WO2019054970A2 - Method of producing plga fibers used as tissue scaffolds and the plga fibers produced with this method - Google Patents

Method of producing plga fibers used as tissue scaffolds and the plga fibers produced with this method Download PDF

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Publication number
WO2019054970A2
WO2019054970A2 PCT/TR2018/050482 TR2018050482W WO2019054970A2 WO 2019054970 A2 WO2019054970 A2 WO 2019054970A2 TR 2018050482 W TR2018050482 W TR 2018050482W WO 2019054970 A2 WO2019054970 A2 WO 2019054970A2
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Prior art keywords
plga
syringe
coagulation bath
solution
wet spinning
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PCT/TR2018/050482
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French (fr)
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WO2019054970A3 (en
Inventor
Görke GÜREL PEKÖZER
Nergis ABAY
Gamze Torun Kose
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Yeditepe Universitesi
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Publication of WO2019054970A2 publication Critical patent/WO2019054970A2/en
Publication of WO2019054970A3 publication Critical patent/WO2019054970A3/en

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/56—Porous materials, e.g. foams or sponges
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/14—Macromolecular materials
    • A61L27/18—Macromolecular materials obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/58—Materials at least partially resorbable by the body
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/30—Macromolecular organic or inorganic compounds, e.g. inorganic polyphosphates
    • A61K47/34—Macromolecular compounds obtained otherwise than by reactions only involving carbon-to-carbon unsaturated bonds, e.g. polyesters, polyamino acids, polysiloxanes, polyphosphazines, copolymers of polyalkylene glycol or poloxamers

Definitions

  • the present invention relates to a fiber production method, which is developed for the purpose of obtaining tissue scaffolds with fibrous structure used in tissue engineering for repair of critical size bone defects, and this method is realized such that the PLGA (poly(lactic-co-glycolic acid)) polymer is introduced into the wet spinning apparatus in a syringe with three tips that enters directly into the coagulation bath and the polymer is precipitated into fibers in the horizontal plane.
  • PLGA poly(lactic-co-glycolic acid)
  • Tissue engineering is a branch of science that deals with producing organs and tissues in laboratory conditions for transplantation to patients. Tissue engineering has emerged due to failure to meet the increasing demands of tissue and organ transplantations. Tissue engineering utilizes a 3 dimensional scaffold, cells that will form the tissue and biochemical molecules that will direct the cells towards the desired tissue. The purpose is to produce the tissue scaffold using biomaterials to provide mechanical support to the defected tissue and supply a 3 dimensional structure to the defect site and at the same time to provide regeneration by producing the target tissue with a cell seeded scaffold and to ensure enough space for regenerated tissue by means of biodegradation of scaffolds. For this reason, mostly biodegradable natural or synthetic polymers are used when producing tissue scaffolds.
  • tissue scaffolds having a porous structure, fibrous structure or gel form can be produced.
  • Methods used when forming the tissue scaffold are solvent evaporation, solvent casting-particulate leaching, electro spinning and rapid prototyping etc.
  • the problems generally encountered in the methods for production of tissue scaffolds used in the state of the art are the inability to increase the surface area / volume ratio sufficiently, failure to provide adequate porosity, failure of compliance of the rate of degradation to the regeneration rate of the target tissue, inability to form resistant structures in terms of mechanical properties and inability in reproducibility of the scaffolds.
  • the wet spinning method is based on the fact that the polymer dissolved in a solvent is coagulated in another liquid which can be mixed with the solvent that is not the solvent of the polymer.
  • the solvent is separated from the solution, and phase separation occurs by the introduction of the non- solvent liquid in the coagulation bath and the polymer precipitates as a fiber.
  • the parameters such as the solvent of the polymer, the concentration of the polymer, types and ratios of the liquids forming the coagulation bath, and the rate of spinning of the solution significantly affect the properties of the resulting fibers. These properties that are affected are fiber structure and dimensions, surface properties of fibers, and the mechanical properties and the rate of biodegradation of the fabricated scaffold. These properties also indirectly affect the cell adhesion and proliferation on the fibrous biomaterial obtained.
  • the patent document no. KR20110045895 discloses a technique for preparing a fibrous material which enables gene delivery in gene therapy by means of a wet spraying and emulsion coating method.
  • the polymer from which the fibrous material is fabricated is dissolved (at a concentration of 0.5% weight/volume) and introduced into a single tipped syringe with a diameter of 0.5 to 1.2 mm and pumped at a flow rate of 50 to 150 ⁇ 7 ⁇ .
  • the polymer pumped from the vertical plane first contacts the air and enters into the coagulation bath and upon coagulation, it is collected within a rotary cylinder. Fibers with a diameter of 50-100 ⁇ are produced with this method.
  • the coagulation bath can be comprised of ethyl alcohol or methyl alcohol.
  • the objective of the present invention is to combine the fibers obtained by the wet spinning method in different concentrations of PLGA polymer and in 60:40 (IP: DS) (IsoPropanol: Distilled Water) coagulation bath to form the tissue scaffold.
  • Another objective of the present invention is to obtain uniformly sized fiber structures in horizontal plane by means of a syringe system designed with three tips that directly enters into the coagulation bath.
  • a further objective of the present invention is to obtain fibers for fabrication of tissue scaffolds having diameters of 50-100 ⁇ , which can maintain 80% of their weights over a period of 120 days without decreasing cumulative pH value of the medium below 5, and have a pressure modulus of an average of 2000-3000 GPa and tensile modulus of 60-80 GPa and support cell proliferation.
  • Another objective of the invention is to obtain fibers which enable fabrication of tissue scaffolds that affect differentiation of the cells into bones and have fibrous structures with high pressure modulus that allow easy adherence and proliferation to the cells by increasing the cells' adhesion surface.
  • Figure 1 is a schematic view of the wet spinning apparatus comprising a syringe pump and rotary table.
  • Figure 2 shows the scanning electron micrographs of 20% PLGA concentration spun in 60:40 (IP:DW) coagulation bath, ((a) 100X magnification, (b) 200X magnification) (the measured sizes of the fibers are denoted on the photographs.)
  • Figure 3 shows the scanning electron micrographs of 25% PLGA concentration spun in 60:40 (IP:DW) coagulation bath, ((a) 100X magnification, (b) 200X magnification) (the measured sizes of the fibers are denoted on the photographs.)
  • Figure 4 shows the scanning electron micrographs of 30% PLGA concentration spun in 60:40 (IP:DW) coagulation bath, ((a) 100X magnification, (b) 200X magnification) (the measured sizes of the fibers are denoted on the photographs.)
  • Figure 5. is a column chart representation of the comparison of the pressure moduli exhibited by the PLGA solutions of 20%, 25% and 30% concentrations after being spun in 60:40 coagulation bath.
  • Figure 6. is a column chart representation of the comparison of the tensile moduli exhibited by the PLGA solutions of 20%, 25% and 30% concentrations after being spun in 60:40 coagulation bath.
  • Figure 7 is a column chart representation of the comparison of contact angle measurements exhibited by the PLGA solutions of different concentrations after being spun in different coagulation baths.
  • Figure 8. is a graphical representation of the weight loss % of PLGA tissue scaffolds over 120 days.
  • Figure 9. is a graphical representation of the pH change of PLGA tissue scaffolds over 120 days.
  • Figure 10 is a graphical representation of the number of cells proliferated over a 21 day incubation period on PLGA tissue scaffolds with 20%, 25% and 30% concentrations that were spun in different coagulation bath compositions.
  • the components shown in the figures are each given reference numerals as follows:
  • the present invention is a method of producing PLGA fibers, which is developed in order to form a fibrous tissue scaffold in tissue engineering wherein the adhesion surface of the cells is increased and the differentiation of them to form a bone is facilitated, and is applied by means of a wet spinning apparatus (1) comprising
  • an injection apparatus which is positioned perpendicular to the ground plane and is fixed in vertical plane and comprises at least one hollow cylindrical syringe (14) having at least one syringe needle (16), at least one syringe pump (12) connected to the syringe (14) and regulating the flow rate of the fluid in the syringe (14) with the amount of pressure exerted thereon, and a motor providing the power necessary for the operation of the syringe pump (12), • a test table (30), which is in the form of a plate parallel to the horizontal plane beneath the injection apparatus (10), and has a coagulation beaker (20) positioned with coagulation bath (22) filled therein such that the syringe needle (16) can go in and out of it, and a rotary table (32) which rotates the solution within the coagulation beaker (20),
  • a control unit (42) which is connected to the rotary table (32) via a electrical connection of table (44) and controls the rotational speed of the rotary table (32), and which is connected to the injection apparatus (10) via the injection electrical connection (46) and controls the operation of the syringe pump (12),
  • IP:DW IsoPropanokDistilled Water
  • the PLGA fibers obtained by the production method of the present invention and used as a tissue scaffold for repair of critical size bone defects are the ones which can provide long-term mechanical support, lose about 20% or less of their weights over a 120-day period, and have a molar ratio (in percentage) of 75:25 (%) PLA: PGA (poly(lactic acid): poly(glycolic acid)).
  • the PLGA polymer in the step of production of the PLGA solution, is dissolved in dichloro methane at a concentration of 20% w/v. In a preferred embodiment, this concentration is prepared by dissolving 1.2 g of PLGA polymer in 6 ml of dichloromethane.
  • the mixture of IP: DW (IsoPropanol: Distilled Water), which is filled in the coagulation beaker (20) and used as a coagulation bath (22), has a ratio of 60:40 (IP: DW) by volume.
  • the solution in the step of pumping the PLGA solution, the solution is pumped by the syringe pump (12) in the vertical plane at a flow rate of 10-15 ⁇ / ⁇ .
  • the wet spinning apparatus (1) used in the scope of the invention and shown in Figure 1 is comprised of an injection apparatus (10) fixed in the vertical plane and a syringe pump (12) that fixes the flow rate of the solution, and a test table (30) having a rotary table (32) that rotates the solution which is the coagulation bath (22) within a coagulation beaker (20).
  • the syringe needle (16) portion of the syringe (14) in the injection apparatus (10) includes at least one and preferably three syringe needles (16).
  • the wet spinning apparatus (1) comprises a control unit (42) which controls the rotational speed of the rotary table (32) via the electrical connection of table (44), and which controls the operation of the syringe pump (12) via the injection electrical connection (46). All results are transferred to the respective programs on the computer (40). As a result of the study, the material with fibrous structure (24) to be used for the tissue scaffold is produced. In Figure 1, fibrous structure is introduced to the PLGA solution in the coagulation bath (22) in the wet spinning apparatus (1) comprising the syringe pump (12) and the rotary table (32).
  • the PLGA polymer is dissolved in dichloromethane at a concentration of 20% w/v (1.2 g PLGA polymer / 6 ml dichloromethane) and is drawn into a syringe (14) with three syringe needles (16) with a diameter of 6 mm and is pumped by the syringe pump (12) in the vertical plane at a flow rate of 10-15 ⁇ / ⁇ .
  • dichloromethane 1.2 g PLGA polymer / 6 ml dichloromethane
  • the syringe pump (12) in the vertical plane at a flow rate of 10-15 ⁇ / ⁇ .
  • a more uniform homogeneous fibrous structure is provided in the coagulation bath (22) by means of the syringe (14) with three syringe needles (16).
  • fibrous PLGA poly(lactic-co-glycolic acid)
  • PLA poly(lactic acid): poly(glycolic acid)
  • the invention is realized by having different concentrations (20%, 25%, 30%) of the PLGA (poly(lactic-co-glycolic acid)) polymer pumped by means of the syringe with three needles (16) at flow rates of 10-15 ⁇ / ⁇ and then entering directly into the 60:40 (IP: DW) coagulation bath (22) without coming into contact with air and being spun therein.
  • PLGA poly(lactic-co-glycolic acid)
  • the wet spun PLGA poly(lactic-co-glycolic acid) fibers with a molar ratio (in percentage) of 75:25 (%)
  • PLA PGA
  • poly(lactic acid): poly(glycolic acid) having fibrous structure and low biodegradation rate, and providing long-term mechanical supportare aimed to be used as a tissue engineering scaffolds for repair of critical size bone defects.
  • the invention has been developed for the fields of Bioengineering and Materials Engineering and is intended for use in the Biotechnology and Medical sectors.
  • the method of production of the present invention enables to fabricate a tissue scaffold with fibrous structure which can be used in hard tissue engineering supporting cell adhesion, proliferation and differentiation, providing mechanical support to the tissue until tissue formation is achieved and having a biodegradation rate suitable for healing speed of the tissue.
  • the PLGA polymer containing a ratio of 75:25 lactic acid: glycolic acid-obtained in the scope of the present invention is dissolved in dichloromethane at a concentration of 20% w/v, and drawn into three syringe needles (16) of a syringe (14) with a diameter of 6 mm.
  • the PLGA polymer is pumped by the syringe pump (12) in the vertical plane at a flow rate of 10-15 ⁇ .
  • tissue scaffolds having diameters of 50-100 ⁇ , maintaining 80% of their weights over a period of 120 days without decreasing cumulative pH value of the medium below 5, and having a pressure modulus of an average of 2000-3000 GPa and tensile modulus of 60-80 GPa are fabricated.
  • the coagulation bath (2) is comprised of isopropanol (IP) and distilled water (DW) and is optimally prepared at a ratio of 60:40 IP:DW.
  • IP isopropanol
  • DW distilled water
  • the obtained scaffolds meet the criteria of being used as an implant with or without cells in hard tissue engineering because they support cell adhesion and proliferation, have good mechanical properties and low biodegradation rate.
  • the fact that they have a fibrous structure increases the cell adhesion surface thereby enabling the cells to easily attach and proliferate.
  • a high pressure modulus is also effective on the differentiation of cells into bone.
  • Biodegradable natural or synthetic polymers can be used in the fabrication of fibrous tissue scaffolds by the same method.
  • poly(lactic acid) PLA
  • poly(glycolic acid) PGA
  • PLA poly(lactide-co-glycolide)
  • PCL poly ( ⁇ - caprolactone)
  • the coagulation bath (22) consists of liquids in which the polymer is not dissolved but can mix with the solvent in which the polymer is dissolved. Accordingly, in the preferred embodiment of the invention, the coagulation bath (22) comprising distilled water and isopropanol is used. Alternatively, in different embodiments of the invention, ethyl alcohol, methyl alcohol, or their mixtures or the mixture of water and at least one of the solvent in which the polymer is dissolved can be used.
  • a fibrous PLGA scaffold fabrication whose fiber properties can easily be adjusted by the wet spinning method can be provided. Due to the fibrous structure of the tissue scaffold prepared by wet spinning method, the surface area/volume ratios of the tissue scaffolds are about 2-3 times higher than the non-fibrous biomaterials. In addition, it is possible to form strong and durable scaffolds in terms of mechanical properties, as the thicknesses of fiber can be adjusted by increasing the polymer concentration and by varying the types and proportions of the liquids used in the coagulation bath (22).
  • the tissue scaffolds that are composed of fibrous wet-spun PLGA obtained within the scope of the invention have porous, smooth fiber structures with appropriate mechanical properties and low degradation rate and they are reproducible.
  • PLGA polymer is provided with a fibrous structure by wet spinning method at different concentrations and different coagulation baths (22) in order to determine the optimum values, and then frozen at -80°C and dried in a freeze drier for 2 days and cut into a diameter of 8 mm.
  • a number of analyses are applied:
  • Degradation analysis is carried out after selecting 3 different concentrations (20%, 25%, 30%) of PLGA in 60:40 (IP:DW) coagulation bath according to the results of contact angle measurements and mechanical analysis,
  • MTS test is applied to PLGA polymers having different concentrations to observe cell proliferation.
  • PLGA is degraded by hydrolysis of the ester bonds in an aqueous medium.
  • the rate of degradation of PLGA varies depending on the ratio of PLA and PGA to each other. Apart from the 50:50 PLA: PGA ratio, which exhibits the fastest degradation rate, the rate of degradation increases as the PGA ratio increases (Makadia and Siegel, 2011).
  • the wet spinning apparatus (1) consists of a syringe pump (12) which is fixed in the vertical plane fixing the flow rate of the solution, and a rotary table which rotates the coagulation bath (22) in the horizontal plane ( Figure 1).
  • Each sample shows wettability, i.e. they are hydrophilic, since their contact angle is below 90 degrees.
  • the hydrophilicity of the surface is important for cell seeding, adhesion and cell migration.
  • the 20%-60-40, 25%-60-40 and 30%-60-40 samples are selected from the compositions of the wet spun PLGA concentration and coagulation bath (22), and their degradation profiles are determined for 120 days ( Figures 8 and 9). On days 7, 15, 30, 60, 90 and 120, the pH values and weights of the PLGA tissue scaffolds are measured, and the percentages of weight losses according to their initial weights are calculated.
  • the tissue scaffolds containing PLGA retains at least 80% of their weight in the first 90-day portion of the degradation analysis, regardless of the concentration and coagulation bath (22) composition (Figure 8). However, 30% PLGA concentration shows a rapid degradation process after day 90 and is completely degraded within 120 days. The tissue scaffolds do not cause a significant pH change during the first 90 days, but decreases in the pH value occur due to the degradation observed after day 90 ( Figure 9). A significant decrease in pH value is undesirable as it causes necrosis in cells and tissues. It can be observed that in the first 60-day period, although 20% of the tissue scaffolds degrade, there is not too much change in pH value.
  • the seeded cells adhere to the PLGA scaffold ( Figure 10).
  • Cell proliferation can significantly be observed in 20%-60-40 sample. Since the samples of 30% PLGA concentration are prone to rapid degradation according to the degradation analysis results; 20% -60-40 sample is selected according to the MTS cell proliferation, degradation and mechanical analyses due to easier and more practical preparation of the samples.
  • the syringe (14) with three syringe needles (16) is enabled to directly enter into the coagulation bath (22), whereby the polymer is precipitated as fibers in the horizontal plane.
  • the obtained fibers with diameters of 50-100 ⁇ that are capable of retaining 80% of their weights over a period of 120 days without decreasing the cumulative pH value of the medium below have an average compressive modulus of 2000-3000 GPa and tensile modulus of 60-80
  • GPa and can be used as tissue scaffolds.
  • ⁇ S According to these properties, it supports cell proliferation. While its fibrous structure increases adhesion surface of the cells enabling them to easily adhere and proliferate, its high pressure modulus is effective on differentiation of the cells to bone.

Abstract

The present invention relates to the production of PLGA (poly(lactic-co-glycolic acid)) fibers obtained by wet spinning method from PLGA polymer be used as tissue engineering scaffolds for repair of critical size bone defects. The objective of the invention is to provide PLGA fibers having a fibrous structure by wet spinning method that can provide long-term mechanical support and low biodegradation rate.

Description

METHOD OF PRODUCING PLGA FIBERS USED AS TISSUE SCAFFOLDS AND THE PLGA FIBERS PRODUCED WITH THIS
METHOD
DESCRIPTION
Field of the Invention
The present invention relates to a fiber production method, which is developed for the purpose of obtaining tissue scaffolds with fibrous structure used in tissue engineering for repair of critical size bone defects, and this method is realized such that the PLGA (poly(lactic-co-glycolic acid)) polymer is introduced into the wet spinning apparatus in a syringe with three tips that enters directly into the coagulation bath and the polymer is precipitated into fibers in the horizontal plane.
Background of the Invention
Tissue engineering is a branch of science that deals with producing organs and tissues in laboratory conditions for transplantation to patients. Tissue engineering has emerged due to failure to meet the increasing demands of tissue and organ transplantations. Tissue engineering utilizes a 3 dimensional scaffold, cells that will form the tissue and biochemical molecules that will direct the cells towards the desired tissue. The purpose is to produce the tissue scaffold using biomaterials to provide mechanical support to the defected tissue and supply a 3 dimensional structure to the defect site and at the same time to provide regeneration by producing the target tissue with a cell seeded scaffold and to ensure enough space for regenerated tissue by means of biodegradation of scaffolds. For this reason, mostly biodegradable natural or synthetic polymers are used when producing tissue scaffolds. These biodegradable polymers are formed according to the characteristics of the target tissue. For example, tissue scaffolds having a porous structure, fibrous structure or gel form can be produced. Methods used when forming the tissue scaffold are solvent evaporation, solvent casting-particulate leaching, electro spinning and rapid prototyping etc. The problems generally encountered in the methods for production of tissue scaffolds used in the state of the art are the inability to increase the surface area / volume ratio sufficiently, failure to provide adequate porosity, failure of compliance of the rate of degradation to the regeneration rate of the target tissue, inability to form resistant structures in terms of mechanical properties and inability in reproducibility of the scaffolds. The wet spinning method is based on the fact that the polymer dissolved in a solvent is coagulated in another liquid which can be mixed with the solvent that is not the solvent of the polymer. When the polymer solution is introduced into the coagulation bath, the solvent is separated from the solution, and phase separation occurs by the introduction of the non- solvent liquid in the coagulation bath and the polymer precipitates as a fiber. The parameters such as the solvent of the polymer, the concentration of the polymer, types and ratios of the liquids forming the coagulation bath, and the rate of spinning of the solution significantly affect the properties of the resulting fibers. These properties that are affected are fiber structure and dimensions, surface properties of fibers, and the mechanical properties and the rate of biodegradation of the fabricated scaffold. These properties also indirectly affect the cell adhesion and proliferation on the fibrous biomaterial obtained.
The patent document no. KR20110045895, one of the state of the art applications, discloses a technique for preparing a fibrous material which enables gene delivery in gene therapy by means of a wet spraying and emulsion coating method. In the apparatus used in this invention, the polymer from which the fibrous material is fabricated is dissolved (at a concentration of 0.5% weight/volume) and introduced into a single tipped syringe with a diameter of 0.5 to 1.2 mm and pumped at a flow rate of 50 to 150 μΙ7ηιίη. The polymer pumped from the vertical plane first contacts the air and enters into the coagulation bath and upon coagulation, it is collected within a rotary cylinder. Fibers with a diameter of 50-100 μιη are produced with this method. The coagulation bath can be comprised of ethyl alcohol or methyl alcohol. Summary of the Invention
The objective of the present invention is to combine the fibers obtained by the wet spinning method in different concentrations of PLGA polymer and in 60:40 (IP: DS) (IsoPropanol: Distilled Water) coagulation bath to form the tissue scaffold.
Another objective of the present invention is to obtain uniformly sized fiber structures in horizontal plane by means of a syringe system designed with three tips that directly enters into the coagulation bath. A further objective of the present invention is to obtain fibers for fabrication of tissue scaffolds having diameters of 50-100 μιη, which can maintain 80% of their weights over a period of 120 days without decreasing cumulative pH value of the medium below 5, and have a pressure modulus of an average of 2000-3000 GPa and tensile modulus of 60-80 GPa and support cell proliferation.
Another objective of the invention is to obtain fibers which enable fabrication of tissue scaffolds that affect differentiation of the cells into bones and have fibrous structures with high pressure modulus that allow easy adherence and proliferation to the cells by increasing the cells' adhesion surface.
Detailed Description of the Invention
"Method of Producing PLGA Fibers Used As Tissue Scaffolds" developed to fulfill the objective of the present invention is illustrated in the accompanying figures, in which; Figure 1. is a schematic view of the wet spinning apparatus comprising a syringe pump and rotary table.
Figure 2. shows the scanning electron micrographs of 20% PLGA concentration spun in 60:40 (IP:DW) coagulation bath, ((a) 100X magnification, (b) 200X magnification) (the measured sizes of the fibers are denoted on the photographs.)
Figure 3. shows the scanning electron micrographs of 25% PLGA concentration spun in 60:40 (IP:DW) coagulation bath, ((a) 100X magnification, (b) 200X magnification) (the measured sizes of the fibers are denoted on the photographs.)
Figure 4. shows the scanning electron micrographs of 30% PLGA concentration spun in 60:40 (IP:DW) coagulation bath, ((a) 100X magnification, (b) 200X magnification) (the measured sizes of the fibers are denoted on the photographs.)
Figure 5. is a column chart representation of the comparison of the pressure moduli exhibited by the PLGA solutions of 20%, 25% and 30% concentrations after being spun in 60:40 coagulation bath. Figure 6. is a column chart representation of the comparison of the tensile moduli exhibited by the PLGA solutions of 20%, 25% and 30% concentrations after being spun in 60:40 coagulation bath.
Figure 7. is a column chart representation of the comparison of contact angle measurements exhibited by the PLGA solutions of different concentrations after being spun in different coagulation baths.
Figure 8. is a graphical representation of the weight loss % of PLGA tissue scaffolds over 120 days.
Figure 9. is a graphical representation of the pH change of PLGA tissue scaffolds over 120 days.
Figure 10. is a graphical representation of the number of cells proliferated over a 21 day incubation period on PLGA tissue scaffolds with 20%, 25% and 30% concentrations that were spun in different coagulation bath compositions. The components shown in the figures are each given reference numerals as follows:
I. Wet Spinning Apparatus
10. Injection Apparatus
12. Syringe Pump
14. Syringe
16. Syringe needle
20. Coagulation Flask
22. Coagulation Bath
24. PLGA fibers with fibrous structure
30. Test table
32. Rotary table
40. Computer
42. Control unit
44. Injection Electrical Connection
46. Electrical Connection of Table
The present invention is a method of producing PLGA fibers, which is developed in order to form a fibrous tissue scaffold in tissue engineering wherein the adhesion surface of the cells is increased and the differentiation of them to form a bone is facilitated, and is applied by means of a wet spinning apparatus (1) comprising
• an injection apparatus (10) which is positioned perpendicular to the ground plane and is fixed in vertical plane and comprises at least one hollow cylindrical syringe (14) having at least one syringe needle (16), at least one syringe pump (12) connected to the syringe (14) and regulating the flow rate of the fluid in the syringe (14) with the amount of pressure exerted thereon, and a motor providing the power necessary for the operation of the syringe pump (12), • a test table (30), which is in the form of a plate parallel to the horizontal plane beneath the injection apparatus (10), and has a coagulation beaker (20) positioned with coagulation bath (22) filled therein such that the syringe needle (16) can go in and out of it, and a rotary table (32) which rotates the solution within the coagulation beaker (20),
• a control unit (42) which is connected to the rotary table (32) via a electrical connection of table (44) and controls the rotational speed of the rotary table (32), and which is connected to the injection apparatus (10) via the injection electrical connection (46) and controls the operation of the syringe pump (12),
• at least one computer (40) that collects all the data received from the control unit (42) via the respective programs loaded therein and enables the operator to observe;
and comprises the steps of
- forming PLGA solution by dissolving PLGA (poly(lactic-co-glycolic acid)) polymer in dichloromethane,
- drawing the obtained solution into a syringe (14) having three syringe needles (16),
- attaching the syringe (14) having three syringe needles (16) to the syringe pump (12) of the wet spinning apparatus (1),
- filling IP:DW (IsoPropanokDistilled Water) mixture into the coagulation bath (22) of the wet spinning apparatus,
- activating the wet spinning apparatus and immersing the three syringe needles (16) of the syringe (14) into the IP:DW mixture,
- pumping the PLGA solution from the three syringe needles (16) of the syringe (14) immersed into the IP:DW mixture in the coagulation bath (22) into the mixture via the syringe pump (12) of the wet spinning apparatus without coming into contact with air,
- obtaining PLGA fibers having a fibrous structure as the final product in horizontal plane. The PLGA fibers obtained by the production method of the present invention and used as a tissue scaffold for repair of critical size bone defects are the ones which can provide long-term mechanical support, lose about 20% or less of their weights over a 120-day period, and have a molar ratio (in percentage) of 75:25 (%) PLA: PGA (poly(lactic acid): poly(glycolic acid)).
In one embodiment of the invention, in the step of production of the PLGA solution, the PLGA polymer is dissolved in dichloro methane at a concentration of 20% w/v. In a preferred embodiment, this concentration is prepared by dissolving 1.2 g of PLGA polymer in 6 ml of dichloromethane.
In one embodiment of the invention, the mixture of IP: DW (IsoPropanol: Distilled Water), which is filled in the coagulation beaker (20) and used as a coagulation bath (22), has a ratio of 60:40 (IP: DW) by volume.
In one embodiment of the invention, in the step of pumping the PLGA solution, the solution is pumped by the syringe pump (12) in the vertical plane at a flow rate of 10-15 μί/ηιίη. The wet spinning apparatus (1) used in the scope of the invention and shown in Figure 1 is comprised of an injection apparatus (10) fixed in the vertical plane and a syringe pump (12) that fixes the flow rate of the solution, and a test table (30) having a rotary table (32) that rotates the solution which is the coagulation bath (22) within a coagulation beaker (20). The syringe needle (16) portion of the syringe (14) in the injection apparatus (10) includes at least one and preferably three syringe needles (16). The wet spinning apparatus (1) comprises a control unit (42) which controls the rotational speed of the rotary table (32) via the electrical connection of table (44), and which controls the operation of the syringe pump (12) via the injection electrical connection (46). All results are transferred to the respective programs on the computer (40). As a result of the study, the material with fibrous structure (24) to be used for the tissue scaffold is produced. In Figure 1, fibrous structure is introduced to the PLGA solution in the coagulation bath (22) in the wet spinning apparatus (1) comprising the syringe pump (12) and the rotary table (32). In the PLGA fibers obtained by the wet spinning method of the present invention, the PLGA polymer is dissolved in dichloromethane at a concentration of 20% w/v (1.2 g PLGA polymer / 6 ml dichloromethane) and is drawn into a syringe (14) with three syringe needles (16) with a diameter of 6 mm and is pumped by the syringe pump (12) in the vertical plane at a flow rate of 10-15 μί/ηιίη. Compared to the syringe with single syringe needle (16), a more uniform homogeneous fibrous structure is provided in the coagulation bath (22) by means of the syringe (14) with three syringe needles (16). The three syringe needles (16) of the syringe (14) directly enters into the coagulation bath (22) without coming into contact with air and fiber precipitation is enabled upon coagulating in the coagulation bath (22) that rotates at a certain speed in the horizontal plane. By using this wet spinning method, fibrous PLGA (poly(lactic-co-glycolic acid)) structures with a molar ratio (in percentage) of 75:25 (%) PLA: PGA (poly(lactic acid): poly(glycolic acid)) which can be used as tissue scaffolds for repair of critical size bone injuries by providing long-term mechanical support, and losing about 20% or less of their weights over a 120-day period are obtained.
The invention is realized by having different concentrations (20%, 25%, 30%) of the PLGA (poly(lactic-co-glycolic acid)) polymer pumped by means of the syringe with three needles (16) at flow rates of 10-15 μί/ηιίη and then entering directly into the 60:40 (IP: DW) coagulation bath (22) without coming into contact with air and being spun therein. Within the scope of the invention, the wet spun PLGA (poly(lactic-co-glycolic acid)) fibers with a molar ratio (in percentage) of 75:25 (%) PLA: PGA (poly(lactic acid): poly(glycolic acid)) having fibrous structure and low biodegradation rate, and providing long-term mechanical supportare aimed to be used as a tissue engineering scaffolds for repair of critical size bone defects. The invention has been developed for the fields of Bioengineering and Materials Engineering and is intended for use in the Biotechnology and Medical sectors.
The method of production of the present invention enables to fabricate a tissue scaffold with fibrous structure which can be used in hard tissue engineering supporting cell adhesion, proliferation and differentiation, providing mechanical support to the tissue until tissue formation is achieved and having a biodegradation rate suitable for healing speed of the tissue. The PLGA polymer containing a ratio of 75:25 lactic acid: glycolic acid-obtained in the scope of the present invention is dissolved in dichloromethane at a concentration of 20% w/v, and drawn into three syringe needles (16) of a syringe (14) with a diameter of 6 mm. The PLGA polymer is pumped by the syringe pump (12) in the vertical plane at a flow rate of 10-15 μυηιίη. Since the syringe needles (16) of the syringe (14) are within the coagulation bath (22), the polymer enters into the coagulation bath (22) without coming into contact with air and precipitates as fibers upon coagulating in the coagulation bath (22) that rotates at a certain speed in the horizontal plane. By means of this method, tissue scaffolds having diameters of 50-100 μιη, maintaining 80% of their weights over a period of 120 days without decreasing cumulative pH value of the medium below 5, and having a pressure modulus of an average of 2000-3000 GPa and tensile modulus of 60-80 GPa are fabricated. The coagulation bath (2) is comprised of isopropanol (IP) and distilled water (DW) and is optimally prepared at a ratio of 60:40 IP:DW. The obtained scaffolds meet the criteria of being used as an implant with or without cells in hard tissue engineering because they support cell adhesion and proliferation, have good mechanical properties and low biodegradation rate. The fact that they have a fibrous structure increases the cell adhesion surface thereby enabling the cells to easily attach and proliferate. A high pressure modulus is also effective on the differentiation of cells into bone. Biodegradable natural or synthetic polymers can be used in the fabrication of fibrous tissue scaffolds by the same method. Among the natural polymers, collagen and poly(hydroxybutyric acid-co-hydroxyvaleric acid), which is a bacterial polyester; and among the synthetic polymers, poly(lactic acid) (PLA) and poly(glycolic acid) (PGA) poly(lactide-co-glycolide) (PLGA), poly (ε- caprolactone) (PCL), their blends and ceramic-containing composites can be used.
The coagulation bath (22) consists of liquids in which the polymer is not dissolved but can mix with the solvent in which the polymer is dissolved. Accordingly, in the preferred embodiment of the invention, the coagulation bath (22) comprising distilled water and isopropanol is used. Alternatively, in different embodiments of the invention, ethyl alcohol, methyl alcohol, or their mixtures or the mixture of water and at least one of the solvent in which the polymer is dissolved can be used.
In the present invention, in which fibrous tissue engineering scaffolds with desired physical and mechanical properties are intended to be obtained, a fibrous PLGA scaffold fabrication whose fiber properties can easily be adjusted by the wet spinning method can be provided. Due to the fibrous structure of the tissue scaffold prepared by wet spinning method, the surface area/volume ratios of the tissue scaffolds are about 2-3 times higher than the non-fibrous biomaterials. In addition, it is possible to form strong and durable scaffolds in terms of mechanical properties, as the thicknesses of fiber can be adjusted by increasing the polymer concentration and by varying the types and proportions of the liquids used in the coagulation bath (22). The tissue scaffolds that are composed of fibrous wet-spun PLGA obtained within the scope of the invention have porous, smooth fiber structures with appropriate mechanical properties and low degradation rate and they are reproducible.
Experimental Study In the scope of the invention, PLGA polymer is provided with a fibrous structure by wet spinning method at different concentrations and different coagulation baths (22) in order to determine the optimum values, and then frozen at -80°C and dried in a freeze drier for 2 days and cut into a diameter of 8 mm. In order to characterize the PLGA polymers, a number of analyses are applied:
1. Morphological properties of the fibrous PLGA samples prepared at different concentrations and precipitated in the 60:40 (IP:DW) coagulation bath (22) are analyzed by the scanning electron microscope,
2. Compressive and tensile stress analyses of PLGA samples are performed in order to be able to choose a scaffold with appropriate mechanical properties,
3. Contact angle of the PLGA films produced under the same conditions (in term of PLGA concentration and coagulation bath (22) composition) are measured,
4. Degradation analysis is carried out after selecting 3 different concentrations (20%, 25%, 30%) of PLGA in 60:40 (IP:DW) coagulation bath according to the results of contact angle measurements and mechanical analysis,
5. MTS test is applied to PLGA polymers having different concentrations to observe cell proliferation.
PLGA is degraded by hydrolysis of the ester bonds in an aqueous medium. The rate of degradation of PLGA varies depending on the ratio of PLA and PGA to each other. Apart from the 50:50 PLA: PGA ratio, which exhibits the fastest degradation rate, the rate of degradation increases as the PGA ratio increases (Makadia and Siegel, 2011).
Within the scope of the invention, since the tissue engineering scaffold is intended to be used for repair of critical size bone injuries, PLGA which has a ratio of 75:25 PLA: PGA having low degradation rate and providing long-term mechanical support in the body is used. The wet spinning apparatus (1) consists of a syringe pump (12) which is fixed in the vertical plane fixing the flow rate of the solution, and a rotary table which rotates the coagulation bath (22) in the horizontal plane (Figure 1). For fabrication of the fibrous tissue engineering scaffold, 20%, 25% and 30% concentrations of the PLGA (PLA: PGA 75:25) (Mw = 66.000-107.000) polymer is dissolved in dichloromethane, and the solutions are spun in the coagulation bath (22) by the syringe (14) and syringe pump (12) in which isopropanol (IP) and distilled water (DW) ratio (IP: DW) is 60:40. The most significant fiber structure is obtained at the concentration of 20% and the rest of the studies are conducted under these conditions.
In the scanning electron micrographs, it can be observed that formation of fiber structure is provided when 20%, 25% and 30% PLGA concentrations are spun in coagulation baths (22) with 60:40 (IP:DW) composition. According to the scanning electron micrographs, it can be concluded that coagulation bath (22) with 60:40 (IP: DW) composition is successful in wet spinning, regardless of the 20%, 25% and 30% PLGA concentrations (Figure 2 (a, b), Figure 3 (a, b), Figure 4 (a, b)). Compressive and tensile tests of the PLGA samples of different concentrations (20%, 25%, 30%) spun in the 60:40 (isopropanol: distilled water) (IP:DW) coagulation bath (22) are carried out. The results are given in the graphics in Figures 5 and 6. According to the compressive modulus analysis, 20% PLGA concentration exhibits a higher pressure modulus in 60:40 (IP:DW) coagulation bath (22) composition (Figure 5).
The tensile modulus analysis reveals that PLGA tissue scaffolds spun in 60:40 (IP:DW) coagulation bath (22) are more resistant in terms of tensile strength (Figure 6). In addition, 20%-60-40 sample exhibits the highest tensile modulus. As the contact angle is measured on flat and smooth surfaces, PLGA solutions are prepared in 20%, 25% and 30% concentrations and poured onto l x l cm lamellae, and dried at room temperature and allowed to take the form of a film. Each of the samples in the form of a film is allowed to rest in the coagulation bath (22) with a composition of 60:40 (IP: DW). The samples, which are incubated overnight at 4°C, are washed with distilled water and allowed to dry, and then used for contact angle measurement. The contact angles of the samples are shown in Figure 7.
Each sample shows wettability, i.e. they are hydrophilic, since their contact angle is below 90 degrees. The hydrophilicity of the surface is important for cell seeding, adhesion and cell migration. When the measured contact angles are evaluated, it can be observed that there is no significant change in the contact angles of different PLGA concentrations spun in the 60:40 (IP: DW) coagulation bath (22). The reason for this is that although the ratio of the materials used is changed, the chemical content stays the same.
The 20%-60-40, 25%-60-40 and 30%-60-40 samples are selected from the compositions of the wet spun PLGA concentration and coagulation bath (22), and their degradation profiles are determined for 120 days (Figures 8 and 9). On days 7, 15, 30, 60, 90 and 120, the pH values and weights of the PLGA tissue scaffolds are measured, and the percentages of weight losses according to their initial weights are calculated.
The tissue scaffolds containing PLGA retains at least 80% of their weight in the first 90-day portion of the degradation analysis, regardless of the concentration and coagulation bath (22) composition (Figure 8). However, 30% PLGA concentration shows a rapid degradation process after day 90 and is completely degraded within 120 days. The tissue scaffolds do not cause a significant pH change during the first 90 days, but decreases in the pH value occur due to the degradation observed after day 90 (Figure 9). A significant decrease in pH value is undesirable as it causes necrosis in cells and tissues. It can be observed that in the first 60-day period, although 20% of the tissue scaffolds degrade, there is not too much change in pH value. However, due to degradation of 30% PLGA spun in the 60:40 coagulation bath (22) composition at the end of 120 days, the pH value dropping below 5 with a rapid change after day 90 may cause necrosis in the cells and tissues. On the other hand, 20% and 25% PLGA tissue scaffolds spun in 60:40 coagulation bath (22) compositions do not cause a significant pH change.
According to the MTS cell proliferation assay, the seeded cells adhere to the PLGA scaffold (Figure 10). Cell proliferation can significantly be observed in 20%-60-40 sample. Since the samples of 30% PLGA concentration are prone to rapid degradation according to the degradation analysis results; 20% -60-40 sample is selected according to the MTS cell proliferation, degradation and mechanical analyses due to easier and more practical preparation of the samples.
Advantages obtained by means of the present invention can be listed as follows:
V In the prepared wet spinning apparatus (1), a system comprising a syringe
(14) with three syringe needles (16) is designed so that uniformly sized fiber structures can be obtained.
■S The syringe (14) with three syringe needles (16) is enabled to directly enter into the coagulation bath (22), whereby the polymer is precipitated as fibers in the horizontal plane.
■S The obtained fibers with diameters of 50-100 μιη that are capable of retaining 80% of their weights over a period of 120 days without decreasing the cumulative pH value of the medium below have an average compressive modulus of 2000-3000 GPa and tensile modulus of 60-80
GPa and can be used as tissue scaffolds.
■S According to these properties, it supports cell proliferation. While its fibrous structure increases adhesion surface of the cells enabling them to easily adhere and proliferate, its high pressure modulus is effective on differentiation of the cells to bone.

Claims

A method of PLGA fiber production, which is developed to fabricate a fibrous tissue scaffold in tissue engineering wherein the adhesion surface of the cells is increased and differentiation of them to a bone is facilitated, is applied by means of a wet spinning apparatus (1) comprising
• an injection apparatus (10) which is positioned perpendicular to the ground plane and is fixed in vertical plane and comprises at least one hollow cylindrical syringe (14) having at least one syringe needle (16), at least one syringe pump (12) connected to the syringe (14) and regulating the flow rate of the fluid in the syringe (14) with the amount of pressure exerted on it, and a motor providing the power necessary for the operation of the syringe pump (12),
• a test table (30), which is in the form of a plate parallel to the horizontal plane beneath the injection apparatus (10), and has a coagulation beaker (20) positioned with coagulation bath (22) filled therein such that the syringe needle (16) can go in and out of it, and a rotary table (32) which rotates the solution within the coagulation beaker (20),
• a control unit (42) which is connected to the rotary table (32) via a electrical connection of table (44) and controls the rotational speed of the rotary table (32), and is connected to the injection apparatus (10) via the injection electrical connection (46) and controls the operation of the syringe pump (12),
• at least one computer (40) that collects all the data received from the control unit (42) via the respective programs loaded therein and enables the operator to observe;
and characterized in that it comprises the steps of
- preparing PLGA solution by dissolving PLGA (poly(lactic-co-glycolic acid)) polymer in dichloromethane, - drawing the obtained solution into a syringe (14) having three syringe needles (16),
- attaching the syringe (14) having three syringe needles (16) to the syringe pump (12) of the wet spinning apparatus (1),
- filling IP:DW (IsoPropanohDistilled Water) mixture into the coagulation bath (22) of the wet spinning apparatus,
- activating the wet spinning apparatus and immersing the syringe needles (16) of the syringe (14) with three syringe needles (16) into the IP:DW mixture,
- pumping the PLGA solution from the syringe needles (16) of the syringe (14) with three syringe needles (16) immersed into the IP:DW mixture in the coagulation bath (22) via the syringe pump (12) of the wet spinning apparatus without coming into contact with air,
- obtaining PLGA fibers having a fibrous structure as the final product in horizontal plane.
Method of producing PLGA fibers according to Claim 1, characterized in that, in the step of forming the PLGA solution, PLGA polymer is dissolved in dichloromethane at a concentration of 20% w/v.
Method of producing PLGA fibers according to Claim 1 and 2, characterized in that, in the step of forming the PLGA solution, PLGA solution is prepared by dissolving 1,2 g PLGA polymer in 6 ml dichloromethane .
Method of producing PLGA fibers according to Claim 1, characterized in that the mixture of IP: DW (IsoPropanol: Distilled Water), which is filled in the coagulation beaker (20) and used as the coagulation bath (22), has a ratio of 60:40 (IP: DW).
5. Method of producing PLGA fibers according to Claim 1, characterized in that, in the step of pumping the PLGA solution, the solution is pumped by the syringe pump (12) in the vertical plane at a flow rate of 10-15 μΙ7πώι.
6. The fibers, which are obtained by the PLGA fiber production method according to any one of the preceding claims, characterized in that PLGA (poly (lactic-co-glycolic acid)) fibers with a molar ratio (in percentage) of 75:25 (%) PLA: PGA (poly(lactic acid): poly(glycolic acid)) which can provide long-term mechanical support, and lose about 20% or less of their weights over a 120-day period are used as a tissue scaffold for repair of critical size bone defects., .
PCT/TR2018/050482 2017-09-13 2018-09-12 Method of producing plga fibers used as tissue scaffolds and the plga fibers produced with this method WO2019054970A2 (en)

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CN110540404A (en) * 2019-10-17 2019-12-06 广州润虹医药科技股份有限公司 calcium phosphate bone cement with hollow through structure, preparation method and application thereof
JP7370029B1 (en) 2022-05-23 2023-10-27 国立大学法人 名古屋工業大学 Cotton-shaped bone regeneration material produced using wet spinning method, and method for producing cotton-shaped bone regeneration material using wet spinning method

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US7531503B2 (en) * 2005-03-11 2009-05-12 Wake Forest University Health Sciences Cell scaffold matrices with incorporated therapeutic agents
CN105297153B (en) * 2015-11-19 2017-12-12 暨南大学 A kind of wet spinning device and wet spinning process of electrostatic auxiliary

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CN110540404A (en) * 2019-10-17 2019-12-06 广州润虹医药科技股份有限公司 calcium phosphate bone cement with hollow through structure, preparation method and application thereof
JP7370029B1 (en) 2022-05-23 2023-10-27 国立大学法人 名古屋工業大学 Cotton-shaped bone regeneration material produced using wet spinning method, and method for producing cotton-shaped bone regeneration material using wet spinning method
WO2023228905A1 (en) * 2022-05-23 2023-11-30 国立大学法人名古屋工業大学 Method for continuously producing biodegradable fiber material containing inorganic filler particles using wet spinning, and cotton-like bone regeneration material produced with said method
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