OKLAHOMA STATE UNIVERSITY- TULSA
HELMERICH ADVANCED TECHNOLOGY RESEARCH CENTER (HATRC)
Synthesis and antibacterial activity of gelatin\nanosilver scaffolds intended for soft
tissue engineering applications
Mostafa YazdiMamaghani 1, Gerwald A. Köhler 2,3, Masoud Mozafari1, Senait Assefa 2, Vahid Shabafrooz 1, Daryoosh Vashaee 4, Lobat Tayebi 1,5
1
Helmerich Advanced Technology Research Center, School of Material Science and Engineering, Oklahoma State University, OK 74106, USA
2
Department of Biochemistry and Microbiology, Oklahoma State University Center for Health Sciences, Tulsa, OK 74107, USA
3
Department of Cell and Tissue Biology, University of California, San Francisco, CA 94143, USA
4
Helmerich Advanced Technology Research Center, School of Electrical and Computer Engineering, Oklahoma State University, OK 74106, USA
5
School of Chemical Engineering, Oklahoma State University, Stillwater, OK 74078, USA
Abstract:
In this study, the addition of silver nanoparticles into the gelatin-based scaffolds
has been investigated in terms of microstructural property and antibacterial activity. The
obtained results indicated that by further addition of silver into the scaffolds the pore size
has been significantly increased from 300 to 700 micrometer, which is appropriate for
cell ingrowth and penetration into the pores. The antibactrerial activity observations
indicated that all the silver contained samples inhibited the bacterial growth on the
surface of the scaffolds. The successful incorporation of silver nanoparticles into the
gelatin-based scaffolds extends the usage of scaffolds from natural polymers, exhibiting
excellent antibacterial activity, which allows a broader application and a more complex
tissue engineering strategy for soft tissues.
4. Scanning electron microscopy of the polymeric scaffolds:
It is worth mentioning that the pores were obviously interconnected as shown in
Fig. 2. As can be seen in SEM micrographs, by further addition of silver concentration in
the samples from 0 mMol to 40 mMol, the pore size of the scaffolds dramatically
increased. It seems that presence of nanosilver can cause a significant increment in the
pore size from 300 micrometers for silver free gelatin sample to about 700 micrometers
for the sample containing 40mM silver nanoparticles.
1.
Preparation of Ag containing solutions:
The nanosilver/gelatin nanocomposites were typically prepared as follows. First,
different concentrations of AgNO3 solution (0–40mM) were prepared by dissolve AgNO3
powders in deionized water. Next, gelatin powders with total concentration of 10 wt%
were mixed homogeneously in AgNO3 solution. The obtained solutions stirred and
maintained for 48 hours at 60°C. These prepared solutions injected into a mold, which
was developed in our laboratory (diameter: 10 mm, thickness: 5 mm), to obtain the
scaffolds in an desirable size. Afterward, the solutions were frozen at -20°C for 4h and 50°C for 24h. After treating by freeze-lyophilization at -53°C and 0.05 mbar for 24 hours,
the scaffolds were immersed in genipin crosslinking solution at room temperature for 48
h. The residual genipin of scaffolds were replaced thrice by deionized water. Finally, the
cross-linked scaffolds were freeze-dried again under the same condition.
2.
Fourier Transform Infrared Spectroscopy:
As can be seen in Fig. 1, the Fourier transform infrared spectrum of plain gelatin
showed vibration bands at around 3000 cm−1 for N–H stretch coupled with hydrogen
bonding and alkenyl C–H stretch, around 2900 cm−1 for CH2 asymmetrical stretching,
around 1600 cm−1 for C=O stretch/hydrogen bonding coupled with COO−, band at around
1550 cm−1 for N–H bend coupled with CN stretch [1].
The cross-linking of gelatin with genipin was observed by the band shifting of
1550 cm-1 and 2900 cm-1 to the lower wavenumbers attributed to the nucleophilic
substitution by the amino groups on the olefinic carbon atom at C-3 of genipin followed
by opening the dihydropyran ring to form heterocyclic amine. The band shifting of 1600
cm-1 was revealed about the formation of secondary amide group with gelatin as a result
of nucleophilic substitution of the ester group on genipin. The association of these
intermediate compounds leads to the formation of cross-linked networks with short
chains of cross-linking bridges [2].
The spectrum of Ag containing samples showed a blue shift of the gelatin peaks at
around 1600 cm−1 to 1700 cm−1, and also displayed a high intensity peaks. According to
Bin Ahmad et al. [3], the vibration band of Ag containing samples at around 1400 cm−1
could indicate the interaction between Ag nanoparticles with gelatin molecules.
Fig. 2. SEM micrographs of the synthesized scaffolds of Gelatin (GA0), Gelatin/Ag 5mMolar (GA5),
Gelatin/Ag 10mMolar (GA10), Gelatin/Ag 20mMolar (GA20) and Gelatin/Ag 40mMolar (GA40)
4.
Antibacterial activity:
To examine the bacterial growth behavior of the scaffolds, 0.02gr of each sample
was immersed in 20ml of germ-containing nutrient solution with a bacterial concentration
of approximately 105 CFU/ml. Herein, we investigated antibacterial activity of the
scaffolds against E. coli as a Gram-negative and S. aureus as a Gram-positive bacteria.
The incubation was performed at 37℃ and oscillated at a the frequency of 150 rpm for 24
hours. After 24 hours the scaffolds were fixed in 3.7% formaldehyde for 30 min at room
temperature. The samples were dried using ethanol followed by a brief vacuum drying.
As shown in Fig. 3, the bacterial colonies are predominant on the surface of the silver
free scaffolds, but not on the surface of scaffolds containing silver nanoparticles,
indicating that E. coli and S. aureus can grow relatively well and rapidly on the former
surface but can hardly survive on the latter surface.
Fig. 1. The FTIR spectra of different samples.
3.
Porosity measurement
The porosity of the scaffolds was measured by liquid displacement method [4].
Ethanol was selected as the displacement liquid as it permeates easily through the
scaffolds without swelling or shrinking of the matrix. The scaffolds were immersed in
absolute ethanol under vacuum for 20 min, and weighed after the excess ethanol was
removed from the surface using a filter paper. The porosity was calculated from the
following equation:
Porosity= [(W1−W2/ρV] ×100%
where W1 and W2 are the weights of the scaffolds before and after immersion in absolute
ethanol, respectively, ρ is the density of absolute ethanol and V is the volume of the
scaffolds. The obtained results indicated that the porosity of the scaffolds increased from
~ 72% to ~ 88% with the increase of silver amount from 0 to 40mM.
Fig. 3. SEM of E. coli bacteria on the silver free scaffolds (A), the scaffold with 40mMol nanosilver (B)
and S. aureus bacteria on the silver free scaffolds (C), the scaffold with 40mMol nanosilver (D)
5.
Summary:
In conclusion, the incorporation of silver nanoparticles into the scaffolds caused
significant changes in microstructural property and antibacterial activity. In addition,
FTIR analysis showed effective cross-linking of the polymer matrix with genipin
molecules. The excellent antibacterial behavior of the samples proved the potential of the
scaffolds for soft tissue engineering applications.
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325-332.
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[4] J.L. Li, J.L. Pan, L.G. Zhang, X.J. Guo, Y.T. Yu, J. Biomed. Mater. Res., 67 (2003), pp. 938–943