(554a) Design of a Two-Phase System for the Sustained Delivery of Growth Factors for Bone Tissue Engineering Applications

In recent years,
bone tissue engineering has emerged as a promising solution to the limitations
of current gold standard treatment options for bone related-disorders such as
bone grafts.  Bone tissue engineering provides a scaffold design that mimics
the extracellular matrix, providing an architecture that guides the natural
bone regeneration process.  During this period, a new generation of bone tissue
engineering scaffolds has been designed and characterized, that explores the
incorporation of signaling molecules in order to enhance cell recruitment and
ingress into the scaffold, as well as osteogenic differentiation and
angiogenesis, each of which is crucial to successful bone regeneration.  In the
different phases of bone healing, a variety of growth factors are involved in
bone regeneration.  Generally, they can be divided into three main categories:
(i) inflammatory factors which promote the invasion of cells into the
scaffold/fracture site, (ii) angiogenic factors which are essential for the
vascularization of the new bone, and (iii) osteogenic factors which trigger
osteogenic differentiation of mesenchymal stem cells. 

A key challenge
in growth factor delivery is that the growth factors must reach the site of
injury without losing bioactivity and remain in the location for an extended
time.  Three primary methods have recently been studied in literature.  Physical
entrapment or adsorption of the growth factors to a scaffold leads to a large burst
release, but has shown diminished levels of sustained delivery.  Covalent
binding of the protein to a scaffold allows for sustained constant release, but
does not protect the growth factors from rapid degradation and loss of
bioactivity.  Incorporation of protein into particles can offer both protection
and a combined delivery profile (an initial burst release followed by a shorter
sustained release).  However, the particles can easily diffuse out of the
scaffold and traverse to other areas of the body. 

Here, we have
developed a two-phase system for the sustained delivery of growth factors.  The
system is comprised of growth factors encapsulated in nanocarriers, which are
then covalently bound to a degradable scaffold.  These nanoparticles incorporate
a hydrolytically degradable crosslinker that can be tuned to enable the desired
sustained release profile.  By incorporating chemically-conjugated nanocarriers,
our two-phase system protects the growth factor from rapid degradation while
also improving the release kinetics.  

The scaffolds
were prepared using a high molecular weight chitosan.  The chitosan solutions
were cast into molds and allowed to incubate overnight in order to allow for
physical crosslinking of the chitosan chains.  The scaffolds were then
lyophilized, and the sublimation of the solvent introduced an interconnected
porosity as well as a directionality to the pores.  We fabricated scaffolds as
a function of chitosan weight percent in solution, and subsequently analyzed
the difference in mechanical strength, structure, and porosity.  Our 2 wt%
chitosan scaffolds were relatively high strength (1000s of Pa) when compared to
similar materials used for bone tissue engineering in the literature, with
chitosan-collagen hydrogels being closer to 100s of Pa.

The nanocarriers
were synthesized using an aqueous, one-pot UV-initiated emulsion polymerization. 
The nanocarriers were comprised from methyl methacrylate, methacrylic acid, and
a customizable poly(lactic acid)-b-poly(ethylene glycol) dimethacrylate
crosslinking agent.  Tunability of the nanoparticle degradation was
investigated through systematic variation of PLA and PEG chain length in the
crosslinker.  The physical properties of the resulting nanoparticles were
compared using dynamic light scattering, zeta potential, FTIR, and NMR to
elicit the influence of polymer composition on swelling ratio, surface charge,
and degradation kinetics.  The delivery capacity was analyzed using trypsin as
a model for bone morphogenetic proteins, and the biocompatibility of the
nanocarrier was analyzed using HUVEC cells.

The optimized
nanocarriers were then covalently bound to the optimized scaffold backbone.  Carbodiimide
crosslinker chemistry was used to bind the carboxylic groups on nanoparticles to
the primary amines on the chitosan backbone.  Fluorescent microscopy was used
to compare the ability of our two-phase system to enable sustained retention of
the nanoparticles.  All three components of the system were labeled with a distinct
fluorescent dye, and three systems were compared: (system I) free protein
adsorbed to the scaffold, (system II) free nanoparticles simply entrapped in
the scaffold, and (system III) nanoparticles covalently bound to the scaffold. 
The labeled systems were incubated in phosphate buffered saline for up to 4
weeks, and images were obtained for both the scaffold and surrounding fluid. 
In both systems I and II, the free protein and particles diffused away from the
scaffold within 72 hours.  In contrast, the covalently bound particles in
system III were still highly present in the scaffold for 4+ weeks. 

Ultimately, we
developed the tunability of our platform system for a variety of growth factor
delivery applications.  Our work demonstrates that the incorporation of
two-phase systems consisting of growth factor-loaded nanoparticles embedded
into scaffolds shows great promise, both by providing sustained release over a
therapeutically relevant timeframe and the potential to sequentially deliver
multiple growth factors.