(314f) Mussel Inspired Protein Mediated Mineralization of Electrospun PCL Fibers

Mussel Inspired Protein Mediated
Mineralization of

Electrospun PCL Fibers

Jingwei
Xie^1*,
Bing Ma¹, Shuler D. Franklin²

¹Marshall Institute for Interdisciplinary
Research and Center for Diagnostic Nanosystems, Marshall University,
Huntington, WV, 25755 USA

²Department of Orthopaedic
Surgery, Joan C. Edwards School of Medicine, Marshall University, Huntington,
WV 25701 USA

^*Correspondence should be addressed to: xiej@marshall.edu

Introduction

A
great advancement in development of bone scaffolds with various compositions
and structures have been achieved using many techniques. Among them,
electrospinning has been attracted much attention for fabrication of nanofiber
scaffolds for use in bone tissue regeneration in that a non-woven mat of
electrospun nanofibers can serve as an idea scaffold to mimic the extracellular
matrix for cell attachment and nutrient transportation owing to its high
porosity and large surface-area-to-volume ratio.¹Recent efforts have been focused on the development of
composite nanofiber scaffolds which can better mimic the composition and
further match the mechanical property of natural bone.^2-4 There are
two approaches for developing composite nanofiber scaffolds -  incorporating
inorganic phase materials (e.g., hydroxyapatite, octacalcium phosphate), which
are one of the compositions of natural bone and precursors, to organic phase
materials (e.g., biodegradable polymeric nanofibers): i) encapsulating
inorganic phase materials (e.g., hydroxyapatite nanoparticle, nanorods) inside
polymeric nanofibers; ii) depositing inorganic phase materials on the
surface of polymeric nanofibers. The encapsulation of inorganic materials could
improve the mechanical property of fibrous materials. However, the fiber
surface is not optimized being a support to maintain desirable cell-substrate
interaction. Direct deposition of inorganic materials on the nanofiber surface
can not only enhance its mechanical property but also provide favorable
substrate for cell proliferation and osteogenic conduction.

Minerals
attachment to polymeric materials in micro-/nanoscale is a big challenge as
they represent inorganic phase materials and organic phase materials,
respectively, having significant difference in mechanical properties.  Prior
studies demonstrated the control of morphology and grain size of minerals
deposited on the electrospun polymeric fibers to enhance mechanical properties
(e.g., stiffness) to a certain extent.^3,4 However, none of them
attempted to enhance the mechanical property by attaching minerals to polymeric
fibers with a ?filler? or ?glue?. In the present work, we aim to develop novel
composite nanofiber scaffolds by attaching minerals (inorganic phase) to
polymeric fibers (organic phase) through an adhesive material mussel-inspired
protein polydopamine as a ?glue?. We hypothesized that these hybrid fiber
scaffolds could have superior mechanical properties.

Materials
and Methods

The
electrospinning setup used in the present work was similar to those described
in our previous publications. Poly(ε-caprolactone) (PCL) (Mw=80,000 g/mol;
Sigma-Aldrich, St. Louis, MO) was dissolved in a solvent mixture consisting of
dichloromethane (DCM) and N, N-dimethylformamide (DMF) (Fisher Chemical,
Waltham, MA) with a ratio of 4:1 (v/v) at a concentration of 10% (w/v). Poly(_L-lactide)
(PLA) (Mw=130,000  g/mol; Sigma-Aldrich, St. Louis, MO) was dissolved in a
solvent mixture consisting of DCM and DMF with a ratio of 3:2 at a
concentration of 5% (w/v). Polymer solution was loaded into a 10 mL plastic
syringe with a 22 gauge needle attached and pumped at a flow rate of 0.5 mL/h
using a syringe pump. The working distance between the tip of the needle and
the collector was about 15 cm and a voltage of 12 kV was applied. Alignment of
electrospun fibers was achieved by making use of a high-speed rotating mandrel
as a collector.

Composite
fibers were fabricated in two different ways. One was to coat mussel inspired
protein on the surface of plasma-treated, electrospun fibers and subsequently
perform biomineralization of polydopamine-coated fibers. Specifically, nanofiber materials were treated with plasma for 8 min and
then immersed in 0.2 mg/mL dopamine·HCl  in Tris buffer (pH 8.5) for 4 h.
Polydopamine-coated nanofiber materials were then washed with DI water to
remove excess monomer. Subsequently, the fiber mat was immersed in a
supersaturated solution of 10×SBF which was prepared from NaCl, CaCl₂,
and NaHPO₄·H₂O in the presence of different amounts of
NaHCO₃. The composition of 10×SBF was shown in Table 1. The ion
concentrations in 10×SBF solution were 1 M of Na⁺, 2.5×10^-2
M of Ca²⁺, and 1.0×10^-2 M of HPO₄^-.
The other is to directly biomineralize fibers in the mineralized solution in
the presence of different amounts of dopamine and NaHCO₃.

Fiber mats were cut into
sections and fixed onto a paper frame. Gauge length and width was set to 10 mm
and 5 mm according to the frame size and the thickness of each sample,
pre-determined by light microscope. Samples were mounted on a nano tensile
tester (Nano Bionix, MTS, USA) and the edge of frame was
cut before testing the fiber samples. Ten samples were stretched to failure at
a low strain rate of 1%/sec at room temperature while related displacement and
force values were recorded. 

Results
and Discussion

In this work, we chose PCL
as a model material because it is a biocompatible and biodegradable polymer
that has been approved by FDA for certain human clinical applications. We
firstly coated electrospun PCL fibers with polydopamine following our recent
study. Specifically, PCL fibers were plasma treated and immersed in 0.2 mg/mL
dopamine solution at pH 8.5 for 4 h. Morphology was similar between
polydopamine-coated and un-coated fibers, with both fiber populations demonstrating
consistent fiber diameters and limited surface roughness. Subsequently, we
immersed polydopamine-coated PCL fibers in 10×SBF solutions at 37^oC
for 24 h in the presence of 0.01 M and 0. 04M NaHCO₃. We examined
the influence of NaHCO₃ concentration on the mineralization of
polydopamine-coated PCL fibers. At a low concentration (0.01 M), large, thin,
and plate-like minerals tended to be formed and loose structure of minerals was
observed on the fibers. At a high concentration (0.04 M), smaller grain size of
minerals displayed on the fibers and denser coating of minerals was seen
compared to the samples fabricated in a low concentration of NaHCO₃.
The diameter of fibers after coating for 24 h was around 1.2 µm. We also
investigated the mineralization of polydopamine-coated PCL fibers at these two
concentrations for longer period time (72 h). At low concentration, more
plate-like minerals were seen on the surface of fibers and the fibrous
morphology was still visible.  In contrast, minerals coated on the fiber
surface in the presence of higher concentration of NaHCO₃ were dense
and smooth. The fiber diameter was around 2.8 µm after 72 h coating. 

In order to examine the influence of
dopamine, we explored the mineralization of PCL fibers for 24 h in the presence
of 0.04 M NaHCO₃ and a series of concentrations of dopamine (Figure 1).

Figure
1.
SEM images of PCL fibers which were mineralized in the 10×SBF solutions
containing 0.04 M NaHCO₃ and 0 mg/mL (A), 0.02 mg/mL (B), 0.1 mg/mL
(C), 0.2 mg/mL (D), 0.4 mg/mL (E), and 1 mg/mL (F) dopamine at 37^○C
for 24 h.

With increasing the dopamine
concentration from 0.02 mg/mL to 0.2 mg/mL, there was no evident variation of
mineral morphology and grain size (Figure 1, A-D). When the dopamine
concentration reached 0.4 mg/mL and 1 mg/mL, the mineral morphology changed
from plate-shape to nanorod-shape to particle-shape (Figure 1, E and F). We
also examined the influence of dopamine on the mineralization in the presence
of low concentration of NaHCO_3. Plasma-treated PCL fibers were
mineralized in 10×SBF solution containing 0.01 M NaHCO₃ and
different amounts of dopamine for 2 h. In the absence of dopamine, few
plate-like minerals were deposited on the PCL fibers. With increasing dopamine
concentration, the morphology of minerals changed significantly. Also, more
minerals were deposited on the fibers and the grain size of minerals decreased
dramatically.  

We also examined the mineralization of
PCL fibers in 10×SBF solutions containing 0.04 M NaHCO₃ and 0.2
mg/mL dopamine at 37^oC for different times. The dense and smooth
mineral coatings were observed. The fiber diameters increased with increasing
the mineralization time.

Conclusion

We
have demonstrated the biomineralization of electrospun PCL fibers by making use
of polydopamine as a filler or ?bioglue? to bridge the minerals and polymeric
fibers. We found that the morphology, grain size, and thickness of CaP mineral
coating on PCL fibers can be readily controlled by adjusting the composition of
mineralized solution, surface property of fibers, and duration of
mineralization. The mineral coating was found to enhance the mechanical
property of fibers to large extent.

References

1. J. Xie, X. Li, and Y. Xia, Macromol. Rapid
Commun., 2008, 29, 1775.

2.
X. Li, J. Xie, X. Yuan, Y. Xia, Langmuir 2008; 24: 14145.

3.
W. Liu, Y. C. Yeh, J. Lipner, J. Xie, H. W. Sung, Y. Xia, Langmuir 2011;
27: 9088.

4.
X. Li, J. Xie, J. Lipner, X. Yuan, Y. Xia, Nano Lett. 2009; 9:
2763.