DESCRIPTION

Technical Field

The present invention relates to a new tissue scaffold production method using a natural polymer, chitosan and polycaprolactone which is a synthetic biocompatible polymer that can be used for the treatment of tissue losses occurring due to various reasons in the human body.

The Prior Art

The cell anlage made of the cells with the same purpose, aggregated to construct a specific function, which come together to constitute the organs is called a tissue. Tissue losses may occur during the surgeries performed for the treatment of various injuries and illnesses. In cases where these losses are not recovered, problems such as loss of function and/or aesthetic defects that affect human health and living standards arise. The tissues are capable of self-renewal, but there is a limit of this capability. There are two important elements for the self- renewal of the tissues. The first of these is the requirement to be in contact with neighboring cells by means of bio-signals. The other important element is the requirement of adsorption for the growth of animal cells. In cases where there is a considerable loss of tissue and the said two important elements are lacking and/or insufficient, tissue regeneration cannot occur .

The body provides self-healing without outside intervention, in cases when tissue loss is below the limit values. In case the loss of tissue is above the limit values, external support is absolutely needed. In cases when the tissue losses are above the limit value, the method of transferring pieces taken from a region of the patient's body with a similar tissue or from a region of another species with a genetically similar tissue, to the damaged area, is widely used. During the treatment, in cases when pieces taken from a region of the patient's body with a similar tissue are used, a new injured area is formed on the patient's body because of a second surgery, thus causing a prolonged healing process, as well as a loss of function and/or aesthetic defects at the region where tissue is taken. There may be cases when the dimensions of the tissue required for renewing the lost tissue, to be transferred are too large to be taken from the patient's body. In case the tissue to be transferred has to be taken from a healthy person, tissue compatibility problems may arise. Due to such tissue compatibility problems, it becomes more difficult to find a donor. There is also a risk of tissue transplant rejection although there is biocompatibility . In the event of transferring the lost tissue from another living body, different problems may be faced. There is a possibility that the tissue taken from a different species may be pathogenic, i.e. carrying disease. Despite the fact that animals used for tissue transplantation were raised specifically to have genetics close to human genetics, there is a possibility of tissue transplant rejection for this transplant too.

Due to all these reasons, biomaterials resembling the extracellular matrix structure in natural human tissue, which is called the tissue scaffold have started to be used. Tissue scaffolds are placed in the lost tissue area to provide the structural basis necessary for enabling the adsorption and reproduction of the cells to rebuild the missing tissue. In the design of te said scaffolds, the selection of the material is the most important criterion. Materials must be biocompatible, in other words it should not lead to adverse tissue reactions when implanted in the body and must also have a surface chemistry that enhances cell adsorption and function. Biodegradability is another desired feature. However during degradation, the material should not lose its biocompatibility, rendering toxic products and must have mechanical properties that will provide the appropriate environment for the new tissue. Furthermore, it should have a porous structure that allows passage of cells and nutrients. Among materials with these features, polymers, ceramics, metals and combinations of them can be considered.

For tissue scaffold applications, biodegradable natural polymers, such as alginate, gelatin, starch, collagen, chitin and chitosan, as well as biodegradable synthetic polymers such as polycaprolactone, polylactic-co-glycolic acid, polyethylene glycols, polyvinyl alcohol and polyurethane are used. Natural polymers are preferred as they are bioactive/biofunctional and biologically and chemically similar to natural tissues. However biomaterials made of natural polymers such as collagen and chitin, have poor mechanical resistance and generally cannot be melted by heat and often a special solvent is required. On the other hand, in spite of the disadvantage that they cannot be obtained from natural sources, the synthetic polymers used in the field of tissue engineering, have such advantages as that they can be easily processed, unlike the natural polymers, that they are degradable by means of chemical hydrolysis and that the degree of their degradation does not change from one patient to the other as they are not affected, by enzymatic processes. While a limited number of manufacturing techniques such as freeze drying in aqueous solution/crosslinking are used to produce tissue scaffolds from natural polymers, numerous techniques such as solvent casting, particle removal, phase separations, electrospinning are used to produce tissue scaffolds from synthetic polymers.

The electrospinning technology has been developed in the last decade, for tissue engineering applications to manufacture biodegradable and/or biocompatible filamentary structures with a fiber diameter of micrometer or nanometer size. The polymer solution is directed along the capillary in such a manner that a polymer droplet is formed at the head/end section. High voltage is applied to a section between the end portion and the grounded collection zone. If the electric field tension is greater than the surface tension of the droplet, the fountain of polymer solution is accelerated towards the collection zone. As the fountain proceeds in the air, the solvent moves away from the structure and a non-woven fabric structure is formed in the collection zone. In case routed and tubular structures are desired to be prepared, the grounded rotary cylinder may be used as the collection zone. It is possible to obtain non-woven fibrous structures for tissue engineering purposes by using natural macromolecules such as collagen and fibrinogen and synthetic polymers as polycaprolactone, polyglycolic acid, polylactic-co-glycolic acid, and synthetic polypeptides.

In practice natural polymers are preferred for tissue scaffold applications, due to their bioactive/biofunctional properties. However, biodegradable synthetic polymers, with the disadvantage that they cannot be obtained from natural sources, are preferred, as they can easily be processed and unlike the natural polymers, are degradable by chemical hydrolysis and they have advantages of their rate of degradation being able to change from one patient to other as they are affected by enzymatic processes. Such features of the tissue scaffold as having the similar physical structure as the three dimensional structure of the living tissue, providing mechanical characteristics that are suitable to the region it is used and having a porous structure to allow a material exchange, may vary depending on the technique used for production. While a limited number of manufacturing techniques such as freeze drying in aqueous solution/crosslinking are used to produce tissue scaffolds from natural polymers, numerous techniques such as solvent casting, particle removal, phase separation, electrospinning are used to produce tissue scaffolds from synthetic polymers. Tissue scaffolds obtained in a single stage do not show sufficient mechanical strength and flexibility.

In prior art there are structures wherein synthetic polymers and natural polymers are used in a combination for the production of biocompatible tissue scaffolds with desired properties.

Chitosan being one of the natural polymers, has such advantages as being cheaper and the second most abundant, thus easily obtainable natural polymer as it is obtained by deacetylation of chitin and as it is positively charged being able to interact with the glycosaminoglycans existing in the ECM, being biocompatible, having anti-microbial activity, being soluble in weak acids, can be easily processed as a film and porous tissue scaffold. However, in terms of mechanical properties, it is very low degree of flexibility and biodegradability limits are among its most important disadvantages. Chitosan films are quite fragile when wet.

On the other hand PCL, being a synthetic polymer, can be processed easily because of its low melting temperature. Its mechanical properties and non-enzymatic degradability (decomposition through hydrolysis) can be regulated by PCL's crystalline structure. However PCL has such disadvantages as its limited bioactivity, being hydrophobic and uncharged, having sensitivity to via bacteria-degradability .

In prior art, a tissue scaffold obtained by dissolving simultaneously polycaprolactone and chitosan in acetic acid is described in application No. CN201310327715. The poration process of the tissue scaffold is achieved by the creation of air gaps by suspending polycaprolactone-chitosan polymer in water and drying it under vacuum.

Also, in the application No. CN20091199316, the polycaprolactone and chitosan copolymer is obtained. Tissue scaffold is obtained by integrating polycaprolactone with chitosan in a single phase using methane sulfonic acid as the reactive solvent, amino as a preservative agent and chitosan as the initiator.

In the application No. CN2013168447, polycaprolactone and chitosan, are integrated as a separate layer by using a process of high pressure static spinning on the polylactic acid layer. Polycaprolactone, and chitosan are mixed and spun under high pressure statically.

In the application No. CN2007185930, the tissue scaffold is obtained by using acetic acid as a solvent and dissolving chitosan and polycaprolactone at the same time. The porous structure is obtained by adding a NaOH solution and drying it.

The application No. KR20060014449 discloses a method of culturing the fibroblast and keratinocyte cells together. For the culturing process to be applied, the improvement of a tissue scaffold material by using a plurality of different polymers also including chitosan and polycaprolactone, has been disclosed .

The application No. WO2006CN01027 discloses the production of nano-sized scaffolds by using the self-assembly approach, wherein numerous polymers including polycaprolactone and chitosan, as well as a plurality of anionic or cationic charges also including chitosan, are used.

As disclosed in the application No. CN102605466 (A), polyelectrolyte solution is used together with the polymer solution to form a core-shell structure and these two solutions are used in the electrospinning process after being mixed at a certain ratio, to provide a single solution.

The application No. CN101653624 discloses the production of a fibrous structure obtained by preparing 3 polymers including PCL and chitosan as a single solution and applying it onto a rotating roller to apply electro-spinning.

In similar studies seen in the literature, the polymers are used as a single solution, often by being dissolved with a common solvent or dissolved separately and then mixed together. However, as the polymers used for this kind of approach are mixed together homogeneously, their physical and chemical properties cannot be sufficiently reflected in the final product.

It is necessary that, the scaffolds should be biocompatible, should not be rejected by the immune system, should have a physical structure similar to the extracellular matrix, should show sufficient mechanical strength and flexibility, should remain in place until the tissue repair process begins and as the tissue takes shape, disappear not to create any toxic substances. It is seen that the biomaterials known in the prior art do not appear to offer all of these conditions.

Considering the disadvantages faced at the end of the explanations and studies seen above, by using the production process of the subject invention, tissue scaffolds with a good mechanical resistance and higher biocompatibility/bioactivity, thanks to the chitosan/PCL hybrid structure, have been obtained. Furthermore, the electrospinning technique has been selected as the production technique and the 3-dimensional (3D), similar to natural ECM, produced by this method provided high-density cell growth/migration in 3-dimensional (3D), non-woven reticulated structures .

Problems That The Invention Aims To Solve

The aim of the invention is to develop a new tissue scaffold suitable for use in the treatment of tissue losses with a critical flaw size, which is biocompatible, that is not rejected by the body, having chemical and biological activity, not generating any toxic substance, having sufficient mechanical strength and flexibility, with sufficiently porous structure for the diffusion of nutrients to enable the growth/reproduction of cells, and its manufacturing process.

Another object of the invention is to develop a tissue scaffold that is superior in terms of mechanism and porosity, due to the structure obtained by the solvent cast-freeze drying methods, wherein a natural polymer chitosan and a synthetic polymer, polycaprolactone are used in the composite structure. Another object of the invention is to obtain a tissue scaffold having a good mechanical strength, obtained by eliminating its poor physical property which is the disadvantage of chitosan.

Description of Figures Figure 1 : Scanning Electron Microscopy-SEM images obtained by scanning at 20 micrometers (μπι) scanning size, of the material freeze dried for 12 hours after pre-dried in the refrigerator at -20°C for 24 hours. Figure 2 : Scanning Electron Microscopy-SEM images obtained by scanning using 5 micrometers (μπι) scanning size, of the material freeze dried for 12 hours after pre-dried in the refrigerator at -20°C for 24 hours.

Figure 3: SEM images obtained by scanning using 20 μπι scanning size, of the material freeze dried for 24 hours after pre-dried in the refrigerator at -20°C for 24 hours.

Figure 4: SEM images obtained by scanning using 5 μπι scanning size, of the material freeze dried for 24 hours after pre-dried in the refrigerator at -20°C for 24 hours. Figure 5: SEM images obtained by scanning using 20 μπι scanning size, of the material freeze dried for 24 hours after pre-dried in the refrigerator at -20°C for 48 hours.

Figure 6: SEM images obtained by scanning using 5 μπι scanning size, of the material freeze dried for 24 hours after pre-dried in the refrigerator at -20°C for 48 hours.

Figure 7: SEM images obtained by scanning using 20 μπι scanning size, of the material freeze dried for 12 hours after pre-dried in the incubator at 40°C for 5 hours.

Figure 8: SEM images obtained by scanning using 5 μπι scanning size, of the material freeze dried for 12 hours after pre-dried in the incubator at 40°C for 5 hours.

Figure 9: SEM images obtained by scanning using 20 μπι scanning size, of the material freeze dried for 24 hours after pre-dried in the incubator at 40°C for 5 hours Figure 10: SEM images obtained by scanning using 5 μπι scanning size, of the material freeze dried for 24 hours after pre-dried in the incubator at 40°C for 5 hours

Figure 11: SEM images obtained by scanning using 20 μπι scanning size, of the material freeze dried for 48 hours after pre-dried in the incubator at 40°C for 5 hours Figure 12: SEM images obtained by scanning using 5 μπι scanning size, of the material freeze dried for 48 hours after pre-dried in the incubator at 40°C for 5 hours

Disclosure of the Invention The present invention relates to a tissue scaffold which has a good biocompatibility and can degrade without causing toxic effects, that has a physical structure similar to the three- dimensional structure of the living tissue, which can provide mechanical properties suitable to the region used and which has a porous structure to allow the exchange of substances, and it also relates to its graduated production process.

The production process for the tissue scaffold that feature macro/micro-sized spherical porosity and a structure consisting of fibers in-between the same, presenting adequate mechanical strength and flexibility, having a composite PCL/chitosan material structure comprises of two stages, namely three- dimensional integration of the PCL fibrous structure with chitosan polymer and drying at the oven followed by freeze- drying. In order to produce the composite PCL/chitosan tissue scaffold, first of all the structure consisting of PCL fibers known in the prior art should be achieved.

Achieving a structure consisting of PCL fibers comprises of the process steps set forth hereunder;

• Preparing a chloroform/methanol solvent with a volumetric ratio of three to one,

• Forming a solution by adding 5-25% by weight of polycaprolactone (PCL) to the prepared solvent at 5-60°C by stirring at 100-450 rpm for 20-60 minutes, • Spraying, by employing the electro-spinning method, the prepared solution fed through a syringe onto a flat surface from a distance of 10-30 cm at a rate of 5-30 μΐ/min, under 10-30 kV voltage, and

Obtaining a fibrous structure comprising randomly oriented PCL fibers with a thickness of 0.1 to 2.5 mm.

The three dimensional integration is achieved after obtaining a fibrous structure consisting of randomly oriented PCL fibers at thickness in the range of 0,1-2,5 mm for the tissue scaffolding that feature developed composite PCL/chitosan material structure of the invention and is freeze-dried after being dried in an oven .

1. The three-dimensional integration of the PCL fibrous structure with chitosan polymer comprises of the process steps set forth hereunder;

• Preparing a solution by stirring 1-3% by weight of chitosan in 0.1-0.3M acetic acid solvent for 1-2 h, at a rate of 100 to 500 rpm, at a temperature of 10-50°C,

• Pouring the solution into a petri dish,

• Immersing the fibrous structure consisting of randomly oriented PCL fibers with a thickness of 0,1-2,5 mm into the solution,

• As PCL presents a hydrophobic nature, applying pressure on the PCL to ensure integration of the chitosan solution with PCL fibers,

• Removing the resulting PCL/chitosan structure from the solution, and

• Drying in oven at a temperature of 10-60°C for 2-5 hours. 2. Freeze-drying;

• Consists of the process step wherein dried PCL/chitosan structure is freeze-dried at a temperature range of -65°C to -80°C under a vacuum in the range of 10 ^_1 -10 ^-3 mbar for a period of 6 to 48 hours.

The resulting PCL/chitosan structure, is the tissue scaffold obtained in a similar manner to the extracellular matrix (ECM) which performs the functions of natural scaffold in animal tissues .

The poly ( ε-caprolactone ) (PCL) ; being one of the synthetic polymers used in the first stage of the above-described production process, is a hydrophobic, biodegradable aliphatic polyester and has a vitric transition temperature of -60°C and a melting point at 60°C.

PCL is generally prepared by ring-opening polymerization of ε- caprolactone . It is used as substrate for biodegradability studies and as matrix for controlled drug emission systems. As the degradation of the PCL in an in vivo medium is slower than that of poly ( -hydroxy acid), its use as a long-term, up to 1-2 years, controlled release device is more preferred. PCL is widely used in such areas as artificial skin production (in plain film) with tissue engineering, coating of urethral stents, musculoskeletal tissue engineering applications and most importantly, as a support material, i.e. tissue scaffold for osteoblast and fibroblast cell growth.

Furthermore, in the used electrospinning method, depending on the process parameters such as voltage applied, polymer flow rate and depending on the solvent parameters such as polymer concentration, the volatility of the solution, the fiber orientation (arrayed regularly and randomly) , the porosity/pore diameter (cellular infiltration) and fiber diameter (nanometers/micrometer size) can be changed.

Chitosan (poly [ β- ( 1 , 4 ) -2-amino-2-deoxy-D-glucose ] ) , used in the second stage of the above-described production process and is one of the natural polymers, can be obtained as the result of the partial deacetylation of chitin.

Chitosan is a linear polysaccharide. It has a high potential for tissue engineering applications both because it contains chemically modifiable reactive amino and hydroxyl groups and it is easy to be physically manipulated for various pore structures. Chitosan is located in the exoskeletons of crustaceans and in the cell walls of fungi and is located in the cell wall is composed of different numbers of randomly located N-acetyl- glucosamine and D-glucosamine residues. Therefore it shares similar characteristics as glycosaminoglycan and hyaluronic acid found in articulation cartilage. The properties of chitosan such as being biologically renewable; biodegradable, biocompatible, non-antigenic, nontoxic and bio-functional properties of it allow the use of this polymer and complexes obtained using it in biomedical applications such as wound dressing material, drug delivery systems. Chitosan can be dissolved in weak acids and, consequently, a cationic polymer having a high charge density. Because of this property, chitosan can form polyelectrolyte complexes with numerous anionic polymers. Its degradation by lysozyme in the body provides the formation of macrophage activating oligomers. Fragmentation contributes to the healing of the wound tissue by providing the formation of N- acetylglucosamine, an important component of the dermal tissues. Moreover, it is also used as means of non-viral gene transport because of its ability to form complexes with DNA. Particularly it is preferred for bone tissue engineering studies, because of its osteo-compatibility and osteo-inductivity properties.

In the second phase of the above-described production process, a tissue scaffold is obtained by providing a three-dimensional integration of the PCL fibrous structure with chitosan polymer.

The biocompatibility and biodegradability properties of the chitosan/PCL hybrid tissue scaffold formed by chitosan, a biodegradable natural polymer and PCL, a biodegradable synthetic polymer vary depending on the components used during production. The inventive tissue scaffold with enhanced biocompatibility and mechanical properties is used in such a manner that the natural polymer chitosan and the synthetic polymer with a good biocompatibility polycaprolactone, form a composite structure.

Whereas such features of the tissue scaffold as having a physical structure similar to the three dimensional structure of the living tissue will allow to providing mechanical characteristics suitable to those of the region used and having a porous structure to allow material exchange, may vary depending on the technique used for production. While a limited number of manufacturing techniques using natural polymers, such as freeze- drying in aqueous solution/crosslinking for the production of tissue scaffolds, for the production of the same but using synthetic polymers numerous methods, such as solvent casting, particulate removal, phase separation, electrospinning are used. The use of PCL tissue scaffolds produced by freeze-drying technique, in the Chondrocyte cell cultures is one of the tissue engineering applications that draw attention. PCL fibers are produced by electrospinning technique for use in soft tissue engineering applications. It has been observed that the adsorption and growth of the L929 cell on PCL films in the technique of thermo-printing were very good. It is well known that porous, micro/patterned PCL tissue scaffolds are prepared with thermo-printing and particle removal techniques and these tissue scaffolds are used in vascular tissue engineering applications .

With the method according to the present invention, chitosan/PCL hybrid tissue scaffolds are fabricated using chitosan, a biodegradable natural polymer which is often preferred for the production of tissue scaffolds and a biodegradable synthetic polymer PCL. Chitosan, has many advantages, namely being cheaper, can be obtained by deacetylation of chitin which is the second most abundant natural polymer and is easily obtained, being positively charged able to provide interaction with negatively charged glycosaminoglycans in the ECM, being biocompatible, its anti-microbial activity, is soluble in weak acid, can be easily processed as film and porous tissue scaffold. However, having very little flexibility in terms of mechanical properties occur and biodegradability limits are among the most important disadvantages. Chitosan films are quite fragile in wet state. On the other hand, PCL can be processed easily because of its low melting temperature. Its mechanical properties and non- enzymatic degradability (decomposition through hydrolysis) can be regulated by the crystalline structure of PCL. However PCL has such disadvantages as having limited bioactivity being hydrophobic and uncharged, sensitivity to via bacteria- degradability . According to the present invention, tissue scaffolds with a good mechanical strength and a high biocompatibility/bioactivity are obtained thanks to the chitosan/PCL hybrid structure. Moreover, the technique of electrospinning has been selected as the production technique and a high-density cell growth/migration is provided in 3- dimensional (3D), nonwoven reticulated structures that are similar to natural ECM, produced by this method. The tissue scaffolds according to the present invention are achieved by a three-stage production process. Contact angle measurements are made for the characterization of the tissue scaffold obtained by using the inventive method and the results of the measurement are shown in Table 1. The values shown below the title R + F are the contact angles obtained according to the freeze-drying time after pre-drying for 24 hours in the refrigerator at -20°C. The values shown under the title I + F are the contact angles obtained according to the freeze- drying time after pre-drying in the incubator at 40°C for 5 hours .

Table 1. Contact Angle Measurements

Tensile testing experiments have been made to determine the mechanical properties of the tissue scaffolds obtained using the inventive method. The Young's modulus and yield strength results according to the freeze-drying time after pre-drying for 5 hours at 40°C in the incubator, are shown in Table 2.

Table 2. Mechanical properties of PCL/Chitosan example Freeze Drying Young ' s Modulus Yield Strength

Time (hours) (MPa) (MPa)

12 2,68 ± 0,76 0, 19 ± 0, 003

24 3,25 ± 0,78 0,26 ± 0, 002

48 1,98 ± 0,23 0, 13 ± 0, 003

Implementation of the Invention to the Industry:

As the subject of the invention is a tissue scaffold with enhanced biocompatibility and mechanical properties and its production method; a tissue scaffold being biocompatible, with a physical structure similar to the three-dimensional structure of the live tissue, having a high mechanical strength, with a porous structure that enables the material exchange, providing an alternative treatment to the autograft, allograft and xenograft tissue transport and also eliminating some undesired negative aspects of this method and a tissue scaffold production method can be used in the treatment of tissue loss, the critical flaw size and has superior properties as compared to other similar products and methods.