Enhanced Osteogenic Differentiation of Stem Cells in Novel Bioactive Cold Plasma Treated Nanostructured Bone Scaffolds
Mian Wang1; Xiaoqian Cheng1; Benjamin Holmes1; Wei Zhu1, Michael Keidar1; Lijie Grace Zhang1,2*
1 Department
of Mechanical and Aerospace Engineering, 2 Department of Medicine;
The George Washington University, Washington, DC 20052.
Introduction
Figure 3, Emission spectrum of helium plasma.
(a)
800
(b)
700
500
400
300
200
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Figure 12, Greatly enhanced total calcium deposition on CAP modified
nHA/Chitosan scaffold after 3 weeks of culture. Data are mean ±SEM; n=9.
*p<0.01 when compare to all other substrates. **p<0.05 when compare to
nontreated substrates.
O/N2+ ra o
0.25
600
0.2
N2+ (391 nm)
O (777 nm)
N2 (337 nm)
OH (309 nm)
O/N2+
Materials and Methods
(A)
Emission Intensity (a.u.)
As an interdisciplinary field, regenerative medicine and tissue engineering
aim to recreate living and functional tissues via the use of biomaterials,
growth factors, and stem cells. To date, they hold huge potential for bone
regeneration. Specifically, autologous human mesenchymal stem cells
(MSCs) from bone marrow are readily accessible and harvested, i.e. from
the iliac crest, and display several key biological characteristics. However,
current stem cell-based bone regeneration still has many limitations such
as low levels of stem cell engraftment and transplant survival,
uncontrollable osteogenic differentiation and decreased production of
extracellular matrix proteins, which have greatly inhibited the full clinical
potential. Native stem cells reside in a nanostructured bioactive
extracellular matrix (ECM) environment and are extremely sensitive to
minute changes to their surroundings. Thus, the objective of this study is to
create a biomimetic and bioactive cold atmospheric plasma (CAP)
modified three-dimensional nanocomposite scaffold to address the
aforementioned limitations and current challenges to further improve MSC
osteogenic differentiation and bone regeneration in situ.
(A)
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0.05
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Fabrication of chitosan/nHA scaffold
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9.8
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14.8
Biomimetic nanocystalline hydroxyapatites (nHAs) were synthesized via a
Output voltage (kV)
Input voltage (V)
wet chemistry precipitation method plus a special hydrothermal treatment.
3D porous chitosan scaffolds with 10% hydrothermally treated nHA were Figure 4, (a) Emission intensity as function of output voltage; and (b) O (777 nm)/N2+ (391 nm)
ratio as a function of output voltage.
fabricated via a simple lyophilization procedure.
CAP modification
The prepared hydrated nHA/Chitosan scaffolds were placed in a 48-well
plate. The CAP generator had a 1 mm diameter central powered electrode
and a grounded outer electrode wrapped around a 4.5 mm diameter quartz
tube. Different CAP treatment conditions (i.e., output voltage: 3.4, 3.6, and
3.8 kV) were investigated. The distance between the CAP jet and samples
was 20 mm.
Figure 8, Low and high magnification SEM images of unmodified nHA/chitosan scaffold (A, a1
and a2), and CAP modified nHA/chitosan scaffolds with different treatment time (B, b1 and b2
is 3 min; C, c1 and c2 is 5 min; D, d1 and d2 is 10 min) under the 3.6 kV output voltage.
Figure 13, Total collagen synthesis on CAP modified nHA/chitosan scaffold after 1,
2 and 3 weeks of culture. Data are mean ±SEM; n=9. *p<0.01 when compared to
controls after 3 weeks of culture; **p<0.01 when compared to all other substrates at
week 1 and 2.
Figure 5. Transmission electron microscopy images of nHA with (A) low magnification and (B)
high magnification.
(a)
(B)
(b)
(C)
(c)
Figure 9. 3D surface topography images of nHA/chitosan obtained using white light
interferometry, without CAP treatment (A) and 3 min CAP treatment
Figure 1, (a) Configuration of CAP, probe, and holder (b) Schematic diagram of probe
and holder of emission intensity of output voltage.
Scaffold Characterization
Surface topographies of the untreated scaffold and CAP modified scaffolds
were imaged via a scanning electron microscopy (SEM) and white light
interferometry. Surface wettability of scaffolds was measured using a
contact angle analyzer (DSA4, Krüss). Specific proteins absorptions were
evaluated via ELISA assays.
MSC adhesion study and osteogenic differentiation study in vitro
For adhesion study, MSCs were seeded at a density of 50,000 cells per
scaffold on different CAP modified nHA/chitosan scaffolds and cultured for
4h. For differentiation study, MSCs were seeded at a density of 150,000
cells/cm2 in 0, 3, and 5 minute 3.6 kV output voltage CAP treated
nHA/chitosan scaffolds and were cultured in an osteogenic medium for 1, 2
and 3 weeks. Total protein, collagen synthesis and calcium deposition were
quantified via biochemistry assay.
MSC growth morphology under a confocal microscope
The MSCs were cultured in CAP treated scaffold for 3 days, then stained
with DAPI and Rhodamine dyes. Cell growth morphology was imaged
under a confocal microscope.
(A)
Figure 6, Increased surface hydrophilicity of nHA/chitosan after 3, 5 and 10min CAP
treatments. Data are mean ± standard deviation, n=3. *p<0.05 when compared to all other
samples; and **p<0.05 when compared to 5 min CAP treated sample.
Figure 10. Vitronectin and fibronectin absorptions on nHA/chitosam after 3 and 5 min CAP
treatment by ELISA, n=3. *p<0.01 when compared to 0 min CAP treated group; and **p<0.1
and ***p<0.05 when compared to 0 min CAP treated group.
Figure 14, Confocal microscopy images of MSC growth in different CAP modified
bone scaffold. (A) and (a) control scaffolds; (B) and (b) 3 min CAP treated
scaffolds; (C) and (c) 3 min CAP treated scaffolds. (a), (b) and (c) are cell only
images.
Conclusion
Results
Figure 2, Plasma appearance
as function of output voltage.
Figure 7, MSC cell adhesion on modified scaffold with different CAP conditions. Data are
mean ±SEM; n = 9. Output voltages of Conditions 1, 2, 3 are 3.4, 3.6, and 3.8 kV,
respectively.
Figure 11, Enhanced total protein synthesis on CAP modified nHA/Chitosan scaffold after 1 and
2 weeks of culture. Data are mean ±SEM; n=9. *p<0.01 when compare to all other substrates.
In summary, biomimetic and bioactive nHA/chitosan scaffolds were
fabricated via a lyophilization method and were surface modified via
a CAP treatment technique. Significantly improved MSC adhesion,
migration and osteogenic differentiation were observed in 3 and 5
min CAP modified nHA/chitosan scaffolds. A fibrous morphology,
more open pores and hydrophilic surface can be formed on
scaffolds after CAP treatment, which can create a more biomimetic
microenvironment for specific protein absorption, cell attachment,
infiltration and differentiation. Our results show the potential of CAP
modified nHA/chitosan scaffolds for bone regeneration. In particular,
as an emerging technique for medicine, the CAP treatment can be
easily used for many other complex tissue scaffold modification.