Is quercetin an alternative natural crosslinking agent to
genipin for long-term dermal scaffolds implantation?
Karin V. Greco
, Lydia Francis
, Honglei Huang
, Rutger Ploeg
, Aldo R. Boccaccini
Northwick Park Institute for Medical Research (NPIMR), Watford Rd, Harrow, Middlesex, UK
Nufﬁeld Department of Surgical Sciences, Medical Sciences Division, University of Oxford, Oxford, UK
Department of Materials Science, Engineering, Institute of Biomaterials, University of Erlangen, Nuremberg 91058, Erlangen, Germany
As biocompatible matrices, porcine dermal scaffolds have limited application in tissue engineering due to rapid degradation
following implantation. This study compared the physical, chemical and biomechanical changes that occurred when genipin
and quercetin were used to crosslink dermal scaffolds and to determine whether quercetin could be used as an alternative to
genipin. Physicochemical changes in the collagen were assessed using spectroscopic methods [X-ray diffraction analysis
(XRD) and nuclear magnetic resonance (NMR) analysis]. The crosslinking reaction was evaluated by quantiﬁcation of amino
acids and the degree of this reaction by ninhydrin assay. Because the mechanical behaviour of the collagen matrices is highly
inﬂuenced by crosslinking, the tensile strength of both sets of scaffolds was evaluated. The highest mechanical strength,
stiffness, degree of crosslinking and changes in the packing features of collagen (measured by XRD) were achieved using
genipin. Some of the results found in the quercetin-crosslinked scaffolds were possibly due to hydration and dehydration effects
elicited by the solvents (phosphate-buffered saline or ethanol), as seen in the NMR results. In the quercetin-ethanol-crosslinked
scaffolds, possible reorientation of the amino groups of the collagen molecule may have taken place. Therefore, depending on
their proximity to the crosslinking reagent, different types and numbers of interactions may have occurred, inducing a higher
crosslinking degree (as evidenced by the ninhydrin assay) and reduction in the free amino acids after reaction. Both crosslinking
agents and solvents interfere in the physicochemical properties of collagen thereby inducing variations in the matrix structure.
Quercetin-crosslinked scaffolds may have broader clinical application where a lower degree of crosslinking and stiffness is
required. Copyright © 2016 John Wiley & Sons, Ltd.
Received 31 August 2016; Accepted 3 October 2016
Keywords quercetin; genipin; cross-linked; dermis; implantation
Tissue engineering has attempted to repair and regenerate
a variety of tissue defects and organs through a
combination of cells, scaffolds and biomolecules for the past
20 years. Designed as a skin substitute, both epidermal and
dermal acellular scaffolds have been under investigation for
over a decade (Castro-Bras et al., 2010; Gentile et al., 2013;
Kirsner, 1998; Shevchenko et al., 2009). In our previous
work, a non-enzymatic de-cellularisation method was
developed to produce an acellular dermal scaffold in less
than 2 days. The extracellular matrix (ECM) components
of this scaffold were preserved, and supported cell
migration, proliferation and differentiation (Greco et al.,
2015). The ECM is highly conserved among species, and
consists of molecules such as collagen, ﬁbronectin, laminin,
vitronectin, glycosaminoglycans and growth factors
(Agrawal et al., 2011). Collagen is the primary
mechanostructural element for dermis, conferring tensile
strength and proteolytic resistance to the tissue
(Sundararaghavan et al., 2008).
Although natural crosslinking of collagen occurs, reagents
used during the de-cellularisation process may loosen the
collagen ﬁbrils and disrupt the microstructure of the ECM
(Ott et al., 2010), making it liable to degradation by
collagenase in the host organism. Hence, it is often necessary
to confer structural stability and collagenase resistance to
implanted materials by the introduction of exogenous
crosslinking into the molecular structure (Greco et al.,
2015), which can be modiﬁed with respect to intensity and
degree depending on the ﬁnal biomedical application.
Exogenous crosslinking agents stabilise the collagen molecule
by forming covalent and hydrogen bonds between the ﬁbres.
Many crosslinking agents are available (Elder et al., 2011; Qiu
et al., 2013), and the choice that is not detrimental to the
biomechanics and biocompatibility of the ECM, such as
natural-derived ﬂavonoids genipin (Qiu et al., 2013) and
quercetin (Yang et al., 2009), may represent good options.
Genipin is an iridoid glycoside, one of the main
ingredients extracted from the gardenia fruit (Gardenia
jasminoides ELLIS), and has previously been used to
crosslink biological matrices (Chang et al., 2005; Somers
et al., 2008). Genipin has certain advantages over other
chemical crosslinking agents, for example, it is
cytocompatible (Greco et al., 2015) as well as resistant
to biodegradation (Qiu et al., 2013). It is capable of
*Correspondence to: Tahera Ansari, Northwick Park Institute for Medical
Research (NPIMR), Watford Rd, Harrow, UK. E-mail: firstname.lastname@example.org
K. V. G. and L. F. contributed equally to this manuscript.
Copyright © 2016 John Wiley & Sons, Ltd.
JOURNAL OF TISSUE ENGINEERING AND REGENERATIVE MEDICINE
J Tissue Eng Regen Med 2018; 12:e1716–e1724.
Published online 18 April 2017 in Wiley Online Library (wileyonlinelibrary.com) DOI: 10.1002/term.2338