DEGRADABLE MAGNESIUM AND BIOPOLYMER COMPOSITES FOR BONE TISSUE
ENGINEERING
Emma McBride, John Ohodnicki, Abhijit Roy, Prashant N. Kumta
University of Pittsburgh
INTRODUCTION
In tissue engineering scaffolds are placed in the patient’s
body to regenerate lost tissue after the patient’s own cells have
been cultured on the scaffolds. The scaffolds are important
because they provide support to the tissue surrounding the
injury site. The scaffolds are usually highly porous which
promotes cell growth. Depending on the material, some
scaffolds degrade over time which allows the native tissue to
replace the scaffold and eliminates the need for a second
removal surgery. [1] The biocompatibility of a material is its
ability to give the appropriate and favorable host response for
an application. This depends mostly on the material and its
associated byproducts. Biopolymers, such as Polycaprolactone
(PCL) and Poly(lactic-co-glycolic acid) (PLGA), are very
biocompatible and have been approved for clinical use by the
FDA. However, as they degrade, biopolymers generate acidic
byproducts that may be harmful in a concentrated area and
damage growing cells. [2] One approach to neutralizing this
acidity would be to create a composite polymer.
Scaffolds for bone tissue engineering can either be loadbearing or non-load bearing. Materials such as polymers are
easy to deform so they are usually used in surface and
craniofacial applications.
Magnesium is an important nutrient for osteoblast
development because it aids in the metabolism of calcium. [3]
Magnesium compounds are slightly basic and previous studies
have shown that adding them to biopolymers stabilizes the pH.
[3] This exploratory study also determined that the magnesium
compounds supported new bone cell growth, more than the
pure polymer scaffolds. [3] Furthermore higher calcium content
and a greater cell attachment were found in the polymer
scaffolds that contained the magnesium compound. [3]
OBJECTIVE
The objective of this project was to synthesize 5, 15, 25,
and 50 weight percentages of both Mg(OH)2:PCL and
MgO:PCL composites, as well as a pure PCL scaffold. Then
the degradation profiles of all the composites and controls
would be characterized. Lastly, the cell viability would be
studied for all three systems.
HYPOTHESIS
Since magnesium is a basic substance and important for
the growth and development of osteoblast cells, the hypothesis
for this study is that the addition of magnesium compounds to
PCL will increase the pH of the degradation solution to a
physiological level while encouraging more cell growth
compared to PCL alone.
METHOD
To meet the first part of the objective, the composite
polymers were synthesized using commercially obtained PCL,
Mg(OH)2, and MgO. Dichloromethane (10 mL) is added to the
PCL pellets (3 g) in order to liquefy it while on a shaker. Then
the correct weight percentage (5, 15, 25 or 50 wt %) of the
magnesium compound is mixed in. The dichloromethane
evaporates at room temperature in the hood after 24 hours and
leaves a sheet of the composite polymer. This sheet is cut up
into approximately 1 cm2 pieces. About 0.2 g of the pieces is
heated to 80 °C and pressed into uniform disks about 10 mm in
diameter using a die.
The degradation study of the composites lasted 91 days. A
sample size of n=4 for each weight percentage of each
composite was submerged in phosphate buffer solution (PBS)
in separate scintillation vials. One day each week the disks
were removed from the solution, dried and weighed. The pH of
the PBS was also measured. The PBS was changed every few
days as well as 24 hours before the measurements in order to
prevent the build-up of degradation products. One disk from
each weight percentage of each composite, not included in the
measurement samples, was removed at each time point for
further material characterization testing.
For characterization of the material, the samples from the
different time points of each composite were analyzed with XRay Diffraction. The resulting spectra were compared to
standard spectra of the parent compounds: PCL, Mg(OH)2, and
MgO. Peaks unique to certain compounds were identified in the
composites.
For a cell viability study, MC3T3 pre-osteoblast cells were
seeded onto each composite as well as the pure PCL scaffold.
The live/dead cell staining technique was used as a qualitative
measure of cytotoxicity. Calcein stained the live cells green and
ethidium homodimer stained the dead cell nuclei red. The
samples were imaged at 1 and 4 days after staining.
RESULTS
From the degradation study, information was obtained
about the pH and percent weight change over time. The pH was
initially high but converged to a more physiological level (7.4)
over the three month time period as shown in Figure 1. The pH
of each of the different composites was higher than that of PCL
alone.
Figure 1. The change in pH over the degradation study.
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For most of the composites, the weight decreased slightly
as shown in Figure 2. However the higher weight percentages
of the MgO:PCL composites showed an accelerated weight
gain.
Figure 2. The change in weight over the degradation study.
The X-Ray Diffraction spectra for the lower weight
percentages of each compound mostly resembled the spectra
for PCL. Peaks unique to Mg(OH)2 and MgO appeared in the
higher weight percentages of their respective composites. Peaks
characteristic of Mg(OH)2 were also observed in the spectra for
the higher weight percentages of the MgO:PCL composites.
For the cell viability study, images of the composites
presented more cell growth and more widespread attachments
when compared to the images of the pure PCL scaffold, shown
in Table 1. The results were similar for both Day 1 and Day 4
time points.
Table 1. Images taken of the cells at different days in the study.
Day 1
Day 4
PCL
5%
Mg(OH)2:PCL
5% MgO:PCL
DISCUSSION
The results confirm that the magnesium compounds
increased the pH of the degradation solution of the composites
compared to PCL alone which would neutralize the acidity of
the degrading biopolymer scaffolds. Although the pH was
initially basic, this was due to the magnesium ion release and
the pH converged to a physiological level over time. Most of
the composites decreased in weight slightly during the
degradation study, but the increase in weight of the higher
weight percentages of the MgO:PCL composites was
unexpected and much greater than was predicted to account for
any absorption of PBS. Further analysis by X-Ray Diffraction
attributed this weight gain to the formation of Mg(OH)2 on the
surface of the composites through a hydrolysis reaction with the
surrounding PBS. Since the molecular weight of Mg(OH)2 is
greater than the molecular weight of MgO, the weight of the
composites increased. Finally, greater cell proliferation was
observed on the composites compared to the pure PCL scaffold
which confirms the positive effects magnesium ions have on
the growth of osteoblast cells.
This study was limited to in vitro analyses of the
composites so the results may not be extended to the in vivo
response. In the future, in vivo studies would be appropriate for
determining the biocompatibility of the composite materials, as
opposed to the cytocompatibility that was obtained from the in
vitro studies, which is the reaction of cells to the material.
Furthermore, PCL is a very slow-degrading polymer and it
would be beneficial to explore other polymers that degrade
more quickly to gain information on the time point when the
pH reaches a physiological level for the higher weight
percentages of the magnesium compounds without performing
the degradation study for longer than three months. Lastly
further in vitro studies should be performed to obtain
quantitative results on the cell viability of the composite
polymers.
ACKNOWLEDGMENTS
Authors gratefully acknowledge the financial support of NSFERC, Grant # EEC-0812348, America Makes, and all of the
ERC-RMB team members.
REFERENCES
[1] Peng, W., et al. (2014). "Fabrication and evaluation of
bioresorbable PLLA/magnesium and PLLA/magnesium
fluoride hybrid composites for orthopedic implants."
Composites Science and Technology 98: 36-43.
[2] Varde, N. K., et al. (2007). “Influence of particle size and
antacid on release and stability of plasmid DNA from uniform
PLGA microspheres.” Journal of Controlled Release 124: 172180.
[3] Guarino, V., et al. (2014). “MgCHA particles dispersion in
porous PCL scaffolds: In vitro mineralization and in vivo bone
formation.” Journal of Tissue Engineering and Regenerative
Medicine 8: 291-303.
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