A mechanically stiff 3D printed structure combined with a chondrogenic hydrogel for cartilage defect repair
Elizabeth Aisenbrey1, Andrew Tomaschke1, Callie Fiedler1, Cecilia Pascual-Garrido2, Virginia Ferguson1, Robert McLeod1,
Stephanie J. Bryant1.
1
University of Colorado, Boulder, CO University of Colorado Anschutz Medical Campus, Denver, CO 2
Statement of Purpose: Hydrogels laden with
physiochemical cues arising from cartilage-ECM analogs
mesenchymal stem cells (MSCs) are a promising treatment
of chondroitin sulfate and cell adhesion peptides and with
for cartilage defects in vivo. To design a successful
dynamic loading, to enhance chondrogenesis, and with
hydrogel strategy, the ideal hydrogel will promote
MMP-7 crosslinks to facilitate degradation and tissue
chondrogenesis of MSCs, facilitate growth of a newly
growth. After 9 weeks, encapsulated MSCs had formed
synthesized extracellular matrix (ECM), and support
their own cartilage ECM made of collagen II. Hydrogel
physiological loads in vivo that restore joint function.
degradation was confirmed using an antibody against PEG.
However, hydrogels suitable for cell encapsulation and
The compressive modulus measurements showed an initial
tissue growth require a low modulus; thus, achieving a
drop indicative of cell-mediated degradation, followed by
design that embodies all three attributes is challenging. To
a steady increase that is attributed to ECM deposition and
overcome this limitation, we are developing an approach
growth (Fig. 1B). The stiff structure was designed with a
that decouples the cellular hydrogel environment from the
cathedral-like arch structure to capture the orientation of
structurally stiff component. Herein we present our
the collagen fibers natively. As initial proof of concept, a
approach whereby a 3D digitally printed stiff hydrogel
3D printed structure of continuous columnar pillars
structure is created and backfilled with a MSC-laden
(~200µm) was implanted in a cartilage defect and infilled
cartilage biomimetic and biodegradable hydrogel.
with a soft hydrogel (Fig. 1C). We are extending this initial
Methods: An ex vivo model of a cartilage defect (3 mm
study to a stiff printed PEG-based hydrogel. Studies are
diameter, ~2-3 mm deep) in porcine osteochondral plugs
currently underway to combine the soft MSC-laden
(8.5 mm diameter, ~10 mm height) was created. Cartilage
hydrogel with the 3D printed to create a mechanically stiff,
defects were filled with a high modulus (E=1MPa) 40wt%
but chondrogenic hydrogel.
poly(ethylene glycol) (PEG) dimethacrylate hydrogel or
left empty and subjected to dynamic compressive loading
(20% offset strain, 2% amplitude strain) for 1 hr/day for 28
days. Sulfated glycosaminoglycan (sGAG) retention was
determined by safranin O staining. A soft hydrogel was
formed from 9wt% 8-arm PEG norbornene (10kDa), 1wt%
thiolated chondroitin sulfate (16% conjugated), and 0.1
mM CRGDS, with a matrix metalloproteinase (MMP)-7
sensitive peptide (GCRDPLELRADRCG) at 1:1 thiol:ene
ratio. Human MSCs (25 year old female) were
encapsulated at 50 million cells/ml via photopolymerization (8 minutes, 352 nm, 5mW/cm2). Cell-laden
hydrogels were cultured in chondrogenic differentiation
media with TGFβ3 (2.5 ng/ml) for 9 weeks. The cell-laden
constructs were analyzed for collagen II and for PEG
(courtesy of Dr. Steve Roffler of Anti-PEG and the
Institute of Biomedical Sciences at Academia Sinica) by
immunohistochemistry and by compressive modulus
Figure 1. A) Cartilage defects empty and filled with a 1MPa PEGDM
(unconfined compression at a constant strain rate of
hydrogel in porcine osteochondral explants after 4 weeks of
0.1mm/s; linear region of stress-strain curves at 10-15%
physiological loading. Safranin O staining (100x) shows retention of
strain). A structural component (2.06mm(l), 2.06mm(w),
GAGs (red) of tissue surrounding defect. B) Collagen II and PEG
2.0 mm(h)) of continuous columnar pillars (200µm) was
(green) and nuclei (blue) IHC (scale bar=20µm) and the compressive
modulus (n=3) of an MSC-laden MMP7 degradable hydrogel. C) 3D
3D printed via open-source stereolithography (SLA) using
printed pillar structure implanted in a cartilage defect of porcine
an Autodesk Ember printer. The 3D printed structures were
osteochondral explants that is infilled with the soft hydrogel.
implanted into cartilage defects of osteochondral explants
and backfilled with a soft PEG hydrogel.
Conclusions: We demonstrate that a mechanically
Results: Our ex vivo cartilage defect model backfilled with
supportive matrix protects the surrounding cartilage from
a 1 MPa stiff hydrogel, similar in modulus to native
degeneration when subjected to physiologic loading.
cartilage, protected the surrounding cartilage evident by
Furthermore, we demonstrate our approach in developing
maintenance of sGAGs. However when left unfilled,
a 3D printed structure that can be backfilled with a soft
degeneration was evident after 28 days suggesting the
chondrogenically promoting and biodegradable hydrogel
necessity of a mechanically stiff hydrogel to maintain the
to achieve cartilage tissue growth and simultaneously
health of the surrounding cartilage (Fig. 1A). To develop
support joint loads.
our approach, we first identified a soft cartilage biomimetic
Acknowledgments: Funded by NIH 1R01AR069060.
hydrogel. Specifically, the hydrogel was designed with