CRYOGEL / NANOFIBER HYBRID BIOMATERIAL

Technical Field

The relates to cryogel I nanofiber hybrid biomaterial and manufacturing method thereof for use as soft tissue implants in biomedical fields such as bone tissue engineering, nerve regeneration, dental and eye applications.

Prior Art

Biomaterials are natural or synthetic materials that are used to perform or support the functions of living tissues in the human body, and that come into contact with body fluids (e.g. blood) continuously or at regular intervals. Common uses of these biomaterials are implants, prostheses, extracorporeal devices (devices placed outside the body but interacting with the body) and diagnostic kits (Pasinli, 2004).

The most important feature expected from biomaterials is biocompatibility. Biocompatibility is defined as whether the physiological consequences of the chemical interaction of the materials and the body fluids causes harm the body and refers to the fact that the material does not cause toxic, allergic, mutagenic and carcinogenic effects when in contact with living tissues. Biocompatibility of the material means that it is accepted in the physiological environment of the living being it is placed in. Biocompatibility is made possible by placing compatible material in the compatible area of the body (Wintermantel et al., 1996).

Even though other properties expected from ideal biomaterials vary according to their areas of use, they are expected to be non - toxic and carcinogenic, have adequate mechanical strength, not to cause reactions other than those occurring in the body, and not to become corroded (Guven, 2014).

Biomaterials can be grouped under 4 main headings according to the material type as metals, ceramics, polymers and composites (Guven, 2014). Examples of metallic biomaterials are stainless steel, cobalt, chromium, and cobalt - chromium alloys. Metallic biomaterials can be used in orthopedic surgery, cardiovascular surgery and facial and maxillofacial surgery. Its superior mechanical properties are among its important advantages. Metallic biomaterials have disadvantages such as being corroded by body fluids and having a very hard structure compared to tissues. For example, it has been found out as a result of research in patients with hip prosthesis made of cobalt and chromium, that metals that stay in the body in for increased periods of time cause various cancer types of urinary, lymph, skin and blood tissue (Pasinli, 2004).

Examples of ceramic biomaterials can be listed as zirconium, alumina and carbon. Ceramic biomaterials have a wide range of uses in dentistry, hip prostheses and implant coatings. It has advantages such as high biocompatibility, lightness and resistance against abrasion, rusting, oxidation and corrosion. However, along with its advantages, it also has disadvantages such as hardness, brittleness, weak resilience and weak fatigue strength (Guven, 2014).

Examples of polymeric biomaterials are polyhydroxyethyl methacrylate (PHEMA), polyvinyl alcohol (PVA), polylactic acid (PLA), poly(lactic acid - co - glycolic acid) (PLGA), polyglycolic acid (PGA), polymethyl methacrylate (PMMA), polyethylene (PE), polyurethane (PU), polytetrafluoroethylene (PTFE), polyethylene terafthalate (PET), polyvinylchloride (PVC), polypropylene (PP), polystyrene (PS), chitin, chitosan, gelatin and collagen. Polymeric materials can be used in soft tissue implants, cardiovascular systems, surgical sutures and plastic surgery implants. It has advantages such as being prepared in different ways such as fiber, film, gel and nanoparticle, being in natural or synthetic form, being easy formed, offering the possibility of producing complicated parts, biodegradability and flexibility (Akdemir, 2009). Despite these advantages, it is not possible to provide the properties expected from biomaterials with a single polymer type and a single morphology.

The importance and use of polymeric biomaterials in the biomedical field is increasing day by day. Polymeric biomaterials can be prepared in different shapes and compositions such as fiber, gel and film (Bronzino, 2000). Cryogel and nanofibrous structures are of great interest in the biomedical field. Cryogels are gel matrices consisting of interconnected pores ranging between 10 - 200 pm, prepared with partially frozen monomer or polymer solutions. The term cryogel is formed by combining the Greek words "cryo", which means freezing or ice, and "gel", (Plieva et aL, 2011 Jespersen, 2014 Nayak et aL, 2018).

In cryogel synthesis, the monomer or polymer precursors are usually dispersed in a suitable solvent such as water. Interconnected ice crystals are formed by freezing a large part of the solvent below the freezing temperature of the solvent (Lozinsky, 2002). The polymer precursors in the unfrozen part around the ice crystals are polymerized using an appropriate initiator, thus forming a polymeric network around the ice crystals. When the reaction is completed and the frozen mixture is brought to the room temperature, the ice crystals melt and a three - dimensional interconnected macroporous polymeric network is obtained (Lozinsky, 2003). Cryogels can be prepared in different geometric shapes such as monolith, disc, sphere or membrane.

Cryogels are successfully used in many areas such as bioseparation, chromatography and biomedical treatment. Structural properties such as the size and interconnection of the pores, the surface area of the pores, the wall thickness and density of the pores are very important for cryogels developed for biotechnological, medical and environmental applications. In recent years, the use of cryogels has attracted attention, especially in biomedical applications due to their interconnected macroporous structure (Osman et aL, 2019). Cryogels provide suitable environments for the reproduction and development of cells. Their interconnected porous structure makes cryogels suitable materials for cell reproduction, development and tissue formation.

Another type of material which is often preferred in biomedical applications is nanofibers. Nanofibers are fibers with diameters below 1 micrometer. There are various techniques for the production of nanofibers. One of them is the electrospinning method. This method involves a continuous, economical and easy process. Electrospinning devices consist of a high voltage power supply, a syringe pump, a needle and a collector. The power supply is responsible for converting the loaded polymer solution into fiber form. The flow rate of the polymer solution is controlled by a syringe pump. The needle is used to distribute the charge on the polymer jet. The collector is responsible for the collection of electrospun fibers (§afak, 2016 Kanmaz 2018). In the electrospinning process, there are some parameters that directly affect the process of obtaining fiber from the polymer solution. The said parameters are given in the table below. These parameters can be grouped under three headings as solution parameters, process parameters and ambient parameters (Kanmaz 2020).

Parameters that affect the electrospinning system

Solution parameters Process parameters Ambient Parameters

Concentration Voltage Temperature

Viscosity Feed rate Humidity

Molecular weight Tip to collector distance Pressure

Solvent Needle diameter Atmospheric composition

Surface tension Collector type

Conductivity

Nanofibers produced with electrospinning method have large specific surface areas, very small fiber diameters and surface adhesion properties. They are also very suitable for cell growth and formation of three - dimensional cellular colonies. Morphologically, the surfaces produced with electrospinning are very similar to the natural human extracellular matrix (ECM). For this reason, biomedical applications are one of the areas where nanofibrous mats are used the most. In order to accelerate the formation of new cellular structures instead of damaged cells or to start such formation, nanofibrous mats produced from various polymers can be used in many areas such as medical prostheses, artificial vascular and organ applications, wound dressings, drug delivery systems, tissue scaffolds, skin care products (§afak, 2016).

Even though the advantages of nanofibers and cryogels are utilized separately in the biomedical field, there is no product that provides the advantages of both materials at the same time.

In the patent and literature search conducted for the prior art, the application numbered IN 2019 21015266 A was found. In the said document, fiber membranes containing cryogel coating are discussed. The membranes consist of a polymer and cryogel functionalized with graphene oxide. Cryogels functionalized with graphene oxide cover polymer - based membranes. However, a method for the preparation of hollow fiber membranes and a bioreactor obtained in this manner are also disclosed in the document. However, there is no explanation in the document that the cryogel is based on PHEMA, and there is no mention of nanofiber structures. In this sense, there is no hybrid structure.

Therefore, due to the drawbacks described above and the inadequacy of the existing solutions on the subject, it has become necessary to make an improvement in the relevant technical field.

Brief Description of the Invention

The invention relates to a cryogel I nanofiber hybrid biomaterial that meets the requirements mentioned above, eliminates all the disadvantages and provides some additional advantages.

Inspired by the present situations, the invention aims to solve the disadvantages mentioned above.

The main purpose of the invention is to produce hybrid biomaterials by combining nanofiber and cryogel structures. In this way, it is possible to benefit from the known advantages of two different materials, nanofiber and cryogel, simultaneously. The hybrid biomaterial, which is the subject of the invention, creates a buffering effect by showing swelling by means of the cryogel layer in the area where it is implanted, while the ECM imitation will be provided by means of the nanofibers and the drug loaded on the matrix will be released in a controlled manner.

It is possible to implant the material developed within the scope of the invention in any damaged or voided area formed in the organ or tissue in a living organism where it is to be used. The material must be sterilized with ethylene oxide gas before use.

The invention proposes the production of cryogel structures and nanofibrous mats in a layered formation (hybrid), different from the applications in prior art. In addition, superposition of PHEMA and PVA nanofibers in nanofibrous mat production is another characterizing aspect of the invention. Thus, a PHEMA - based nanofibrous mat is crosslinked with glutaraldehyde (GA) for the first time. The structural and characteristic features of the invention and all its advantages will be understood more clearly by means of the figures provided below and the detailed explanation written with reference to these figures. Therefore, the evaluation should be made by taking these figures and detailed explanation into account.

Figures to Help Understanding the Invention

Figure 1 illustrates the SEM images ((A) 500x, (B) 3000x) of the PHEMA - PVA nanofibrous mat, which is the subject of the invention.

Figure 2 illustrates the SEM images ((A) 500x, (B) 3000x) of the crosslinked PHEMA - PVA nanofibrous mat.

Figure s illustrates the SEM images ((A) 100x, (B) 500x) of the cryogel I nanofiber hybrid biomaterial.

Detailed Description of the Invention

In this detailed description, preferred embodiments of the cryogel I nanofiber hybrid biomaterial of the invention are explained only for the purpose of providing a better understanding of the subject.

The invention relates to cryogel I nanofiber hybrid biomaterial and manufacturing method thereof for use as soft tissue implants in biomedical fields such as bone tissue engineering, nerve regeneration, dental and eye applications. In the method of the invention, firstly, polymer solutions are prepared to be used in the production of nanofibrous mats. The PHEMA solution is prepared by dissolving the PHEMA polymer in an ethanol / distilled water mixture containing sodium chloride. In this process, the weight of the PHEMA (Mv 1 .000.000) polymer in the solution volume is kept constant at 8 % (w I v). Ethanol I distilled water mixture (4:1 ) is used as the solvent. In the preparation of the solutions, PHEMA polymer is added to the ethanol I distilled water mixture in an inert glass bottle, and the polymer is dissolved by stirring continuously for 24 hours, preferably in a magnetic stirrer, at 45 °C. To improve the electrospinnability, the weight of the PHEMA polymer in the solution volume is preferably kept constant at 8 % (w I v) and sodium chloride (NaCI) is added to the solution at the ratio of 0.1 % (w I v) of the solution volume.

Afterwards, the PVA polymer (Mw 85,000 - 124,000) is dissolved, preferably in distilled water, to prepare the PVA solution. Preferably, a heated magnetic stirrer is used in the preparation of the solutions mentioned. A homogeneous solution of preferably 12 % by weight (w I v) is obtained by mixing the PVA polymer in distilled water at a temperature of 80 <0 in a magnetic stirrer, preferably for 24 h ours, and the prepared solution is let to rest overnight at room temperature. Measured viscosity, conductivity, surface tension and pH properties of the prepared polymer solutions are given in the table below.

Polymer solution properties

Polymer Polymer Viscosity Conductivity Surface tension pH concentration (cp) (ps / cm) (mN / m)

PHEMA 8% 553.6 10.25 30.97 4.79

PHEMA - NaCI 8% 588.8 419 31.38 4.82

PVA 12% 250. 650 45.76 5.56

At the next process step, PHEMA and PVA nanofibers are produced in layers, preferably using an electrospinning device. In accordance with this purpose, the prepared PHEMA and PVA solution is subjected to electrospinning with a solution feed rate of preferably 0.5 mL I h and preferably with 17 cm distance between the needle and the collector. The voltage applied in the production of the nanofibrous mat is preferably kept constant at 20 kV, and the speed of the rotating collector roller is preferably kept constant at 100 rpm. With these parameters, the orientation of PHEMA nanofibers is also provided. Preferably, after every 60 minutes of PHEMA solution, preferably 15 minutes of PVA solution is fed to the collector. Thus, PVA nanofibers are formed between PHEMA nanofibers and a layered nanofibrous mat is obtained. With the PVA nanofibers fed to the interlayers, mechanical strength is provided in the next step in the crosslinking process and structural degradation is prevented. Production is carried out in room conditions. SEM images of the resultant surfaces are illustrated in Figure 1 .

GA, HCI and acetone are used for preparing the crosslinking solution and the PHEMA I PVA nanofibrous mat is crosslinked. PHEMA and PVA polymers used in nanofibrous mat production exhibit hydrophilic properties due to the hydroxyl groups in their structures. Crosslinking process is applied to the surfaces to improve the resistance of the produced nanofibrous mats against water and water vapor. At this stage, GA is used as a crosslinker. Acetal bonds are formed between the aldehyde (CHO) groups in the structure of GA and the hydroxyl (OH) groups in the structure of PHEMA and PVA polymers, and water is released. Thus, OH groups, which that provide hydrophilic properties in the structure of polymers, are blocked. In this process, acetone, which does not dissolve polymers, is used as a solvent. Since the crosslinking process with GA takes place in acidic conditions, HCI is added to the crosslinking solution. This way, the crosslinking solution is prepared by dissolving preferably 0.2 mL of HCI and 0.5 mL of GA in 39.3 mL of acetone. The nanofibrous mat is kept in the crosslinking solution preferably for 10 minutes. For the removal of GA residues, the nanofibrous mat is preferably treated with ethyl alcohol for 5 minutes and washed in phosphate buffer solution (pH 7.0). Then, the crosslinked nanofibrous mat is dried at room conditions. The SEM image of the crosslinked surfaces is illustrated in Figure 2.

At the next process step, separate solutions are prepared for 2 - hydroxyethyl methacrylate and N,N’ - methylene - bisacrylamide. For this purpose, 2 - hydroxyethyl methacrylate monomer is dissolved in distilled water. Likewise, the N,N’ - methylene - bisacrylamide crosslinker is dissolved in distilled water. The hydroxyethyl methacrylate solution and the N,N’ - methylene - bisacrylamide solution are mixed with each other. A magnetic stirrer is used in these process steps. Ammonium persulfate (APS) and N,N,N',N' - tetramethyl ethylenediamine (TEMED) are added to the mixture solution and the resulting solution is poured onto the PHEMA / PVA nanofibrous mat to polymerize it forming cryogel. For this purpose, preferably 1.3 mL of HEMA is dissolved in 3.7 mL of distilled water. Preferably, the solution prepared by dissolving 0.283 g of N - N’ methylene bisacrylamide in 7 mL of distilled water is mixed with the HEMA solution. APS and TEMED are added to the resulting mixture in an ice bath. The prepared mixture is poured between two glass surfaces. Before the mixture is poured, the prepared PHEMA - PVA nanofibrous mat is placed on the glass surfaces. The nanofibrous mat can be placed on a single glass surface or on two opposite glass surfaces. The thickness of the cryogel can be determined by varying the distance between the glass surfaces. The solution between the glass surfaces is preferably polymerized at -12 <0 for 24 hours. As a result of the polymerization of the reaction mixture by cold free radical polymerization, macroporous PHEMA cryogel - nanofibrous mat is prepared. The resulting structure is preferably kept at room temperature for 4 hours and removed from between the glass surfaces. Then, the structure is washed abundantly with distilled water to remove the unreacted monomers. SEM image of the obtained cryogel I nanofiber hybrid biomaterial is illustrated in Figure 3.

Usable amounts and preferred amounts of the ingredients used in the invention are given as percentages. Percentages are by weight unless stated otherwise. Ingredients used as volume are indicated in parentheses. In the technical field, nanofibers are defined as fibers with a diameter of less than 1 micrometer (Deitzel et aL, 2001 Supaphol et aL, 2012). Nanofibers have advanced performance properties such as very large specific surface area, very small pore size, flexible surface functionality, and superior mechanical properties (Bhat and Uppal, 2010 Cengiz Qalhoglu, 2013 Greiner and Wendorff, 2007). Nanofibrous mats have an increasing importance especially in the biomedical field. There are various techniques for nanofiber production. One of them is the electrospinning method. Nanofibers produced by electrospinning method have large surface areas, very low fiber diameters and surface adhesion properties. At the same time, electrospun nanofibers are very suitable for cell growth and the formation of three - dimensional cellular colonies. Morphologically, the mats produced by electrospinning are very similar to the natural human extracellular matrix (ECM).

For this reason, biomedical applications are one of the areas where nanofibrous mats are used the most. In order to accelerate the formation of new cellular structures instead of damaged cells or to start such formation, nanofibrous mats produced from various polymers can be used in many areas such as medical prostheses, artificial vascular and organ applications, wound dressings, drug delivery systems, tissue scaffolds, skin care products (Ustundag et al,, 2010 Zhong et al,, 2010 Kumar, 2004 Yoo et al,, 2009 Kenawy et al,, 2009 Venugopal et al,, 2006 Fang et al,, 2008 Patanaik et al,, 2007 Jian et al„ 2008).

Cryogels are gel matrices composed of interconnected pores ranging from 10 - 200 pm, prepared with partially frozen monomer or polymer solutions (Plieva et aL, 2011 Jespersen, 2014 Nayak et aL, 2018). 90 % of the cryogel completely swollen with water consists of the water in the pores. By means of its flexible morphology, 70 % of the water in its structure can be physically removed from the structure. This is evidence that the cryogel structure has interconnected macropores (Plieva et aL, 2005). Cryogels can maintain their porous structure even when they are in dry state (Okay, 2009). Cryogels are used successfully in many fields such as bioseparation, chromatography and biomedical treatment. In recent years, the use of cryogels has attracted attention especially in biomedical applications due to their interconnected macroporous structures (Osman et aL, 2019). PHEMA, which is used as a polymeric matrix in the composition of the invention, is a synthetic biopolymer obtained by polymerizing hydroxyethyl methacrylate monomer in the presence of suitable initiators (azobisisobutyronitrile, redox couples, ammonium persulfate, etc.). It can be obtained as a result of emulsion, suspension and bulk polymerization. Its chemical structure is given below. It exhibits hydrophilic character due to the hydroxyl groups in its structure. PHEMA is chemically stable and biocompatible. Its biostability is very high, so it is difficult to self - degrade (Bayrak 2019 Qakal 2004). PHEMA is a polymer approved for clinical uses by the US Food and Drug Administration. Therefore, PHEMA has a wide range of use in the medical sector. The fields of use of PHEMA are contact lenses (Wang et al., 2020), drug delivery systems (Lu et al., 1999), orthopedic implants (Zhang et al., 2008), nanofibrous scaffolds for skin tissue engineering (Zhang 2011 ), dental applications (Examples include Kronman et al., 1979), wound dressing materials (Micic et al., 2013), and anticancer implants (Rao and Rajiv 2014).

Chemical structure of PHEMA polymer

PVA, which is used as another polymeric matrix in the composition of the invention, is produced by first polymerizing vinyl acetate into polyvinyl acetate (PVAc) molecule and then hydrolyzing the PVAc polymer. The chemical structure of the PVA polymer is given below. The degree of hydrolysis (the amount of acetate groups in the PVA structure) and molecular weight affect the chemical properties and solubility of the PVA polymer. PVA polymer, which is a biocompatible and water - soluble polymer, is characterized with having chemical resistance, flexibility, mechanical strength, biocompatibility and biodegradability (Safi et al. 2007, Lee et al. 2007, Mahmoudifard et al. 2011 , Seringay et al. 2013). Due to its chemical stability at room temperature, good physical and mechanical properties, it has the ability to form very good fibrous materials by itself or mixed with other polymers. Wound dressings (Shalumon et al. 2011 , Abdelgawad et al. 2014), tissue scaffolds (Alhosseini et al. 2012), drug delivery systems (Li et al. 2013) can be given as examples of the fields of use for PVA.

OH OH

Chemical structure of the PVA polymer

GA, a crosslinking agent, is a biologically functional compound and is frequently used in the crosslinking of biopolymers. Improvements in the mechanical properties of biopolymers crosslinked with GA can be recorded (Reddy et al. 2015). The chemical structure of GA is given below.

Chemical structure of crosslinking agent GA

In different embodiments of the invention, PHEMA / polyethylene glycol (PEG) or PHEMA I cellulose acetate or PHEMA I pullulan nanofibers can be produced, instead of PHEMA / PVA nanofiber production. It is also possible to use methanol instead of ethanol.

It is possible to makes changes in the parameters of the process steps that make up the method in different embodiments of the cryogel / nanofiber hybrid biomaterial production method, which is the subject of the invention. Applicable ranges of the mentioned parameters are given below.

• Preparation time of PHEMA electrospinning solution: 20 - 24 hours,

• Preparation temperature of the PHEMA electrospinning solution: 25 - 45 ^< C,

• Preparation time of the PVA electrospinning solution: 20 - 24 hours, • The preparation temperature of the PVA electrospinning solution is 60 - 80 <0,

• The distance between the nozzle and the collector during the electrospinning of the solution: 10 - 22 cm,

• Voltage to be applied during electrospinning of the solution: 12 - 25 kV, • Feed rate of the solution during electrospinning: 0.2 - 1.5 mL / hour,

• Ratio of PHEMA in the unit layer (PHEMA I PVA I PHEMA) of the nanofibrous mat: 80 % - 90 %,

• PVA ratio in the unit layer (PHEMA I PVA I PHEMA) of the nanofibrous mat: 10 % - 20 %, • Cryogel formation time on the nanofibrous mat: 24 - 26 hours,

• Temperature during cryogel formation on the nanofibrous mat: - 18 ^< C.

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