(275d) Composite Hydrogel From Chitosan and Hemicellulose for Musculoskeletal Tissue Engineering

A thermally-responsive composite
hydrogel has been developed and synthesized from the natural polymers chitosan
and xylan.  The
new material is a viscous liquid at room temperature, but turns to a solid gel
at physiological temperature (37 °C).
Rate of gellation is controlled with addition
of a salt solution.  Applications are for
tissue engineering and local delivery of therapeutic agents including proteins
and other drugs.  The specific
application that has been tested is for delivery of the protein BMP-2 for
creation of new bone and 2D cell culture for osteogenic
stimulation of mesenchymal cells.

The polymer composite remains a
viscous liquid for more than 2 hours at room temperature after the salt
solution is added.  This allows for
addition of the desired mass of growth factors and ample time to complete the
surgical procedures.  Once the polymer
solution reaches physiological temperature it undergoes a phase change to
become a solid gel in less than 10 minutes.
This system allows for a known mass of therapeutic agent(s) to be
accurately delivered to injuries of complex shape.  In situ
gelling lets the material match the complex geometries of injuries that
prefabricated scaffolds and carriers are unlikely to match.  The material is delivered with commonly used
techniques, requiring only a common syringe to administer the liquid solution
to the correct target area.  This gelling
material can be used in combination with current surgical techniques without
disrupting the procedure.  For example, a
non-union fracture is usually fixed in place with titanium rods or fixation
plates.  This thermally-responsive
composite hydrogel can be injected after placement of the more traditional
healing aids, filling in the areas of likely non-union to accurately provide a precise
dose of growth factor and medium in which the cells can move and bridge the
defect.

A thermally-sensitive hydrogel (thermogel) graft substitute made from ultrapure chitosan
has been previously used to treat critical sized unicortical
defects in a rat femur.¹  The thermogel successfully delivered osteogenic
peptides that induced healing over an 8 week time period.  The pure chitosan thermogel
was successful at delivery of osteogenic factors, however it did not promote ingrowth of tissue or
cells before complete degradation.  The
hydrogel material did not allow cells to penetrate into the interior or allow
tissue growth into the area until the chitosan was completely removed from the
defect site.  One reason for this is that
the chitosan only displays positive charges in the thermogel
matrix.  This has a large potential to
impede the healing at an injury site as cell movement is blocked at the
boundary of the hydrogel.

A multi-ion environment is more
conducive to cell migration within biomaterials.  To address this issue, chitosan was blended
with another natural polymer, xylan, which is a
hemicellulose that displays negative charges on its side chains.  We have developed this hydrogel to behave as
a thermally-sensitive hydrogel, or thermogel, based
on the results from the pure chitosan thermogel.  Natural polymers were chosen for this
hydrogel composite because of a large emphasis in engineering circles to
increase sustainability in all areas of research from biofuels to
biomaterials.  Chitosan is already a
renewable material as it is found in crustacean shells.  Hemicellulose can be up to 90% of the biomass
material from plants used as feed stock in other applications (sugarcane, switchgrass, wood, algae, etc.).  However, as hemicellulose is only efficiently
degraded by enzymes, it is not an easy product to use for further
conversion.  Use of this material in
biomedical technology applications provides added value for the hard to digest
byproducts of biomass production as well as provides tissue engineering
applications with an abundant source of renewable and biocompatible material
readily available in most areas of the world.

The success of this chitosan/xylan composite hydrogel has been seen both in vivo and in vitro.  This new chitosan/xylan composite hydrogel has been used to deliver 0.25 µg/µL BMP-2 to an ectopic site in a rat where bone was seen to
form.  With a chitosan/xylan ratio of 3/1, BMP-2 was successfully delivered to the
surrounding tissue in rat muscle and created mineralized bone (Figure 1).  PLGA microsphere scaffolds were filled with
either the pure chitosan thermogel or the new
chitosan/xylan composite.  The scaffolds were placed into the thigh
muscle of a rat without any other stimulating factors.  After 2 weeks the scaffolds were removed, cut
into 8 micron sections and stained with H&E.  When compared to the pure chitosan thermogel, the composite implants showed greater
integration of the surrounding tissue into the scaffold (Figure 2).

In
vitro cell culture experiments have also revealed benefits of the composite
material over a pure chitosan hydrogel.  Real
time PCR measurement of the 18s gene expression in D1 mouse mesenchymal
stem cells (a common gene used as a control that can reveal the difference in
relative cell numbers between cultures) showed that cells cultured on the
chitosan/xylan composite had much higher expression
of this gene compared to the cultured on the pure chitosan material (real time
PCR results:  composite 18s: 1.08x10-4 ±
7.42x10-6; pure chitosan 18s:  3.54x10-8
± 8.89x10-9).  This indicates that there
are many more cells living on the chitosan/xylan
hydrogel after 3 days than on the pure chitosan hydrogel.

Real Time PCR measurement of the
expression of the osterix and runx2 genes in D1 stem
cells exposed to chitosan/xylan for 3 days, or cells in
monolayer with differentiation medium for 7 days, or cells in monolayer with
growth medium for 7 days (Figure 3).
Gene expression levels are normalized with respect to cell numbers in
each culture.  The results show that
after only 3 days (chitosan/xylan 3 day), the cells
grown on the xylan/chitosan composite match the bone
related gene expression level of stem cells grown in osteogenic
media (ODM) for an entire week (monolayer/ODM 7 day).  DMEM is normal cell culture media which does
not promote any specific cell changes.
The third group (labeled monolayer/DMEM 7 day) which shows very low
levels of osteogenic markers indicates the results of
cells in normal media grown as a monolayer on tissue culture plastic.  The left bar of each of the three groups
(black) represents osterix expression, and the right
bar represents runx2 expression.  The
ordinate represents normalized gene expression, based on gene/18s.

The results of these experiments
show this chitosan/xylan composite hydrogel to be a
biocompatible material that provides marked improvement in host tissue response
when compared to pure chitosan hydrogels.

Reference:

1. Bush JR, Madhu V, Balian
G, Laurencin CT, Nair LS.  Thermosensitive
chitosan as a matrix for the controlled delivery of biologically active
molecules for accelerated bone repair. American Institute of Chemical
Engineers Annual Meeting, Salt Lake City, Utah, November 4-9, 2007

Figure 1.  Mineralized
bone formed in the thigh muscle of a rat images with µCT at 4 weeks.  BMP-2 was delivered from the chitosan/xylan composite thermogel at this
ectopic site.  0.25 µg/µL
was delivered in a 40 µL injection of the thermogel.  A) ectopic bone in
thigh muscle, B) enlarged image with higher threshold value to focus on hard
tissue.

Figure 2.  PLGA
microsphere scaffolds were filled with A) pure chitosan thermogel, and B) the chitosan/xylan
thermogel and placed in the thigh muscle of a
rat.  The chitosan/xylan
composite allowed much more tissue penetration (B) than the pure chitosan thermogel (A).  Both
images are magnified at 4x.

Figure 3.  Real Time PCR measurement of the expression of the osterix and runx2 genes.  Gene expression levels are normalized with
respect to cell numbers in each culture.
The results show that after only 3 days, the cells grown on the xylan/chitosan composite match the bone related gene
expression level of stem cells grown in osteogenic
media (ODM) for an entire week.  DMEM is
normal cell culture media which should not promote any specific cell changes.