Biomedical Engineering Reference
In-Depth Information
environment, and affects intracellular signaling downstream (Schiller et al. 2011).
Despite its importance, the vast majority of published studies on cell-matrix inter-
actions have not quantified the surface chemistry of the matrix used in a study due
to lack of appropriate methods. A few studies have reported the mass of adsorbed
matrix biomolecules on a flat cell culture dish (Maheshwari et al. 2000; Engler
et al. 2004; Valenick et al. 2006). Other methods measure quantities related to the
density of RGD (arginine-glycine-aspartic acid) ligands in artificial biomaterials
that contain a single ligand type (Barber et al. 2005; Harbers et al. 2005; Kong
et al. 2006; Huebsch and Mooney 2007; Hsiong et al. 2008). Certain spectroscopic
techniques quantify chemical groups on the surface of biomaterials (Ma et al. 2007;
Kingshottet al. 2011); however, data from these measurements cannot be converted
straightforwardly to density of ligands recognized by particular adhesion receptors.
A novel methodology has been described for quantifying the density of ligands of a
particular adhesion receptor on the surface of a 3D matrix in situ (Tzeranis et al. 2010,
2014). It consists of: (1) developing soluble fluorescently labeled markers whose bind-
ing properties mimic those of particular adhesion receptors, (2) using a binding assay
of the markers, based on 3D microscopy to detect the fluorescent markers bound on
the matrix, and (3) estimating the density of adhesion ligands using a novel model that
describes binding of soluble receptors on ligands present on an insoluble surface.
The methodology has been used to measure the adhesion ligand density for the
two major collagen-binding integrins (α1β1, α2β1) in two kinds of porous collagen
scaffolds (named here “active” and “inactive”) that were utilized in peripheral nerve
(PN) regeneration research (Soller et al. 2012). The active scaffold had previously
shown strong regenerative activity in a peripheral nerve study while the inactive
control was minimally active (Soller et al. 2012). The two scaffolds had identical
pore geometry and chemical composition (having been fabricated by freeze-drying
microfibrillar type I collagen using the same protocol) but differed in the cross-
linking treatment used to introduce intermolecular bonds. The active scaffold was
crosslinked by dehydrothermal treatment (DHT), a physicochemical treatment that
forms peptide bonds between chains without using a crosslinking agent (Yannas
and Tobolsky 1967). The inactive scaffold was crosslinked chemically using the
reagents EDAC and NHS (Hermanson et al. 2008). It is known that EDAC-NHS
crosslinking agents react with carboxylic groups, identified in key acidic residues
in all major ligands of α1β1 and α2β1 (Leitinger et al. 2011). Used as baseline in
this study, a scaffold was prepared by freeze-drying process and had not been cross-
linked; this scaffold had previously shown negligible regenerative activity. The two
nearly identically structured, crosslinked scaffolds were selected after showing re-
markably different ability to induce peripheral nerve regeneration in the transected
rat sciatic nerve model (Soller et al. 2012).
To prepare fluorescent labels for ligands for each integrin (α1β1 or α2β1) two
kinds of soluble markers were expressed in
E. coli
, and purified by affinity chro-
matography: the I domain of the integrin α subunit (a nonfluorescent marker), and
the same I domain tagged with a tetracysteine (TC) motif. It has been reported that
I domains recognize and bind to the same adhesion ligands as does the correspond-
ing integrin (Calderwood et al. 1997; Hynes 2002). When the TC tag binds the
