(511f) Gradient Hydrogels for Osteochondral Differentiation of Synovial Mesenchymal Stem Cells

Gradient
Hydrogels for Osteochondral Differentiation of Synovial Mesenchymal
Stem Cells

Tanmay Gharat¹, Andrea C. Jimenez-Vergara²,
Dany Munoz-Pinto², Melissa Grunlan³, Mariah S. Hahn²

¹ Department of Chemical Engineering, Rensselaer
Polytechnic Institute, Troy, New York

² Department of Biomedical Engineering, Rensselaer
Polytechnic Institute, Troy, New York

³ Department of Biomedical Engineering, Texas A&M
University, College Station, Texas

Introduction: Tissue engineering is a promising alternative to
conventional treatments such as micro fracture or grafting for the treatment of
osteochondral defects. Traditional tissue engineering approaches to
osteochondral regeneration typically involve a combination of two distinct scaffolds
for cartilage and subchondral bone. However, such scaffolds are generally unable
to recapitulate the native cartilage-to-bone transition needed for long-term mechanical
stability¹. Moreover, these scaffolds are also unable to achieve the
gradual cell phenotypic transition from chondrocyte to osteoblasts found within
the transition zone. Since mesenchymal stem cells (MSCs) have the unique
ability to undergo differentiation under the influence of matrix-mediated cues,
we propose hydrogel constructs incorporating gradient biochemical signals for
stimulating spatially-graded MSC differentiation for osteochondral repair. Specifically,
polyethylene glycol diacrylate (PEGDA)-based
hydrogels were chosen for their modifiable properties and the ability to
incorporate bioactive factors. Within this PEGDA base, we incorporated
spatially decreasing levels of chondroitin sulfate (CSC) and
transforming growth factor-beta1 (TGF-β1) for inducing decreasing levels
of chondrogenesis with increasing construct depth². Simultaneously,
increasing concentrations of the polydimethylsiloxane (PDMS) (a polymer which
has previously been shown to have osteoinductive capacity when combined with
PEGDA) ³ and bone morphogenetic protein-2 (BMP-2) were used to
achieve increasing MSC osteogenesis with increased construct depth. In this
design, growth factors were tethered to the hydrogel network to enable prolonged signaling (allowing for reduced growth
factor concentrations) and to enable localized delivery⁴, minimizing
the potential for undesirable responses in surrounding tissues. To
evaluate the capacity of these gradient hydrogels to drive spatially-specific
osteochondral differentiation, MSCS derived from synovial fluid (SMSCs) ⁵ were encapsulated within: 1) a ?high CSC,
high TGF-β1? hydrogel [for cartilage], 2) a ?high PDMS, high BMP-2?
hydrogel [for bone], and 3) an ?intermediate TGF-β1, PDMS and BMP-2? hydrogel
[for the transition zone].

Methods: Synthesis of Macromers. PEGDA (3.4 kDa), PDMS_star-MA (2.5 kDa)
and CSC-MA (51 kDa) were prepared as previously described³. Acrylate-derivatized TGF-β1,
acrylate-derivatized BMP-2 and acrylate-derivatized cell adhesion ligand RGDS
were synthesized per standard NHS-chemistry. Fabrication of constructs:
Three hydrogel precursor solutions containing 10% wt. PEGDA, 1 mM acryloyl-RGDS
and photoinitiator were prepared as follows: i) 5.0 mg/ml CSC-MA + 5.0 ng/ml
acrylate-derivatized TGF-β1 (designated ?Cartilage?) ii) 0.5 wt% PDMS_star-MA
+ 2.0 ng/ml acrylate-derivatized TGF-β1 + 20 ng/ml acrylate-derivatized
BMP-2 (designated ?Transition zone?) and iii) 2.0 wt% PDMS_star-MA +
50 ng/ml acrylate-derivatized BMP-2 (designated ?Bone?) along with a PEGDA control
containing no additives. Canine SMSCs were encapsulated within these hydrogels
by 6 min exposure to 365 nm UV light. Construct Culture and Cell
Characterization: Following 21 days of culture in media without supplements,
the phenotype of encapsulated MSCs was analyzed using Western blotting.

Results and Discussion: SMSC protein-level expression of various cartilage and
bone markers was analyzed in each hydrogel formulation following 21 days of
culture (Figure 1). The ?Cartilage' formulation showed a 1.2-fold and 2.2-fold
increase for sox9 and collagen II (Col-2), respectively, relative to the PEGDA
control. Furthermore, the levels of sox9 and Col-2 in the ?Cartilage?
formulation were also 1.5-fold and 2.4-fold higher than in the ?Bone? hydrogels.
These data indicate increased chondrogenesis within the ?Cartilage?
formulation. In contrast, the levels of osteogenic markers collagen I (Col-1)
and alkaline phosphatase (TNAP) were 3.3-fold and 1.6-fold greater in the
?Bone? formulation than in the PEGDA control hydrogels. These markers were also
both approximately 2-fold higher in the ?Bone? constructs relative to the
?Cartilage? hydrogels, indicating increase osteogenesis in the ?Bone?
formulation. In the ?Transition zone? hydrogels, levels of sox9 and Col-2 were
intermediate between those observed in the ?Cartilage? and ?Bone? formulations.
Furthermore, levels of TNAP, Col-I, and osterix were similar to or increased
relative to the ?Bone? formulation, suggesting mixed ?Bone?/?Cartilage? cell
behavior in the ?Transition zone? formulation.  

Conclusions: Overall, the results indicate a gradual transition
from chondrocyte-like to osteoblast-like phenotype within the developed
gradient hydrogels, as evident from SMSC neo-matrix deposition. In the
long-term, scaffolds formed with spatial gradients of chondrogenic and
osteogenic mediators may prove beneficial for osteochondral regeneration.

Acknowledgments: The authors gratefully acknowledge funding from the NIH, NIBIB and AKC.

References: 1. Keeney, M., Tissue Eng. Part B 15 (1), 55-73 (2009),
2. Varghese S., Matrix Biology 27, 12-21 (2008) 3. Munoz-Pinto, D. J., Tissue
Eng. Part A 18, 1710?1719 (2012), 4. Chen, C., Knee Surgery, Sport. Traumatol. Arthrosc.
19, 1597?1607 (2011). 5. De Bari, C., Arthritis & Rheumatism 44 (8),
1928-1942 (2001).