascularization
and mass transport. This work sought to develop methods for assembling 3D
constructs by fusing microcapsules reloaded with biopolymer solution with the
long term goal of engineering functional tissues by establishing a technology
foundation for subsequent rapid assembly of three-dimensional, tissue density
constructs.

Methods:
Porcine aortic smooth muscle cells (SMC) and Rat hepatocytes were encapsulated
in microcapsules produced by complex coacervation between chitosan, collagen
and hyaluronan. Briefly, cells were suspended in a polyanion solution (0.5 wt% hyaluronan,
0.1 wt% type I collagen, 3.8 wt% sorbitol). Droplets of this solution were generated
and collected in stirred chitosan solution (0.6 wt% high MW chitosan, 3.8 wt%
sorbitol, 1 wt% acetic acid). Ionic interactions between oppositely charged
polymers at the droplet surface generated the encapsulating membrane. To
explore culture of fused capsule modules, 1 week cultured SMC capsules were equilibrated
with 1 wt% heparin in PBS for 2 hours. They were then fused by briefly rinsing
with 0.06 wt. % chitosan solution and allowing 4-5 capsule layers to sit in
contact for 10 minutes, followed by culture medium equilibration. The capsule
with hepatocytes in addition was spun for 10 seconds in 50G, to take out the
excess solution. Separate cultures explored the external seeding of porcine aortic
endothelial cells (EC) on outer surfaces.

Results/Discussion:
Hyaluronan-collagen
containing capsules exhibited cell-mediated contraction during culture,
resulting in the elimination of internal void space within capsules. H&E
staining of sections from the fused, multilayered capsule constructs revealed a
compact internal structure with inter-capsule channels capable of supporting
promote vasculogenesis (Fig. A). Trichrome staining of these sections showed a
dense collagen matrix inside capsules and also large amount of collagen
integrated into the capsule walls (Fig. B). This irregular collagen rich
outside wall may provide a superior substrate for support of extracapsular
angiogenesis. The sections of the hepatocyte seeded construct showed more densely
packed cell construct with potential vascularizable spaces. The EC seeded onto
SMC containing capsules exhibited extensive proliferation (Fig. D). SMC in
these co-culture capsules also exhibited higher rates of proliferation compared
to SMC-only capsules. Hence, this approach has the potential to make
vascularized tissue constructs with high tissue densities.

AB

CD

Conclusions:
These
results demonstrate that GAG-based microcapsules can be fused to from 3D
constructs with vascularizable interconnected channels. This finding
establishes a technology foundation for subsequent rapid assembly of three-dimensional,
tissue density constructs. When coupled with growth of endothelial cells on the
external capsule surfaces, these scalable systems are a promising platform for
modular tissue engineering of several organ systems.