This study evaluated the use of adipose-derived stem cells (ADSCs) and neonatal articular chondrocytes (NChons) encapsulated in a methacrylated chondroitin sulfate hydrogel to catalyze cartilage formation. The results showed that the chondroitin sulfate hydrogel supported cell proliferation and deposition of cartilage markers like collagen and GAGs when ADSCs and NChons were co-cultured in the presence of TGF-β. Histological analysis and mechanical testing confirmed the formation of cartilage tissue only in the co-culture group with TGF-β. The study demonstrated the potential of a fully biodegradable chondroitin sulfate hydrogel for catalyzed cartilage

1. Harnessing Adipose-Derived Stem Cells to Catalyze Tissue Regeneration
by Neonatal Chondrocytes in Fully Biodegradable Hydrogels
Krista Chew | Heather Rogan | Fan Yang
Stanford University | Bioengineering – Orthopaedic Surgery
Background and Rationale
Methodology
Conclusions and Discussion
• Cartilage injury is a leading cause of disability among adults. Cartilage loss is largely irreversible, due to
its low cellularity and avascular nature.
• Allogeneic neonatal articular chondrocytes (NChons) are a promising cell source for cartilage
regeneration because they are highly proliferative, immune-privileged, and readily produce abundant
cartilage matrix. However, scarce donor availability for NChons greatly hinders their broad clinical
application. Stem cells, aside from their ability to differentiate into different tissue types, may contribute
to tissue regeneration through the secretion of paracrine factors. Our lab has recently reported the
potential to use a minimal number of NChons to catalyze cartilage tissue formation by co-culturing them
with adipose-derived stem cells (ADSCs) in 3D biomimetic hydrogels (5% poly [ethylene glycol] diacrylate
[PEGDA] / 3% chondroitin sulfate [CS] [Lai et. al., Scientific Reports, 2014]).
• The goals of this study are: (1) to evaluate the potential of a CS-based, fully biodegradable,
cartilage matrix–mimicking hydrogel in supporting catalyzed cartilage formation by ADSCs and
NChons; and (2) to determine whether transforming growth factor beta (TGF–β) is required for such
synergy to occur.
• We chose a methacrylated CS hydrogel: a fully degradable
hydrogel made from components of native cartilage matrix. We
chose a 9% (weight/volume) CS concentration for cell
encapsulation, which is comparable to the Young’s modulus of
previously used PEGDA/CS composite hydrogels that supported
catalyzed cartilage formation (30 kPa).
• ADSCs were isolated from human liposuction aspirates.
NChons were isolated from juvenile bovine articular cartilage.
The cells were encapsulated at a density of 13 million cells/mL.
CS was cross-linked under UV light for 5 minutes at 4 mW/cm2.
The cells were cultured in chondrogenic media for 21 days.
ADSC (100A)
NChon (100N)
Co-Culture (75A:25N)
- +
+
+
-
-
TGF-β
Groups
Cell Type
Results
ADSC
Co-Culture
NChon
- TGF-β + TGF-β- TGF-β + TGF-β
A B C
Figure 1 | Co-culture in CS hydrogels led to enhanced cell proliferation and catalyzed collagen deposition; TGF-β is required for
such synergy to occur. (A) Cell proliferation is quantified by DNA content. (B) Hydroxyproline content per wet weight at day 21.
(C) Compressive moduli of cell/hydrogel constructs at day 21. The red line represents the average hydrogel stiffness at day 1.
Figure 2 | CS hydrogels supported catalyzed neocartilage formation, as shown by immunofluorescence
staining of cartilage marker type II collagen (A) and Safranin O staining (B). (A) Distribution of newly-
deposited type II collagen. Green: collagen II; blue: cell nuclei. Scale bar = 100µm. (B) Safranin O imaging.
Red: sGAG; black dots: cell nuclei. The insets portray a 2X image of the hydrogel. See Acellular (right) for
depiction of background (hydrogel and extracellular matrix). Scale bar = 100µm.
A B
Figure 3 | Cell viability assay
at day 21 confirms high
viability and formation of
larger neocartilage nodules
in the co-culture group only
in the presence of TGF-β.
Green: live cells; red: dead
cells. Scale bar = 200µm.
• Outcome analyses: live/dead assays, mechanical testing, histological analysis, and biochemical
analysis (PicoGreen assay for DNA content, hydroxyproline assay for collagen content). Preparation for
biochemical assays included acquisition of wet weight, lyophilization, acquisition of dry weight, and
digestion in papainase solution. Samples for histology were paraffin fixed, embedded in OCT, frozen in
liquid nitrogen, and cryosectioned prior to staining.
NChonCo-Culture
- TGF-β
+ TGF-β
ADSC
kvchew@stanford.edu
• The results demonstrate that fully degradable, CS-based hydrogels are capable of supporting ADSCs in
catalyzing NChons to form cartilage in 3D. Such synergy requires the presence of TGF-β.
• The outcomes of this study justify the feasibility of using fully biodegradable hydrogels to achieve
catalyzed cartilage formation, which is preferable for clinical translations.
• Future studies will test the synergy and efficacy in vivo using the cartilage defect model.
Krista Chew would like to thank Heather Rogan and Professor Fan Yang for mentoring her throughout
the summer. We would like to thank the Stanford Bioengineering REU program for supporting this
work.
Acknowledgements