Patent Description:
It is commonly known that periodontal damage is a frequent occurrence in dentistry and dental surgery, which requires tissue separation using a barrier membrane. This damage is caused by disease, age, injury, or genetic factors, and it leads to physical and aesthetic dysfunction of the masticatory apparatus. One of the causes of periodontal damage is inflammation that destroys tooth-supporting tissue. The tooth-supporting tissue wastes away and the periodontal ligament and its surrounding bone are destroyed. The use of a separation membrane is also indicated in cases of maxilla or mandible damage due to incorrect tooth extraction or the need for screwing a titanium implant into the patient's bone. In such case, after the introduction of an implant, the cavity is sprinkled with a natural or synthetic bone powder, secured with a separation membrane, and immobilized using special pins. In addition, a bone substitute in the form of granules or porous blocks can be introduced into the bone cavity. Separation membrane covering of bone loss inhibits the epithelial downgrowth in place of the bone tissue undergoing regeneration. After about six months the bone cavity is filled while the separation membrane should undergo gradual dissolution. The procedure of using a barrier membrane for tissue separation is called Guided Tissue Regeneration (GTR) or Guided Bone Regeneration (GBR). Separation membranes used for such purposes should be primarily characterized by controlled biodegradation, which enables tissue regeneration on both its sides which is conducive to tissue growth while providing nourishment by enabling the free flow of nutrients and oxygen through the structure of the membrane.

Both non-biodegradable and fully biodegradable membranes are used in therapeutic practice. Biodegradable separation membranes are made of, among other things, animal-derived collagen type I and III, of porcine and bovine origin. They are non-porous or naturally porous and available in many sizes and thicknesses, from <NUM> to <NUM>. Currently, such membranes are the most widespread in clinical use. They are characterized by a mild degradation profile and satisfactory tissue growth. They ensure stable base attachment, although occasionally it is necessary to use fixation stitches/pins to prevent the barrier material sliding over tissues. An inconvenient feature of such separation membranes is the need for animal tissue extraction and the accompanying long process of removing lipids, protein remnants, or potential zoonotic pathogens. In order to eliminate the need for using animal-derived tissue, separation membranes made of synthetic biodegradable polymers were developed. Crystalline polylactide (PLA), semi-crystalline poly-L-lactide (PLLA), amorphous poly-D,L-lactide (PDLLA), polyglycolide (PGA), polylactide-glycolide (PLGA) copolymer, and polycaprolactone (PCL) are typically used for this purpose, producing solid fibrous or mesh separation membranes. They ensure good base attachment and the porosity constitutes a relative barrier for cells and protection against bacterial penetration. Decomposition products of barrier membranes made of aforementioned biodegradable polymers are fully metabolized in the body. There is, however, an increased risk of inflammation during biodegradation due to a local drop in tissue pH. Publication <CIT> discloses a separation material made of PLGA, consisting of a solid layer of this material, coated on both sides with fibres with a diameter from <NUM> to <NUM>. Publication <CIT> describes a barrier material made of a PGA/PLLA composite obtained using the method of evaporating solvent from the solution poured into a mould, which can produce solid plates with a thickness from <NUM> to <NUM>.

Coating medical implants with hydroxyapatite nanoparticles to improve tissue affinity of such implants is also well-known and practised. Fibrous implants with spontaneously oriented fibres obtained from biodegradable polymers using the method of electrospinning are well-known and used in tissue engineering, including implants containing hydroxyapatite nanoparticles attached to their fibres. Information about tissue implants made of hydroxyapatite-coated polymer fibres was disclosed in the publication written by <NPL>(<NUM>)]. This publication discloses a process of producing an electrospun structure from a PLGA polymer and subsequently coated with collagen and nano-hydroxyapatite. The fibrous structure (fabric) is obtained in the process of electrospinning using a <NUM>% PLGA solution in the mixture of DMF/tetrahydrofuran (THF), producing fibres with a diameter of <NUM> (± <NUM>). Coating this fabric with collagen and hydroxyapatite was performed, in turn, using the plasma method and by immersion and leaving the fabric overnight in a <NUM>% water suspension of nano-hydroxyapatite (nHA). The membrane obtained in this manner was characterized by a lattice of PLGA fibres with large collagen and hydroxyapatite agglomerates with significant dispersion.

The publication written by <NPL>] discloses the process of manufacturing a fibrous electrospun structure (fabric) made of a hydroxyapatite-modified PDLLA polymer with a size under <NUM>. This fabric was characterized by an average pre-coating fibre thickness of <NUM>. The modification step involved immersing the polymer fabric in ethyl alcohol containing nano-hydroxyapatite. The fabric's material shrinks under the influence of ethyl alcohol and the obtained structure has polymer PDLLA fibres with loosely embedded large hydroxyapatite agglomerates that fill the fibrous structure's pores. The hydroxyapatite content by weight ranged from <NUM> to <NUM>%. A drop in mechanical strength was observed when attempting to increase the hydroxyapatite content, resulting from the material's shrinkage and exposure to ethyl alcohol. <CIT> discloses nanofibrous membranes comprising a polyester made by e.g. electrospinning for use as periodontal bone implants. The polyester can be polylactide-co-glycolide, polylactic acid, polyglycolic acid or a blend thereof. The fiber thickness is <NUM>-<NUM>. The nanofiber membrane is coated with a calcium phosphate material having a Ca/P ratio between <NUM> and <NUM>, such as a hydroxyapatite.

The purpose of the invention was to obtain a separation membrane with better properties than previously known.

This purpose is met by a membrane according to the invention in the form of a thin, porous, and elastic fibrous structure made of spontaneously distributed polymer fibres made of a biodegradable polymer solution using the method of electrospinning. Within this structure, its constituent polymer fibres have a thickness of <NUM> to <NUM> and are coated with hydroxyapatite nanoparticles with a size under <NUM> and a molar ratio of calcium to phosphorus (Ca/P) within the range of <NUM> to <NUM>. The invention consists in the fact that the polymer fibre material is a mixture of poly-DL-lactide (PDLLA) and polylactide-glycolide (PLGA) while the hydroxyapatite content within the membrane ranges from <NUM> to <NUM>% by weight. The hydroxyapatite coating of polymer fibres has the form of a matrix of these fibres with a thickness ranging from <NUM> to <NUM>, advantageously from <NUM> to <NUM>, and the average size of matrix hydroxyapatite particles does not exceed <NUM>. The membrane's porosity ranges from <NUM> to <NUM>%, advantageously from <NUM> to <NUM>%, and the size of membrane pores ranges from <NUM> to <NUM>, advantageously from <NUM> to <NUM>. Water absorption capacity ranges from <NUM> to <NUM>%, advantageously from <NUM> to <NUM>%. The hydroxyapatite coating rate of polymer fibres is at least <NUM>%. The specific surface area of the membrane material ranges from <NUM><NUM>/g to <NUM><NUM>/g, advantageously from <NUM> to <NUM><NUM>/g.

In one of variants of the membrane according to the invention, the mass content of both polymers in the fibrous structure's fibre material is equal.

In next variant of the membrane according to the invention, the mass content of poly-DL-lactide (PDLLA) in the fibrous structure's fibre material is at least <NUM>%.

In next variant of the membrane according to the invention, the mass content of polylactide-glycolide (PLGA) in the fibrous structure's fibre material is at least <NUM>%. In next variant of the membrane according to the invention, the fibre matrix is made up of two layers of hydroxyapatite, made of hydroxyapatite grains of two different sizes.

In next variant of the membrane according to the invention, the external layer of the matrix is made up of hydroxyapatite grains with a size no larger than <NUM>.

In another variant of the membrane according to the invention, its thickness in the dry state ranges from <NUM> to <NUM>, advantageously from <NUM> to <NUM>.

A method according to the invention includes a first step, which produces a thin, porous, and elastic fibrous structure from spontaneously distributed polymer fibres with a thickness of <NUM> to <NUM> using the method of electrospinning from a biodegradable polymer solution, and a second step, where polymer fibres of the fibrous structure produced in the first step are coated with a layer of hydroxyapatite by immersing the produced fibrous structure in a suspension, which is made up of a liquid dispersing phase and the dispersed phase in the form of grains of synthetic hydroxyapatite with an average size no larger than <NUM> and a molar ratio of calcium to phosphorus (Ca/P) ranging from <NUM> to <NUM>. The invention consists in the fact that the first step uses a solution made of a mixture of poly-DL-lactide (PDLLA) and polylactide-glycolide (PLGA) by dissolving one part by weight of the mixture of these polymers in five parts by weight of a volatile solvent in order to create the fibrous structure. This solution is used to produce a fibrous structure with a porosity ranging from <NUM> to <NUM>%, advantageously from <NUM> to <NUM>%, with pore size ranging from <NUM> to <NUM>, advantageously from <NUM> to <NUM>, and with thickness of its fibres ranges from <NUM> to <NUM>. The second step of this method produces a suspension whose dispersing phase is water and dispersed phase are hydroxyapatite particles with an average size no larger than <NUM>, and the mass content of the dispersed phase in the suspension is at most <NUM>%, and the temperature of this suspension is maintained at the level of at most <NUM>. After immersing the fibrous structure produced in the first step in the prepared suspension, an ultrasound wave with a power intensity ranging from <NUM> to <NUM> W/cm<NUM> and a duration of at least four minutes is generated near the polymer fibres of this structure.

In one of variants of the method according to the invention, the frequency of the ultrasound wave generated in the suspension is <NUM> (± <NUM>).

In next variant of the method according to the invention, the polymer solvent is a mixture of nine parts chloroform (CHCl<NUM>) and one part dimethylformamide (DMF). In next variant of the method according to the invention, the mass content of both polymers (PDLLA and PLGA) in the mixture of polymers, used in the first step to make the solution for producing fibres of the fibrous structure, is equal.

In next variant of the method according to the invention, the mass content of poly-DL-lactide (PDLLA) in the mixture of polymers, used in the first step to make the solution for producing fibres of the fibrous structure, is at least <NUM>%.

In next variant of the method according to the invention, the mass content of polylactide-glycolide (PLGA) in the mixture of polymers, used in the first step to make the solution for producing fibres of the fibrous structure, is at least <NUM>%.

In next variant of the method according to the invention, the second step produces the first and second suspension, in which the fibrous structure produced in the first step is immersed in turn, and an ultrasound wave is generated near the immersed polymer fibres, and the average size of hydroxyapatite particles of the first suspension is different from the average size of hydroxyapatite particles of the second suspension.

The structure and material of the membrane according to the invention is intended to block the penetration of cells of tissues being separated, while enabling their free growth on the surface thanks to the observed free flow of nutrients through it. This membrane exhibits a very high saline wettability (absorbency) exceeding <NUM>% thanks to the obtained porosity and significant growth of the specific surface area compared to polymer fabrics without hydroxyapatite coating. The wetted membrane material has mechanical properties that are almost identical to the properties of natural gum tissue while being susceptible to bending, stitching, rolling, and cutting at a temperature of <NUM> without losing hydroxyapatite coating on fibres. During the fibre degradation process, hydroxyapatite particles embedded on these polymer fibres neutralize the pH drop in surrounding tissue. Because the degradation time of the hydroxyapatite-coated polymer fibre is slower than observed in the case of the same fibres but without coating, and the drop in pH is slower as well, using the membrane according to the invention protects against tissue inflammation for a longer period.

Unexpectedly, it turned out that the method according to the invention preserves the desired fabric structure and does not destroy its very thin fibres while significantly and homogeneously coating them with hydroxyapatite and with a radical shortening of the finished membrane production process. Furthermore, the membrane biodegradation period within the body can be easily controlled by selecting the proportions of both polymers used to make the fibres, selecting the type of hydroxyapatite powder used to prepare the suspension, and selecting the parameters of the ultrasound wave.

The invention is schematically presented in the drawing where <FIG> shows a diagram of the setup for coating polymer fibres with hydroxyapatite, used in first, second, third and fourth examples. <FIG> shows a photo of hydroxyapatite-coated polymer fibres in the first example, obtained from a scanning electron microscope with a magnification of 5000x. <FIG> show an assembly of photos from a scanning electron microscope of the structure from the first example and photos from an optical microscope during the measurement of the contact angle, wherein <FIG> shows a pure fabric obtained during the first step of the membrane production process while <FIG> shows a finished membrane. <FIG> presents a diagram of the setup for coating polymer fibres with hydroxyapatite used in the second example. <FIG> shows a diagram of the bilayer matrix of the polymer fibre from the second example, and <FIG> shows a photo of this matrix obtained from a scanning electron microscope.

Four exemplary embodiments of the invention are described below. In each case, the process of producing four exemplary membranes according to the invention is made up of the fibrous structure production step and the following step of coating this structure with hydroxyapatite (HAp) particles.

In order to produce the four fibrous structures described below using the method of electrospinning, two biodegradable polymers belonging to the group of aliphatic polyesters were used, i.e. poly-DL-lactide (PDLLA), or polylactic acid, and polylactide-glycolide (PLGA), or lactic acid and glycolic acid copolymer. For the described purposes, a PLGA polymer was used with a lactide to glycolide ratio of <NUM>:<NUM> and a molecular mass of <NUM>,<NUM>-<NUM>,<NUM> Da, and a PDLLA polymer was used with a molecular mass of <NUM>,<NUM> Da, both manufactured by Polysciences from the United States. To obtain the solution used to produce polymer fibres, <NUM> of the polymer mixture with the ratio described below was weighed and <NUM> of chloroform (CHCl<NUM>) and <NUM> of dimethylformamide (DMF) were added, after which the mixture was mixed using a magnetic mixer in a closed vessel for about four hours. The obtained <NUM>% solution of the polymer mixture was placed in a <NUM> disposable syringe which was placed in a syringe pump with the flow rate of <NUM>µl per minute. The entire system was placed in a laminar flow cabinet with a temperature ranging from <NUM> to <NUM> and air humidity ranging from <NUM> to <NUM>%. Then a metal needle with a diameter of <NUM> was connected to the syringe and a voltage of <NUM>-<NUM> kV was applied to the needle. The forming fibres were collected on a grounded rotating cylinder collector with a diameter of <NUM> located at a distance of <NUM> from the aforementioned metal needle and rotating with a velocity of <NUM> rpm until a fibrous structure (fabric) with a thickness of approx. <NUM> was produced on the collector. After completing the electrospinning process, the fabric in the form of a tube surrounding the collector was cut along its axis into <NUM> x <NUM> fragments, which were then washed twice with deionized water and left to dry completely for <NUM> hours in a laminar flow cabinet. The dried fragments of the fabric were weighed using an analytical scale with a precision of four decimal places.

During the step of coating the polymer fibres of the produced fabric, synthetic hydroxyapatite powder was used, i.e. a chemical compound with the formula Ca<NUM>(PO<NUM>)<NUM>(OH)<NUM>, where the calcium to phosphorus ratio (Ca/P) is greater than <NUM> and less than <NUM>. In the following embodiments, two varieties of hydroxyapatite powder with the common trade name GoHAP were used, i.e. GoHAP type <NUM> (hereinafter GoHAP3) and GoHAP type <NUM> (hereinafter GoHAP6). The GoHAP3 powder was characterized by an average grain size of <NUM> ± <NUM>, a specific surface area of <NUM><NUM>/g ± <NUM><NUM>/g, and a skeletal density of <NUM>/cm<NUM> ± <NUM>/cm<NUM>, while the GoHAP6 powder was characterized by an average grain size of <NUM> ± <NUM>, a specific surface area of <NUM><NUM>/g ± <NUM><NUM>/g, and a skeletal density of <NUM>/cm<NUM> ± <NUM>/cm<NUM>.

The average size of these nanoparticles was determined by analysing the image obtained using a transmission electron microscope (TEM) using the dark-field method for at least <NUM> particles, and the average size was the diameter of the circle outlining the grain shape. The specific surface area is understood here as a scalar parameter expressing the area of a substance per its amount, measured by analysing the BET adsorption isotherms (Brunauer-Emmett-Teller isotherm).

The following embodiments state the properties of obtained separation membranes defined below:.

During the first step, in order to prepare the polymer solution for electrospinning, <NUM> of the PDLLA polymer and <NUM> of PLGA polymer were weighed and covered with the aforementioned solvent; the produced fibres were collected on a collector in the form of a tube of a diameter of <NUM> and rotated with a velocity of <NUM> rpm.

During the second step, the suspension <NUM> was prepared in which the dispersing phase was water and the dispersed phase was the GoHAP3 powder in the amount of <NUM>% by weight. The dried fragments of the fabric <NUM> were immobilized on the stainless-steel mesh <NUM>, and the mesh <NUM> with the fragment of fabric <NUM> was immersed in the prepared suspension <NUM> with a volume of <NUM>,<NUM> contained in the tank <NUM>. The tank <NUM> also contained mixing parts <NUM> of a magnetic mixer not shown in the drawing in order to maintain a homogeneous suspension. Then the T-shaped sonotrode <NUM> connected to an ultrasound generator (not shown in the drawing) was placed in the suspension <NUM>. The frontal area <NUM> of the sonotrode <NUM> with a length of <NUM> and a width of <NUM> was placed in front of fragments of the fabric <NUM>, which were subjected for ten minutes to the ultrasound wave <NUM> with a frequency of <NUM> (± <NUM>) and a power intensity of <NUM> W/cm<NUM> (± <NUM> W/cm<NUM>). The temperature of the suspension <NUM> was maintained at a level of <NUM>-<NUM> using a flow cooling system within the walls of the tank <NUM>. The temperature of the liquid in the tank <NUM> was constantly controlled using a thermocouple not shown in the drawing. After finishing the process, the hydroxyapatite-coated fragments of the fabric <NUM> were taken out of the suspension <NUM> and washed with deionized water, after which they were left to dry for <NUM> hours. The dried membranes were stored in a desiccator containing moisture-absorbing granules until the obtained material was characterized.

The membrane obtained in this embodiment was characterized by:.

The membrane material was subjected to cellular cytotoxicity tests. In the cytotoxicity test, as per the PN-EN ISO <NUM>-<NUM>:<NUM> standard, NCTC clone <NUM> mouse fibroblast line cells in the MEM (Lonza) medium, and fetal bovine serum FBS (Euroclone) with an antibiotic solution (Lonza) were used. The measurement procedure involved tests on extracts after culturing in nutrients with serum at <NUM> ± <NUM> after <NUM> hours of exposure. Negative, positive, and reagent culture controls were used. According to the research expert report, the sample was deemed not toxic to cells.

During the first step, the fabric was produced from the PLGA and PDLLA polymer solution described in the first embodiment, collecting fibres on a collector in the form of a drum with a diameter of <NUM> and rotating with a velocity of <NUM> rpm. During the second step, the first water suspension <NUM>' was produced, in which the dispersed phase was GoHAP6 in the amount of <NUM>% by weight, and the second water suspension <NUM>" was produced, containing <NUM>% of the GoHAP3 powder by weight, i.e. analogous to the suspension <NUM> from the first embodiment. The dried fragment of the fabric <NUM>' was immobilized on the stainless-steel mesh <NUM>' and immersed in the stainless-steel tank <NUM>'. The tank <NUM>' was equipped with a liquid temperature stabilization system analogous to the one in the first embodiment, containing the mixing part <NUM> of the magnetic mixer and <NUM> of the first suspension <NUM>' (GoHAP6). Then the head <NUM>' of the cylinder sonotrode <NUM>' with a diameter of <NUM>, connected to the ultrasound generator not shown in the drawing, was placed against the fragment of the fabric <NUM>', after which this fragment of the fabric <NUM>' was subjected for five minutes to the ultrasound wave <NUM>' with a frequency of <NUM> (± <NUM>) and a power intensity of <NUM> W/cm<NUM> (± <NUM> W/cm<NUM>). Then the fragment of the fabric <NUM>' already coated with the first type of hydroxyapatite was taken out of the suspension <NUM>' and washed with deionized water, after which ultrasound coating of the membrane being produced was applied again in the second suspension <NUM>" (GoHAP3) in the same conditions for another five minutes. After taking the finished membrane out of the second suspension <NUM>", it was washed once with deionized water and left to dry for <NUM> hours. The obtained membrane was stored in a desiccator with moisture-absorbing granules until its properties were tested.

The separation membrane produced in this example had a bilayer matrix on polymer fibres <NUM>, which was made up of an internal layer <NUM> of the GoHAP6 hydroxyapatite and an external layer <NUM> of the GoHAP3 hydroxyapatite. This membrane was characterized by the following properties:.

During the first step, a <NUM>% solution of polymers was produced from their mixture containing <NUM>% (i.e. <NUM>) of the PDLLA polymer and <NUM>% (i.e. <NUM>) of the PLGA polymer. The production of this fabric solution using the method of electrospinning took place in the same conditions as in the second embodiment.

During the second step, the obtained fragments of the fabric <NUM> were coated using ultrasound in the same conditions (GoHAP3 suspension) as in the first embodiment, and after washing the finished membrane with deionized water, it was left to dry for 24hours. The membrane produced in this example was characterized by the following properties:.

During the first step, a fabric was produced from a mixture containing <NUM>% (i.e. <NUM>) of the PLGA polymer and <NUM>% (i.e. <NUM>) of the PDLLA polymer. The production of this fabric solution using the method of electrospinning took place in the same conditions as in embodiments two and three.

Claim 1:
A biological barrier membrane in the form of a thin, porous, and elastic fibrous structure made of spontaneously distributed polymer fibres fabricated from a biodegradable polymer solution using the method of electrospinning, in which the constituent polymer fibres are coated with hydroxyapatite nanoparticles with a size under <NUM> and a molar ratio of calcium to phosphorus (Ca/P) within the range of <NUM> to <NUM>, characterized in that:
- the thickness of the polimer fibres ranges from <NUM> to <NUM>;
- the material of the polymer fibres (<NUM>) is a mixture of poly-DL-lactide (PDLLA) and polylactide-glycolide (PLGA);
- the hydroxyapatite content in the membrane is <NUM> to <NUM>% by weight;
- the coating of polymer fibres (<NUM>) with hydroxyapatite has the form of a homogeneous coating on these fibres (<NUM>) whose thickness ranges from <NUM> to <NUM>, advantageously from <NUM> to <NUM>;
- the average size of hydroxyapatite particles is <NUM> at most;
- the membrane's porosity ranges from <NUM> to <NUM>%, advantageously from <NUM> to <NUM>%, and the size of membrane pores ranges from <NUM> to <NUM>, advantageously from <NUM> to <NUM>;
- water absorption capacity ranges from <NUM> to <NUM>%, advantageously from <NUM> to <NUM>%;
- the hydroxyapatite coating rate of polymer fibres is at least <NUM>%;
- the specific surface area of the membrane material ranges from <NUM><NUM>/g to <NUM><NUM> g, advantageously from <NUM> to <NUM><NUM>/g.