WO2018056937A2 - Nanofibrous adhesion barrier - Google Patents

Abstract

The invention relates to adhesion barriers used in the biomedical field. The invention relates in particular to a nanofibrous mat suitable for use as an adhesion barrier in biomedical field and obtained by electrospinning method from a mixture of hyaluronic acid (HA), sodium alginate (NaAlg) and carboxymethyl cellulose (CMC) polymer solutions.

Description

DESCRIPTION NANOFIBROUS ADHESION BARRIER

Technical Field The invention relates to adhesion barriers used in the biomedical field. The invention relates in particular to a nanofibrous mat suitable for use as an adhesion barrier in biomedical field obtained by electrospinning method from a mixture of hyaluronic acid (HA), sodium alginate (NaAlg) and carboxymethyl cellulose (CMC) polymer solutions.

The Prior Art

Adhesions are described as abnormal adhesions that are not normally associated with each other in the intra-abdominal region, and that the organs surrounded by the serous membrane are involved with each other's and / or adjacent organs following injury or surgical operations. The main causes of adhesions are surgical procedures. Adhesions are common after thoracal, heart and abdominal operations. Postoperative intraabdominal adhesion formation rates are between 64% and 97%. Abdominal adhesions, which are one of the most important problems of both surgeons and patients, lead to chronic abdominal and pelvic pain, organ obstructions (bowel, ovarian tubule, kidney drainage channels, etc.) and functional disorders. As a result, it causes new operations to be performed. In surgical studies, it t is seen that adhesions are responsible for one third of all intestinal obstructions and two-thirds of small intestinal obstructions [1 - 5]. For complications due to adhesions, only 400,000 adhesion operations are performed annually in the USA. Adhesion opening operations are long-running operations. During these operations, anesthesia and the length of stay at the hospital are prolonged. Therefore, prevention of intra-abdominal adhesion is a very important issue during surgical interventions [6, 7].

The main approaches proposed in the literature to prevent or reduce adhesion are divided into three categories: the development of surgical techniques, the use of anti-adherence drugs, and the separation of tissues in the healing process. The basic surgical principles that all surgeons must apply in order to prevent adhesion are to reduce surgical trauma as much as possible, to avoid unnecessary and excessive manipulations, to remove foreign bodies and dead tissues, to prevent dryness due to inadequate blood supply and loss of water in the tissues and to keep bacterial invasion to a minimum level. However, considering the adhesion-forming nature of the intra-abdominal region to protect the organism during the healing process, it is suggested that the therapies and technological developments to be made with the surgical technique may not prevent adhesion formation but only reduce it [3, 4]. Drugs used to prevent adhesion are either directed to inflammatory processes or to agents that cause adhesion (infection, endotoxin, exudate, etc.). The drug should be specific to adhesions and not affect normal wound healing. However, the clinical and experimental efficacy of these drugs is questionable and has side effects such as adverse effects on the immune system and delayed wound healing [7]. Another method of separating tissues from each other during the healing process is the use of adhesion barriers. Adhesion barriers allow the surfaces in the injured intraabdominal region to be separated from each other and freely heal and thus prevents the formation of adhesion. Today, physical barriers used as adhesion inhibitors are divided into two main groups as liquid barriers and membrane barriers. These barriers are often used with a mesh material. Composite mesh structures consisting of a combination of mesh and adhesion barriers are also available. However, these materials are very expensive and cannot be used in any part of the body [4, 5].

An ideal adhesion barrier should not affect wound healing, be non-reactive, be effective in the presence of body fluids and blood, be easy to use, and be biodegradable. In addition, it should not cause infection and inflammation, should be antibacterial, be stable in the initial phase of adhesion formation, then metabolize and be economical. Adhesion barriers which have recently been developed and found to be the most widely used in clinical practice are the oxide regenerated cellulose membrane (Interceed®), e- polytetrafluoroethylene membrane (Gore-tex®) and carboxymethyl cellulose / hyaluronic acid membrane (Seprafilm®). The first two are used only in gynecology, and the latter are widely used both in general surgery and in gynecology. However, the existing barriers, it requires special skills to use, despite its beneficial effects in preventing the adhesion, to give rise to complications, they cannot be used in every region and most importantly, their use is limited because they are expensive (one of them is 1 .200 TL on average and 2-3 units are used in each operation). In addition to being expensive, other disadvantages are to lead to the separation of blood vessels, the risk of abscess formation, the tendency to break when sharp edges are bent due to film structures, and the difficulty of applying them to tissue [1 -8]. A lot of material was used to prevent adhesion formation until today but it has not been precisely shown that no one blocks the intra-abdominal adhesion. Studies continue to reduce or prevent intra-abdominal adhesions with used materials and with the products put forward constitute the million dollar health market. The natural polymers hyaluronic acid (HA), sodium alginate (NaAlg) and carboxymethyl cellulose (CMC) are used in mixture with different polymers or purely as a gel, film, membrane or fiber / nanofiber in biomedical field. Previously, however, no nanofibrous mat was produced by electrospinning from the HA/NaAlg/CMC polymer mixture. In addition, no nanofibrous mats have been produced from these polymers, either alone or in admixture, for use as an adhesion barrier.

The developments known in the art concerning the subject are given below.

The patent with the publication number EP2598180B1 relates to "Hyaluronic acid based hydrogel and its use in surgery". The present invention relates to hydrogels based on hyaluronic acid-based derivatives which are more resistant to chemical and enzymatic degradation than hyaluronic acid alone. Hyaluronic acid based hydrogel, in various surgeries, has an optimal use for example for injection into bone fractures or cavities and for the production of prosthetic coatings in orthopedic surgery, as fillers in cosmetic and maxillofacial surgery, and as an anti-adhesion barrier in abdominal and abdominal / pelvic surgery and in the general surgery with the prevention of postoperative adhesions. The patent with the publication number EP1975284B1 relates to "the electrospinning apparatus for serial production of nanofibers". The invention refers to an electrophotographic apparatus having electrical stability and an improved nozzle blocks.

The patent with the publication number TR 2013 1341 7 relates to "Coaxial nanofibrous mats with plant extract". For the release of plant extracts, which are natural bioactive agents, mats are produced from coaxial biopolymer nanofibers trapped in plant extracts in their own regions. Nanofibrous mats having remedial effects because they show antioxidant properties, produced from natural or synthetic biopolymers with plant extracts exhibiting antimicrobial properties thanks to the phenolic components they contain, they can be used as tissue scaffold or drug release system. Biologically compatible and degradable nanofibrous mats capable of releasing antioxidant and antimicrobial plant extracts, are produced by coaxial electrophoresis.

As a result, due to the above mentioned negativities and the inadequacy of the existing solutions, an improvement in the technical field has been required. Brief Description of the Invention

The present invention is concerned with a nanofiber adhesion barrier which meets the above-mentioned requirements, removes all disadvantages and adds some additional advantages.

The primary object of the invention is to obtain a nanofibrous mat from a mixture of hyaluronic acid (HA), sodium alginate (NaAlg) and carboxymethyl cellulose (CMC) polymer solutions suitable for use as an adhesion barrier in biomedical field.

The invention aims to provide a nanofibrous mat by means of electrospinning from a mixture of hyaluronic acid (HA), sodium alginate (NaAlg) and carboxymethyl cellulose (CMC) polymer solutions.

One object of the invention is to provide an alternative product that is easier and more efficient to use than commercial adhesion barriers used in the market.

Another object of the invention is to provide a nanofibrous product produced by electrospinning from carboxymethyl cellulose, sodium alginate and hyaluronic acid polymers, having the potential to inhibit / reduce adhesion by being used during intraabdominal surgery, thus reducing post-operative complications and providing less costly alternatives to commercially available products.

The invention is a nanofibrous mat obtained from the hyaluronic acid and carboxymethyl cellulose polymer mixture, suitable for using as an adhesion barrier in the biomedical field in order to fulfill the above-mentioned purposes. Said polymer mixture also comprises sodium alginate.

The method of producing said nanofibrous mat to realize the objects of the invention comprises the steps of; · The preparation of the hyaluronic acid solution in the solvent,

• The preparation of the aqueous carboxymethyl cellulose solution,

• The mixing of the two solutions prepared,

• The addition of surfactant to the resulting mixture solution,

• The application of electrospinning method to the mixture solution,

· The cross-linking of the obtained nanofibrous mat. In an alternative embodiment of the invention, an aqueous sodium alginate solution is prepared and mixed with the previously prepared hyaluronic acid solution and carboxymethyl cellulose solution.

In order to realize the objects of the invention, betaine or Triton X-100 as surfactant, NaOH/Dimethyl sulfoxide or NaOH/Dimethylformamide as solvent are used. In the NaOH/Dimethyl sulfoxide or NaOH/Dimethylformamide solvent, the mixing ratio between NaOH and Dimethyl sulfoxide or Dimethylformamide is 4/1 by volume

In order to realize the objects of the invention, the concentration of hyaluronic acid solution prepared in the solvent is in the range of 8-15% by mass/volume %. The concentration of carboxymethyl cellulose solution prepared in purified water is in the range of 1 -4% mass/mass %. The concentration of sodium alginate solution prepared in pure water is in the range of 1 -4% by mass/mass %.

To accomplish the objects of the invention, said hyaluronic acid solution, carboxymethyl cellulose solution and sodium alginate solution are mixed at a ratio of 3/1 /1 to 5/1 /1 by volume.

In order to realize the objects of the invention, the mixture of the hyaluronic acid solution, the carboxymethyl cellulose solution and the sodium alginate solution is mixed with the surfactant in a ratio of 5/1 to 9/1 by volume.

In order to realize the objects of the invention, the cross-linking process comprises the steps that,

• 1 -ethyl-3-(3-imethylaminopropyl) carbodiimide hydrochloride, dissolving in a solvent,

• The dissolution of N-hydroxysuccinimide or divinyl sulfone in a solvent,

• The mixing of the two solutions prepared by volume of 1 /1 -3/1 ratio,

· Submerging of the nanofibrous mat into the resulting mixture solution and waiting at room temperature for 24 hours,

• Agitation of the nanofibrous mats removed from the mixture solution in ethanol,

• Leave to dry in an incubator at 37°C for 12 hours.

To accomplish the objects of the invention, said 1 -ethyl-3-(3-imethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide or divinyl sulfone is 50-100 imM. Ethanol or methanol is used as the solvent. In order to fulfill the above-mentioned objects, the invention is a nanofibrous mat obtained by electrospinning from a mixture of hyaluronic acid and carboxymethyl cellulose polymer suitable for use as an adhesion barrier in biomedical field. Said polymer mixture also comprises sodium alginate. The structural and characteristic features of the invention and all advantages thereof will be more clearly understood by means of the following figures and detailed description which are given by referring to these figures. For this reason, the evaluation should be done taking these forms and detailed explanation into consideration.

Figures for Better Understanding of the Invention

Figure-1 : Is the Scanning Electron Microscopy (SEM) views of the nanofibrous mats ((a)

3/1 /1 mixture ratio, (b) 5/1 /1 mixture ratio) obtained from the 10% HA / 2% CMC / 2% NaAlg mixture solution.

Figure-2: Is the Scanning Electron Microscopy (SEM) views of the nanofibrous mats ((a)

3/1 /1 mixture ratio, (b) 5/1 /1 mixture ratio) obtained from the 12% HA / 2%

CMC / 2% NaAlg mixture solution.

Figure-3: Is the EDC/NHS cross-linking process views, ((a) the samples given to the

EDC/NHS solutions, (b) the samples waiting for 24 hours, (c) after the incubator) Figure-4: Is the view of the water resistance test of the nanofibrous mat before cross- linking process.

Figure-5: Is the view of the water resistance test of the nanofibrous mat after cross- linking process.

Figure-6: Is the view of Scanning Electron Microscopy (SEM) views of the nanofibrous mat after the cross-linking process with EDC/NHS. ((a) 50 mM/100 imM, (b)

70 mM/100 mM, (c) 80 mM/100 mM, (d) 100 mM/100 mM)

Figure-7: It is a schematic view showing the insertion of the adhesion barrier (such as mesh) into the tissue [81 ].

Figure-8: It is a schematic view of the electrospinning process. Description of Parts Reference

1 . High voltage power supply

5. Feeding unit

10. Grounded collector

15. Liquid polymer

20. Cone form

Detailed Explanation of the Invention

In this detailed explanation, the inventive nanofibrous adhesion barrier and its preferred embodiments are described only for a better understanding of the subject and without forming any restrictive effect.

The invention relates to a nanofibrous mat obtained by electrospinning a mixture of hyaluronic acid (HA), sodium alginate (NaAlg) and carboxymethyl cellulose (CMC) polymer, suitable for use as an adhesion barrier in the biomedical field. In Table 1 below, the raw materials used to obtain the inventive nanofibrous mat are given.

Table 1 : Raw materials used to obtain the nanofibrous mat

Raw material

Hyaluronic acid (HA)

Carboxymethyl cellulose (CMC)

Sodium alginate (NaAlg)

Cocamidopropyl betaine (Betain)

Sodium hydroxide (NaOH)

Dimethyl sulfoxide (DMSO)

Pure water

1 -ethyl-3-(3-imethylaminopropyl) carbodiimide hydrochloride (EDC)

N-hydroxy succinimide (NHS)

Ethanol

Hyaluronic acid (HA) is a high molecular weight, highly water-absorptive, antitoxic, biocompatible and biodegradable polymer. It is used in cosmetics, biomedical and food industries. Its ability to absorb water, high viscoelasticity and non-toxic properties due to high molecular weight and negatively charged nature make it possible to use HA in cosmetic, biomedical and food industries. Due to its biocompatibility and biodegradability, HA polymer has been especially found in tissue engineering applications in gel and/or film [26, 28-31 ]. Due to the water retention properties of the HA polymer, it is not possible to produce nanofibers in a conventional electrospinning unit. There are various studies in the literature regarding the usability of HA nanofibrous mats produced by different methods as tissue scaffolds and wound covers [32-38].

The hyaluronic acid (HA) from the glucose aminoglycan polymer is a long chain polysaccharide first isolated from the transparent fluid of the retina in the eye. It is composed of repeating disaccharide units occurred by glycosidic linkage β-1 ,3 and β-1 ,4 of the N-acetyl-D-glucosamine and D-glucuronic acid. The molecular structure of hyaluronic acid is given below.

Molecular structure of hyaluronic acid [27]

Hyaluronic acid (HA) is one of the major components of the extracellular space between cells in various living organisms such as cartilage, joint fluid, skin and umbilical cord. It can be purifed from rooster swallow, baby cord and some other animal sources. In addition, it can be obtained from bacteria by fermentation and isolation methods. It has been noted that hyaluronic acid does not cause any allergic reaction in any way [25-27].

Carboxymethyl cellulose (CMC) is an inert, water-soluble, biocompatible polymer with high water retention capacity, resistant to bacterial growth. This polymer is used as a binder, blowing agent, gelling agent, adhesive and stabilizer agent in textile, paper, pharmaceutical, paint, cosmetic, ceramic and food industries. The main reasons for preferring CMC as an additive in the fields of use are that it is physiologically inert, its solubility in water, resistance to certain pH, high water-holding capacity, compatibility with other colloids and resistance to bacterial growth due to high sodium content. It is also known that carboxymethyl cellulose is transparent, flexible and forms films resistant to oils and organic solvents [39-42]. There are two main differences between carboxymethyl cellulose (CMC) and cellulose: These differences are due to the fact that the carboxymethyl cellulose contains hydrophilic carboxymethyl groups and the hydrogen bonds are very weak due to not the homogeneous settling of these groups. These two conditions cause the CMC polymer to absorb too much water [39].

While the sodium salt of CMC is generally produced, other salts such as potassium, calcium, and ammonium are also produced for special uses. CMC molecules have a flat structure at low concentrations. Due to their ionic carboxyl groups, they have negatively charged (anionic), long and rigid molecules and these molecules push each other in solution. For this reason, CMC solutions show a highly viscous stable structure [39-42].

Carboxymethyl cellulose (CMC) is a cellulose-derived polysaccharide formed by the attachment of carboxymethyl groups (CH2-COOH) to hydroxyl groups of glucopyranose monomers which constitute the cellulose chain, resulting from the reaction of alkali groups and chloroacetic acid. In other words, it's produced with carboxylation of the cellulose and enters to the group of cellulose ethers. The re-unit that forms the CMC molecule chains consists of β-(1 - 4) -D-glucopyranose linked to the cellulose group [39-42]. The molecular structure of carboxymethyl cellulose is given below.

Molecular structure of carboxymethyl cellulose [42] Because of its rigid structure and high surface tension it does not allow chain complexity and therefore the aqueous solutions cannot form a stable jet, the electrospinning of CMC polymer is quite hard. For this reason, in the limited studies in the literature, it has been possible to produce CMC nanofibrous mats by using co-polymers such as polyvinyl alcohol (PVA) and polyethylene oxide (PEO) or by adding additives such as aluminum and lithium [43-45]. The CMC nanofibrous mats produced in this way were used in lithium ion batteries [45-49] and drug delivery systems [50].

Sodium alginate (NaAlg) is a biocompatible polymer that has the ability to absorb wounds, stop the bleeding, and have high moisture absorption and gelling ability. The specific properties of sodium alginate that facilitate wound healing, high moisture absorption and ion exchange abilities, excellent biocompatibility and bleeding inhibitor properties make it a unique raw material in the production of high absorbent wound dressing [54]. Alginates have been used in food, pharmaceutical, medical, textile and paper industries for many years. Recently, its use has increased, especially in biomedical and medical fields.

Alginate is a natural polysaccharide derived from brown seaweed and on the cell walls of these mosses it is present as calcium, magnesium and sodium salts of alginic acid. While the calcium and magnesium salts of alginate are insoluble in water, the sodium salt is soluble in water. Sodium alginate is defined as a block copolymer because its polymeric structure is formed by the incorporation of two types of monomeric acids as blocks into a-L-guluronic acid (G) and β-D-mannuronic acid (M). The molecular structure of sodium alginate is given below.

Molecular structure of sodium alginate [53]

In the chemical formula of alginate, which is structurally similar to cellulose, unlike cellulose, the COOH group is replaced by the CH2OH group [51 , 52]. The most important feature of alginates is the reaction with polyvalent cations to convert them into hydrogels resulting in ion exchange. In particular, a three-dimensional networked gel structure is formed, in which the Ca+2 ions are replaced by Na+1 ions in the alginate molecule and attached to the carboxylate groups [54].

The alginate polymer has a long and rigid chain structure. Due to rigid chain structure and high electrical conductivity, it is quite difficult to subject aqueous sodium alginate solutions to electrocution alone. In the literature, alginate nano particles produced by electrospinning in the presence of ancillary polymers are used as wound dressing [55- 57], tissue scaffold [58-60] and drug release system [61 ], there are some studies about its use. Nanofibrous mats are the materials proven in tissue engineering due to its flexibility, large surface area, nano-porous structure, oxygen permeability, non-bacterial permeability, similarity to natural tissue, favorable cell growth and be inexpensive of its production.

The concept of nanofiber refers to fibers with diameters less than one micron. Biomedical applications are one of the most applied areas of nanofibers. Nanofiber materials are used in many places such as medical prostheses, artificial veins and organ applications, wound covers, drug distribution systems, tissue scaffolds, skin care products. The large surface area and nano-porous structure of the nanofibrous mats provide oxygen and air permeability while exhibiting barrier properties against bacteria [9-15]. Morphologically, the mats obtained by electrospinning are very similar to the natural human extracellular matrix (ECM). Therefore, it can be used as a tissue scaffold in cell culture and tissue engineering applications. Electrospinning method is the most studied nanofiber production technique in recent years. Nanofibrous mats obtained by the electrospinning method are very convenient for cell growth and for the formation of three-dimensional cellular colonies because of the attainment of very low fiber diameters. Thus, in order to accelerate or promote the formation of new cellular structures, nanofibrous mats produced by electrospinning from various polymers are used as wound covers [16, 17], drug release systems [18-20], tissue scaffolds [21 -24].

The electrospinning process is based on the principle that the electrically charged liquid polymer (15) is positioned in continuous fiber form on a grounded surface [62, 63]. There are basically 4 main elements in an electrospinning mechanism.

• High voltage power supply (1 )

• Feeding unit (jet, syringe, metal needle, etc.) (5)

• Grounded collector (plates, cylinder, disc, drum, etc.) (10)

· a viscous polymer in liquid form (melt or solution) (15)

In Figure 8, a schematic representation, components, and steps of the electrospinning process are given.

The liquid polymer (15) in melt or solution is fed from a capillary tube. By means of a high voltage power supply (1 ), very high voltages are applied to the polymer solution. Thus, the surface of the solution droplet suspended at the tip of the needle is electrically charged. As the applied voltage increases, the polymer droplet receives the cone form (Taylor cone) (20). When the voltage reaches a critical value and the push forces of the charges in the droplet absorb the surface tension forces a thin jet is launched from the tail of the Taylor cone (20) and the jet travels from one end of the same electrical charge to the next to the grounded collector (10). During the course of this process, this polymer jet follows prior stable then unstable (spiral) track path. During this time, the solvent in it evaporates and leaves behind a charged polymeric fiber having diameters in the nano scale. The resulting continuous nanofibers are randomly positioned on the collector plate (10) and form a nonwoven mat [65-67].

Electrospinning is an inexpensive and simple method of producing nanofibres, while controllability is a very difficult process. Because there are many technical parameters affecting the process [9]. The electrospinning process and the structure and morphology of the nanofibers obtained from this process are directly related to the parameters collected in the three main headings as solution properties, process conditions and ambient conditions. The properties of the polymer solution are the most important parameters affecting the electrospinning process and the morphology of the formed fiber. Surface tension plays an important role in the formation of beads, one of the most common problems on nanofibrous mats. The solution viscosity and electrical properties are influential on the formation and movement of the polymer jet. All have an effect on the diameter of electrospinning fibers [68]. Another important parameter that affects the electrospinning process is the various external factors that influence the electrospinning jet. These factors include the voltage applied, the feed rate, the solution temperature, the collector type, the nozzle diameter and the distance between the nozzle and the collector. Although not as much as the solution parameters, process parameters also have a significant effect on the resulting fiber diameter and morphology [69, 70].

During the electrospinning process, when the polymer jet is separated from the Taylor cone (20) formed at the tip end, the polymer jet is extended and stretched by the influence of the electrostatic forces, the Coulomb repulsion forces etc. as they travel towards the collector (10). In the process of stretching the solution, it is the complexity of the molecular chains that prevents breaks in the electrically moving polymer jet and thus allows the formation of a continuous solution jet. The increase in solution viscosity causes the polymer chain complexity to increase and overcomes the surface tension forces, thereby ensuring jet continuity during the electrospinning process. With increase in viscosity, the bead shape changes from spherical to spindle-like structure while the formation of beads in the nanofibrous mat decreases. With increased viscosity, fiber diameters also increase due to the decrease of the jet path. However, too high viscosity will make it difficult to pump the solution from the nozzle. In very low viscosities, bead formation occurs along the nanofibers, there is low chain complexity and the surface tension forces on the polymer jet are dominant. Electrospray occurs instead of electrospinning, and polymer particles are formed on the mat instead of fibers [54, 67, 69, 71 ]. The electrically charged solution must come from above the surface tension so that the electrospinning can begin. That is, surface tension is a factor that makes electrospinning difficult. Depending on the surface tension, when the concentration of free solvent molecules is high, the tendency of the solvent molecules to aggregate and take up a global shape will increase. In this case, surface tension may cause beads to form along the jet as the polymer jet travels toward reservoir plate (10). High viscosity means more interaction between solvent and polymer molecules, so that when the solution is stretched by the action of the charge, the solvent molecules will diffuse into the complex polymer molecules, thereby reducing the tendency of the solvent molecules to aggregate under the influence of surface tension [67-69].

In the electrospinning process, the polymer jet is stretched by pushing the loads on the surface together. If the electrical conductivity of the solution is increased, more charge may be carried in the electrospinning jet. If the solution is not fully stretched, pilling will occur. Another effect of the increased load is to increase the collection area of the fibers to produce finer fibers. There is a correlation between the pH and conductivity of the solution. The negatively charged OH ions present in a solution prepared in basic conditions have a significant effect on the increase of electric conductivity and jet tension. Although the electrical conductivity is advantageous for the electrospinning process, it has a decisive effect, which makes the process more difficult, even after a certain limit. At very high conductivity values, it is very difficult to maintain the load on the droplet surface at the tip of the needle in electrospinning and this affects the formation of the characteristic cone (20). As conductivity increases, the classical cone-jet pattern changes and multijet formation can be seen [72, 73].

The applied high voltage ensures that the polymer solution with a certain electrical conductivity is electrically charged and the electrostatic forces which cause the solution to travel in a thin jet towards a grounded collector (10). Electrospinning processes start when the electrostatic forces acting on the resistors absorb the surface tension forces of the solution. When voltage is applied, the resulting electric field affects the jet tension and acceleration. That is, both the voltage and the electric field obtained have an influence on the morphology of the fiber obtained. When a higher voltage is applied, due to the higher Coulomb power in the jet and the stronger electric field, the solution will stretch further. As this situation leads to a reduction in fiber diameter at the same time it causes the solvent to evaporate more rapidly, resulting in more dry fibers [69, 74]. The feed rate defines the amount of solution available for electrospinning. To keep the Taylor cone (20) stable, there is a corresponding feed rate for a given voltage. As the feed rate increases, the volume of the solution coming from the gland increases, resulting in an increase in fiber diameter or bead size. Due to the greater volume of solution coming out of the nozzle, the drying of the jet takes longer. As a result, the solvent in the fibers collected during the same flight does not have enough time to evaporate. Some solvent remaining after evaporation will evaporate after the fibers are placed on the collector (10), so that sticking can occur at points where the fibers are in contact with each other [69, 70]. The distance between the collector (10) and the nozzle is the area where the jetting occurs, the jet is incinerated and the solvent evaporates and forms solid fibers. That is, the electrospinning process takes place at this distance. The flight time and the electric field strength of the polymer jet in the air until reaching the collector (10) are factors affecting the electrospinning process and formed fibers. By changing the distance between the nozzle and the collector (10), both the flight time and the electric field strength are changed. When the distance is shortened, this time will be shortened and since the solvent does not evaporate completely, the sticks and bead formations will be seen at the contact points of the fibers. When the distance is increased, the electric field strength will increase and the jet speed will increase and the fiber diameters will decrease [69, 71 ].

CMC, HA and NaAlg used to form the nanofibrous mat subject of the invention are water- soluble polymers. The resulting nanofibrous mats have low resistance to water and water vapor. This would lead to problems in practical applications of nanofibrous mats that are planned to be used as adhesion barriers it is necessary to make an appropriate cross- linking treatment on the produced mats to increase the water resistance. Cross-linking is the process of attaching two or more molecules together by covalent bonding with a chemical method. Cross-linking of the adhesion zone nanofibrous mat is carried out in the presence of 1 -ethyl-3- (3-dimethylaminopropyl) carbodiimide hydrochloride (EDC) and N-hydroxysuccinimide (NHS). EDC is a water-soluble, biocompatible and non-toxic cross-linking agent used to couple carboxyl groups to primary amines. The non-inclusion of EDC in the cross-linked structure, i.e., not binding to polymer molecules, is particularly recommended for materials used in the biomedical field [75, 76]. The chemical structure of EDC is given below.

Chemical structure of EDC [77] EDC binds by activating carboxyl groups on polysaccharide molecules and forming ester bonds between hydroxyl and carboxyl groups. In the reaction mechanism exemplified below, the carboxyl groups of the HA polymer are activated with carbodiimide and form an O-acylurea labile intermediate product. This unstable intermediate product is shortlived and breaks down to link the hydroxyl and carboxyl groups of the HA polymer [78, 79].

cross-Miiking HA

The cross-linking reaction mechanism of EDC to HA [78]

NHS is a cross-linking agent used to activate carboxylic acid groups. When a normal carboxylic acid forms a salt with amines, the acids which are activated in the presence of NHS react with amines to give amides [79]. The chemical structure of NHS is given below.

Chemical structure of NHS [77]

EDC productivity increases in NHS presence. NHS is a homo-bifunctional crosslinker. This cross-linker is used in conjunction with EDC to inhibit the formation of irreversible N- acylic compounds. The combined use of EDC and NHS provides intermediate formulations that are hydrolytically resistant and cannot be regenerated. NHS has no toxic properties and imparts properties such as biocompatibility to the material, resistance to enzymatic degradation. NHS provides cross-linking by activating the esters of the glucuronic acid moiety present in the HA polymer [80]. Below, the EDC/NHS cross-linking reaction mechanism of HA is given.

O-acviarea

product

The cross-linking reaction mechanism of EDC/NHS to HA [80]

In general, the method of producing the nanofibrous mat of the present invention comprises;

• The preparation of hyaluronic acid solution in NaOH/Dimethyl sulfoxide (DMSO)

• The preparation of the aqueous carboxymethyl cellulose solution,

• The preparation of aqueous sodium alginate solution,

• The mixing of the three solutions prepared, • The addition of betaine to the resulting mixture solution,

• The application of electrospinning method to the mixture solution,

• The cross-linking of the obtained nanofibrous mat.

Nanofibrous Mat Production

First, HA solutions were prepared in 2% aqueous CMC, 2% aqueous NaAlg, 1 0% and 1 2% NaOH/DMSO. The measured properties of the prepared solutions are given in Table 2-4.

Table 2. Properties of aqueous CMC polymer solution

Subsequently, mixtures of 1 0% HA / 2% CMC / 2% NaAlg and 12% HA / 2% CMC / 2% NaAlg solutions were prepared at ratios of 3/1 /1 and 5/1 /1 . Betain was added to the prepared solutions, and a nanofibrous mat was produced by electrospinning. Solution properties are shown in Table 5, and Scanning Electron Microscopy (SEM) views are given in Figures 1 and 2. Table 5. HA/CMC/NaAlg polymer blend solution and production parameters

Cross-linking Process 50, 70, 80, and 100 mM EDC and 100 mM NHS to provide cross-linking were dissolved in ethanol. Into four EDC/NHS mixture solutions prepared in a volume ratio of 1 /1 , nanofibrous mat samples were immersed and allowed to stand at room temperature for 24 hours. After this time, the nanofiber cross-linked without any structural degradation, gelling or dissolution were removed from the solutions, rinsed in ethanol and allowed to dry for 12 hours at 37 ^QC in the incubator. Figure 3 shows the cross-linking images made with EDC/NHS.

Following the drying process, in order to understand whether the cross-linking agent used improves the water resistance of the mats, and the mats that cross-linked and had not been cross-linked were left to stand in purified water at room temperature for 24 hours. It is seen that the non-crosslinked mat is rapidly dispersed and entered into the gelling process from the first seconds after it was thrown into the water. In the 15th second following the moment when it was thrown in the water, the mat is completely gelated. In the Figure 4, the water resistance test of the nanofibrous mat is given before cross-linking. After the cross-linking methods employed, it has been observed that the cross-linked mats do not dissolve in water and maintain their dimensional stability. Figure 5 shows the water resistance test of the nanofibrous mat after cross-linking with 80 imM/100 mM EDC/NHS cross-linking agent. Furthermore, it is understood that the fibrous structure is maintained in the mat after crosslinking SEM images in Figure 6.

In the production of the nanofibrous mat subject of the invention; it is possible to use;

• Instead of the HA/CMC/NaAlg mixture solution, the HA/CMC mixture solution,

• Dimethyl formamide (DMF) instead of DMSO,

• Instead of Betain; Triton X-100,

· Instead of ethanol; methanol,

• Instead of NHS, divinyl sulfone (DVS).

In the production of the nanofibrous mat subject of the invention; it is possible that the polymer, the polymer mixture solution and the production parameters are within the following ranges. -HA concentration to be prepared in NaOH/DMSO; 8-15% (w/v %)

-CMC concentration to be prepared in pure water; 1 -4% (w/w%)

-NaAlg concentration to be prepared in pure water; 1 -4% (w/w%)

-The mixing ratio of the three polymer solutions (HA/CMC/NaAlg); 3/1 /1 -5/1 /1 (v/v/v)

-The amount of betaine to be added into the mixture solution ((HA/CMC/NaAlg)/Betaine); 5/1 -9/1 (v/v)

-Distance between the nozzle and the collector during electrospinning of solution; 7-15 cm

-Voltage to be applied during the electrospinning of the solution; 15-25 kV -The feed rate of the solution during electrospinning; 0,2-0,8 ml/h -The amount of EDC to dissolve in ethanol during cross-linking; 50-100 mM -The amount of NHS to dissolve in ethanol during cross-linking; 50-100 imM -EDC/NHS mixture ratio during cross-linking; 1 /1 -3/1

A lot of material was used to prevent adhesion formation until today but it has not been precisely shown that no one blocks the intra-abdominal adhesion. Studies continue to reduce or prevent intra-abdominal adhesions with used materials and with the products put forward constitute the million dollar health market.

The invention includes a low cost nanofibrous product that can reduce postoperative complications and has an alternative to commercial products used in the prior art, having potency to inhibit / reduce adhesion by using electrospinning from carboxymethyl cellulose, sodium alginate and hyaluronic acid polymers during intra-abdominal surgical operations.

Different polymers have been used for the first time for nanofibrous mats recommended as adhesion barrier. Some of these polymers (carboxymethyl cellulose and hyaluronic acid) are also polymers used in the production of commercial barriers. However, the polymer advantages of commercial barriers (gel, membrane, etc.) in different constructions are combined with the advantages of a nanofibrous mat.

The natural polymers HA, NaAlg and CMC are used in mixture with different polymers or purely as a gel, film, membrane or fiber/nanofiber biomedical field. Previously, however, no nanofibrous mat was produced by electrospinning from the HA/NaAlg/CMC polymer mixture. In addition, no nanofibrous mat have been produced from these polymers, either alone or in admixture, for use as an adhesion barrier.

Nanofibrous mats are the materials proven in tissue engineering due to its flexibility, large surface area, nano-porous structure, oxygen permeability, non-bacterial permeability, similarity to natural tissue, favorable cell growth and be inexpensive of production. According to the invention, the adhesion barrier formed of a nanofibrous mat is an easier, cheaper and more effective alternative to commercial adhesion barriers used in the market.

The insertion of the adhesion barriers (mesh) into the tissue is carried out as follows.

In practice, hernia or gingival adhesions are liberated if there are organs that stick after the application area is opened. With intraperitoneal onlay technique, the adhesion is placed between the pubic peritoneal and intra-abdominal or between the rectus posterior sheath and the peritoneum. Then the wound is closed by applying simple continuous stitching with a surgical thread which can be absorbed 2/0 from the wound edges on the wall of the abdomen. Care must be taken to ensure that there are no stresses while the stitches are passing. The operation thread material and the sewing method to be used during the operation must cause a minimum reaction. The barrier placement process with intra-peritoneal onlay technique is schematically shown in the Figure 7.

REFERENCE LIST

1 . Sumer, A. 2005. Effects of Oxidized Regenerated Cellulose, Polyethylene Glycol and Hylan Gf-20 on Adhesions after Prolene Mesh Application. Specialization Thesis, Ministry of Health Haydarpasa Numune Training and Research Hospital, Istanbul. 2. Akgil, A. M. 2008. Creation of Tissue Barrier with Sodium Hyaluronate and Carboxymethylcellulose Membrane (Seprafilm®) to Prevent Adhesion After Surgery in Rabbit Mediastinum. Specialization Thesis, Istanbul University, Istanbul.

3. Sahiner, i . T. 201 1 . Comparison of Intraabdominal Adhesions in the Repair of Abdominal Wall Defects with Simvastatin Loaded Polypropylene Patch. Specialization Thesis, Kirikkale University, Kirikkale.

4. Yegenoglu, A. 2006. Comparison of the Activities of Heparin, Seprafilm, Heparin and Seprafilm in the Prevention of Postoperative Intraperitoneal Adhesions. Specialization Thesis, Ministry of Health Dr. Lutfi Kirdar Training and Research Hospital, Istanbul. 5. Altinel, Y. 201 1 . Comparison of Two Different Polypropylene Mesh and Chitin Coated Forms in the Treatment of Abdominal Wall Hernia in Rats. Specialization Thesis, Uludag University, Bursa.

6. Ensari, N. 2008. Its Application to the Middle Ear in Order to Investigate the Neurotoxic Effect of Polylactic Acid Bioabsorbable Adhesion Barrier Film in Guinea Pig Specialization Thesis, Gazi University, Ankara.

7. Gunaydm, M., GCiveng, D., Yildiz, L., Aksoy, A., Tander, B., Bigakci, LI., Ayyildiz, H.

S., Sunter, A. T., Bernay, F. 2012. "Comparison of the Materials Used to Prevent Abdominal Adhesion: An Experimental Study in Rats", Turkish Clinics Journal of Medical Sciences, 32(2), 337-345. 8. Malgik, H. 2009. The Effect of Proanthocyanidine on the Development of Experimental Intraabdominal Adhesions in Rats. Specialization Thesis, Ministry of Health Taksim Training and Research Hospital, Istanbul.

9. Cengiz, F., Krucinska, I., Gliscinska, E., Chrzanowski, M., Goktepe, F. 2009.

"Comparative Analysis of Various Electrospinning Methods of Nanofibre Formation", Fibres & Textiles in Eastern Europe, (17) 13-19. 10. Jian, F., HaiTao, N., Tong, L., XunGai, W. 2008. "Applications of Electrospun Nanofibers", Chinese Science Bulletin, 15, 2265-2286.

1 1 . Huang, C, Chen, S., Lai, C, Reneker, D. H., Qiu, H., Ye,Y., Hou, H. 2006.

"Electrospun Polymer Nanofibres With Small Diameters", Nanotechnology, 17, 1558- 1563.

12. Kriegel, C, Arrechi, A., Kit, K , Mcclements, D. J, Weiss, J. 2008, "Fabrication, Functionalization, and Application of Electrospun Biopolymer Nanofibers", Critical Reviews in Food Science and Nutrition, 48(8), 775-797.

13. Kumar, A. 2010. Nanofibers. Croatia:lntech. 14. Patanaik, A., Anandjiwala, R. D, Rengasamy, R. S, Ghosh, A, Pal, H. 2007.

Nanotechnology in Fibrous Materials- A New Perspective. London:Taylor & Francis.

15. Huang, Z. M., Zhang, Y. Z., Kotaki, M., Ramakrishna, S. 2003. "A review on Polymer Nanofibers by Electrospinning and their Applications in Nnanocomposites", Composites Science and Technology, 63(15), 2223-2253. 16. Llstundag, C. G., Karaca, E., Ozbek, S., Cavusoglu, L, 2010. "In Vivo Evaluation of Electrospun Poly (Vinyl Alcohol)/Sodium Alginate Nanofibrous Mat as Wound Dressing", Tekstil ve Konfeksiyon, 20, 290-298.

17. Zhong, S. P., Zhang, Y. Z., Lim, C. T., 2010. "Tissue Scaffolds for Skin Wound Healing and Dermal Reconstruction", WIREs Nanomed Nanobiotechnology, 2, 510- 525.

18. Yoo, H. S., Kim, T. G., Park, T. G. 2009. "Surface-Functionalized Electrospun Nanofibers for Tissue Engineering and Drug Delivery", Advanced Drug Delivery Reviews, 61 (12), 1033-1042.

19. Kenawy, E. R., Abdel-Hay, F. I., El-Newehy, M. H., Wnek, G. E. 2009. "Processing of Polymer Nanofibers Through Electrospinning as Drug Delivery Systems", Materials

Chemistry and Physics, 1 13(1 ), 296-302.

20. Zeng, J., Xu, X., Chen, X., Liang, Q., Bian, X., Yang, L., Jing, X. 2003. "Biodegradable Electrospun Fibers for Drug Delivery", Journal of Controlled Release, 92(3), 227-231 .

21 . Li, C, Vepari, C, Jin, H.J., Kim, H. J., Kaplan, D. L. 2006. "Electrospun Silk-BMP-2 Scaffolds for Bone Tissue Engineering", Biomaterials, 27, 31 15-3124. 22. Min, B. M., Lee, G. N., Kim, S. H., Nam, Y. S., Lee, T. S., Park, W. H. 2004."Electrospinning of Silk Fibroin Nanofibers and Its Effect on the Adhesion and Spreading of Normal Human Keratinocytes and Fibroblasts in vitro", Biomaterials, 25, 1289-1297. 23. VanugopaL, J. R., Zhang, Y., Ramakrishna, S. 2006. "In Vitro Culture of Human Dermal Fibroblasts on Electrospun Polycaprolactone Collagen Nanofibrous Membrane", Artif Organs 30, 440-446.

24. Zhang, X., Reagan, R. M., Kaplan D. L. 2009. "Electrospun Silk Biomaterial Scaffolds for Regenerative Medicine", Advanced Drug Delivery Reviews, 61 , 988-1006 25. Diragoglu D. 2007. "Intraarticular Hyaluronic Acid Treatment in Osteoarthritis- Education", Turkish Journal of Physical Medicine and Rehabilitation, 53,154-159.

26. Aytar, P., Buruk, Y., Cabuk, A. 2013. "Determination of Optimum Conditions of Hyaluronic Acid Production with Streptococcus Equi by Plackett-Burman Method", Electronic Microbiology Review , 1 1 (1 ) 28-35. 27. Wang, X., Urn, I. C, Fang, D., Okamoto, A., Hsiao, B. S., Chu, B. 2005. "Formation of Water- Resistant Hyaluronic Acid Nanofibers by Blowing-Assisted Electro-Spinning and Non-Toxic Post Treatments", Polymer, 46(13), 4853-4867.

28. Ji, Y., Ghosh, K., Shu, X. Z., Li, B., Sokolov, J. C, Prestwich, G. D., Rafailovich, M.

H. 2006. "Electrospun Three-Dimensional Hyaluronic Acid Nanofibrous Scaffolds", Biomaterials, 27(20), 3782-3792.

29. Schante, C. E., Zuber, G., Herlin, C, Vandamme, T. F. 201 1 . "Chemical Modifications of Hyaluronic Acid for the Synthesis Of Derivatives for a Broad Range of Biomedical Applications", Carbohydrate Polymers, 85(3), 469-489.

30. Collins, M. N., Birkinshaw, C. 2013. "Hyaluronic Acid Based Scaffolds for Tissue Engineering-A Review", Carbohydrate Polymers, 92(2), 1262-1279.

31 . Price, R. D., Berry, M. G., Navsaria, H. A. 2007. "Hyaluronic Acid: The Scientific and Clinical Evidence", Journal of Plastic, Reconstructive & Aesthetic Surgery, 60(10), 1 1 10-1 1 19.

32. Li, J., He, A., Han, C. C, Fang, D., Hsiao, B. S., Chu, B. 2006. "Electrospinning of Hyaluronic Acid (HA) and HA/Gelatin Blends", Macromolecular Rapid

Communications, 27(2), 1 14-120 33. Li, J., He, A., Zheng, J., Han, C. C. 2006. "Gelatin and Gelatin-Hyaluronic Acid Nanofibrous Membranes Produced by Electrospinning of their Aqueous Solutions", Biomacromolecules, 7(7), 2243-2247.

34. Liu, Y., Ma, G., Fang, D., Xu, J., Zhang, H., Nie, J. 201 1 . "Effects of Solution Properties and Electric Field on the Electrospinning of Hyaluronic Acid",

Carbohydrate Polymers, 83(2), 101 1 -1015.

35. Uppal, R., Ramaswamy, G. N., Arnold, C, Goodband, R., Wang, Y. 201 1 . "Hyaluronic Acid Nanofiber Wound Dressing-Production, Characterization, and in Vivo Behavior", Journal of Biomedical Materials Research Part B: Applied Biomaterials, 97(1 ), 20-29. 36. Ji, Y., Ghosh, K., Li, B., Sokolov, J. C, Clark, R. A., Rafailovich, M. H. 2006. "Dual- Syringe Reactive Electrospinning of Cross-Linked Hyaluronic Acid Hydrogel Nanofibers for Tissue Engineering Applications", Macromolecular Bioscience, 6(10), 81 1 -817.

37. Kim, T. G., Chung, H. J., Park, T. G. 2008. "Macroporous and Nanofibrous Hyaluronic Acid/Collagen Hybrid Scaffold Fabricated by Concurrent Electrospinning and

Deposition/Leaching of Salt Particles", Acta Biomaterialia, 4(6), 161 1 -1619.

38. Yao, C, Li, X., Song, T. 2007. "Fabrication of Zein/Hyaluronic Acid Fibrous Membranes by Electrospinning", Journal of Biomaterials Science, Polymer Edition, 18(6), 731 -742. 39. Kirci, H. 2001 . "Cellulose Derivatives and Usage Locations", Science and Engineering Magazine, 4(2), 1 19-130.

40. Qiu, X., Hu, S. 2013. "Smart" Materials Based on Cellulose: A Review of the Preparations, Properties, and Applications", Materials, 6(3), 738-781 .

41 . Klemm, D., Heublein, B., Fink, H. P., Bohn, A. 2005. "Cellulose: Fascinating Biopolymer and Sustainable Raw Material", Angewandte Chemie International

Edition, 44(22), 3358-3393.

42. http://www.sigmaaldrich.com/catalog/product/sial/c9481 ?lang=en&region= TR,2014 Last Access Date 10 March 2016.

43. Zhang, X. X., Sun, J., Wang, J. J., Dai, L. X. 2013. "Preparation and Characterization of pH-Responsive Poly (Vinyl-Alcohol)/Sodium Carboxymethylcellulose Nanofibers",

Advanced Materials Research, 796, 132-135. 44. Xie, L, Shao, Z. Q., Lu, S. Y. 2012. "Preparation of AINPs/CMCN Composite Nanofibers by Electrospinning", Applied Mechanics and Materials, 130, 1266-1269.

45. Qiu, L, Shao, Z., Liu, M., Wang, J., Li, P., Zhao, M. 2014. "Synthesis and Electrospinning Carboxymethyl Cellulose Lithium (CMC-Li) Modified 9, 10- Anthraquinone (AQ) High-Rate Lithium-Ion Battery", Carbohydrate Polymers, 102, 986-992.

46. Qiu, L, Shao, Z., Yang, M., Wang, W., Wang, F., Wan, J., Duan, H. 2014. "Study on Effects of Carboxymethyl Cellulose Lithium (CMC-Li) Synthesis And Electrospinning on High-Rate Lithium Ion Batteries", Cellulose, 21 (1 ), 615-626. 47. Qiu, L., Shao, Z. Q., Liu, M. L., Liu, Y. H. 2014. "Electrospinning Carboxymethyl Cellulose Lithium (CMC-Li) Nano Composite Material for High-Rate Lithium-Ion Battery", Advanced Materials Research, 924, 69-72.

48. Qiu, L, Shao, Z., Xiang, P., Wang, D., Zhou, Z., Wang, F. , Wang, J. 2014. "Study on Novel Functional Materials Carboxymethyl Cellulose Lithium (CMC-Li) Improve High- Performance Lithium-Ion Battery", Carbohydrate Polymers, 1 10, 121 -127.

49. Qiu, L, Shao, Z., Wang, D., Wang, F., Wang, W., Wang, J. 2014. "Carboxymethyl Cellulose Lithium (CMC-Li) as a Novel Binder and its Electrochemical Performance in Lithium-Ion Batteries", Cellulose, 21 (4), 2789-2796.

50. Frenot, A., Henriksson, M. W., Walkenstrom, P. 2007. "Electrospinning of Cellulose- Based Nanofibers", Journal of Applied Polymer Science, 103(3), 1473-1482.

51 . Qin, Y. 2008. "Alginate Fibres: An Overview of the Production Processes and Applications in Wound Management", Polymer International, 57, 171 -180.

52. Seventekin, N. 2001 . Chemical Fibers. Izmir: Ege University Textile and Apparel Research and Application Center Publications. 53. http://www.fao. org/docrep/W6355E/w6355e0x.htm,2014.

Last Access Date: 18 August 2014.

54. Llstundag, C. G. 2009. Nanofibrous Mat Preparation and Its Application for Biomedical Application by Electrospinning Method. Master's Thesis, Uludag University, Bursa. 55. Shalumon, K. T., Anulekha, K. H., Nair, S. V., Nair, S. V., Chennazhi, K. P., Jayakumar, R. 201 1 . "Sodium Alginate/Poly (vinyl alcohol)/Nano ZnO Composite Nanofibers for Antibacterial Wound Dressings", International Journal of Biological Macromolecules, 49(3), 247-254.

56. Tarun, K., Gobi, N. 2012. "Calcium Alginate/PVA Blended Nano Fibre Matrix for Wound Dressing", Indian Journal of Fibre & Textile Research, 37, 127-132. 57. Coskun, G., Karaca, E., Ozyurtlu, M., Ozbek, S., Yermezler, A., Cavusoglu, i. 2014.

"Histological Evaluation of Wound Healing Performance of Electrospun Poly (Vinyl AlcohoiySodium Alginate as Wound Dressing in Vivo", Bio-medical Materials and Engineering, 24(2), 1527-1536.

58. Jeong, S. I., Krebs, M. D., Bonino, C. A., Samorezov, J. E., Khan, S. A., Alsberg, E.

2010. "Electrospun Chitosan-Alginate Nanofibers With In Situ Polyelectrolyte

Complexation for Use as Tissue Engineering Scaffolds", Tissue Engineering Part A, 17(1 -2), 59-70.

59. Jeong, S. I., Krebs, M. D., Bonino, C. A., Khan, S. A., Alsberg, E. 2010. "Electrospun Alginate Nanofibers with Controlled Cell Adhesion for Tissue Engineering", Macromolecular Bioscience, 10(8), 934-943.

60. Ma, G., Fang, D., Liu, Y., Zhu, X., Nie, J. 2012. "Electrospun Sodium Alginate/Poly (Ethylene Oxide) Core-Shell Nanofibers Scaffolds Potential for Tissue Engineering Applications", Carbohydrate Polymers, 87(1 ), 737-743.

61 . Yang, D., Li, Y., Nie, J. 2007. "Preparation of Gelatin/PVA Nanofibers and Their Potential Application in Controlled Release of Drugs", Carbohydrate Polymers, 69(3),

538-543.

62. Andrady, A. L. 2008. Science and Technology of Polymer Nanofibers. New Jersey:Wiley Pres.

63. Chronakis, I. S. 2005. "Novel Nanocomposites and Nanoceramics Based on Polymer Nanofibers Using Electrospinning Process-A Review", Journal of Materials

Processing Technology, 167(2-3), 283-293.

64. http://nano.mtu.edu/images/Electrospinning_alt.jpg, Son Erisim Tarihi 8 Kasim 2009.

65. Supuren, G., Kanat, Z. E., Cay, A., Kirci, T., Gulumser, T., Tarakgioglu, I. 2007. "Nano Fibers (Part 2)", Textile and Apparel, 2, 83-89. 66. Doshi, J., Reneker, D. H. 1995. "Electrospinning Process and Applications of Electrospun Fibers", Journal of Electrostatics, 35(2-3), 151 -160. Llstundag, G. C, Karaca, E. 2009. "Investigation of Nanofibrous Mats Obtained by Electrodeposition Method of Poly (Vinyl Alcohol) / Sodium Alginate Mixtures", Uludag University Journal of Engineering and Architecture, 14 (1 ), 159-172. Mit-uppatham, C, Nithitanakul, M., Supaphol, P. 2004. "Ultrafine Electrospun Polyamide-6 Fibers: Effect of Solution Conditions on Morphology and Average Fiber

Diameter", Macromolecular Chemistry and Physics, 205, 2327-2338. Ramakrishna, S., Fujihara, K., Teo, W. E., Lim, T. C, Ma, Z. 2005. An Introduction to Electrospinning and Nanofibers. Singapore:World Scientific Publishing Co. Deitzel, J. ., Kleinmeyer, J., Harris, D, Becktan, N. C. 2001 . "The Effect of Processing Variables on the Morphology of Electrospun Nanofibers and Textiles", Polymer, 42,

261-272. Kozanoglu, G. S. 2006. Nanofiber Production Technique by Electrospinning Method. Master's Thesis, Istanbul Technical University, Istanbul. Angammana, C. J., Jayaram, S. H. 2008. "Analysis of the Effects of Solution Conductivity on Electro-Spinning Process and Fiber Morphology", Industry

Applications Society Annual Meeting, Canada. Li, Z, Wang, C. 2013. One-Dimensional Nanostructures Electrospinning Technique and Unique Nanofibers. New York:Springer. Ascioglu, B. 2005. Manufacturing and Heat Transfer Analysis of Nano-Micro Fiber Composites. Ph.D Thesis, Auburn University, Auburn, Fischer, R. L., McCoy, M. G., Grant, S. A. 2012. "Electrospinning Collagen and Hyaluronic Acid Nanofiber Meshes", Journal of Materials Science: Materials in Medicine, 23(7), 1645-1654. Xu, S., Li, J., He, A., Liu, W., Jiang, X., Zheng, J., Han, C. C, Hsiao, B. S., Chu, B., Fang, D. 2009. "Chemical Crosslinking and Biophysical Properties of Electrospun

Hyaluronic Acid Based Ultra-thin Fibrous Membranes", Polymer, 50(15), 3762-3769. https://www.thermofisher.com/order/catalog/product

Last Access Date: 12 April 2014. Lu, P. L, Lai, J. Y., Ma, D. H. K., Hsiue, G. H. 2008. "Carbodiimide Cross-linked Hyaluronic acid Hydrogels as Cell Sheet Delivery Vehicles: Characterization and Interaction with Corneal Endothelial Cells", Journal of Biomaterials Science: Polymer Edition, 19(1 ), 1 -18.

79. Tomihata, K., Ikada, Y. 1997. "Crosslinking of Hyaluronic Acid with Water-soluble Carbodiimide", Journal of Biomedical Materials Research, 37(2), 243-251 . 80. Svldronova, P. B. B., Vojtova, L. 2014. Crosslinking of Polysaccharide Microfibers.

Master Thesis, Brno University of Technology, Brno.

81 . Dilek, O. N, Turel, K. S. 2009. "The Use of Mesh in Hernia Repair and It's Complications". Journal of Surgical Arts, 2, 1 -9.

Claims

1. A nanofibrous mat obtained from the hyaluronic acid and carboxymethyl cellulose polymer mixture, suitable for using as an adhesion barrier in the biomedical field.

2. The nanofibrous mat according to Claim 1 , and it is characterized in that, the said polymer mixture comprises a sodium alginate.

3. A production method of the nanofibrous mat according to Claim 1 , and it is characterized in that, it comprises following process steps;

• preparation of the hyaluronic acid solution in a solvent,

• preparation of the aqueous carboxymethyl cellulose solution,

· mixing of the two solutions prepared,

• addition of surfactant to the obtained mixture solution,

• application of electrospinning method to the mixture solution,

• cross-linking of the obtained nanofibrous mat.

4. A method according to Claim 3, and it is characterized in that, it comprises following process steps;

• preparation of aqueous sodium alginate solution,

• process of mixing the prepared hyaluronic acid solution and the carboxymethyl cellulose solution.

5. A method according to Claim 3, and it is characterized in that, the said surfactant is betaine or Triton X-100.

6. A method according to Claim 3, and it is characterized in that, the said solvent is NaOH/Dimethyl sulfoxide or NaOH/Dimethyl formamide.

7. A method according to Claim 6, and it is characterized in that, the ratio of the mixture of said NaOH with Dimethyl sulfoxide or NaOH to Dimethyl formamide is 4/1 by volume.

8. A method according to Claim 3, and it is characterized in that, the concentration of hyaluronic acid solution prepared in the solvent is 8-15% by mass/volume %.

9. A method according to Claim 3, and it is characterized in that, the concentration of carboxymethyl cellulose solution prepared in distilled water is 1 -4% by mass/mass %.

10. A method according to Claim 4, and it is characterized in that, the concentration of sodium alginate solution prepared in distilled water is 1 -4% by mass/mass %.

11. A method according to Claim 4, and it is characterized in that, the said hyaluronic acid solution, the carboxymethyl cellulose solution and the sodium alginate solution are mixed at a ratio of 3/1 /1 -5/1 /1 by volume.

12. A method according to any of Claim 3, 4 or 1 1 , and it is characterized in that, the mixture solution is mixed with the surfactant at a ratio of 5/1 -9/1 by volume.

13. A method according to Claim 3, and it is characterized in that, the said cross- linking treatment comprises following process steps; · dissolving of 1 -ethyl-3-(3-imethylaminopropyl) carbodiimide hydrochloride in a solvent,

• dissolving of N-hydroxysuccinimide or divinyl sulfone in a solvent,

• mixing of the two solutions prepared,

• submerging of the nanofibrous mat into the resulting mixture solution and waiting at room temperature for 24 hours,

• agitation of the nanofibrous mats removed from the mixture solution in ethanol,

• leaving in an incubator at 37°C for 12 hours to dry.

14. A method according to Claim 13, and it is characterized in that, the said 1 -ethyl- 3-(3-imethylaminopropyl) carbodiimide hydrochloride, N-hydroxysuccinimide or divinyl sulfone is 50-100 imM.

15. A method according to Claim 13, and it is characterized in that, it comprises the mixing of the two solutions prepared in a volume of 1/1 -3/1 by volume.

16. A method according to Claim 13, and it is characterized in that, the said solvent is ethanol or methanol.

17. A nanofibrous mat obtained from the hyaluronic acid and carboxymethyl cellulose polymer mixture with electrospinning method, suitable for using as an adhesion barrier in the biomedical field.

18. A nanofibrous mat according to Claim 17, and it is characterized in that, the said polymer mixture comprises a sodium alginate.