(453d) A Multiscale Approach to Stem Cell-Based Chondrogenesis for Cartilage Repair

Tissue
engineering is a promising method for long-term repair of cartilage lesions;
however, current tissue-engineered cartilage constructs have inferior
mechanical properties compared to native cartilage. Other researchers attribute this problem to a lack of
an oriented structure in the constructs at the microscale that is present in
the native tissue.  In this
study, we utilized contact guidance to develop constructs with microscale
architecture for improved chondrogenesis and function. In this system, we used
microscale channels on the scaffold surface to guide mesenchymal stem cells
(MSCs) undergoing chondrogenesis to produce oriented extracellular matrix
(ECM).  Stable channels of varying microscale dimensions were formed in
collagen-based and polydimethylsiloxane membranes via a combination of
microfabrication and soft-lithography. Human MSCs were selectively seeded in
these channels. The chondrogenic potential of MSCs seeded in these channels was
investigated by culturing them for 3 weeks under differentiating conditions
with transforming growth factor β1 and then evaluating the subsequent
synthesized tissue for mechanical function and by type II collagen
immunohistochemistry. We demonstrated selective seeding of viable MSCs within
the channels (Fig. 1). MSC aligned and produced mature collagen fibrils
along the length of the channel in smaller linear channels of widths 25-100 µm
compared to larger linear channels of widths 500-1000 µm. Further, substrates
with microchannels that led to cell alignment also led to superior mechanical
properties compared to constructs with randomly seeded cells or selectively
seeded cells in larger channels. The ultimate stress and modulus of elasticity
of constructs with cells seeded in smaller channels increased by as much as
four folds (Fig. 2). We demonstrated that microscale guidance is useful
to produce 2-dimensional oriented cartilage structures with improved mechanical
properties. Furthermore, we extended the 2-dimensional construct findings to a
3-dimensional construct for preclinical experiments. We successfully created
3-dimensional constructs for in vivo applications by rolling up 2-dimensional
MSC-seeded collagen based scaffolds and formed 3-dimensional rolled-up large scale (3.5 mm in diameter × 18 mm in length) cartilage constructs with microscale
architecture to guide the differentiating MSCs to produce oriented ECM.
Histology and immunohistochemistry indicated extensive glycosaminoglycan and
collagen type II production in the 3-dimensional constructs, which are both indicative of chondrogenesis (Fig. 3). Our
results show that the microscale guidance channels incorporated within the
3-dimensional cartilage constructs led to the production of aligned cell-produced
collagenous matrix and enhanced mechanical function. The tissue modulus of elasticity of the 3-dimensional cartilage constructs
containing guidance channels (1.16-2.07 MPa) increased by as much as six times compared to constructs without
channels, which are higher
than most current tissue-engineered constructs (Fig. 4). Overall, these findings offer new insight into how
microscale guidance channels can regulate matrix deposition and long term
construct development. Furthermore, our results provide new parameters which
help optimize the design of functional MSC-based tissue-engineered cartilage
using collagen-based substrates and can be used to fabricate large clinically
useful MSC-based cartilage constructs with superior mechanical properties.

Fig.
1 Fluorescent images of cells
in linear channels of widths 25 μm (A), 50 μm (B), and
100 μm (C) show selective seeding compared to untreated channel
surfaces (D). Scale bars: 50 μm

Fig.
2 Effect of microscale
guidance on mechanical properties. Collagen membranes were subject to tensile
testing until failure. Mean values of elasticity modulus (A), and ultimate stress (B) are shown. BK
and EDC represent respectively uncrosslinked and EDC crosslinked
collagen membranes without microchannels or MSCs. C25 and C100
represent collagen membranes consisting of 25 μm and 100 μm linear
channels only, without MSCs. All other channels were seeded with MSCs. Ran:
EDC crosslinked collagen membranes without channels; 25, 50, 100,
250, 500 and 1000 represent widths (μm) of linear
channels in the EDC crosslinked collagen membranes (A)
* indicates statistically significant difference (p < 0.05) compared to data
with no channels (RAN). # indicates statistically significant difference
(p < 0.05) compared to data with 1000 μm channels (1000). (B) *
indicates statistically significant difference (p < 0.05) compared to data
with no channels (RAN), 250 (250), 500 (500) and 1000
μm (1000) -wide channels.

Fig.
3 Cartilage tissue constructs formed
by the role up process. Three different microchannel designs were used. A-C
indicate longitudinal sections and a-c indicate cross sections. Scale Bar=100 μm
(A-C), 1 mm (a-c)

Fig.
4 Mechanical properties of cartilage
constructs with microchannel guided ECM. Compressive modulus of the whole
construct (A) and the chondrogenic tissue (B) are shown.