1. Researchers prepared crosslinked poly(aspartic acid) nanofibers using electrospinning for applications in tissue engineering and as hernia meshes. Electrospinning produced non-woven fibers from a polymer solution of poly(succinimide) modified with cysteamine. Crosslinking occurred during spinning via disulfide bond formation between thiol groups. 2. Fiber characterization with light microscopy and atomic force microscopy found uniform, beadless fibers with an average diameter of 88±30 nm, similar to collagen fibers. This nanofiber structure mimics the extracellular matrix and could support cell growth as a biodegradable scaffold. 3. The fibers have properties required for biomedical applications

1. The International Istanbul Textile Congress 2013
May 30th to June 1th 2013, Istanbul, Turkey
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ELECTROSPUN CROSSLINKED POLY(AMINO ACID) BASED
NANOFIBERS FOR TISSUE ENGINEERING
K. Molnar1
, A. Jedlovszky-Hajdu1
,M. Czobel2
, Gy. Weber2
and M. Zrinyi1
1
Semmelweis University, Departement of Biophysics and Radiation Biology, Nanochemistry
Research Group, Budapest, Nagyvarad ter 4
1089, Hungary
2
Semmelweis University, Departement of Surgival Research and Techniques, Budapest,
Nagyvarad ter 4.
1089, Hungary
mikloszrinyi@gmail.com
Abstract: The importance of biodegradable and biocompatible polymers is increasingly recognized, and
extensive studies have been conducted on their uses in various biomedical applications. The commercial
hernia meshes are not biodegradable, usually made from poly(propylene). The main aim of the researchers
is to find a material with controllable degradability in the living systems, in line with the refurbishing of the
tissue. Until now, the search for biomaterials has been essentially limited to a very narrow subset of all
available poly(amino acids). In this work we have prepared polymer fibres from anhydrous form of
poly(aspartic acid) (poly(succinimide)) with the electrospinning method. Electrospinning is a fast, efficient,
and inexpensive polymer processing method for the formation of special structures (nonwoven), which can
be suitable for applicability as a hernia mesh. During the experiments concentrated polymer solutions were
used in organic solvent under high voltage. The crosslinking reaction took place during the electrospinning.
The mean value and distribution of the fibre diameter were determined after the sample preparation.
Keywords: poly(succinimide), nanofibers, crosslinking, hernia mesh
1. Introduction
The importance of nanotechnology in our days is well recognized in the area of biomedical applications. The
interest in the biocompatible and biodegradable polymer matrices increased with their usability in wide range
of industrial uses.
In the biomedical, pharmaceutical and cosmetic applications neither the polymers nor their derivatives
should be toxic. Therefore, a convenient choice for basic materials for polymers would be poly(amino acids),
since their protein like (polymer molecule containing peptide bonds), structure should be compatible with the
human body. It is difficult to synthetize poly(amino acids) with long chain and large molecular weight.
Therefore, the usage of an amino acid derivative is a simpler way to create artificial polymers. For example
linear poly(aspartic acid) (PASP) of high molecular weight can be prepared in a two-step way. First creating
poly(succinimide) (PSI) by the thermal polycondenzation of L-aszpartic acid, than the alkaline hydrolysis of
the previously created PSI provides PASP molecules. The advantage of this method is that the PSI molecule
can be easily reacted with mono amines in nucleophile reaction at room temperature, thus it is relatively
easy to functionalize and graft the polymer.
Nucleophile reaction of poly(succinimide) (PSI), which is the derivative of aspartic acid, with amines results in
the formation of imide groups. As a consequence, PSI molecules can be cross-linked by diamines to yield a
network, and after hydrolysis a functionalized polymer chain can be obtained [1]. If functionalizing cross-
linkers and side chains are also amino acids and/or natural diamines (for example, putrescin, lysine or
cystamine), the biocompatibility does not change after the modification of the polymer chain, and the
biodegradability becomes controllable [2].
The electrospinning technique has large historical background; the first patent was submitted in the mid-30’s
[3]. However, because of the great opportunities provided by nanotechnology, in particular, in the field of
biomedical applications, this method is now a promising tool in synthetic nanochemistry [4; 5].
The synthetized poly(amino acid) based fibres can be suitable for acting as a scaffold for the cell growing or
regeneration/differentiation [6]. This two or three dimensional network, prepared with electrospinning
technique, is applicable for lesion treatment, or it develops special tissue structure, such as the hernia mesh.
Presently the most common hernia meshes are based on polypropylene polymers. The common property of
the meshes is the non-degradable woven polymer fibre, characterized with a well-defined pore size, which is
sometimes modified with different molecules. These commercial meshes are not biodegradable at all; hence,

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May 30th to June 1th 2013, Istanbul, Turkey
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the main aim of the researchers is to find a material with controllable degradability in the living systems, in
line with the refurbishing of the tissue. In the course of the tissue recovery process the mesh should be the
scaffold. The ideal mesh has the following criteria for the application: suture maintenance, high tensile
strength, ideal porosity, minimum shrink tendency, controllable degradability and three-dimensional
structure.
In this work we focus on creating a special hernia mesh, which can be used as a scaffold in the regeneration
of the abdominal tissue in hernia surgeries. The mesh should be biocompatible, having controllable
degradation in time. The most important parameter is the tensile strength of the artificial mesh.
We have prepared polymer fibres from anhydrous form of poly(aspartic acid) (poly(succinimide)) with the
reactive electrospinning method. Electrospinning is a fast, efficient, and inexpensive polymer processing
method for the formation of special structures (nonwoven), which can be suitable for applicability as a hernia
mesh. During the experiments concentrated polymer solutions were used in organic solvent under high
voltage. The crosslinking reaction took place during the electrospinning. The mean value and distribution of
the fibre diameter were determined after the sample preparation.
2. Materials and methods
2.1 Materials
L-Aspartic acid (puriss, 99.0% Aldrich), phosphoric acid (Aldrich, 99%), methanol (p.a. 99.8%),
dimethylformamide (DMF) (Fluka, purum, 99%), cysteamine (CYSE) (Sigma 98%),DL-dithiothreitol (DTT)
(Fluka, 99%) from Sigma–Aldrichwere used. All reagents and solvents were used without further purification.
2.2 Preparation of poly(succinimide)
The poly(succinimide) (PSI) (30kDa) was prepared by the thermal polycondenzation of L-Aspartic acid in the
presence of o-phosphoric acid in a 1 l flask connected to a rotary evaporator (Rotadest IKA RV10). The
detailed description can be found in our previous paper [1]
2.3 Modification of PSI with cysteamine
The five member rings of PSI could be easily reacted with primer amines at room temperature without any
catalyst [régebbi saját cikk]. For creating a reactive, functional polimer of PSI for reactive-electrospinning, we
modified the PSI chains with cysteamine (CYSE). The amino groups of CYSE connected to the PSI chains in
a ring opening reaction (Fig).
Figure 1. Modification of PSI with cysteamine
The thiol groups of the modified polymer (PSICYSE) forms dissulfide bonds between the polymer chains in
the presence of oxygen therefore the grafting reaction was performed under nitrogenous atmosphere. In a
glass reactor 0.04 g cysteamine was disolved in 1.3 g DMF. 2 g, 25 m/m % PSI-DMF solution was added to
the reaction mixture and was stirred for an hour. The molar ratio of succinimide monomer units to moles of
cysteamine was 10, which means that - on average – every 10
th
of PSI monomer units reacts with
cysteamine respectively, if the stoichiometry holds. This polymer is denoted as 15PSI10CYSE where the first
number stands for the polymer concentration and the number between PSI and CYSE represents the degree
of graftage.
2.4 Reactive-electrospinning
For the creation of fibers a basic home-made instrument was used. The 15PSI10CYSE right after the
preparation was poured into a glass syringe (Fortuna Optima 7.140-33) with a Hamilton metal needle.
0.4 ml/h flow rate was regulated by a syringe pump (KD Scientific KDS100). The 15 kV voltage was provided
by a high voltage DC power supply (STATRON TYP4211). For the collection of fibers, a wooden plank
covered with alumina foil was placed 15 cm far from the tip of the needle. Reactive electrospinning is a
special class of the electrospinning technique where a chemical reaction takes place during the fiber

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May 30th to June 1th 2013, Istanbul, Turkey
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formation. In case of PSICYSE as we previously described in section 2.3 the thiol side groups on PSI chains
form intermolecular dissulfid-bonds in the presence of O2.
2.5 Characterization of the fibers
For the observation of the fibers we used a HUND-WETZLAR H500 light microscope with a Sony Hyper HAD
CCD-IRIS/RGB Color Video Camera. Photos were taken by the Scope Photo software.
The diameter and surface properties of the polymer fibers were imaged in air-oscillation mode (resonance
frequencies of about 0.2-0.5 Hz and 0.3-0.5 V target value) with a Molecular Force Probe 3D (MFP3D)
Atomic Force Microscope (AFM) instrument (Asylum Research, Santa Barbara, CA, USA). For the sample
moving an OlympusIX81invert microscope was used. For the visualization and diameter measurement
IgorPro 6 software (Wavemetrics, Lake Oswego, OR) was used. For the diameter distribution 50 fibers were
measured.
3. Results
The synthesis of the PSI polymer resulted in a hard, brown foam which after the cleaning and drying
processes turned into a white powder. To make the required 25 m% DMF solution for the modification of the
polymer, it took several hours for the PSI to completely dissolve.
For the electrospinning method we used a concentrated polymer solution (15PSI10CYSE) mentioned above.
The fiber preparation took place under high electric voltage which induces electrostatic charge on the
surface of the polymer solution droplet appearing at the tip of the needle. If a critical electric field strength is
reached, the electrically charged droplet elongates and a polymer jet ejects from it heading to the grounded,
alumina foil covered target. One of the main advantages of this technique is that during the sample
preparation the solvent evaporates as the jet reaches the target creating a non-woven fibrous matrice.
It turned out, that during the synthesis of the modified polymer, the crosslinking reaction between the thiol
groups was only slowed down by the inert nitrogenous atmosphere. Therefore the viscosity of the polymer
solution inside the syringe used for the electrospinning process constantly rose to an extent where by
clogging the needle, the fiber formation stopped. The spinnable window of the grafted polymer, where the
viscosity of the solution was in an optimal range was sustainable for 1 to 1.5 hours depending on the
synthesis time. The spinnable time could be expanded by shortening the synthesis time of PSICYSE or
reducing the amount of grafting CYSE but in that case the risk of electrospraying due to the low viscosity and
weak interactions of the polymer chains would rise. The polymer beads inside the electrospun matrice
causes inhomogeneity in the fiber structure, which can be a problem in future applications, because they can
lower the mechanical properties of the system or pollute its surroundings. Therefore the synthesis and
processing parameter provided in section 2.3 and 2.4 are the result of a long optimization trial.
After preparation, the dry, solution free PSICYS fiber matrices were easily removed from the collectors
alumina foil for further treatment and microscopic studies. Each time small part of the matrice was dipped
into DMF, the matrices could not dissolve in the solution thus proving evidence for the presence of crosslinks
between the polymer chains inside the fibers. The size of the matrices changed slightly and the color turned
from clear white to opaque which means the so called gel fibers in took solution, thus swelled during
solvation.
This highly wet fibrous structure resembles to that of the extracellular matrix of the human body which is a
similarly wet and fibrous structure providing a structural scaffold for cells, regulates cellular functions and
provides a transportation channel for information and nutrients. This similarity provides a good background
for biomedical applications but to completely copy the native extracellular matrix there are several other
conditions to meet. The artificial matrices should be biocompatible which means it should not cause
inflammation inside the body, and biodegradable, which means after fulfilling its role it should degrade to
biocompatible fragments which excrete from the body, and so on.
Poly(succinimide) is an anhydrous form of the poly(aspartic acid). In a bio relevant media, which pH is
around 7.5, a slow hydrolyzation reaction will take place [2] and the PSI based polymer fibers transform into
poly(aspartic acid) (PASP) based fibers. The transformed chemical structure contains only peptide-bonds
therefore it should be biocompatible and biodegradable. In the case of hernia meshes, as mentioned in the
introduction, there are other demands to be met. The needed tensile strength could be achieved by
thickening the artificial matrice and because of the ECM like structure, it would be easier to incorporate to the
human body and also easily penetrable for nutrients and other chemicals thus improving tissue regeneration
in the damaged area.
To meet the previously mentioned demands of ECMs and hernia meshes, tuning of the matrices and
optimization of the processing parameters was needed. For fast feedback for the success of fiber formation,
and to gain basic information about the fiber matrices such as layout, uniformity, beads between fibers etc.
we used light microscopy. In the case of the optimized fiber formation these investigations showed the
expected non-woven, beadless fibrous structure containing fairly uniform fibers of diameters less than the

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microscopes resolution (Fig 1. 1). Therefore by using this technique we were not able to measure the
diameter of the fibers.
In order to gain more accurate information about the diameter and surface properties of the fibers we used
AFM technique. The surface of the fibers was fairly uniform without any surficial defects (Fig 1. 2). We found
the average diameter of our sample to be 88 ± 30 nm which gives the system a very high surface area for
the regenerating cells to attach to. Also the collagen fibers creating the fibrous backbone of the native
extracellular matrix have a similar diameter of 60 nm [7].
Figure 1. Light microscopic (a) and AFM (b) pictures of the PSICYS, fiber diameter distribution by AFM (c).
The artificial extracellular matrices could be a useful material in biomedical applications, such as replacing
the commercially available non degradable hernia meshes. Because of its poly(amino acid) based structure
it is biodegradable, and the degraded fragments are nutrients for the regenerating cells. By the thickening of
the meshes the obtainable tensile strength could be obtained for hernia application. In the future, we would
like to investigate the biological response of animals to our hernia mesh.
4. Conclusion
A novel method has been developed for the preparation of crosslinked poly(succinimide) based nanofibers
via a reactive electrospinning technique. The processing parameters were optimized to gain uniform beadles
fibers without surficial defects. Our system disposes of the most important properties for biomedial
applications such as artificial extracellular matrices for tissue engineering and hernia meshes such as
possible biocompatibility, biodegradability elasticity and tensile strength. The average fiber diameter of our
samples was found to be 88 ± 30 nm which resembles to the average diameter of collagen fibers in native
extracellular matrice. This artificial matrice could be useful material for biomedical applications such as the
replacement of non-biodegradable hernia meshes or for improving cell based regenerations in damaged
bodily areas. For further improvement of the system cell growth factors and actuators can be attached to the
base polymer of our fibrous system.
Acknowledgements
This research was supported by OTKA NK 101704 and OTKA K 105523.
References
[1] Zrinyi, M., Gyenes, T., Juriga, D., Kim, JH.: Volume change of double cross-linked poly(aspartic acid)
hydrogels induced by cleavage of one of the crosslinks, Acta Biomaterialia, 9. (2013), 5122–5131.
[2] Varga, Zs., Molnár, K., Torma V., Zrínyi, M.: Kinetics of volume change of poly(succinimide) gels during
hydrolysis and swelling Phys. Chem. Chem. Phys., 2010, 12, 12670–12675
[3] Frenot A., Chronakis I. S.: Polymer nanofibers assembled by electrospinningCurrent Opinion in Colloid
and Interface Science, 8. (2003), 64–75.
[4] Kim, T. G., Park, T. G.: Biomimicking Extracellular Matrix Cell Adhesive RGD Peptide Modified
Electrospun Poly(D,L-lactic-co-glycolic acid) Nanofiber Mesh, Tissue Engineering, 12. (2006), 221-233
[5] Bakera, S. C., Atkin, N., Gunning, P. A., Granville, N., Wilson, K., Wilson, D., Southgate, J.:
Characterisation of electrospun polystyrene scaffolds for three-dimensional in vitro biological studies,
Biomaterials, , 27. (2006), 3136–3146.
[6] Ramakrishna, S., Fujihara, K., Teo, W. E., Yong, T., Ma, Z., Ramaseshan, R.: Electrospun nanofibers:
solving global issues, Materialstoday, 9. (2006), 40-50.
[7] Shultz, G. S.; Ladwig, G.; Wysocki, A.: World Wide Wounds, Available from
http://www.worldwidewounds.com/2005/august/Schultz/Extrace-Matric-Acute-Chronic-Wounds.html
Accessed: 2005-08