(678a) Collagen-Mimetic Hydrogels for Elucidating Mesenchymal Stem Cell Fate Decisions | AIChE

(678a) Collagen-Mimetic Hydrogels for Elucidating Mesenchymal Stem Cell Fate Decisions


Becerra, S. - Presenter, Texas A&M University
Munoz-Pinto, D., Texas A&M University
Rivera, J., Texas A&M Health Science Center
Russell, B., Texas A&M Health Science Center
Hook, M., Texas A&M Health Science Center

Introduction: A number of obstacles remain before
engineered tissues based on mesenchymal stem cells (MSCs) can be considered
viable clinical alternatives. In particular, rational design of scaffolds to
elicit desired MSC lineage progression is problematic due, in part, to our
incomplete understanding of MSC responses to extracellular matrix
(ECM)-mediated stimuli. In the present work, we begin to address this challenge
by probing MSC responses to collagen-based biochemical motifs using novel
hybrid hydrogels. These hydrogels were generated by covalently crosslinking
diacrylate-derivatized poly(ethylene glycol) (PEGDA) and a collagen-mimetic
protein, termed  Scl2-1. Scl2-1 is unique in that it contains the GXY repeats
and stable triple helical structure of native collagen but lacks collagen's
array of cell adhesion, cytokine binding, and enzymatic degradation sites.
Thus, Scl2-1 provides a ?blank-slate? into which desired collagen adhesion
sequences can be programmed by site-directed mutagenesis while maintaining the
triple helical context natively associated with these motifs. The current work
employs modified Scl2-1 proteins to investigate the impact of α1β1
and α2β1 integrin signaling on human
bone-marrow derived MSC (hMSC) fate decisions. Specifically, the influence of Scl2-2
(Scl2-1 containing an α1β12β1
binding motif) on hMSC lineage progression was compared to that of Scl2-3 (Scl2-1 containing an α1β1
binding motif).

and Methods: Expression and Mutagenesis of Scl2
. Scl2-2 and Scl2-3 were generated by
encoding for sequences GFPGER and GFPGEN, respectively, using site-directed
mutagenesis of the plasmid encoding for Scl2-1. Associated proteins were
recombinantly expressed in E.coli JM101 and were purified by affinity
chromatography. Scl2 Conjugation to a PEG
. Scl2 proteins were
functionalized with photoreactive crosslink sites by reaction with
acrylate-PEG-hydroxysuccinimide (Ac-PEG-NHS, MW 3400) per standard
protocols. Fabrication of PEGDA-Scl2 Hydrogels. PEGDA-Scl2
gels were fabricated by combining 10 wt% PEGDA (3.4 kDa) with photoinitiator
(Irgacure 2959), 1 mg/mL of Ac-PEG-Scl2, Ac-PEG-Scl2-2, or Ac-PEG-Scl2-3,
and 1x106 hMSCs (Lonza) per mL. The solutions were then crosslinked
via 90 s exposure to 365 nm UV light. In fabricating these gels, a weight ratio of PEGDA to Scl2 of
100:1 was used to ensure that the elastic
modulus, mesh size, and degradation rate of each hydrogel network would be
dominated by PEGDA. Constructs were cultured in DMEM supplemented with 10%
heat-inactivated fetal bovine serum (FBS) for 7 days.

and Discussion:

Following confirmation of the ability of Scl2-2 and Scl2-3 to stimulate
distinct focal adhesion kinase (FAK) signaling (Fig. 1A), hMSC-laden
PEGDA-Scl2 hydrogels were prepared and cultured for 7 days. Media without
differentiation supplements was utilized in order to focus on the influence of
integrin-mediated signaling on hMSC fate decisions. Endpoint samples were
harvested and transcription factors indicative of early lineage progression
were analyzed by competitive ELISA. No differences in myoD or Runx2 levels were
noted among hydrogel formulations, suggesting that the selected integrin
adhesion motifs did not significantly stimulate myogenic or osteogenic
differentiation, at least for the gel formulations probed. However, significant
differences in PPARg (adipogenic) and sox9 (chondrogenic)
levels were observed (Fig. 1B). Specifically, PPARg
expression appeared to be elevated by α2β1
signaling but not by α1β1 adhesion (Scl2-2 vs
Scl2-3). In contrast, α1β1 signaling (Scl2-3)
was associated with increased sox9 expression, but not when in the presence of
simultaneous α2β1 adhesion (Scl2-2). Given that
α2β1 binding activates p38 whereas α1β1
does not (Fig. 1A), the p38 MAPK signaling cascade may play an
important role in hMSC adipogenic differentiation.

The present results
demonstrate our ability to achieve a controlled 3D environment that can be used
to probe MSC responses to highly defined collagen-based adhesion signals while
maintaining collagen's triple helical context. This controlled hydrogel
platform enables more precise examination of the signaling underlying observed
cell responses and should significantly advance our understanding of the
processes associated with MSC lineage progression. Future studies will
incorporate gene silencing to elucidate causative cell signaling.

See more of this Session: Biomimetic Materials

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