Harnessing Cell–Biomaterial Interactions for Osteochondral Tissue Regeneration - Tissue Engineering: Cell-Surface Interactions for Tissue Culture - page 83

markers, including type II collagen, aggrecan, and SOX9, as well as increased
production of sulfated GAG and total collagen than a PhaP scaffold without RGD
and a blank scaffold.
RGD peptides immobilized on macroporous alginate scaffolds have also been
shown to stimulate chondrogenic differentiation of human MSCs [ 128 ]. Specifi-
cally, the positive effect of RGD modification of scaffolds upon chondrogenesis was
illustrated in the observation that the TGF-induced Smad signaling pathway
involved in chondrogenic differentiation of MSCs was more activated in the
presence of RGD peptide under the conditions studied [ 128 ]. A western blot and
its densitometric analysis showed that the phosphorylation of both SMAD2 and
ERK1/2 was significantly higher in the RGD-incorporating scaffolds than in the
control alginate scaffolds. This result is consistent with the observed upregulation
of chondrogenic maker gene expression, including type II collagen and SOX9.
A study using human articular chondrocytes also demonstrated that bioactive RGD
incorporation on a copolymer substrate of polystyrene/poly( L -lysine)/PEG
improved GAG production and type II collagen messenger RNA (mRNA)
expression compared with a blank polystyrene substrate [ 129 ]. Another study
sought to mimic native RGD release by combing MMP-13 cleavage sites and
demonstrated the importance of temporal regulation of integrin-binding peptides in
chondrogenic differentiation [ 130 ]. In this study, human MSCs were encapsulated
in PEG hydrogels with either an uncleavable RGD tether (CRGDSG) or a cleavable
RGD tether (CPENFFGRGDSG). Both tethers were designed with MMP-13-
specific cleavage sites. Once MMP-13 had been produced by encapsulated cells,
RGD was released from the hydrogel system via the MMP-13 enzymatic cleavage
of the tether. Released RGD induced greater chondrogenic differentiation of the
MSCs than gels with uncleavable sequences, as indicated by higher GAG deposi-
tion and type II collagen staining. It has been suggested that RGD also functions as
a mechanotransducer [ 131 ]. Under the mechanical loading environment, RGD
ligands stimulated cartilage-specific gene expression and ECM protein synthesis.
When dynamic compressive strains were applied to bovine chondrocytes encap-
sulated in a PEG hydrogel, the chondrocyte phenotype index (the expression ratio
of collagen II and collagen I) and the proteoglycan synthesis were enhanced in
RGD-incorporating gels relative to those without RGD incorporation.
ADSCs obtained from rats have also been used to investigate the influence of
the integrin-binding peptides on chondrogenic differentiation [ 132 ]. RGD-
chimeric protein with a cellulose binding domain in alginate beads resulted in
increased gene expression of type II collagen, SOX9, aggrecan, and fibronectin, as
well as the accumulation of chondrogenic matrix during TGF-b 3 -induced differ-
entiation. The results also demonstrated that the mechanism of RGD-chimeric
protein stimulation of chondrogenic differentiation might be associated with
suppressed RohA activity in the early differentiation stage. Furthermore, human
ESC-derived cells could also be encapsulated in RGD-incorporating hydrogels
[ 79 , 133 ]. Human ESC-derived MSCs were positive for MSC surface markers
including

CD29, CD44, CD109, and platelet-derived growth factor a [ 79 ].

These cells exhibited in vitro neocartilage formation with basophilic ECM