Dodge, G.R.

Farran, A.J.

Mauck, R.L.

Mohanraj, B.

Elsevier - Publisher Connector 

Temporal evaluation of scaffold-free cartilage tissue analogs: mechanical properties and cell and matrix content 

Abstracts / Osteoarthritis and Cartilage 21 (2013) S63–S312S306Fig. 3. Histological analyses in transduced human normal (A,B) and OA (C, D)
cartilage (day 30): safranin O/H&E staining and type-II collagen immunoreactivity
(insets) All at magniﬁcation x40.
Fig. 4. Immunohistochemical detection of BrdU in transduced human normal and
OA cartillage (day 30). Magniﬁcation x10.Fig. 5. Immunohistochemical detection of type-X collagen in transduced human
normal and OA cartilage (day 30). Magniﬁcation x20.
596
TEMPORAL EVALUATION OF SCAFFOLD-FREE CARTILAGE TISSUE
ANALOGS: MECHANICAL PROPERTIES AND CELL AND MATRIX
CONTENT
B. Mohanraj, A.J. Farran, R.L. Mauck, G.R. Dodge. Univ. of Pennsylvania,
Philadelphia, PA, USA
Purpose: Cartilage tissue engineering aims to mimic the architecture
and function of native cartilage. Although many approaches rely on the
use of cells seeded-biocompatible materials, a number of alternative
approaches use scaffold-free platforms. We have previously established
a material-free cartilage tissue analog (CTA), and successfully demon-
strated this model can maintain chondrocyte phenotype in vitro for
extended periods. In the absence of a scaffold, cells in CTAs self-
aggregate and elaborate matrix, therefore mechanical properties take
longer to develop than in a scaffold-based system. Here, we evaluated
the temporal development of CTA mechanical properties, and deﬁned
the relationship between construct mechanics and cell and matrix
content. This study will not only help to determine patterns of growth
and maturation of an engineered cartilage with the potential to repair
defects, but also identify time periods at which construct properties
most closely mimic juvenile or adult cartilage for the study of age-
related cartilage pathology and underlying mechanisms.
Methods: Cartilage was harvested from juvenile bovine knees, minced,
and digested overnight in DMEM with 1mg/mL collagenase-II. The cell
suspension was ﬁltered, washed and seeded at 1x106 cells/well in our
self-aggregating suspension culture model using hydrogel coated 96
well plates. CTAs were cultured in DMEM with 10%FBS 1 to 24 weeks,
during which time the mechanical properties, biochemical content,
histological features, and molecular aspects were evaluated. CTA and
cartilage equilibrium (10% strain, stress relaxation) and dynamic (1%
strain, 1Hz) compressive properties were tested in unconﬁned
compression. GAG and DNA content was determined using the DMMB
and PicoGreen assays, respectively. Histological sections were stained
with Alcian blue to identify proteoglycan and nuclear fast red; cell:-
matrix ratios were quantiﬁed by analyzing micrographs with ImageJ.
Results: Over the ﬁrst 24 hrs a cell-rich aggregate formed, which
matured via the production of cartilage ECM with time in culture.
Between 4 and 12 weeks, the equilibrium modulus remained low (<50
kPa), but increased by>500% by 16-24 weeks [from 23 (+/-9) kPa to 123
(+/-88) kPa]. These values were greater than 20% of that of native adult
cartilage [560 (+/-252) kPa] (Fig 1A). The dynamic modulus likewise
increased over this time period, though the changes were not as
dramatic (w200%) (Fig 1B).
Abstracts / Osteoarthritis and Cartilage 21 (2013) S63–S312 S307Figure 1. (A) Equilibrium and (B) Dynamic modulus of CTAs mechanically tested over
24 weeks.
CTA mechanical properties strongly correlated with GAG content across
all donors, but not correlate with DNA content. The increase in
mechanics over 16 weeks of culturewas consistent with the histological
analysis. At early times of culture, the cell:matrix ratio was quite high
(0.13+/-0.03), however by 16 weeks of culture it decreased to 0.06+/0.01
matching that of native adult cartilage (0.05+/-0.01) (Fig 2A). Histo-
logical sections illustrate the increased amount of matrix per cell at 16
weeks and its similarity to native cartilage (Fig 2B).
Figure 2. (A) Cell:matrix ratio and (B) representative histological sections of CTAs and
native cartilage.
Conclusions: Our results show that as CTAs mature, chondrocytes
produce cartilage-like ECM, with cell:matrix ratios similar to native
cartilage. This transformation generates engineered cartilage constructs
with near-native mechanical integrity via a scaffold-free mechanism.
Taken together with our previous studies using this model, our results
suggest that mechanical outcomes are more closely related to proteo-
glycan/GAG content, while early growth involves initial cellular estab-
lishment of the CTA. Having established the CTAs baseline mechanical
properties, future studies may employ dynamic loading, which we have
shown increases matrix production and may further improve
mechanical properties.
597
AGE-DEPENDENCE OF ZONAL CHONDROCYTE/MSC CO-CULTURES IN
HA HYDROGELS FOR CARTILAGE TISSUE ENGINEERING
M. Kim, J.A. Burdick, R.L. Mauck. Univ. of Pennsylvania, Philadelphia, PA,
USA
Purpose: To determine whether zonal chondrocyte identity inﬂuences
MSC fate decisions, and whether this inﬂuence is age-dependent.
Methods: Adult (AbMSC) or juvenile bovine MSC (JbMSC) were isolated
from bone marrow, and juvenile zonal chondrocytes (JbCH) were iso-
lated from full thickness articular cartilage (Fig 1). MSC only, zonal CH
only, or a mixed population (MSC:CH ¼ 4:1) were seeded at 60 x 10^6
cells/mL in 1% w/v photo-crosslinkable HA hydrogel. Cylindrical
constructs (Ø4 x 2.25mm) were cultured in a deﬁned medium con-
taining 10ng/mL TGF-ß3. Cell viability and the distribution and prolif-
eration of mixed populations of MSCs and CHs were tracked using
CellTracker. Unconﬁned compression testing was carried out to deter-
mine mechanical properties. Sulfated glycosaminoglycan (s-GAG) and
hydroxyproline contents were determined. Parafﬁn embedded sections
(8mm) were stained with Alcian Blue for proteoglycan, and underwent
immunostaining for collagen I, II, X and chondroitin sulfate. Signiﬁcance
was determined by two-way ANOVA with Tukey's post hoc test
(p<0.05).
Results: JbMSCs were viable in 3D HA gels, both alone and in co-
culture with CHs, and retained the cell tracker signal (Fig 2A-C) over
56 days. While AbMSCs were also viable with stably labeled in co-
culture (Fig 2D-F), AbMSCs alone were less viable and aggregated
together, resulting in the formation of a single mass (Fig 2G).Mechanical and biochemical properties for pure CH and co-culture
groups increased with time and depended on the zonal origin of the
CHs; lower properties were achieved with superﬁcial CHs and higher
properties with deep zone CHs (Fig 3). Constructs containing JbMSCs
alone grew well, matching the properties of deep zone CHs (633 kPa
and 6.1% WW GAG) (Fig 3A-B) while construct formed with only
AbMSCs resulted in poor properties (5 kPa and 1.2%WWGAG) (Fig 3C-
D). Interestingly, properties in constructs seeded with AbMSCs were
markedly improved when co-cultured with zonal JbCHs (Fig 3C-D).
Analysis of the efﬁcacy of co-culture for JbMSC-JbCH showed inde-
pendent contributions of each cell type, while for AbMSC-JbCH co-
cultures, there was a marked synergistic effect in both EY (Fig 3E) and
GAG content (Fig 3F).
Discussion: This study examined the impact of age on the chondrogenic
capacity of bovine MSCs and how aging might alter response to co-
culture with zonal articular CHs in a 3D platform. Zonal origin of CHs
inﬂuenced construct properties both alone and in co-culture groups.
The lowest properties observed were with constructs containing
superﬁcial CHs and the highest properties were with deep zone CHs,
suggesting that CHs retain their zonal characteristics, even after
extensive culture expansion and seeding in this HA system. While
JbMSCs grew quite well on their own in HA gels, AbMScs failed to
mature. While JbCHs had no impact on the growth of co-cultures with
JbMSCs, a synergistic enhancement of construct properties was
observed in co-cultures of AbMSCs and JbCHs. Future work will deter-
mine the mechanism by which CHs inﬂuence MSCs in this co-culture
system. Using these co-cultures, wewill also determine (1) the minimal
density of CHs needed to elicit the synergist growth response, (2) the
potentially reduced need for TGF with co-culture, (3) the resistance of
co-cultures to hypertrophic challenge in/ex vivo, and (4) co-culture
mediated in vivo cartilage formation for functional and stable
restoration.
Fig 1. Co-culture of Zonal CHs and MSCs.Ă
Fig 2. Visualization of CHs (Red) and MSCs (Green) (mixture ratio; CH:MSC [ 1:4)
and geometric changes of AbMSC- based constructs after 56 days of culture. (A-C)
JbMSC-JbCH, (D-F) AbMSC-JbCH (10X; Scale bar[100 mm), (G) Co-cultures of
AbMSCs and JbCHs at day 56 (Marking [ 1 mm).


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Temporal evaluation of scaffold-free cartilage tissue analogs: mechanical properties and cell and matrix content

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