CN118284440A

CN118284440A - Bacterial nanocellulose and preparation method thereof

Info

Publication number: CN118284440A
Application number: CN202280076580.5A
Authority: CN
Inventors: A·拉扎尼; V·尼奥派克; B·亨塞尔
Original assignee: Biotronik AG
Current assignee: Biotronik AG
Priority date: 2021-11-17
Filing date: 2022-11-17
Publication date: 2024-07-02

Abstract

The present invention relates to bacterial nanocellulose and shaped elements made of bacterial nanocellulose and to a method for the production thereof.

Description

Bacterial nanocellulose and preparation method thereof

Technical Field

The present application relates to a bacterial nanocellulose shaped element according to claim 27 and a method for the production thereof as claimed in claim 1. The application also relates to a medical implant comprising a bacterial nanocellulose shaped element to be implanted in a patient and to a method for producing such an implant. The application also describes the bacterial nanocellulose itself and methods for producing bacterial nanocellulose, dried bacterial nanocellulose, stable dried bacterial nanocellulose and locally swellable bacterial nanocellulose. Nanocellulose. The use of bacterial nanocellulose, dried bacterial nanocellulose or stabilized dried bacterial nanocellulose in medical implants is described.

Background

Cellulose may be produced by microorganisms such as plants, animals or bacteria. Bacterial Nanocellulose (BNC) is a special type of cellulose produced by bacteria. The cellulose fibers of bacterial nanocellulose have diameters in the nanometer range, compared to cellulose produced by plants having cellulose fibers with diameters in the micrometer range. Bacterial nanocellulose has high purity. The size of the fibers depends on the particular bacterial strain and the culture conditions selected. For example, BNC fibers synthesized from kapok have diameters of 50nm to 80nm, 100 times finer than plant cellulose fibers. The crystalline structure of natural cellulose is known as cellulose I, which is the homopolymer of β -D glucose monomers linked by β -1, 4-glycosidic bonds, and cellulose I is primarily observed when treated with sodium hydroxide solution, resulting in a thermodynamically more stable structure.

Methods for producing BNC are classified into static and dynamic (stirring or shaking) culture. Static culture is a widely used method. The main objective of all process variants is to obtain the highest reproducibility possible and to provide the best performance for the respective application. The medium is placed in a petri dish, inoculated with bacteria and cultured for 5 to 20 days. After a bacterial adaptation period of the start of the culture, the formation of the first layer can be seen after about two days. Over the next several days, cellulose fluff grows, becoming thicker and denser. The supply of carbon source in the medium limits the activity of the bacteria and thus also the growth of the bacterial film. Once spent, the initially rapidly growing fluff stagnates. The shape of the resulting fluff is determined by the geometry of the culture vessel and the interface with the surrounding oxygen. In the case of static culture, cellulose fluff is formed which sticks together, whereas in the case of stirred synthesis, a separate, loose bacterial film is formed. In contrast, a regularly shaped aggregated cellulose film was formed during dynamic culture. The shape and structure will vary depending on the bacterial strain selected. X-ray diffraction studies have shown that in stirred culture, the degree of polymerization is low and the degree of crystallinity is also low. For example, stirred tank reactors, airlift reactors, aerosol reactors are known for the cultivation of BNCs. In addition to the selected cultivation method and synthesis time, the main conditions during the cultivation, such as temperature and relative humidity, determine the yield and properties of cellulose. Furthermore, the bacterial strain selected, the composition of the nutrient medium used and the ratio of bacteria to nutrient medium during inoculation have a significant effect on the properties of the BNCs.

For the synthesis of cellulose, gram-negative microorganisms (gluconacetobacter, azotobacter, rhizobium, pseudomonas, salmonella, alcaligenes) and gram-positive microorganisms (sarcina gastro-octacoccus) can be used. The most commonly used bacteria are acetobacter gluconate: acetobacter gluconate (Acetobacter xylinum, also known as Acetobacter xylinum) and Acetobacter pastoris (Pasteurella pastoris).

A problem with gram negative bacteria is that they produce endotoxin. These endotoxins can cause fever in the human body. The bacterial cellulose layer may contain residual bacteria that may not be effectively removed by conventional methods using detergents, such as sodium dodecyl sulfate described in EP 1 660 670A.

Materials for medical implants require very high standards in terms of purity and reliable physical properties. It is therefore an object of the present invention to provide bacterial nanocellulose with improved physical properties and shaped elements made of bacterial nanocellulose and a method for the preparation thereof.

This object is achieved by a method having the features of claim 1. Preferred examples of the various aspects of the invention are described below and set out in the respective dependent claims.

Disclosure of Invention

A process for producing a shaped element made of bacterial nanocellulose is disclosed, comprising the steps of

-Providing a shaped article of manufacture,

Providing a growth medium for bacterial nanocellulose and a nutrient solution for said bacteria, the medium comprising bacteria of the family Acetobacter hansenii (Komagataeibacter hansenii) or the species Formica Fusca (K.hansenii) of the genus Gluconobacter coltsfoot (Komagataeibacter), preferably in the form of a bacterial suspension,

-Contacting a portion of the shaped article with a bacterial nanocellulose growth medium, and

-Rotating the shaped article to obtain a shaped element made of bacterial nanocellulose.

Preferably, komagataeibacter are Komagataeibacter hansenii bacteria of the family Acetobacter aceti are Komagataeibacter hansenii, american Type Culture Collection (ATCC) code 53582.

The nutrient solution may comprise at least one monosaccharide and/or one disaccharide, at least one peptone and a yeast extract, and wherein the growth medium has an acidic pH. The nutrient solution may comprise or consist of glucose, peptone, yeast extract, disodium hydrogen phosphate and citric acid. The peptone may be soy peptone. The ratio of bacterial suspension to nutrient solution may be 1:18.

The process may be carried out in an oxygen-containing environment, preferably in air.

The culturing of the growth medium may be performed at a temperature between 23 ℃ and 30 ℃ for at least 30 hours to obtain bacterial nanocellulose. The cultivation of the growth medium may be carried out at a temperature between 26 ℃ and 30 ℃ for 48 hours to 114 hours, preferably at a temperature between 26 ℃ and 28 ℃. The culturing may be performed in the dark.

The rotational molding may be performed at a rotational speed of up to 60 rpm. The rotational molding may be performed at a rotational speed of 10 to 60 rpm. The shaped article may be made of a polymer. The polymer may not contain Si-O groups. The polymer may have a polymer backbone containing alternating ketone and ether groups. The polymer may comprise polyetheretherketone. For example, 40% to 60%, preferably 50% of the surface of the shaped article may be contacted with the growth medium. The rotation of the shaped article may be carried out at a temperature between 23 ℃ and 30 ℃ for at least 30 hours and at a temperature between 26 ℃ and 30 ℃ for 48 hours to 114 hours, preferably at a temperature between 26 ℃ and 28 ℃.

The method may further comprise the step of drying the obtained shaped element made of bacterial nanocellulose to obtain a dried shaped element made of bacterial nanocellulose. The drying step may be performed in air. The drying step may be performed in air during rotation of the shaped article. The drying step may be performed in air during rotation of the shaped article at less than 10 rpm.

An additional step of treating the obtained bacterial nanocellulose with at least one structural stabilizer may be performed before the drying step, in order to obtain a stable shaped element made of bacterial nanocellulose. At least one of the structural stabilizers comprises or consists of glycerol and/or polyethylene glycol, preferably comprises from 5% to 50% by weight of glycerol and/or polyethylene glycol.

The method may further comprise the step of treating the obtained bacterial nanocellulose with a hydroxide solution before or after the drying step.

The shaped article is a rod, a rotationally symmetrical body or a medical implant. The shaped article may be covered by a stent, a prosthetic heart valve, a polymer frame, a metal frame, or a metal alloy frame. The shaped article may be removed from the stent, prosthetic heart valve, polymer frame, metal frame, or metal alloy frame.

Also disclosed is a method of producing bacterial nanocellulose, comprising the steps of:

-preparing or providing a growth medium for bacterial nanocellulose, comprising:

Komagataeibacter genus Komagataeibacter hansenii or k.hansenii species, preferably in the form of a bacterial suspension, and a nutrient solution for said bacteria, wherein said nutrient solution comprises at least one monosaccharide and/or one disaccharide, at least one peptone and a yeast extract, and wherein said growth medium has an acidic pH, and

-Culturing the growth medium to obtain bacterial nanocellulose.

The Komagataeibacter hansenii species of Acetobacter bacteria of Komagataeibacter may be Komagataeibacter hansenii, american Type Culture Collection (ATCC) code 53582.

The nutrient solution may comprise at least one monosaccharide and/or one disaccharide, at least one peptone and a yeast extract, and wherein the growth medium has an acidic pH. The nutrient solution may comprise or consist of glucose, peptone, yeast extract, disodium hydrogen phosphate and citric acid. The ratio of bacterial suspension to nutrient solution may be 1:18.

The growth medium may be in contact with at least a portion of the shaped article. The shaped article may comprise or consist of a polymer. The polymer may not contain Si-O groups (and thus is not a silicone). The polymer may comprise a polymer backbone containing alternating ketone and ether groups. The polymer may comprise polyetheretherketone. The shaped article may be a rod, a rotationally symmetrical body or a medical implant, such as a stent or a heart valve prosthesis.

The method may further comprise the step of drying the obtained bacterial nanocellulose to obtain a dried bacterial nanocellulose. The drying step may be performed in air. An additional step of treating the obtained bacterial nanocellulose with at least one structural stabilizer may be performed before the drying step to obtain a stabilized bacterial nanocellulose. The at least one structure stabilizer may comprise or consist of glycerol and/or polyethylene glycol, preferably comprises 5 to 50% by weight of glycerol and/or polyethylene glycol.

The method may further comprise the step of treating the bacterial nanocellulose with a hydroxide solution before or after the drying step.

The method may further comprise the step of compacting the bacterial nanocellulose before, during or after the drying step, for example by applying a pressure of 2N/mm ² to 40N/mm ², preferably 10N/mm ², to obtain compacted bacterial nanocellulose. The pressing step may be carried out for more than 5 minutes, preferably 15 minutes the pressing step may be carried out at a temperature between 20 ℃ and 90 ℃, preferably 50 ℃.

Further disclosed is a method of producing bacterial nanocellulose, described as comprising the steps of:

-providing a shaped article of manufacture,

Providing a growth medium for bacterial nanocellulose comprising a bacterial suspension comprising bacterial producing bacterial nanocellulose and a nutrient solution comprising mono-and/or disaccharides, peptones, yeast extract, wherein the medium has an acidic pH value,

-Contacting a portion of the shaped article with a medium for bacterial nanocellulose-producing bacteria, and

Rotating the shaped article, preferably at a speed of up to 60rpm, and

-Wherein the shaped article is made of a polymer, preferably the polymer does not contain siloxane groups.

BNCs are formed at the interface of air and nutrient medium. On the first day of synthesis, a gelatinous, loosely branched fibrous structure is initially formed. As the synthesis time increases, the fiber network becomes more and more compact and the already formed BNCs are transferred to the nutrient medium.

As bacterial nanocellulose-producing bacteria, gram-negative aerobic bacteria (e.g., acetobacter gluconate or acetobacter) such as acetobacter gluconate (also referred to as acetobacter) may be used. In the present application, it is preferable to use a bacterium belonging to the genus Komagataeibacter, the genus Komagataeibacter hansenii or the species K.hansenii, belonging to the family Acetobacter.

The shaped article may be rotated, preferably at a rotational speed of up to 60 revolutions per minute (rpm). Rotation speeds below about 10rpm result in non-uniform thickness of the bacterial nanocellulose (along the longitudinal axis of the shaped article, e.g., rod). A rotation speed higher than about 10rpm results in a uniform thickness of bacterial nanocellulose (along the longitudinal axis of the shaped article, e.g. rod). The shaped article may be rotatably mounted, preferably in an apparatus as described below. The shaped article may be of any shape, and the method does not require symmetry. It may be a stem of a tubular element, a rotationally symmetrical body or have the shape of a heart valve prosthesis. The diameter of the rod may be from 1mm to 10mm, preferably from 5mm to 8mm, most preferably 7.6mm. The length of the rod may be from 5mm to 200mm, preferably from 20mm to 90mm, most preferably 87mm. In principle, the polymer may be any chemically inert, mechanically processable but stable and sterilizable polymer. The shaped article may be made of a thermoplastic (polymer). Preferably, the polymer does not contain Si-O groups, i.e. chemical groups containing silicon atoms bonded to oxygen atoms, such as siloxane groups (Si-O-Si groups) or silanol groups (Si-OH groups). Thus, the shaped article may not be made of silicone (polysiloxane). The shaped article is made of, for example, an organic polymer, and the organic polymer does not contain si—o groups, such as siloxane groups (si—o—si groups) or silanol groups (si—oh groups). Preferably, the shaped article is made of a polymer having a polymer backbone containing alternating ketone (R-CO-R) and ether groups (R-O-R). For example, the molded article is made of Polyetheretherketone (PEEK). PEEK is an organic thermoplastic polymer. PEEK is a chemically inert, machinable but stable and sterilizable polymer.

Contacting a portion of the shaped article with a culture medium for bacterial nanocellulose-producing bacteria may mean that 40% to 60%, preferably 50% of the surface of the shaped article is in contact with the culture medium for bacterial nanocellulose-producing bacteria. This is advantageous in that during rotation the part not in contact with the medium for the bacterial nanocellulose-producing bacteria is in contact with oxygen, which enables an improved bacterial nanocellulose to be obtained.

The growth medium for the bacterial nanocellulose may comprise a bacterial suspension comprising bacteria that produce the bacterial nanocellulose and a nutrient solution comprising. The bacterial nanocellulose growth medium may consist of a bacterial suspension.

The nutrient solution contains a carbon source, peptone and yeast extract. The latter provides nitrogen and ensures good cell growth. The choice of carbon source (glucose, fructose, mannitol, etc.) significantly determines the yield and properties of cellulose films. Monosaccharides and/or disaccharides as carbon sources. The mono-and/or disaccharides may be glucose, fructose or sucrose.

The acidic pH can be obtained by using citric acid. Aerobic gram-negative bacteria ferment efficiently at pH 3 to 7 and at a temperature in the range of 25 ℃ to 30 ℃. Some carbohydrate metabolism can result in gluconic acid as a byproduct, which can lower the pH of the medium, thereby negatively affecting cellulose production. However, the presence of antioxidants and polyphenolic compounds inhibits the formation of gluconic acid and is achieved by adding disodium hydrogen phosphate and citric acid as buffers to the medium.

The nutrient solution may be composed of glucose, peptone, yeast extract, disodium hydrogen phosphate, citric acid and solvent. The solvent of the bacterial suspension and/or the nutrient solution may be (purified) water. Preferably, the growth medium for bacterial nanocellulose comprises a bacterial suspension comprising bacteria of the family Acetobacteriaceae, preferably of the genus Komagataeibacter, komagataeibacter hansenii or K.hansenii, and a nutrient solution comprising 20g/l glucose, 5g/l peptone, 5g/l yeast extract, 2.7g/l disodium hydrogen phosphate and 1.5g citric acid. The ratio of bacterial suspension to nutrient solution may be between 1:16 and 1:20, preferably 1:18.

Peptone was used as nitrogen source. The peptone may be soy peptone.

The process may be carried out in a dark environment or under red or yellow light.

The process may preferably be carried out for 48 hours to 114 hours.

The process may be carried out at a temperature of between 26 ℃ and 30 ℃, preferably 28 ℃.

In a further process step, the bacterial nanocellulose obtained may be dried and/or pressed. The (partial) dewatering by drying or pressing changes the morphology of the biological material.

The drying is preferably carried out in air, optionally at 3rpm, for 24 hours. Bacterial nanocellulose can be removed from the shaped article before or after drying.

In order to obtain swellable bacterial nanocellulose, the obtained bacterial nanocellulose may be preserved by at least one structure-stabilizing substance prior to drying. Thus, the method may for example comprise the further step of treating the obtained bacterial cellulose with a solution comprising glycerol and/or polyethylene glycol before drying the bacterial nanocellulose.

The method may further comprise the step of rehydrating the dried bacterial nanocellulose. The obtained (dried and/or re-hydrated) bacterial nanocellulose may be cut into the desired pieces, preferably using laser cutting, for example by CO ₂ laser cutting.

The method may further comprise the step of sterilizing the (dried) bacterial nanocellulose obtained.

The method may further comprise the step of treating the obtained bacterial nanocellulose with an alkaline solution or an acid to remove cell residues and thus endotoxins having toxic effects on the human or animal body. To overcome the endotoxin problem generated by gram-negative aerobic bacteria, the bacterial nanocellulose obtained can be purified with sodium hydroxide (NaOH) solution, preferably 0.1M NaOH solution. This can reduce endotoxin content below 0.1EU/ml (endotoxin units per ml).

The shaped article may be covered with a metal frame, preferably a stent or nitinol frame. In this way, a stent graft can be obtained, wherein the graft material is the bacterial nanocellulose obtained. Furthermore, a positive fit of the metal framework and the bacterial nanocellulose may be obtained, i.e. the metal framework is embedded in the bacterial nanocellulose.

According to the above method, a molded element made of bacterial nanocellulose can be produced. The profiled element is an element having a (macroscopic) geometry, such as a hollow tube. Rectangular sheets of bacterial nanocellulose can also be obtained if the hollow tube obtained is cut, for example, along its longitudinal axis. Solutions containing only bacterial nanocellulose fibres are not to be understood as forming elements. Molded elements made from bacterial nanocellulose are also described herein. The profiled element may have a tubular shape, for example a hollow cylindrical shape. The profiled element may be a planar or curved sheet. This may be obtained, for example, when the hollow cylinder of bacterial nanocellulose is cut along its longitudinal axis.

The shaped element made of bacterial nanocellulose may have a wall thickness of less than 70 μm, preferably 40 μm to 60 μm. The profiled element may have a length of 5mm to 200mm, preferably 20mm to 90mm, most preferably 80 mm. The profiled element may have a diameter of 1mm to 10mm, preferably 5mm to 8mm, most preferably 9mm, for example in the case where it is a hollow tube. The shaping element may have the shape of the outer contour of the shaped article, for example the outer contour of a heart valve prosthesis or a venous valve prosthesis.

The bacterial nanocellulose obtained by the method has different properties than conventional bacterial nanocellulose grown on the surface of an oxygen permeable silicone mold. The tensile strength of the bacterial nanocellulose obtained by the method (8.40±0.40n;5mm sample width) is higher than the tensile strength of the bacterial nanocellulose obtained in the silicone mould in the prior art method (see example 1) (4.61±1.23n;5mm sample width). The bacterial nanocellulose obtained by the method has a fiber density that is about five times higher than the fiber density of the bacterial nanocellulose obtained in the silicone mold.

Also described herein is a medical implant comprising nanocellulose, preferably obtained by the aforementioned method, having at least one of the above-mentioned properties. The medical implant may be a vascular graft, preferably a stent graft; medical scaffolding, preferably a scaffold; a cardiac pacemaker; a leadless pacemaker; prosthetic valves, preferably prosthetic heart valves, more preferably transcatheter prosthetic heart valves; or a prosthetic venous valve comprising bacterial nanocellulose. The bacterial nanocellulose may be a tissue patch. The bacterial nanocellulose may have the shape of an envelope of a cardiac pacemaker or a leadless pacemaker.

Also described herein is the use of bacterial nanocellulose (preferably produced by the above method) in biomedical applications, for vascular grafts, medical implants, medical scaffolds, cardiac pacemaker covers, leadless pacemakers, prosthetic valves, prosthetic heart valves, prosthetic venous valves, transcatheter heart valve prostheses, covered stents or stent grafts, as tissue patches, as drug coatings, for antimicrobial films, or for biosensors.

An apparatus for producing bacterial nanocellulose is also disclosed. The apparatus comprises at least one culture vessel for receiving a culture medium for a bacterial nanocellulose-producing bacterium, wherein bacterial nanocellulose can be produced, and for receiving at least one shaped article. The culture vessel may be adapted to accommodate more than one shaped article simultaneously. However, it is advantageous to have a single culture vessel for accommodating only one shaped body, since thus the growth of bacterial nanocellulose can proceed undisturbed. The apparatus further comprises at least one rotation unit for rotatably mounting at least one shaped article. The apparatus further comprises at least one shaped article rotatably mounted on the rotary unit. The rotation unit may be driven by a motor.

The following apparatus (bioreactor) for producing bacterial nanocellulose is disclosed, comprising

At least one reactor vessel for receiving and culturing a growth medium of bacterial nanocellulose and for accommodating a rotating profile and/or at least one shaped body,

A rotary unit for rotatably mounting at least one rotary profile and/or a shaped body,

At least one rotating profile and/or at least one shaped body rotatably mounted on the rotating unit is rotatably mounted,

At least one drive unit with a gearmotor,

At least one rotary unit driven by a gearmotor,

At least one gear unit for transmitting the motor torque of the gearmotor to at least one rotary profile and/or at least one molded body,

Optionally at least one detection unit for detecting the rotational speed of at least one rotating profile and/or at least one molded body, preferably comprising at least one hall sensor,

At least one evaluation unit and/or control unit for the rotational speed of at least one rotating profile and/or at least one molded body.

The gear unit may include at least one shaft, at least one toothed belt, and at least one gear. The apparatus may further comprise at least one detection unit for detecting the rotational speed of the at least one rotating profile and/or the at least one shaped body. The at least one detection unit may include at least one hall sensor.

Advantageously, each reactor vessel receives only one rotating profile and/or one shaped body, since then the growth of bacterial nanocellulose can proceed undisturbed (via other rotating profiles and/or other shaped bodies).

Bacterial nanocellulose (obtained from k.hansenii), consisting of nanocellulose fibres with a diameter of 30nm to 60nm and/or a density of 1.100g/cm ³ to 1.500g/cm ³, preferably 1.30±0.10g/cm ³.

The compressed BNCs can have a density of between 100mg/cm ³ and 250mg/cm ³. The density of the dried (except freeze-dried) BNC can be between 500mg/cm ³ and 1200mg/cm ³. The density of freeze-dried BNC is 19mg/cm ³ to 30mg/cm ³. The rehydrated BNC can have a density of 500mg/cm ³ to 600mg/cm ³.

Stable and dry bacterial nanocellulose can also be obtained, with a density between 1.100g/cm ³ and 1.500g/cm ³, preferably 1.30±0.10g/cm ³, and/or a refractive index between 1.30 and 1.40 and/or a tensile strength of more than 30MPa. Furthermore, stable and dried bacterial nanocellulose having a breaking strength of 40N to 63N and/or a tensile strength of more than 30MPa and/or an elongation at break of 30% to 45% and/or an F-modulus of 130N to 200N (for stable and dried bacterial nanocellulose flakes having a width of 10mm and a length of 50 mm) and/or a density of 1.140g/cm ³ to 1.215g/cm ³ can be obtained.

Molded elements made from natural, rehydrated or stabilized and dried bacterial nanocellulose with the aforementioned properties can also be obtained.

Example 1 Prior Art

The bacterial nanocellulose obtained in EP 3 572 043 A1 uses a growth medium consisting of: (a) Bacterial suspension containing acetic acid bacteria (bacterial nanocellulose grown in 25ml of growth medium in 50ml tubes, suspended by Turrax) and (b) nutrient solution containing 20g/l glucose, 5g/l peptone, 5g/l yeast extract, 2.7g/l disodium hydrogen phosphate and 1.5g citric acid. (a) and (b) are mixed in a ratio of 1:12. A silicone hose with a stent is immersed in the growth medium. In such growth media, bacterial cellulose is typically formed in an incubator at 26 ℃ to 30 ℃ over a period of 6 to 8 days. A cellulose layer thickness in the range of 0.5 to 10mm or more may be produced.

Example 2a

The growth medium used in this example consisted of the following components: (a) Bacterial suspensions containing Komagataeibacter species Komagataeibacter hansenii or k.hansenii bacteria of the family aceraceae, preferably American Type Culture Collection (ATCC) code 53582; and (b) a nutrient solution containing 20g/l glucose, 5g/l peptone, 5g/l yeast extract, 2.7g/l disodium hydrogen phosphate and 1.5g citric acid. (a) and (b) are mixed in a ratio of 1:18. The process of example 2a was performed under static conditions. However, the process may also be performed under dynamic conditions (as in example 2b for rotation).

Regular maintenance of the strain ensures the continued presence of the active bacterial culture. Bacterial strain Komagataeibacter hansenii%53582 The maintenance of the strain ends in a period of 7 days. A sterile polypropylene (pyrogen-free) laboratory vessel with a capacity of 50ml was filled with 25 standard medium. The medium was then inoculated with 2ml of bacterial suspension. This consisted of nutrient medium and crushed cellulose fluff, which had been cultured for seven days. The mixture of bacterial suspension and nutrient medium was then placed in a cooled incubator at 27 ℃ and a relative humidity of about 90% for synthesis. To ensure constant conditions during cultivation, the temperature and relative humidity in the incubator are monitored. According to this, even if the incubator is opened for a short time, the temperature remains almost unchanged, the relative humidity rapidly decreases, and then returns to around 90% in about six hours. In general, the parameters selected, such as the duration of the culture, depend on the geometry of the culture vessel and the volume of nutrient solution. In the context of this work, these parameters are therefore particularly suitable for the materials used. The harvested fluff was finally placed on a horizontal shaker in ultrapure water for three days to remove the residue of the nutrient medium. Ultrapure water is ultrapure water that is replaced after about 8 hours each. Hereinafter, the state of the cellulose nonwoven fabric after the winding process of the cellulose fluff is referred to as "natural". The sample geometry used in each case was cut with a CO ₂ laser (30W,Epilog Zing 24,Epilog).

Example 2b

In this example, a growth medium for bacterial cellulose-producing bacteria according to the present invention was prepared as described in example 1, except that a ratio of bacterial suspension to nutrient solution of 1:18 was used instead of 1:12. Shaped articles, preferably PEEK rods, made of polymers having a polymer backbone with alternating ketone and ether groups, are rotatably mounted in a medium for bacterial cellulose producing bacteria. The shaped article, preferably a PEEK rod, is rotated in air at a speed of at least 10rpm for 3 days at 28 ℃. Excess bacterial nanocellulose was removed every 12 hours.

The bacterial nanocellulose obtained in the form of a hollow cylinder is mounted on a shaped article (preferably a PEEK rod), which may be further processed according to at least one of the following steps:

a) Rinsing with ultrapure water, for example for 2 hours, to obtain clean bacterial nanocellulose, b) drying (clean) bacterial nanocellulose, for example in air at a speed of 3rpm for 24 hours, to obtain dry bacterial nanocellulose,

C) Rehydrating the dried bacterial nanocellulose with ultrapure water,

D) The (dried or rehydrated) bacterial nanocellulose is contacted with 0.1M NaOH solution for e.g. 3 days,

E) Removing bacterial nanocellulose from the PEEK rods before or after steps a), b), c) or d),

F) The bacterial nanocellulose is cut into the desired form e.g. by CO ₂ laser or surgical knife,

G) Optionally drying the bacterial nanocellulose,

H) The (dried) bacterial nanocellulose is sterilized.

It will be apparent to those skilled in the art that other high purity water may be used instead of ultrapure water, such as distilled or deionized water. The resistivity of the high purity water at 25℃is preferably 18.2 M.OMEGA.cm or less (total organic content < 10ppb, endotoxin content < 0.03 EU/ml). The wall thickness of the nanocellulose obtained is less than 70 μm, preferably 40 μm to 60 μm (depending on the reaction time). Thus, a thinner wall thickness as in example 1 was obtained. The bacterial nanocellulose according to example 2 has higher internal mechanical stability, higher mechanical strength than the bacterial nanocellulose obtained in example 1. The tensile strength of the bacterial nanocellulose obtained in example 1 was 8.40±0.40N, whereas the tensile strength of the bacterial nanocellulose obtained in example 2 was 4.61±1.23N. The bacterial nanocellulose obtained in example 1 had a density four times higher than the bacterial nanocellulose according to example 2.

Example 2c

For the cultivation of cellulose fluff, standard stainless steel trays (e.g. 300mm x125mm x 60mm size) are used. The vessels were filled with 480ml of standard medium (also called nutrient solution) containing 20g/l glucose, 5g/l peptone, 5g/l yeast extract, 2.7g/l disodium hydrogen phosphate and 1.5g of citric acid and 40ml of bacterial suspension. The ratio of bacterial suspension to medium was 1:12. The filled dishes were kept at a temperature of 27℃and incubated in a Peltier-cooled incubator for seven days. After cultivation, BNC fluff was harvested to a thickness of 7mm to 8 mm. The level of excess medium after cultivation was 5mm, which is a limiting factor in fluff synthesis. However, subsequent addition of nutrient medium results in interruption of the formation of the continuous layer of cellulosic fluff.

Example 3 implantation envelope of pacemaker

According to this example, the implant capsule is produced from bacterial nanocellulose, preferably manufactured by the method of example 2, and is used to house a cardiac pacemaker or leadless pacemaker. The bacterial nanocellulose sheet is dried. The layers were bonded or stitched together using a polymer wire (PTFE, 5-0 size) and a 0.3mm suture needle to form an implant capsule for receiving a pacemaker. The pacemaker may be inserted into an implant capsule made of bacterial nanocellulose, which may be further closed by gluing or suturing. Pacemakers covered with an implant envelope made of bacterial nanocellulose may be sterilized and packaged. Pacemakers covered with an implant envelope made of bacterial nanocellulose may be stored in a dry state. Shortly before implantation, the pacemaker may be rehydrated by a sterile (isotonic) saline solution. The bacterial nanocellulose used has a wall thickness of less than 70 μm, preferably 40 μm to 60 μm.

Example 4-transcatheter heart valve prosthesis

According to this example, the components of the transcatheter heart valve prosthesis, such as the inner skirt and/or the outer skirt, are made of bacterial nanocellulose, preferably by the method of example 2. The leaflets may be made of bacterial nanocellulose or pericardial tissue. A transcatheter heart valve prosthesis is a heart valve prosthesis that is used for implantation to replace a native mitral valve. The transcatheter heart valve prosthesis is brought to the implantation site by a catheter system and anchored there. Anchoring in the vessel wall is achieved by a support structure for the actual heart valve, for example by designing and selecting a metal mesh similar to the stent, so the stent is also referred to as stent matrix in the following. The stent matrix may be self-expanding or may be swellable using a balloon catheter. A transcatheter heart valve prosthesis includes a stent matrix capable of swelling from a first size configured for minimally invasive insertion to a functional second size. The actual heart valve is secured to the stent matrix, wherein the heart valve initially assumes a first shape configured for minimally invasive insertion and may swell into a functional second shape during implantation. The stent matrix includes metallic struts, such as nitinol struts. The outer and/or inner skirt, which at least partially covers the stent matrix, may be made of bacterial nanocellulose and may be secured to the stent matrix (e.g., by gluing or suturing using wires such as polytetrafluoroethylene wires), wherein the outer and/or inner skirt is small She Linjie with the heart valve. Since bacterial nanocellulose can be processed and stored in a dry state, and since bacterial nanocellulose can be produced with different layer thicknesses, different swelling capacities and mechanical strengths, it is possible to construct an entire transcatheter heart valve prosthesis, such as a percutaneous aortic valve, from bacterial nanocellulose. The bacterial nanocellulose used has a wall thickness of less than 70 μm, preferably 40 μm to 60 μm.

Example 5-covered stent

According to this example, the scaffold is covered with bacterial nanocellulose preferably prepared by the method of example 2. The bacterial nanocellulose used has a wall thickness of less than 70 μm, preferably 40 μm to 60 μm. A stent (also known as a vascular stent) is a medical implant that is inserted into hollow organs to keep the organs open. Stents are typically small tubular-shaped lattice frames composed of a metal or plastic mesh, also referred to as stent matrices in this example. The stent matrix is covered with a layer of bacterial nanocellulose.

EXAMPLE 6 stent graft

According to this example, the inner shell and/or envelope of the stent graft is made of bacterial nanocellulose, preferably by the method of example 2. The bacterial nanocellulose used has a wall thickness of less than 70 μm, preferably 40 μm to 60 μm. A stent graft is a combination of a stable support frame (hereinafter also referred to as a stent) and a vascular prosthesis (vascular prosthesis). Implantation of a stent graft is an endovascular procedure. Stent grafts are used, inter alia, to exclude aneurysms from the blood stream. In this case, the scaffold has an inner shell made of bacterial nanocellulose. According to the method of the invention, the tube may be made of bacterial nanocellulose. The (self-expanding or self-expanding) scaffold is fixed to an inner shell, which is made using bacterial nanocellulose, for example by surgical suture material or gluing. Then, a strip, which is likewise tubular and preferably has a width of 1 to 2cm, is fastened on the outside at both ends, for example by stitching or gluing. Such strips made of bacterial nanocellulose have a greater swelling capacity than the inner shell, and thus can seal the leak after implantation without significantly increasing the diameter of the implant during implantation.

Example 7-vascular patch

According to this example, a tissue patch, preferably a vascular patch, comprising bacterial nanocellulose, preferably prepared by the method of example 2, is disclosed. The bacterial nanocellulose used has a wall thickness of less than 70 μm, preferably 40 μm to 60 μm. In medicine, a tissue patch, preferably a vascular patch, is understood to be a piece of foreign material used in surgery to close unwanted openings. The patch is always used whenever the opening cannot be closed without complications by simple suturing. One example of conventional use is cardiac surgery, where a septal defect is closed, for example, with a vascular patch. Patches are also used for vascular surgical dilation of blood vessels (arteries and veins) or for covering defects on blood vessels. The patch is sutured into the open vessel, for example, to prevent stenosis caused by the seam, or for widening purposes. In this case, the patch is made of bacterial cellulose treated in the manner described above. The tissue patch, preferably a vascular patch, may be supported by a biodegradable or non-biodegradable support structure, such as a mesh.

Example 8

The following characteristics, if not stated otherwise, refer to the BNCs obtained in example 2 a:

Thickness measurement

To determine the uniformity of the production of the cellulosic nonwoven, its thickness was measured after a separate processing step. A GT2 smart series touch sensor tactile thickness gauge (Keyence Deutschland GmbH, germany) was used for this purpose. A hydraulically controlled contact piston of circular area 10mm in diameter exerts a force of 0.3N on the material to be measured for 2 seconds. The displacement measurement system of the sensor converts the distance between the previously initialized base surface and the measured sample into a thickness value. In addition to the absolute values, the percent reduction in thickness (DR) between the natural and rehydrated states of the sample is shown.

Mechanical properties

BNCs are characterized by high mechanical stability and plastic material properties. For biomedical applications, the integrity of the fibrous material after various processing variants is indispensable. Unless otherwise specified, the mechanical tensile test is performed in the fully processed, rehydrated state of the sample. Using a test stand, both unidirectional and bi-directional tensile tests were allowed. This consists of four drive units, each equipped with a stepper motor and a position encoder, and can be controlled individually. The force is recorded by the platform load cell and transmitted to the software, thereby effecting displacement and time dependent force measurements. The material sample is clamped in a clamping jaw, which is attached to a drive device in a roller rail and protrudes into a tank filled with ultra pure water. The geometry of the samples was designed in accordance with DIN EN ISO 527-2 Standard 1 BA. Since complex fiber composite materials are employed, rectangular geometries with symmetrically arranged grooves corresponding to semi-elliptical shapes are employed to ensure continuous loading of the sample center. When the sample width is changed, the elliptical grooves will scale accordingly. The thickness of each sample was determined by tactile sensation prior to measurement. Once a preload of 2g is reached, the measurement data of the tensile test is recorded. The sample length in this state is automatically transferred to the software as the initial sample length. Unless otherwise specified, during the measurement, the jaws were moved at a speed of 12mm/min per drive unit until the sample failed completely. When 1% of the maximum detection force is reached, the measurement is stopped. To characterize the material, the breaking force Fmax and the breaking f_max and the slope in the linear range of the curve are used on the one hand at a small strain of up to 5% (referred to as initial modulus or F-modulus 5%) and on the other hand shortly before failure of the sample (F-modulus). Detailed description of complex mechanical behavior of BNCs under uniaxial tensile load. In addition, suture Retention Strength (SRS) is part of the suture tear test. The implant material may need to be sutured to the tissue elements or sutured into existing structures, such as in a biological heart valve prosthesis. Suture pullout strength provides information of the maximum possible load of the suture before it is pulled out of the material. To this end, the wire was threaded through the material a distance of 1mm on the short side of a 6.5mm x 32mm rectangular sample and wound onto a screw. As with the mechanical tensile test, the other side of the sample is secured in the jaw. The wire diameter (D _Faden) was 0.1mm. Before each measurement, the thickness of the puncture was determined by tactile sense (d _BNC). The seam tear strength SRS is derived from the maximum force measured during the test at the time of tearing the line (seam tear force F _max) which is related to the thickness of the sample at the puncture point and the diameter of the line, according to equation 3.1.

Water content and water retention properties

The Water Content (WC) describes the percentage of water in the BNC fiber network. Circular rehydration samples For the determination. The surface of the sample was cleaned of adhered moisture by wiping the grid and then wet weight (m _nass) was measured using a precision balance (mertrer-tolidol GmbH) exceeding the series XP 204. The samples were freeze-dried in vacuo for 24 hours and their dry weights were determined ((m _trocken) then WG was calculated using equation 3.2 and expressed as a percentage.

The Water Retention Value (WRV) describes the ability of a fibrous material to retain moisture by capillary forces and adhesion within the fibers and their interstices. According to standard DIN53814: 1974-10. According to DIN53814: 1974-10) at 2380U/min at 20 ℃Centrifuge for 20 minutes (centrifuge 5920R,Eppendorf GmbH). After centrifugation, the weight of the sample (m _{zentrifugiert}) was determined. To determine the dry weight (m _trocken), the centrifuged samples were dried in an oven at 100 ℃ for 24 hours. WRV is finally calculated according to equation 3.3.

With WRV, keratinization can be designated as another parameter characterizing the structure of a substance. This describes the irreversible change of the cellulose fibres that occurs as a result of drying. The fiber network is compressed during drying, resulting in the formation of hydrogen bonds (WBB). The percentage of keratinization is measured as the reduction in WRV and is measured by the public

Formula 3.4.

Here, WRV _native represents WRV of BNC in a natural state, WRV _rehy represents WRV in a dry-rehydration state.

Structural analysis by scanning electron microscopy

Scanning Electron Microscope (SEM) EVO MA15 (Carl Zeiss Microscopy AG, germany) was used to analyze surface properties. With excellent resolution and depth of field, SEM can conduct down to nanometer scale structural studies on BNCs. Images from surface topography, internal microstructure to individual fibers, and qualitative and quantitative information about the properties of the biological material. It is possible to. Due to the sampleIs scanned under high vacuum and thus vacuum dried in advance. (2.0X10 ^- ¹ mbar,. Epsilon.1-4LSC plus,Martin Christ Gefriertrocknungsanlagen GmbH). To produce a conductive surface, the dried sample was then coated with a gold layer under nitrogen (Agar Sputter Coater, plano GmbH, germany).

Characteristics of Natural cellulose nonwoven fabrics

The morphological characteristics of the biological material have been determined during the synthesis of the nonwoven material. Various factors affect the yield and structure of cellulose at the micro-and nano-scale. These factors include the ratio of bacteria to medium at a constant total volume, the duration of fluff synthesis, and the pH of the medium. Based on standard culture, when using medium ATCC 1765, reproducible nonwoven synthesis was produced with respect to pH. Depending on the incubation time, the fiber morphology is investigated hereinbelow with the aid of SEM images. Furthermore, the fiber volume and density of BNCs were experimentally determined using the buoyancy method according to archimedes' principle.

Determination of fiber diameter Using scanning Electron microscope

The size and structure of BNC fibers varies from bacterial strain to bacterial strain. For example, the microfibrils of acetobacter xylosoxidans form fibrous bands having a width between 40 and 100 nm. Other strains such as Acetobacter bovini have fibers on the order of 20 nm. To determine the Fiber Diameter (FD) of bacterial strain k.hansenii used in this study, four series of experiments were analyzed, eight samples each. The synthesis time of samples from one experimental series varied (2 days to 9 days). For the cultivation, 15 ml-capacity test tubes (SARSTEDT AG & Co.KG,) 1Ml of bacterial suspension and 12ml of nutrient medium are contained. After incubation, the samples were rinsed in ultrapure water, freeze-dried and checked by SEM. For quantitative analysis, the image is first converted to a grey-scale image (ImageJ software), then the contrast is adjusted and the structure boundaries are detected (threshold function: threshold ISO 50%). After digital extraction of the desired fiber, its diameter is measured using a software length measuring tool. To obtain a representative value for the diameter analysis, ten different fibers were measured for each sample. The average of ten fiber diameters for each sample is shown in table 3.2. Ten measurements were also made per fiber. From the recordings of FD and the measured values, it is apparent that FD is independent of incubation time. The finest fiber diameter was about 30nm, which was found in both the very short synthesis time samples and the samples incubated for more than one week.

Overall, the results show that in each case the thinnest fibers on the sample surface can be classified in the range of 30nm to 60 nm. However, it is often not possible to clearly define whether the fibers under consideration are single fibers or fiber composites. In addition, undefined aggregation of the fibers can occur during the freeze-drying process.

The samples of test series V4 were additionally washed in 0.1mol sodium hydroxide solution to remove any endotoxin present prior to rehydration.

Table 3.2: fiber diameter of three test series (V1-V3) and one test series (V4) washed with sodium hydroxide solution of natural BNC. One sample was analyzed per culture period, with an average of 10 fibers.

Determination of fiber volume and Density by buoyancy weighing

The thickness and dry mass of the nonwoven material, as well as the characteristics of fiber volume and density, are determined as a function of the duration of nonwoven material synthesis and the ratio of bacterial volume to nutrient medium. The incubation time varied between 2 and 10 days with a bacterial to nutrient medium ratio of 1:12. At a constant total volume (520 ml) and a culture time of 7 days, this ratio was further changed to 1:6 or 1:96.

The dimensions of the samples used were 3.5cm by 3.5cm. To determine the thickness of the nonwoven, a tactile measurement is first used. Wet samples stored in ultrapure water are weighed (m _W) in a liquid of known density in specially constructed equipment. Ultrapure water is used as the liquid, and its temperature is determined for densitometry. Subsequently, the samples were freeze-dried for 48 hours and their dry mass (m _T) was determined. The difference between the two weighings (m _T-m_W) represents the mass of the displacement liquid (archimedes principle). This gives the volume of liquid displaced, taking into account the density of the liquid (ρ _W), giving the volume of displaced body, which in this case corresponds to the volume of cellulose fibres (V _Fasern):

The same applies for the density of BNCs (BNCs):

Along with the extension of the culture time, the fluff thickness and dry mass steadily increased during the synthesis. Only after 6 and 7 days, respectively, there was no significant change in web thickness. In contrast, dry mass also increased to a duration of 10 days. This suggests that as synthesis time is extended, more glucose is metabolized by the bacteria, producing more cellulose fibers. Thus, over time, the fiber network becomes denser. SEM images of samples at different synthesis times also confirm this. In the 10 day cultured samples, the fiber network was more dense compared to the 2 day synthesis. The ratio of bacterial volume to nutrient medium showed no significant differences in nonwoven thickness and dry mass. For larger ratios (1:6), the two eigenvalues have a slightly higher tendency than for smaller ratios (1:96). The minor difference is due to proliferation of the bacterial population by cell division. The growth curve of microorganisms is characterized by an exponential growth. Bacteria are best suited to the nutrient medium and environment and reproduce at their maximum rate of division. Based on these results, it can be assumed that the bacteria multiply over a day or several hours. The BNC hydrogels analyzed herein are fiber composites composed of water and cellulose fibers. In such composites, the volume fraction of the total volume occupied by the cellulosic fibers is hereinafter referred to as the Fiber Volume (FV). The fiber volume per cubic centimeter remained almost constant at 0.8% over a 6 day incubation time. The fiber volume increased to 1.5% over a 10 day synthesis time period. This is consistent with the assumption of a larger fiber number with approximately constant fiber diameter as synthesis time increases.

Changing the ratio of bacterial volume to nutrient medium has no effect on fiber volume. The density of BNCs can also be deduced from the fiber volume and dry mass (equation 3.6). On average, the density of BNCs that produced bacterial strain K.hansenii (ATCC 53582) was measured to be (1.3.+ -. 0.1) g/cm ³.

Fiber count measurement

Based on knowledge of the BNC density and the fiber volume per cubic centimeter, the number N of fibers per cm ³ can be determined. First, the fiber volume V _F is determined. It is assumed that the fibers have a cylindrical bottom. Assuming a radius of 30nm for the substrate area, this is derived from the defined fiber diameter of 30nm to 60 nm. The fiber length was assumed to be 10 μm. Thus, the volume of the fiber corresponds to V _F＝2.83 10^-14cm³. The number of fibers per cubic centimeter is determined by the quotient of the fiber volume per cubic centimeter (F _V) and the fiber volume V _F. Taking a cellulose nonwoven fabric with a synthetic time of 7 days as an example, the number of fibers was cm ³:

Or the number of fibers can be derived by calculating the mass of the fibers (m _F) in view of the determined density (ρ _BNC). The quality of the fibers is therefore independent of the incubation time:

Using the dry mass of the 7 day synthetic BNC sample (m _T (7 days)) relative to the total volume of the sample (in cm 3), the method gives N of fibers per cm ³:

Taken together, these considerations indicate that there are about 1011 fibers in a one cubic centimeter fiber composite BNC. To categorize this result, a comparison with another complex fiber composite material having similar fiber geometry was used. Tautz (2008) determined that the comparable number of asbestos fibers was 1012 fibers per cubic centimeter, the geometry was similar to BNC fibers, and the fiber diameter was The average length is 1-3 μm.

Processing and post-treatment of cellulose nonwoven fabrics

The processing of BNC after incubation was divided into different process variables (see FIG. 16). During the culture, the duration of tissue synthesis was studied. The reduction in thickness of natural nonwoven fabrics is achieved by different drying or pressing methods. A combination of drying and pressing is also important. Furthermore, the effect of cleaning on endotoxin removal and rehydration time will be explained. The analytical method is carried out after rehydration.

Culturing

As described above, the thickness of the natural cellulose nonwoven fabric and the related dry matter increase with the extension of the culture time. This presents a problem as to how much this parameter affects the performance in the dry-rehydrated state.

The synthetic time of the cellulose non-woven fabric is 3 to 10 days. Then dried in a climatic chamber at a temperature of 23℃and a relative humidity of 50% for 72 hours. The cellulosic nonwoven is then dried in a drying oven. A detailed description of the drying method is provided. Finally, rehydration was carried out for 24 hours. It can be clearly seen that the thickness increased with increasing incubation time even in the rehydrated state (table 3.3). This is consistent with the increase in fiber volume fraction and dry mass of the natural sample.

Table 3.3: natural and dry-rehydrated nonwoven thickness, moisture content and water retention capacity as a function of incubation time (n=24, n=2).

As the mass of cellulose fibres increases, the water content therefore decreases, as the number of fibres in this volume increases, and the void space available for water storage correspondingly decreases. At the same time, WRV decreases and keratinization increases, indicating that more intermolecular WBBs is formed due to a denser fibrous structure. This assumption is confirmed when considering the mechanical properties of different incubation times. The breaking strength increases significantly with increasing synthesis time, while the elongation at break remains almost unchanged. This indicates that more load-bearing fibers are present with prolonged incubation time, however, this allows the sample to have the same macroscopic elongation. Furthermore, a more rigid material behavior is evident, not only when the slope before the failure point is considered, but also especially at the onset of small strain loading (table 3.4). The keratinization increases with prolonged incubation time, indicating an increase in the stable formation of intermolecular WBB.

Furthermore, it is attractive that after 7 days of culture, fiber synthesis stagnates due to the limited availability of carbon sources in the nutrient medium. The nutrient medium was used to give about 100ml of supernatant. Furthermore, the mechanical strength of the nonwoven material is no longer significantly increased. Therefore, a nonwoven fabric having a synthetic time of seven days was used as a standard for the subsequent study. Subsequent addition of nutrient medium to again provide a carbon source will result in interruption of formation of the continuous layer, and thus failure to form coherent fluff.

Table 3.4: mechanical properties of failure point and force response as a function of incubation time at small strains up to 5% (n=60, n=2).

Drying

The drying of natural cellulosic nonwoven is an important step in the processing. In addition to the reduced thickness, the physical properties of the biomaterial are also significantly affected. Depending on the drying method chosen, dewatering can lead to structural changes in the fiber network. Hereinafter, drying (KS), oven drying (O) and freeze drying (GT) in the climatic chamber are distinguished. The drying of the climatic cabinet is characterized by low temperature and controlled ambient air humidity. On the other hand, in an oven, drying is performed at a high temperature. In both variants, the sample to be dried is placed between filter papers and pressed down with a grid and a stainless steel square block (total weight 300 g). This reduces drying induced wrinkling and achieves a flat sample surface. In the freeze-drying step, the sample is first frozen on a thermally conductive holder at normal pressure. The frozen water then becomes gaseous by reducing the pressure to 0.07 mbar. By reaching the sublimation pressure, a phase change occurs. Finally, the temperature was gradually increased to 20 ℃. After drying, all samples were rehydrated in ultra pure water at 37 ℃ for 24 hours. The percent reduction in sample thickness for the different drying methods did not show any significant difference between climatic chamber drying and oven drying (table 3.5). Both methods achieve a significant thickness reduction (DR) of about 98% based on the original initial thickness. In contrast, after freeze-drying, the thickness was reduced by only about half of the initial state. This demonstrates the structure-retaining properties of sublimation lyophilization.

Table 3.5: thickness as a function of the different drying methods (n=60, n=2).

Table 3.6: moisture content, water retention and keratinization (n=24, n=2) as a function of the different drying methods.

Furthermore, with respect to WG and WRV results, GT shows values comparable to natural samples, whereas in the case of climatic chamber and oven drying WG and WRV decrease significantly with increasing temperature during drying. It is speculated that at higher temperatures, greater compression of the fibers occurs with increased agglomeration of the fibers. When the morphology is observed by SEM images, a very dense network of fibers is evident during oven drying (100 ℃) compared to climatic oven or freeze drying. This was also confirmed by mechanical analysis. A significant increase in initial modulus was observed at higher drying temperatures. The elongation at break and the force modulus did not show any significant difference, whereas the force at break was significantly increased due to the drying process compared to the natural sample (table 3.7). The microstructure of the fibers provides an explanation for the behavior of the dried material. These molecules consist of glucose molecules, each having three free hydroxyl groups. Thus, fibrils are able to form intermolecular and intramolecular WBBs with adjacent dextran chains as well as water molecules. During drying, removal of the water molecules releases additional hydroxyl groups, which again form WBBs with the adjacent free hydroxyl groups. Thus, the removal of water results in densification of the fibers with concomitant collapse of the pore structure. Experimental determination of BNC material density after different drying methods confirm this hypothesis. Even after complete rehydration, the induced keratinization of the material is mainly irreversible. Thus, the formation of additional WBB mainly results in a stiffer material behavior under low loads and a slight increase in breaking strength.

Table 3.7: the point of failure and mechanical properties of force response at small strains up to 5% according to different drying methods (n=25, n=2).

The results of the seam tear strength show that the maximum force until the line is torn is about 5N, independent of the drying method selected. Since the thickness of the samples is indirectly proportional in the calculation of seam tear strength, the dried samples obtained higher strength than the freeze-dried samples. The maximum seam tear strength did not show any significant difference regardless of the thickness of the sample. It can thus be assumed that the tightness of the fiber network of different BNC nonwovens is comparable, thus obtaining almost the same value in terms of seam tearing force.

Pressing

As an alternative to drying, the pressing process in which the initial dewatering is carried out in a natural state is focused on below. Here too, a reduction in thickness accompanied by a structural change of the biological material is achieved, which is desirable for applications in the biomedical field. In particular, a combination of drying and pressing was studied. After rinsing the natural cellulosic fluff, a pressing process is performed using a manual lever press (LaboPress P H, vogt Labormaschinen GmbH). The sample (7 cm x 6 cm) was placed between two heat-conducting platens and the required pressure p=f was set using a lever. The pressing time for each sample was 15 minutes. In the combination of pressing (P) and drying (T), various pressing parameters are first changed. Here, drying was performed in a climatic chamber (23 degrees celsius, 10 degrees celsius, 24 hours). Subsequently, different drying methods were investigated in the combined process at constant pressing parameters (50 ℃,10N/mm 2).

Regarding the thickness of the nonwoven, at higher pressing temperatures, the thickness reduction is significantly greater. On the other hand, the pressing pressure did not show a significant trend, so the thickness reduction was always significant between 5N/mm ² and 20N/mm ² regardless of the temperature (Table 3.8). Both WG and WRV decrease with increasing temperature and pressure, resulting in more severe keratinization. This tendency is particularly pronounced when considering samples pressed at 100 ℃. The results show that the aggregation of the fibers is caused by a significant reduction in thickness, especially when the temperature is increased. The compression of the material reduces the distance between adjacent fibers, making the WBB more and more likely to form. In the literature, a pitch of about 0.25nm to 0.39nm is given for WBB formation. In addition, the high temperature induces an indirect drying process, which favors the formation of stable WBB. SEM images also show significant compression of the fibrous structure at higher compression temperatures. These results relate to oven drying. The pressing removes water molecules from the fiber network, creating free binding sites on the hydroxyl groups of the glucose molecules. With adjacent dextran chains, new WBBs can be formed between the free hydroxyl groups.

Table 3.8: natural nonwoven thickness (8.01±0.23 mm), pressing and rehydration (n=24, n=2); the moisture content, water retention and keratinization of the rehydrated samples with respect to compression temperature and pressure (n=12, n=1).

Analysis of mechanical properties also confirms this hypothesis. Despite the variation in the compression parameters, the breaking force and breaking strain were unchanged, and significant differences in force response were found over a small strain range. Especially at high temperatures of 100 ℃, the difference reaches significant levels of p < 0.01. Again, this demonstrates the dominant role of WBB in terms of the mechanical behavior of the biological material at low strain. In the case of a combination of pressing and drying (P.fwdarw.T or T.fwdarw.P), there was no change in the reduction in thickness in the case of the drying process investigated (appendix A.9). Furthermore, this is independent of the time of the pressing process. However, when considering mechanical properties, a clear dependence on the pressing time is evident. During the initial pressing process (p→t), the fabric becomes significantly stiffer. The initial modulus is significantly higher than that of the drying process alone, and the elongation at break shows a relatively low value. However, in the case of final pressing after drying (t→p), there is no significant change compared to the drying process alone. The variation of the pressing parameters in the combined process has no effect on the time of the pressing process, which is related to the thickness reduction and WG and WRV. The pressing temperature of 100 ℃ again results in the most severe keratinization. The effect of time of the pressing process is mainly manifested when the initial modulus is considered. Compared to initial drying (t→p), greater fabric stiffness was observed for initial pressing (p→t). In summary, the results indicate that the order of drying and pressing methods determines the structural and physical properties of the biomaterial. The subsequent processing method has only a minor effect on performance. For example, the final pressing step (T→P) prior to rehydration will result in a more uniform material thickness. On the other hand, the initial pressing step (p→t) tends to result in greater hardness of the biomaterial.

Purification of

Since the fiber is synthesized by gram-negative bacteria, endotoxin is a component of biological fiber complex BNC. For use as biological materials for medical implants, removal of these endotoxins is critical to avoid toxic reactions in the body. For this purpose, the endotoxin present in the bacterial cell wall is removed by means of an acid or a base. The 72 hour purification period was chosen to ensure that the implant material in the cardiovascular domain did not exceed the maximum limit of 0.5EU/ml prescribed by the United states food and drug administration. The purification takes place after drying (T), pressing (P) or a combination of both processes (t→p). The cleaning solution is 0.1M sodium hydroxide solution or 1M potassium carbonate. The dried samples (7 cm. Times.11 cm) were placed in 200ml of the corresponding cleaning solution and stored in a hot mixer at 80℃and a rotational speed of 350rpm for 72h. The cleaning solution was changed after 24 hours each. After the cleaning process, the samples were rinsed in ultrapure water until the pH was neutralized. Finally, all samples were autoclaved at 121 ℃ for 20 minutes (vario klav T form, thermo FISHER SCIENTIFIC). There was no significant difference in thickness reduction and WG with respect to the cleaning solution used. With respect to WRV, regardless of the cleaning solution used, the cleaned sample tends to have a higher value. Swelling of fibers upon treatment with an alkaline solution is described. The increased fiber diameter after the cleaning process allows a greater proportion of water to remain. Furthermore, the cleaning process has an influence on the mechanical properties of the BNCs. The force and initial modulus were only slightly different compared to the uncleaned samples, and the breaking force and elongation at break showed significantly lower values of the characteristics. Thus, cleaning with alkaline solutions results in lower tensile strength. An explanation can be given by observing the molecular structure of BNCs. The acid or base causes cleavage of glycosidic bonds between the individual AGUs. This results in a reduced degree of polymerization and thus in hydrolytic degradation of the glucan chains. The force transmission in mechanical tensile testing is determined by the length of the adjacent segments, as the tensile strength is dependent on the force transmission of the adjacent molecular chains. Thus, the cleaned sample tends to obtain a lower mechanical force response. The electron micrograph shows successful cleaning of the biological material based on the removed bacterial residues. In both cleaning solutions, the fibrous structure without bacterial cell residues was clearly visible. Overall, the mechanical and structural properties of BNCs are not significantly affected by endotoxin removal, ensuring the integrity of the biological material used as an implant material.

Rehydration

The final step in the process is rehydration in water. The duration of time until complete rehydration on the one hand and the maximum possible water absorption on the other hand are of interest due to the contact of the material as implant material with the blood in the body. The water absorption capacity is the maximum amount of water that can be absorbed by a structure adapted to the normal climate after storage in water, according to DIN 53923. Furthermore, the water absorption of the dried BNC sample during storage is quantified by the ambient air humidity by the adsorption curve. To determine the water absorption capacity, round samplesThe laser cutting is carried out after drying in a climatic chamber at 23 ℃ and a subsequent pressing step. Dry weight (mt) was determined, and then the sample was placed in 100ml of ultrapure water. Rehydration was performed in an incubator at 37 ℃. After various residence times (1 second to 21 days), the samples were removed and the water adhering to the surface was removed by filter paper or centrifugation. When the water was removed with filter paper, each side of the sample was placed on filter paper (MN 615, macherey-Nagel) for 10 seconds, and then its wet weight (MN) was measured. Alternatively, to confirm removal of excess water, the sample was centrifuged in a centrifuge tube at 2380rpm and 20 ℃ for 20 minutes (centrifuge 5920R,Eppendorf GmbH) and then weighed. The percentage of water absorption comes from:

to evaluate the water uptake during storage or further processing, an adsorption curve was also plotted. For this purpose, for round samples A treatment similar to the water absorption capacity was carried out and then stored for 24 hours in a climatic chamber with a relative humidity of 10% and a temperature of 23 ℃. Starting from this initial value, the relative humidity is continuously increased to 90% in 10% steps. The respective environmental conditions were kept constant for 24 hours, and then 10 samples were taken. The moisture content of these samples was determined by weighing, based on dry weight at 10% relative humidity.

The water uptake after various residence times has been shown to saturate after six hours of storage and remains unchanged after three weeks. Rapid water uptake was also clearly visible within the first five minutes of rehydration. In addition, this trend occurs regardless of the method chosen to remove excess water. Measurements indicate that a rehydration time of about 6 hours is sufficient to achieve complete water absorption.

Analysis of the adsorption curve shows a steady trend in water absorption with increasing relative humidity. Surprisingly, however, the water absorption is below 20% even at 90% relative humidity. Thus, dry BNCs only slightly absorb moisture from the surrounding air. This ensures a non-contaminating storage of the biological material in the dry state at room temperature and represents an outstanding indicator of the application as an implant material.

The nature of natural and dry-rehydrated BNCs is further elucidated. Electron microscopic analysis of the natural cellulose nonwoven was used to reduce the cellulose fiber diameter of bacterial strain k. The density of BNC was experimentally determined to be 1.3g/cm ³. These two characteristic values are independent of the selected incubation time and the ratio of bacteria to nutrient medium during incubation. Only the fiber volume increases with the synthesis time of the nonwoven.

By different variants of post-treatment or processing, standard processes for producing biomaterials for use in the field of cardiovascular implantation were established (see fig. 17) and tested for reproducibility.

Due to the mechanical strength and moderate drying in a climatic chamber at 23℃a incubation time of 7 days proved advantageous. Optionally, a pressing process may be performed after drying to improve thickness uniformity. The cell residues are subsequently removed by cleaning with sodium hydroxide solution. Since cleaning has only a small effect on the properties of BNCs, rehydration in ultrapure water is a standard practice in the following section, unless otherwise noted. Here, the rehydration time of 6 hours has reached saturation in terms of water absorption.

Overall, this type of processing produces a very uniform, repeatable material structure due to the low standard deviation of the eigenvalues, which is likely to be used as an implant material in the cardiovascular field.

Uniaxial tensile load

The basis for evaluating deformation behavior in uniaxial tensile tests is the force-elongation curve. The force-elongation plot of the sample made according to the process of fig. 18 can be divided into four sections (a-D). In part a, the load increase starts at a continuously linearly increasing force (initial modulus or F-modulus 5%) until a plateau region is started in part B, which characterizes the necking process and thus the rearrangement process of the fibers. The force increase in this region is significantly lower than the force increase at the beginning of load absorption. In section C, the force (F-modulus) again increases linearly, as the fibers, now parallel to the direction of stretching, may continue to absorb load until the sample eventually fails in section D. Immediately before sample failure, the shrinkage was (78.1±1.7)% (n=13) relative to the initial sample width of 5 mm. As described in chapter 3, the mechanical properties of biological materials are highly dependent on the culture parameters and the chosen post-treatment method. The treated rehydrated BNCs exhibit a stress at break of >30MPa and an elongation at break of about 40%. This makes BNC biomaterials suitable for cardiovascular applications, where the mechanical stress is in the range of 1 MPa.

Microscopic observation of the samples after tensile loading showed failure of several layers. Furthermore, the alignment of the fibers on the surface parallel to the loading direction is clearly visible. However, in deeper layers, the fiber bundles exhibit isotropic orientation. In addition, an increase in sample thickness can be observed.

Effects of humidity and sample geometry

The water content in the fiber network and the number of load-bearing fibers have a significant impact on the mechanical properties of the hydrogels. Thus, the effect of the relative humidity of the different BNC samples and the geometry of the samples on the force response of the material in the uniaxial tensile test will be studied in detail below. The different sample widths were used to systematically analyze the effect of varying the number of load bearing fibers on the mechanical strength of the fiber composite.

To analyze the effect of humidity and geometry on the mechanical behavior of the biomaterial, uniaxial tensile testing was performed according to the method described in the section "mechanical properties". To evaluate the effect of humidity, the samples were not measured in ultrapure water, but in air. These are natural samples with a moisture content of about 98%, dry samples (20% relative humidity) and dry-rehydrated samples (70% relative humidity).

Starting from a standard geometry with an internal width of 5mm, the geometry is then scaled to a thinner internal width (0.3 mm, 0.5mm, 0.75mm, 1mm and 2 mm) by adjusting the elliptical grooves. For samples wider than the clamping area (10 mm), a rectangle with a width of 25mm or 45mm was used. The sample length was always 50mm and the measurement was only performed in the dry-rehydrated state. In addition to the mechanical properties of the breaking force, breaking strain and F-modulus, the work for deforming the sample is given below, the work being determined by the integral of the force-displacement curve.

Different sample conditions result in significantly different force-elongation diagrams. In natural samples of about 7mm to 8mm thickness, little force absorption occurs at 10% elongation. Due to the high moisture content of the porous structure, the fibers are displaced relative to each other at the beginning of load absorption. Only after rearrangement will the applied load cause an increase in longitudinal strain and force response until the sample fails. In the dry sample, fiber rearrangement is almost impossible at microscopic level due to the keratinization induced, which is evident from low elongation at break and almost linear force increase.

The aggregation of the fibers and the additional formation of WBB due to drying resulted in significantly higher breaking strength and greater F-modulus (F-Modul) compared to the natural or rehydrated samples (table 4.1). Table 4.1 shows the characteristic curve of fig. 19 at a relative humidity of about 70%. At the same time, the greater water content allows for significant displacement or rearrangement of the fibers relative to one another as evidenced by the higher elongation at break in the natural or rehydrated sample. Thus, in uniaxial tensile testing, the water content and the density of the fiber network significantly determine the force response.

Table 4.1: mechanical properties and density of BNC samples at different relative humidities (n=25; n=2).

Observations of force-elongation curves of different geometries immediately indicated that the breaking force increased with increasing sample width. This is due to the increasing number of load bearing fibers having larger sample geometries. The total work also illustrates the increase in work consumed by the deformation of the sample. The plateau in the force-elongation curve characterizing the fiber rearrangement process begins with increasing sample width only at higher loads. Since there are more connections in the fiber network as the number of load bearing fibers increases, more force is required to allow for initial displacement of the fibers relative to each other. The expansion of the compressed layered or fibrous structure is also evident in SEM images, for example after uniaxial loading of a 45mm wide sample. The cross section outside the loading zone shows a layered structure in the growth direction of the BNC nonwoven and a dense, gapless surface. In contrast, the cross section loaded by tensile testing shows a cellular structure, which indicates the unfolding of the layer structure and the breaking of the fibers and their interconnections due to the applied load. This is further evidenced by the increase in thickness during the tensile test.

Auxetic behaviour

Materials typically thin under tensile load because they stretch parallel to the direction of stretch. However, there are cases where the cross section becomes large under a tensile load. These materials are known as "auxetic materials" and are characterized by a poisson's ratio v that is negative. They are mainly found in porous materials and composites that allow for volume changes. For isotropic materials, the poisson ratio is between-1 < v < 0.5, while for anisotropic materials the value is not limited. Basically, depending on the anisotropy, high positive or negative values are achievable. Examples are synthetic materials such as foam, ceramic, composite materials or microporous polymers. The auxetic effect is also demonstrated in organic materials. Negative poisson's ratio also occurs in certain directions of the fiber composite. In polymers such as PTFE, the particular microstructure of the node-fibril network provides the auxetic effect. All auxetic materials have non-affine deforming characteristics such as unfolding or untangling. Regarding cellulose, verma et al (2013) reported the auxetic behaviour of paper and TANPICHAI et al (2012) reported the negative poisson's ratio of bacterial nanocellulose. Hereinafter, the thickness of the BNCs during tensile testing is determined experimentally and the poisson's ratio of the materials used herein is deduced therefrom. To determine the thickness of the sample during the tensile test, a simulated laser sensor (IL-S065, keyence Corporation) was mounted perpendicular to the sample surface (see mechanical properties) on the test stand. Recording of the thickness variation was performed simultaneously with the uniaxial tensile test. From the recorded force-elongation curve and the data of the laser sensor, the poisson's ratio v is determined according to equation 4.1.

Where d represents the initial thickness, Δd represents the thickness variation, l represents the initial length, and Δl represents the length variation of the sample during the tensile test. Natural, dry (climatic chamber) and rehydrated BNCs were analyzed. Depending on the different sample conditions (natural, dry and rehydrated), different development of thickness during uniaxial loading was observed. In natural samples, which are characterized by very high moisture content exceeding 98%, water is forced out of the cellular hydrogel structure during the application of the load, resulting in a continuous thinning of the sample. The thickness was reduced by about 70% relative to the initial state until the sample failed (table 4.3). The thickness reduction enters equation 4.1 with a negative sign, resulting in a positive poisson's ratio approaching the value 1. This corresponds to a reduction in volume, which occurs mainly in porous and anisotropic materials, which can be explained by the escape of water. The dry BNC sample showed an increase in maximum thickness of about 730%. Due to the reduced moisture content and the resulting keratinization during drying, additional WBB is formed at the free hydroxyl groups between the fibers, which is considered to be a fixed network point. Verma et al (2013) in the paper considered that these dots of WBB formed at fiber junctions were critical to auxetic behavior. As the paper is stretched, the flexible cellulosic fibers stretch at these network points, moving adjacent fibers perpendicular to the direction of stretch. In dry compressed fibers, this effect is very pronounced, thus resulting in an increased thickness, as no relative sliding between the fibers is possible.

Table 4.3: the thickness is increased: initial and final thicknesses, percent change of natural, dried and rehydrated BNC samples before failure point under uniaxial tensile load (n=6, n=1) and poisson's ratio was calculated.

Also, in the case of rehydrated BNC samples that form irreversible WBBs during drying, this effect can be seen in the same way. Furthermore, the rearrangement process due to the combination of water during rehydration in the plateau region of the force-elongation curve is demonstrated here by a significant, short increase in thickness. Observations of negative poisson's ratio as a function of different strain states of the rehydrated samples also indicate an increase in the expression of thickness increase by 10% at low strain due to the reorientation of the fiber at the beginning of uniaxial tensile load. With greater deformation before failure of the sample, the thickness increase is smaller. Thus, overall, the auxetic material behavior of dried and rehydrated BNCs results from the compressed porous fiber network and its anisotropic material structure under uniaxial tensile loading.

Viscoelastic behavior

Viscoelastic behavior is characterized by the viscosity and elastic deformation of a material. BNCs have amorphous and crystalline regions and a large number of hydroxyl groups connected by WBB. The following experimental analysis of the viscoelasticity of BNC hydrogels composed of BNC fibers and water was performed. Since the biomaterial BNCs are intended for use in complex loading conditions in the human environment, viscoelastic and time-dependent behavior is critical. Multiple cycles of tensile testing, relaxation measurements, and tensile testing as a function of temperature and strain rate were performed for characterization. The multiple cycle tensile test at constant force or stress limit varies greatly depending on the force to which the sample is subjected. At the force limit of 3N, linear viscoelastic behavior is exhibited. however, when the force limit is increased to 8N, a partial plastic deformation can be seen. The same behavior is exhibited for cyclic loads that reach a constant yield strength. Starting from a cyclic load of up to 5% elongation, as the number of cycles increases, less force is required to pull the sample to the corresponding elongation. At the force limit of 3N, the energy absorbed per cycle is significantly continuously reduced. In contrast, at higher force limits, the absorbed energy is significantly suddenly reduced by 60% and 90% between the first and second loading cycles. Most of the energy is absorbed in the first cycle. This demonstrates the energy absorbed per cycle observed under cyclic loading with constant validation stress. It is also worth noting that for a cyclic load of 8N, there is little reduction in absorbed energy between the second and tenth cycles. This is attributable to the microscopic level of fiber rearrangement process that is prevalent in this strain range. One explanation for this phenomenon is that the fibers are redirected or reorganized in the loading direction due to the applied load. Fiber-to-fiber displacement results in irreversible energy absorption. Failure of crosslinking at the microscopic level, which does not lead to macroscopic cracking, ultimately ensures the release of energy and leads to plastic deformation with high ductility of the biomaterial as a whole. The effect of failure of fiber bonds and hydrogen bonds on the microscopic level can also be seen when considering cyclic loads with steadily increasing force limits. The force was increased by 2N per iteration until failure of the sample occurred. The total energy required to fail the sample at the increased cyclic load of the force limit was on average 100mJ lower than the reference without any preload. Thus, the energy required to tear the sample is significantly reduced when considering the elastic component which does not lead to failure. At the higher constant force limit, the absorbed energy steadily increases due to cyclic loading, and the energy correspondingly decreases until the sample eventually fails. The total energy, i.e. the sum of the cyclic preload and the failure measurement, showed only a significant decrease in force from 8N compared to the reference without cyclic loading. The reference measurement and the measurement of the cyclic preload of the sample to 3N force produced the same total energy. Thus, low forces of up to 3N do not result in any significant displacement of the fibers relative to each other and microscopic failure of the associated fiber joint. These findings ensure the use of BNCs as cardiovascular implant materials because no mechanical stress of the extension range (> 8N or 5 MPa) is exerted on them in the human body. For example, according to Zioupos et al (1994), the load on a heart valve prosthesis is below 1MPa.

Relaxation measurements also confirm the viscoelastic character of the biomaterial BNCs.

Relaxation measurement is a method of characterizing the fatigue or recovery of a viscoelastic material by recording the change in recovery force over time of a sample at a constant strain. The material is stretched a specified amount and the stretch remains constant over a period of time. The time course of the initial tension decrease is recorded.

Dynamic fatigue tensile load

In vivo, biological materials are not only subjected to static stress, but also are subjected primarily to cyclic and time-varying loads. This results in fatigue phenomena of the material structure, depending on the type of dynamic load and the repetition rate. Repetition of the same or similar load may result in a significant decrease in intensity compared to a static load. Therefore, the fatigue behavior of the implant material must be studied in terms of the number of load cycles and the subsequent prevailing load in the biological application environment. Therefore, hereinafter, the fatigue behaviour of the biomaterial BNCs under dynamic continuous tensile loading is analyzed. For analysis of fatigue behaviour, an experimental setup of dynamic alternating load was used. The movement is driven by a high performance linear motor (model PS01-23x160H-HP-R, linMot) with linear guide rails (model H01-23x166/180-GF, linMot). For the specimen, a lever and 10:1 from motor to tissue. The ratio from motor to fabric is 10:1. the holder for clamping the sample is connected to a movable aluminium plate, which is connected to the lever by a guiding linear shaft. Eight jigs were each connected to a load cell (U9C type, HBM), and printed using an SLA printer (Form 3, formlabs) made of Resin (Grey Pro Resin, formlabs). The sensor data transmission of the load cell is ensured by a common amplifier (QuantumX MX840B, HBM). To stretch the sample to a prescribed preload, the linear shaft is equipped with a positioning device. The 1.5N preload was manually set before starting the measurement. To prevent the tissue samples from drying out during the test, they were placed in a basin filled with ultrapure water. With the help of software, the sensor data is processed and the motor is controlled. In addition, the measured parameters strain, frequency and repetition rate or duration are determined by software. Samples of the material were prepared according to standard procedures, their fatigue behavior was studied under different parameters (strain, frequency and number of cycles) and finally loaded to failure under uniaxial tensile load. One sample of each test series was analyzed in SEM for changes in fiber network structure caused by dynamic alternating loading. Table 4.7 lists the corresponding mechanical properties. Here, F0 represents the maximum force of the first loading cycle, F _eq represents the saturation force at the end of the measurement period, and the percentage drop represents the decrease in maximum force F0 relative to saturation force F _eq. In addition, the values of the breaking force and work characteristics determined from the uniaxial tensile test are also listed. In each case, a uniaxial tensile test was performed after fatigue tensile loading until the specimen failed. measurements of the different strains (3%, 6% and 10%) were continued for 1 day at a frequency of 5 Hz. The qualitative curves show little difference. However, the eigenvalues obtained indicate a higher percentage of force drop at larger cyclic strains. Similarly, in the tensile test performed later, the breaking force and work are reduced. As expected, greater cyclic strains can cause greater structural damage to the load-bearing fibers. When SEM images are observed, this structural change is revealed by the fiber bundles aligned parallel to the loading direction. Continuous tensile loads at different frequencies (1 Hz, 5Hz and 7 Hz) and 3% constant elongation for 1 day did not produce significant differences. Thus, the speed at which the cyclic load is applied has no effect on the force response of the material. As the number of cycles increases, the measurement length or cycle repetition number (450000, 2000000, and 5000000) results in a lower force response. Similar to the change in strain amplitude, analysis of the mechanical properties showed a greater percentage of force drop with longer loading duration. Also, in the tensile test performed later, the breaking force and work were also decreased (table 4.7). Thus, as the duration of the load is extended, structural damage to the material occurs that is stable and persists.

Table 4.7: the mechanical properties of the cyclic fatigue tensile test (percent decrease in F ₀、F_eq and force response) are a function of strain, frequency and number of cycles and the uniaxial tensile test (breaking force, work) subsequently performed.

Porcine pericardium is a recognized material for implantation applications, showing synchronized force progression under loads of up to 200 ten thousand cycles, as compared to porcine pericardium. Pig pericardium is composed of collagen and elastin and is characterized by a lower percentage of force drop because of the higher recovery of the viscoelastic material under cyclic loading due to the elastic portion of elastin (table 4.7). This comparison shows that the mechanical integrity of the application of BNCs as implant materials is ensured despite the reduced force response at long load durations.

Fracture mechanics

For crack propagation of plant cellulose (load perpendicular to crack propagation), only a small fraction of the work required by BNCs. This may be due to the method of manufacture of the plant cellulose and its associated lower inter-fibre bond formation. In this particular case, the breaking force determining the starting point of crack propagation is about 20N for all samples. Observations of microscopic SEM images clearly show the differences between the loaded and non-mechanically stressed regions of the sample. The deconvolution of the compressed layer structure can be seen by the increase in sample thickness due to the load applied by the uniaxial tensile test. It is apparent that there is a clear transition to the unloading zone. The critical stress intensity factor of bncs gives values of 10MPa to 13MPa vm (yi=2.83) for samples with a notch (5 mm) on one side, according to equation 4.7. Compared to other biological materials, such as wood (kic=10 MPa vm), bone (kic=4 MPa vm) or cartilage (kic=0.08 MPa vm), the fracture toughness of BNCs ensures comparable or in some cases even greater crack propagation resistance. The stress intensity factor for mode I (crack propagation perpendicular to the crack surface) is described by equation 4.7, where σ represents the nominal stress, a represents the crack length, and YI represents the geometric factor.

In addition, lateral shear (i.e., displacement of the crack surface transverse to the crack direction) was studied in addition to vertical crack propagation. The resistance of the incision to further tearing was determined. Hereinafter, according to DIN EN ISO 13937-2:2000-06 a so-called leg tear propagation test was performed to analyze this type of loading of the biomaterial BNCs. Samples of dimensions 38mm x 20mm were used, with an indentation length of 18mm. The legs of the sample were clamped in a special 3D print cradle (Ultimaker, ultmaker) without preload. The tear propagation force FW is determined along the tear propagation distance l _W. This value is always 20mm due to the geometry of the sample. The tear propagation force FW is the tension required to further tear the indentation. The tear propagation force FW of the plants is about 0.5N. The tear force and total energy of the rehydrated BNC tear sample is higher compared to plant cellulose. Furthermore, some layer separation occurs in the rehydrated BNC sample, resulting in a tear propagation of 30% higher than that of plant cellulose. Since in the nano-scale BNC sample the fibers are isotropically distributed in all directions, the fiber connections perpendicular to the forward travel distance affect the material behavior of the neighboring area. The result is a separation of the cellulose layers, rather than a straight further path of travel. SEM images show qualitatively different behavior between linear crack propagation and layer separation. In the case of crack propagation, repositioning and bundling of the fiber bundles is observed in the tensile direction. The effect of the force is locally limited and the beam, which is initially oriented perpendicular to the crack propagation, is oriented parallel to the crack propagation. When layered, the fiber bonds between the continuously formed growth layers during mat synthesis become loose, which is evident by the loose fiber network structure. In areas without tension loading, a tight surface topography is evident, in which individual loose fibers are not contained. Overall, the structure of BNCs exhibits anisotropic material behavior. Fibers in one plane, i.e. perpendicular to the growth direction of the nonwoven, lead to a very uniform crack propagation behaviour. However, parallel to the growth direction, there are structural weak points between the successively formed layers, characterized by detachment of the material from the layers, and this occurs about 60% of the time in the tear propagation test performed.

Puncture behavior

Puncture strength testing is used to determine the puncture or failure characteristics of a material, and thus its strength to withstand point loads. The lancing element is moved at a constant speed in the center of the sample perpendicular to the surface of the sample until a failure occurs. Hereinafter, according to DINISO 7765-2:2009-02 test methods developed for polymer films were modified and various test samples were used to characterize the puncturing behavior of biological material BNCs. The samples were measurement probes with spherical heads of different diameters (0.5 mm, 1mm and 4 mm). Furthermore, surgical needles (40 μm to 280 μm) were used to characterize the penetration behavior in a similar manner. BNCs in the dry state exhibit similar behavior to plant cellulose but have higher failure forces. This can be attributed to the more stable fiber-reinforced structure of BNCs. In the rehydrated BNC samples, the bound water has a significant impact on the lesion deformation. Compared to dry BNCs, water favors larger forces and displacements of the fibers relative to each other, which also results in a significantly smaller increase of the force distribution in the linear region of the force-displacement curve, i.e. lower stiffness. Thus, it is possible for the elongation of the BNCs to a greater extent, so that irreversible structural changes can only be induced after greater deformation. Thus, the total energy required to damage the BNC specimen is much higher than that of plant cellulose. Is very stable to puncture BNCs and exhibits outstanding strength and resistance. The internal microstructure is characterized by an isotropic distribution of nano-scale fibers that facilitates the integrity of the fiber network under point loading. This can be seen when the puncture behavior of the surgical needle is observed with BNCs as compared to aluminum foil. The total energy and failure force of the aluminum foil is an order of magnitude lower. The force steadily drops immediately after the needle is inserted into the aluminum foil. On the other hand, the rehydrated BNCs showed little change after initial insertion. Thus, the internal microstructure of the fiber reinforcement in the punctiform damage areas impedes the propagation of induced failures.

Cut resistance

For cardiovascular implant applications of BNCs combined with metallic stents, the implant is crimped concentrically, e.g., placed in a catheter as part of a minimally invasive implantation. The high cut resistance of BNCs is advantageous compared to stent struts. To characterize this cut resistance, the average behaviour was investigated. The sample was measured as a 9mm long blade striking the sample at 30 ° angle to the center. For further cutting action under a defined preload of the sample, the sample under test (razor blade, length 35 mm) is held in a clamp and moved in a direction perpendicular to the clamping of the sample. For this purpose, a biaxial arrangement is used, allowing simultaneous clamping in x and y directions. The rehydrated BNC samples clamped in the y-direction were prestretched in a defined manner (elongation 10%, 20%, 30%) before measurement. The blade is then moved in a direction perpendicular to the sample. Dry BNCs exhibit similar behavior to plant cellulose in the average test, whereby the total energy of the average titer is higher than the plant cellulose. This represents the high strength of the BNC compact fiber network. The method also shows the effect of water in the rehydrated BNC sample on the behavior of a more flexible material at about the same failure force. This is represented by a damage path that is twice as large. The results of the re-cut test show that as the pretension increases, the total amount of work required to completely cut the sample increases. With greater pre-strain, the cutting of the sample by the blade is induced more rapidly. This is evidenced by a smaller damage path and a steeper force increase at the beginning of the measurement. At greater pre-strain, internal stress in the sample also ensures that less force needs to be applied for initial damage. Rehydrated BNCs exhibit the highest cut resistance compared to plant cellulose and dry BNCs.

Flexural Strength or dimensional stability

Flexural rigidity was compared to porcine pericardium as a bioprosthetic heart valve implant material. BNCs are more resistant to bending than porcine pericardium and can therefore produce a dimensionally stable structure.

To analyze the flexural strength and dimensional stability of BNCs under different process variables, natural (7 day synthesis time), dry BNCs (climate chamber, KS) and dry pressed, rehydrated BNC samples were studied. The high temperature (100 ℃) during drying or pressing indicates an increased formation of hydrogen bonds (keratinization). The additional bonding combined with more compression of the fiber network (i.e., thinner thickness and higher density) results in stiffening of the material. This indicates that the uniaxial tensile load of the material has a higher initial modulus. Similarly, the drop ability of these samples tends to produce a higher drop coefficient in flexural strength studies.

Example 9

The embedding of the hygroscopic exchange material is described as follows:

The functional groups of BNCs allow for changing physical properties by forming hydrogen bonds with other substances. These exchange materials, hereinafter also referred to as stabilizers or plasticizers, ensure a particular protection against keratinization during drying by binding with the free hydroxyl groups of the BNCs. As mentioned above, during the drying process of BNCs, the collapse of the three-dimensional fiber structure and the associated agglomeration of fibers leads to an increase in the formation of inter-fiber hydrogen bonds, which limits the relative movement of the BNC fibers, resulting in stiffer and less flexible material behavior. By introducing the stabilizer before drying, intermolecular forces are reduced and flexibility of the material is improved. The stabilizer not only covers the material surface, but also penetrates into the open-celled BNC matrix. This makes it possible to influence the mechanical force response of the BNCs. Glycerol and polyethylene glycol (PEG) have proven to be effective stabilizers. Both materials are hygroscopic, low toxic and outstanding water-solubility. Both substances have been used as moisturizers and softeners in the cosmetic and textile industries because of their ability to absorb and bind moisture in the environment and thus have a wide range of applications.

Glycerol or glycerol (IUPAC name: propane-1, 2, 3-triol) has one primary hydroxyl group and two secondary hydroxyl groups, and is a trivalent alcohol (formula: C ₃ H₅(OH)₃) from a chemical standpoint. From a physical point of view, glycerol is a clear, viscous liquid, having similar groups due to the hydroxyl groups, with a solubility comparable to that of water and simple fatty alcohols.

PEG (IUPAC name: polyoxyethylene) is a synthetic polymer that is suitable for implantation applications due to its hydrophilicity and biocompatibility. PEG has two terminal hydroxyl groups, and has a molecular formula of H (OCH ₂CH ₂)_n OH. Polyethylene glycol has a molar mass of up to 600g/mol and is a non-volatile hygroscopic liquid at room temperature.

By soaking or rinsing the natural BNC hydrogel in a defined concentration of stabilizer solution, glycerol or PEG molecules diffuse through the pore structure into the BNC fiber matrix. The formation of hydrogen bonds between the stabilizer molecules and the BNC fibers challenges the flowability of the polymer chains. Furthermore, the introduced molecules act as placeholders during drying, thereby increasing the porosity and spacing of the BNC fibers. Depending on the concentration of the selected stabilizer solution and the different molecular weights and hydroxyl content of the material, stable dry BNCs will produce characteristic physical properties upon rehydration.

Hereinafter, the diffusion behavior and binding of hygroscopic exchange material in the native BNCs are analyzed.

Stabilization process and incorporation detection

After static synthesis in an incubator (7 days, 28 ℃), the biomaterials were stabilized with glycerol and PEG400, followed by rinsing with ultrapure water. Thus, the addition of the stabilizer occurs prior to the drying process to prevent hydrogen bonding due to dehydration. Subsequent post-processing follows the standard process (see fig. 20).

Rectangular sample geometry (7 cm. Times.12 cm) was generated and placed in 400ml of stabilizing solution. The solutions were produced from the respective stabilizers glycerol (anhydrous) or polyethylene glycol 400 (PEG 400) and ultrapure water (mil pore Direct-Q5; MERCK CHEMICALS GmbH). BNC samples were rinsed on a shaker (Promax 1020,Heidolph Instruments GmbH&Co.KG) at 140rpm for 24 hours in the respective stabilizing solutions. They are then processed according to standard stabilization procedures (see also fig. 20). After the stabilization process, the virgin square BNC samples were first dried in KS at the respective stabilizer concentrations (1% to 50% by weight) and finally placed in a vacuum dryer (CHRIST ALPHA a-2,Martin Christ Gefriertrocknungsanlagen GmbH) for 24 hours. The samples were then dried in KS. The samples were visually inspected using a transmitted light panel. It can clearly be seen that the stable dried samples show higher transparency than the reference samples which were not previously stable and dried, regardless of the type of stabilizer. The reference sample appeared completely opaque, whereas the transparency was already evident at low stabilizer concentrations. In the literature, the refractive index of BNCs is given as 1.5 to 1.6. The refractive index of the glycerol-water solutions of different concentrations ranges from 1.40 for 50% by weight solutions to 1.30 for very low concentration glycerol solutions. As the concentration of glycerol increases, the refractive index of the solution approaches that of BNC fibers, and thus the composite appears more transparent. Samples treated with 1% by weight stabilizer solution showed incomplete saturation of the material in the center of the sample. There was little difference between the concentrations of 5%, 10% and 20% by weight. At a concentration of 50% by weight of the stabilizer solution, the sample had the greatest thickness, which explains the lower transparency due to the increased interfacial transition. A concentration of greater than 5% by weight gives rise to a softer and more flexible material to the touch than a concentration of less than 1% by weight. The cross section of the sample also shows a very uniform distribution and incorporation of the stabilizer molecules. Electron micrographs of the surface topography showed that the stabilizer at a concentration of 5% by weight had a continuous hydrate shell compared to visual inspection. At a lower concentration of 1% by weight, the individual fiber bundles are clearly visible, which is comparable to the surface topography of the unstabilized reference sample. Thus, the stabilization process of BNCs may occur completely at a concentration of 5% by weight. At concentrations of 10% by weight and above, the stabilizer completely encapsulates the sample even when the cross-section of the sample is viewed, and therefore no individual fibers or fibrils are visible. The same is true for both stabilizers (glycerol, PEG 400). In general, these methods show a uniform incorporation of the substance at a concentration of about 5% by weight, whatever the stabilizer chosen. The diffusion process of substances into the BNC fiber network until the material is saturated is investigated with glycerol and PEG400 solutions having concentrations of 5%, 10% and 20% by weight by means of spectroscopic measurements, such as fourier transform infrared spectroscopy (FTIR). The 95% by weight saturation time (saturation time) of the two stabilizers as a function of the thickness of the natural BNCs is shown in tables 5.1 and 5.2.

Table 5.1: the saturation time of glycerol is a function of the stable solution concentration and the sample thickness at 95% saturation.

Table 5.2: the saturation time of PEG400 is a function of the steady solution concentration and the sample thickness at 95% saturation.

The greater the thickness of the natural BNC sample, the longer the saturation time. Higher concentrations of stabilizing solution do not lead to longer stabilizing times. The saturation time of glycerol is in the range of 200-400 minutes as a function of sample thickness. For PEG400, the saturation time is typically longer (500-700 minutes) than for glycerol. It is apparent that the stabilizer exhibits equal saturation times at about the same sample thickness, but the concentration of the stabilizer solution varies. And thus is independent of the selected concentration of the stabilizer solution. For the same sample thickness. On the other hand, observations of the same solution concentration for different sample thicknesses showed that the saturation time was significantly prolonged as the sample thickness increased. In general, the stabilization of PEG400 results in longer saturation times than the stabilization of glycerol, even though the sample thickness is the same. Glycerol because of the greater molecular weight of PEG400 compared to 92.09g/mol of glycerol. Overall, after extrapolation to the average thickness value of the native BNCs native to BNCs in the range of 6.5nm to 7.5mm, the determined saturation time indicated a stabilization time of 12 hours for glycerol and 24 hours for PEG400, sufficient to ensure complete saturation of the material.

Swelling behavior

The purpose of adding hygroscopic alternatives to virgin BNCs is to prevent keratinization of the material during drying. The embedded stabilizer occupies the free hydroxyl groups of the fibrils, thereby preventing the formation of inter-fibril hydrogen bonds during drying, which are partially washed away during rehydration. This makes it possible for more water to be deposited in the fibrous network structure, thereby giving the material an extraordinary swelling capacity.

Swelling behaviour of stable dry BNCs was quantified by tactile thickness measurement. Stabilization was performed with different concentrations of stabilizer glycerol and PEG400 for 24 hours. After drying at ambient conditions for 72 hours, the BNCs were re-hydrated (24 hours) in ultrapure water. Or a pressing step prior to rehydration. In addition to the absolute thickness value of the sample, the swelling factor (QF) was calculated as the quotient of the rehydration thickness and the dry or pressed thickness. In addition, the water content and water holding capacity are used as indicators of the amount of water stored during rehydration. The stabilization step results in a slight increase in thickness regardless of the concentration of the stabilizing solution and the type of stabilizer. Drying results in a decrease in thickness as the concentration of the stabilizing solution changes. At high concentrations, 100% concentration is only about 10%, while at low concentrations, e.g. 5%, it is as high as 95%. These differences in the dry state have a significant effect on the swelling factor, the higher the concentration of the stabilizing solution, the lower the swelling factor. No difference was observed between the stabilizer glycerol and PEG 400. A significantly higher swelling factor was observed even at very low concentrations compared to the unstabilized reference sample (see fig. 23). For many applications in the biomedical field, such as minimally invasive surgery, low thickness implant materials are useful in a dry, storable state. Thus, after drying, a pressing step is performed to minimize the thickness of the material. The subsequent rehydration shows impressively a change in the swelling factor depending on the stabilizer concentration compared to the stable dry BNCs without the pressing process. The absolute thickness value under compression is in the range of less than 0.2mm, regardless of the concentration considered, and increases by 400% to 1100% due to rehydration. The increase in water binding during rehydration is also reflected in WG and WRV values, which, like the swelling factor, tend to increase with increasing stabilizer concentration (table 5.5).

Table 5.5: the swelling behaviour is changed due to the pressing step after drying to reduce the thickness in the dry state. The thicknesses in the natural, pressed and rehydrated state are shown, as well as the swelling factors (n=16; n=2), WG (n=12; n=1) and WRV (n=12; n=1).

Thus, the stabilizer concentration is selected so that the material can be swelled to a specified thickness by rehydration. This opens up new possibilities in the medical field, such as minimally invasive implants, where due to the limited diameter of the catheter, the implant material is only allowed to have a very small thickness in a dry, storable state. In addition to the swelling behaviour, the incorporation of hygroscopic alternatives into the fiber network of BNCs can also change the mechanical force response in a targeted manner in uniaxial tensile tests. In the following sections, the mechanical properties of stable BNCs after rehydration in glycerol and PEG400 stable dry BNCs at different concentrations were analyzed. The force-strain diagram is analyzed in terms of breaking force and strain, initial modulus and total work analyzed. The analysis was performed in the stabilizers glycerol and PEG400 and selected stabilizing concentrations (3% to 20% by weight) of the stabilizing solution. Stabilization of the material was carried out for 24 hours before drying. Observations of the force-elongation diagram show impressively the change in force profile of the stable dry BNC sample at the beginning of the application of load. The force required to deform the sample is less at a small strain of about the deformation of the sample than an unstabilized reference sample. This manifests itself on the one hand in a much lower initial modulus of 20N to 50N and on the other hand in a generally lower total work required for sample failure. As the concentration of the stabilizing solution increases, both of these eigenvalues tend to decrease. In turn, the breaking force and strain remain almost unchanged. These results can be explained by the greater thickness of the stable dry sample after rehydration. In the preceding section, the thickness variation caused by the stabilization process is explained in the context of swelling behaviour. The fibres can be displaced relative to each other without great force due to leaching of the substance and the resulting increase in moisture in the fibre network. This rearrangement of the fibers is due to the addition of stabilizers during drying which prevent the formation of hydrogen bonds between the fibers. Only after the fibrous structure had been reoriented, starting from an elongation of about 25%, a similar load absorption of the reference and stable dry samples occurred until complete failure. In the case of the reference sample, the keratinization caused by drying limits the realignment or displacement between the fibers, which results in a more rigid material performance at the beginning of the load application. This indicates that its initial modulus is 10 times higher than that of the stable dry sample. Incorporation of hygroscopic exchange material into the fiber network of BNCs prior to drying results in softer or more flexible material behavior over a small load range of up to about 10N. Thus, this concept represents a modification to BNC whereby the biomaterial responds to forces under physiological loading (< 10N).

Example 10

In this example, the biomaterial BNCs are applied to a variety of cardiovascular implants. In addition to being used as a potential vascular graft substitute, a sheath for use as a stent graft is also achieved. With the interest in three-dimensional shaping, a method of producing a seamless tissue composition of BNC for percutaneous aortic valve replacement was developed. By combining locally swellable BNCs, the concept of perivalvular leakage prevention in Transcatheter Aortic Valve Implantation (TAVI) is additionally proposed.

Bacterial nanocellulose as artificial blood vessel implantation material

In vascular surgery, surgical bypass is a potential therapy for treating arterial occlusive disease caused by arteriosclerosis or aortic aneurysm. Vascular grafts replace, bypass, or retain the function of an occluded or diseased vessel. The selected graft is typically taken from the patient (so-called autograft or autograft). However, this requires additional surgery and has limited usability. Thus, the use of synthetic vascular grafts made of polyethylene terephthalate (PET/Dacron) or (swollen) polytetrafluoroethylene (ePTFE) has been established. However, these are only used clinically for vascular grafts with diameters >6mm, as smaller diameters may lead to neointimal hyperplasia or thrombosis due to hemodynamic disturbances. Alternatives to small vessel diameters (< 6 mm) should be provided by natural or synthetic polymers.

In the literature, static synthesis methods, mainly in bioreactors, are used. BNC is formed vertically on a shaped glass cylinder, for example (Klemm et al (2001), yamanaka et al (1990)). However, the length of the graft is limited and a long incubation time (7-14 days) is required. Alternatively, radial synthesis (Hong et al, 2015) was performed on one or more concentrically arranged oxygen permeable silicone membranes, established in (Hong et al 2015,2011, Bao et al 2021). In addition to the long incubation times, low mechanical strength and uneven layer structure strength as well as uneven layer structure should be regarded as disadvantages.

Bioreactor

In the following, BNCs are three-dimensionally shaped in an internal bioreactor under dynamic horizontal rotation of the cylindrical profile. Side writing. For three-dimensional synthesis of BNCs, an exemplary bioreactor consisting of three culture units was developed, rotating the culture surface horizontally under a constant oxygen environment. The bioreactor is based on a modular platform consisting of aluminium profiles and acrylic plates for positioning the individual units. A single unit. The drive device is driven by a gear motor (e.g., 12V, model RB350600-0A101R, reduction ratio 1:600, model load speed 9 rpm). The bearings of the FDM print (Ultimaker 3+, ultimaker) are used as bearings for the motors on the bioreactor module platform. In addition, the motor envelope has a recess, and Hall sensors (Hall-e_ ect Bricklet, tinkerforge) can be used to detect speed. The spring coupling (FKZS, ABP Antriebstechnik GmbH) induces a magnetic field during rotation, which is detected by the hall sensor, thereby cyclically registering each rotation of the shaft. The speed is calculated by software using the data of the hall sensor. A gearbox consisting of a shaft, toothed belt and toothed wheel is used to transmit the motor torque to the individual rotating profiles. The motor is coupled to the main toothed belt shaft by a spring coupling and by brass spur gearsWill be transferred to all three toothed belt shafts. The core of the bioreactor is represented by the rotating profile and the reactor vessel. If not mentioned otherwise, both are made of engineering resins. This is particularly suitable because it has excellent sterilizability. During cultivation, the reaction vessel contains the nutrient medium and bacterial suspension.

Method for manufacturing artificial blood vessel

The production of vascular prostheses is divided into cultivation or synthesis in bioreactors and subsequent post-treatment steps. The cylindrical rotating profile is designed according to the required diameter. For connecting brass gears, the diameter of the two ends of the profile was fixed at 2.8mm. For synthesis, the reaction vessel (100mm x 38mm x 21mm, wall thickness 4 mm) was filled with nutrient medium (36 ml) and bacterial suspension (2 ml) so that the rotating profile was in contact with the medium. As a result of the rotation at 10rpm, BNCs of uniform thickness were formed on the contoured surface. Excess BNCs in the reaction vessel were removed manually at regular intervals after 12 hours. The thickness of the BNC tube depends largely on the duration of the synthesis. In order to obtain a dimensionally stable BNC tube with the smallest possible wall thickness, the cultivation process has proven to be effective with a cultivation time of 3 days. After successful incubation of the BNC tube, it was cleaned in ultrapure water and then, in order to reduce the thickness, placed on a profile in a bioreactor, slowly rotated (3 rpm). Alternatively, the laser cutting is performed in a dry state. After rehydration in ultrapure water for 1 hour, the BNC tube was manually removed from the profile, subjected to a final laser cut, and finally cleaned in 0.1mol NaOH for 24 hours to remove endotoxin.

For end use, BNC tubes were steam sterilized and stored aseptically in 0.5% glutaraldehyde.

Characteristics of

Prototypes of vascular prostheses made from BNCs are described in terms of appearance, mechanical properties, and wall thickness below. In addition, it was also compared with a method of statically synthesizing BNC on a three-dimensional silicon tubular membrane.

In one example, a vascular substitute made of BNC is mounted in a bioreactor on a rotating profile at 10rpmAnd the upper part is carried out for 72 hours. Dimensionally stable tubes with very uniform surface texture are obtained. Another tube with the same parameters was synthesized for 48 hours with the same parameters. This results in a loss of dimensional stability due to reduced cross-linking of the fibrous structure and reduced wall thickness. BNCs synthesized in silicone tubes do not exhibit dimensional stability even after 7 days of synthesis time because the oxygen supply through the silicone membrane is limited in this method. For further evaluation, images were taken using a stereo microscope (SZX 10, zeiss AG) and a scanning electron microscope. In the natural state and in the rehydration state, the BNC oil pipe has a very uniform axial wall thickness. No irregularities are seen in the radial direction. The cross section also shows a uniform and compact fiber structure. Thus, the synthesis of BNC containers disposed in a bioreactor has excellent surface quality and uniformity. In addition to the optical integrity of BNC vascular prostheses, the wall thickness of the tube and its mechanical strength are of decisive importance for their use as vascular prostheses. In the following, BNC tubes were synthesized under different parameters and their wall thicknesses in the natural (Dnative) and rehydrated (Drehy) state were determined. In the natural state, the thickness is measured optically by means of a stereomicroscope image and in the rehydrated state by means of the tactile thickness. For mechanical analysis, a CO ₂ laser was used to generate a tubular ring of 5mm width and loaded in the radial direction until failure. From the force-strain diagram, the failure force (breaking force) and the work required to achieve that force. The corresponding data are shown in Table 6.1. An increase in rotational speed results in a greater natural and rehydrated wall thickness of the BNC tube over the same synthesis time. This is accompanied by an increase in breaking force and mechanical work until failure due to an increase in the number of load bearing fibers. In general, a rotation speed of 10rpm or more is preferable because uneven growth of BNC growth of the BNC tube in the axial direction is observed. The diameter of the rotating profile has no influence on the wall thickness and mechanical properties. This allows the use of BNC tubes regardless of the vessel diameter of the vascular graft and ensures its widespread use in vascular surgery. In addition to the rotational speed, the synthesis time is also a decisive factor in determining the mechanical strength of the BNC tube. As synthesis time increases, natural wall thickness increases, and thus rehydration wall thickness and mechanical properties also increase. Furthermore, starting from a synthesis time of 72 hours, the internal mechanical stability of the fibrous structure leads macroscopically to an exceptional dimensional stability. This was not present during the synthesis time of 48 hours and during the synthesis on the silicone film (7 days). An explanation is provided by considering the fiber density, which is about four times higher in the case of synthesis time of 72 hours in the bioreactor than in the case of synthesis on a silicone membrane. Due to the limited oxygen supply, bacterial activity is reduced, resulting in a reduced density of the fiber network and thus reduced mechanical stability. Less stable fiber networks. Thus, direct contact with oxygen during synthesis in the bioreactor results in significant dimensional stability of the BNC vascular prosthesis. When the synthesis time exceeds 72 hours, wrinkling is evident due to the extremely large natural wall thickness in the rehydrated state. For the synthesis of BNC tubes in a bioreactor, a duration of 72 hours and a rotation speed of 10rpm is preferred at any diameter of the rotation curve.

Table 6.1: the wall thickness of the BNC tubing in the natural state and in the rehydrated state and the mechanical properties (breaking force, work) of the radial tensile test performed in the rehydrated state. N corresponds to the number of synthetic BNC tubes and N is the total number of ring samples produced by these tubes. Annular samples.

EXAMPLE 11 development of stent grafts with BNC Membrane

The term stent graft refers to a stent made of a stable metal framework (stents made of cobalt chrome or nitinol) and synthetic vascular prostheses (typically polymers; PET/ePTFE). In vascular surgery, stent grafts are used to restore blood flow in coronary and peripheral arteries and to treat aneurysms. In addition to bare metal stents and drug eluting stents, stent grafts are widely used for intravascular treatment because the membrane acts as a physical barrier preventing ingrowth of neointimal tissue into the lumen of the vessel, thereby reducing the need for re-intervention due to intimal hyperplasia or stent thrombosis. However, membranes made from most synthetic or electrospun polymers show a tendency to thrombus formation due to lack of blood compatibility, resulting in failure of the prosthesis. Thus, several groups are focusing on methods of making membranes from biological materials, characterized by biocompatibility and the ability to accelerate endothelialization. For example, animal studies using bovine and ovine collagen membranes and stent grafts of pericardial tissue have been conducted. The additional possibility of BNC membranes as antiproliferative drug carriers or to accelerate endothelialization is a promising approach to use BNCs as biomaterials. In the context of this work, the first prototype of a stent graft with BNC membranes was developed by dynamic synthesis in a bioreactor.

Fabrication of stent grafts

To make a stent graft with BNC membrane, a self-swelling nitinol stent with a length of 40mm and a diameter of 8mm was used. The scaffold is placed on the corresponding shaped body prior to synthesis in the bioreactor. In general, in the following, the synthesis of BNC membranes only outside the stent is distinguished from double-sided (i.e. inside and outside) membranes. For the synthesis of the outer film, a cylindrical rotating profile with a diameter of 7.6mm was used. To produce a bilateral membrane, offset profile bodies with smaller diameters (5.6 mm) were designed. Here, the support is in direct contact with the rotating profile only at the ends (contact surface of the rotating profile is 95.5mm ²). The rotating profile for synthesizing the double sided BNC membrane consists of two parts connected by square connectors (2 mm x 2 mm) to allow the removal of the scaffold from the profile immediately after BNC synthesis. The parameters of culture and post-treatment in the bioreactor were based on the parameters from the vascular prosthesis of example 10. Post-treatment drying (spinning in a bioreactor), rehydration and final laser cutting of the stent graft were performed with BNC membranes synthesized externally only on the spinning profile. In this case, the profile body is manually removed at the end of the process before cleaning. In the case of a stent coated with BNCs on both sides, the rotating profile is removed in a natural state, otherwise the BNC film will cause the stent to deform during drying.

Characteristics of

The stent graft synthesized in the bioreactor with BNC membrane was visually evaluated with a stereomicroscope. In addition, a curl test was performed on a film having a diameter of 3mm in a rehydrated state to verify the integrity of the film after curling. In this case, the pore size of the stent will be quantified before and after crimping to exclude the shrinkage of the vessel lumen after implantation. After removal of the rehydration from the stent, the outer BNC membranes in the single-sided sheath stent graft will be subjected to radial mechanical loading and measured by a tensile test. Visual inspection of fully processed, rehydrated stent grafts with BNC film showed an abnormally uniform stent strut coating. There are no discernible irregularities or gaps and this method provides a very uniform wall thickness of the BNC film. In addition, the sheath is stably located on the stent due to radial compression during drying. The double-sided film on the inside and outside is significantly more opaque than the outer film alone. Microscopic images confirm uniform encapsulation of the stent struts. The integrity of BNC membranes is already evident in the as-synthesized natural state and remains intact throughout the process until the final rehydration state. Even radial crimping to a diameter of 3mm does not lead to any damage of the BNC membranes on one and both sides of the stent struts. This was also confirmed by electron micrographs of BNC film morphology. Radial tensile tests were performed on annular samples of BNC outer membranes that were removed from the stent after the treatment was completed. There was no significant difference in breaking force or work compared to tubing made from BNC tubing synthesized without the scaffold in the bioreactor. The direct synthesis of BNC membranes on stent struts does not negatively affect the mechanical strength of BNCs. Pore sizes of scaffolds were determined before crimping (DC 0), immediately after crimping (DC 1) and after one week of storage in a 37℃incubator (DC 2). Six diameter measurements were made for each stent on the opposite stent struts. The reference is a scaffold without BNC membrane. Neither the BNC outer membrane nor both sides result in a significant reduction in pore size. In general, wrapping stent struts with BNC membranes has proven to be a promising concept for achieving a physical barrier to prevent potential restenosis by diffusion into the lumen of a vessel.

Example 12 three-dimensional tissue composition made of BNC for percutaneous aortic valve replacement

Treatment of severe aortic stenosis is typically minimally invasive, i.e., replacement of the native aortic valve. Such catheter-assisted aortic valve implantation (transcatheter aortic valve replacement, TAVR for short) is performed with bioprosthetic heart valves, wherein the tissue component is typically a xenograft of porcine or bovine tissue, attached to a metallic stent. TAVR is largely divided into balloon-expandable prostheses and self-expanding prostheses. In the latter, stents (e.g., nitinol) are used, wherein a tissue element consisting of six sutured individual portions is sutured into the stent. The three leaflets cause the valve function of the implant through the opening and closing process. Three small leaves are fixedly fixed inside the bracket. Traditional manual suturing procedures involve hundreds of individual surgical knots, which are very error-prone, time-consuming and costly. In addition, sutures can form a potential mechanical weakness that results in implant failure. The bioreactor described herein was used to produce seamless three-dimensional tissue components of BNCs for TAVI prostheses. In order to produce three-dimensional tissue elements from BNCs in a bioreactor, modified shaped bodies (also called shaped bodies or shaped articles) are used, on the surface of which synthesis takes place under constant BNCs under constant rotation. To ensure adequate stability of the three leaflets, the synthesis is carried out in a bioreactor for a duration of 4 days. The BNCs of natural three-dimensional shape were dried on the shaped bodies rotated in the bioreactor for 24 hours. After complete drying, the skirt and leaflet edges were three-dimensionally laser cut. The advantage of laser cutting under dry conditions is that the fabric component is prevented from sliding on the profile body. After rehydration, the three-dimensional BNC component is removed from the molded body and fixed in the scaffold after processing steps such as cleaning and sterilization.

To evaluate prototype TAVI with three-dimensional BNC tissue composition, an autolytic nitinol stent was used. The tissue elements are secured by an internal second scaffold. This allows the leaflets to move freely and support the skirt region. The entirely suture-free three-dimensional BNC tissue composition is characterized by an abnormally uniform material structure. The defined shape of the rotating profile allows for a complex small She Jihe shape of the complex leaflet, forming an inwardly directed semilunar capsule. In addition to visual inspection, the closure characteristics of the three-dimensional BNC component were also assessed by a simplified flap closure test. For this purpose, valve closure is produced ex vivo using a fluid column. The prosthesis was placed in a 3D printed stent with a diameter (26 mm) corresponding to the expected diameter of the implanted TAVI prosthesis. A plastic pocket served as a fluid container allowing a fluid column about 10cm above the prototype installed. Complete and symmetrical closure of the valve prosthesis can be observed. Thus, the synthesis of three-dimensional BNC tissue components in a bioreactor represents an innovative, promising concept for the manufacture of functional TAVI prostheses with completely seamless tissue components.

Example 13 prevention of paravalvular leakage in aortic valve prostheses with locally swellable BNCs

One of the most common postoperative complications of percutaneous aortic valve replacement is leakage between the vessel wall and the prosthetic valve due to calcification or incomplete adhesion of the prosthesis to the aortic annulus. Such aortic insufficiency is also known as paravalvular leak (PVL) and results in a2 to 3-fold high mortality rate. PVL causes a non-physiological reflux (regurgitation) of blood from the aorta to the left ventricle of the heart. For the first generation prosthetic valves, the incidence of paravalvular dysfunction after interventional therapy was 15% to 20%. In addition to the goal of smaller catheter diameters and better valve positioning, the design of the new prosthesis also allows for avoiding PVL by components made of plastic (polyester, PET) or biological tissue (porcine tissue) and biological tissue (porcine pericardium). the ratio of moderate to severe shortages has been reduced to 3% to 7%. Even with the new valve design, slight paravalvular regurgitation occurs, however, this occurs in about 30% of cases. In the context of the present invention, a method for manufacturing a TAVI prosthesis with BNC tissue composition, whereby the skirt is characterized by a defined locally swellable region for sealing potential paravalvular insufficiency. To manufacture locally swellable BNCs, stabilization of the material is performed in conjunction with a pressing process. Stabilization with glycerol or PEG400 is performed according to the methods described above to counteract keratinization of the material during drying, thereby ensuring that the material does not swell during rehydration. After complete storage of the stabilizer solution, the stabilizer solution is displaced at defined points during the pressing of the 3D printed molded body, thereby specifically inducing the local keratinization of the BNCs. In these keratinized areas, the swelling process is prevented during rehydration. Subsequent drying is carried out at room temperature (23 ℃) for about 48 hours. During rehydration, the combined stabilizers are rinsed away again partially, which results in a defined swelling of the material according to the geometry of the mould. Finally, cleaning and sterilizing are performed with aqueous sodium hydroxide solution. It should be noted that the process steps of stabilization and pressing may also be performed in the reverse order. In this alternative process variant, the natural BNCs are pressed into a 3D printed PLA mold, and then the stabilizing solution is pipetted into the grooves. The swelling behaviour of the biomaterial BNCs using the stabilizers has been explained above. For stabilization, similar concentrations of 5%, 10% and 20% were used for stabilization. During pressing, BNCs (40 mm x 40 mm) were placed between two platens on the foil. A grid-like 3D stamper (PLA) consisting of struts (1 mm wide) and (5 mm x 5 mm) was placed on the surface of the BNC. To obtain a uniform pressure distribution, additional 3D printed solids (70 mm x 70 mm) and silicon plate (shore 50) were placed on the mold. As a result of the combination of the rigid platen with the partially hollow stamp, BNC regions with stabilizer solution are locally created, which exhibit unidirectional swelling behavior. The applied pressure was increased repeatedly starting from 2N/mm ² for 30 seconds, to 5N/mm ² (5 minutes), and finally to 10N/mm ² (5 minutes) to avoid die damage to the BNC. Observations of the samples after rehydration showed a locally defined, clearly defined swelling in the square groove areas of the stamp. The higher the stabilizer concentration, the greater the swelling capacity. Thickness analysis distinguishing the pillars from squares confirms this visual impression. The swelling factor was calculated from the quotient of the square rehydration thickness and the strut thickness.

It can be seen that the doubling of the stabilizer concentration is accompanied by a doubling of the swelling factor. There was no difference between the stabilizer PEG400 and glycerol. The strut thickness was always below 100 μm, regardless of the concentration of the stabilizer solution. The swelling capacity or thickness of the stabilization square steadily increases with increasing concentration. Thus, depending on the selected concentration of the stabilizing solution, the method of preparing locally swellable BNCs can achieve a defined, locally defined swelling capacity of any geometry. The local swellability of the BNCs explained in the previous section will now adapt to the geometry of TAVI prostheses. To this end, in a first step, a modified 3D compression mold is created, creating localized keratinization in the stent strut areas and ensuring circular swellable areas in the interstices. The latter acts as an extra-stent precaution, since the extra-stent has a ring sealing property, which prevents post-operative paravalvular leakage. PEG400 at a concentration of 20% by weight was used to stabilize the swellable regions. After drying, the BNC component was sutured into the scaffold. Subsequently, the crimping procedure was performed in a dry state for 72 hours. Visual inspection after crimping and rehydration indicated that the material structure was not damaged. The swellable regions are intact and their function is not limited. In addition to visual inspection and assessment of the integrity of the skirt composition after crimping, two aspects related to TAVI prostheses, namely, aspects related to TAVI prostheses, are emphasized. First, the time course of complete swelling is critical to the sealing function, as complications caused by PVL typically occur four weeks before implantation. For this purpose, the swellable regions of the skirt composition have a thickness at different points in time. The thickness increases rapidly after contact with water, reaching a maximum after 1 hour. The thickness of the skirt composition also increased after 21 days of storage. Even after 21 days of storage, the value no longer changed significantly. On the other hand, due to the local swelling of the skirt region, the pore size of the stent should not affect the lumen of the vessel by virtue of the lumen. For this purpose, the diameter of the stent implanted with a diameter of 22mm was determined. The stand is placed in a custom 3D printing device that is filled with water. Furthermore, the device simulates a simple leak. Typically, swelling occurs only in the leakage area, and BNCs swell. Radial force exerted by the stent the radial force exerted by the stent prevents swelling along the simulated aortic annulus in the device. After different points in time, the respective opening diameters of the stents are determined. It can be seen that the orifice diameter is not reduced compared to the dry state. The diameter remained unchanged even after one week of storage. Thus, in general, the application of locally swellable BNCs to skirt components of TAVI prostheses has proven to be an innovative concept in the context of preventing paravalvular leakage in percutaneous aortic valve replacement.

Knowledge gained in the previous section from the simplified skirt composition is now integrated into the conventional skirt geometry of TAVI prostheses. For this purpose, the 3D compression mold is again modified to produce three skirt components, each component having three locally swellable regions. In addition, three leaflets are generated according to a standard procedure. The skirt and leaflet geometry was cut with a laser (CO ₂ laser, epilog Zing, epilog ping) in the dry state. Laser cutting may also be performed in a rehydrated state, if desired. After suturing the six components (skirt and leaflets), the entire BNC fabric component is immobilized in the stent. Thus, TAVI prostheses have BNC tissue components and a locally swellable skirt. The locally swellable regions protrude significantly beyond the diameter of the scaffold and are formed in the prototype by stabilization with 20% by weight PEG 400. The three leaflets were in a natural arrangement to ensure complete closure during the valve closure test. Furthermore, the symmetrical closing behaviour of the three parts is evident. The functionality of the implant is enhanced by sealing the underlying PVL by locally swellable regions. Furthermore, the method allows for a reduction of potential PVL without the use of additional ingredients outside the skirt. Since swelling only occurs after implantation, the catheter diameter is not adversely affected. Overall, prototypes with intact BNC components show a successful integration of the concept of BNC local swelling during the manufacturing process of TAVI prostheses.

In summary, several concepts of using the biomaterial BNC in cardiovascular implants are described. The function of the prototype shows the outstanding potential of the biomaterial BNCs for clinical applications. It has been shown that hygroscopic substances (glycerol, polyethylene glycol) can be introduced into the fiber network such that the mechanical force response of the BNCs systematically changes under load. A method of manufacturing bacterial nanocellulose is described. The described bioreactor is capable of synthesizing BNCs in three-dimensional shapes. This makes possible three-dimensional, seamless tissue compositions made of BNC for transcatheter aortic valve prostheses, vascular prostheses and stent grafts having membranes made of BNC. Furthermore, the production of locally swellable BNCs useful for preventing potential paravalvular leakage of minimally invasive implantable prosthetic heart valves. The application of this concept in aortic valve prostheses is achieved by making the aortic valve prostheses from locally swellable tissue components of BNCs.

The use of bacterial nanocellulose, dried bacterial nanocellulose, pressed dried bacterial nanocellulose, stabilized dried bacterial nanocellulose, rehydrated bacterial nanocellulose in medical, biomedical, cosmetic, packaging industry, paper industry, pharmaceutical industry, water treatment, fluid media filtration, electronic or sensor technology is described.

In particular, the use of bacterial nanocellulose, dried bacterial nanocellulose, pressed dried bacterial nanocellulose, stabilized dried bacterial nanocellulose, rehydrated bacterial nanocellulose in vascular grafts, vascular prostheses, medical implants, vascular implants, stents, stent grafts, cardiac pacemaker covers, heart valves, venous valves, heart valves (prostheses), aortic valves (prostheses), in medical occluders, in tissue patches, as a drug coating or in biosensors is described.

Drawings

Figure 1 shows an electron micrograph of a top view of a bacterial nanocellulose sheet,

Figure 2 shows an electron micrograph of a top view of another bacterial nanocellulose sheet,

Figure 3 shows an electron micrograph of the bacterial nanocellulose sheet of figure 1 in a side view,

Figure 4 shows an electron micrograph of the bacterial nanocellulose sheet of figure 2 in a side view,

Figure 5A shows a photograph of a tubular bacterial nanocellulose,

Figure 5B shows a schematic of a photograph of the tubular bacterial nanocellulose of figure 5A,

Figure 6A shows a photograph comparing two different shaped elements made from bacterial nanocellulose,

Figure 6B shows a schematic view of a shaped element made of the bacterial nanocellulose of figure 6A,

Figure 7 shows a schematic view of a shaped article for a heart valve prosthesis,

Figure 8A shows a schematic side view of a transcatheter heart valve prosthesis comprising bacterial nanocellulose,

Figure 8B shows a schematic diagram of a transcatheter heart valve prosthesis comprising the bacterial nanocellulose of figure 8A in a side view,

Figure 9 shows a schematic of an apparatus for producing bacterial nanocellulose,

Figure 10 shows a detailed schematic of an apparatus for producing bacterial nanocellulose,

Figure 11 shows a schematic view of a stent covered with bacterial nanocellulose,

Figure 12 shows SEM images of plant cellulose fibres and bacterial nanocellulose fibres produced by k.hansenii,

Figure 13 shows the molecular structure of bacterial nanocellulose,

Figure 14 shows the BNC pile and,

Figure 15 shows the sample geometry for a uniaxial tensile test,

Figure 16 shows the force-elongation curve of a dried rehydrated BNC sample,

Figure 17 shows BNC force-elongation diagrams dried with different drying methods,

Figure 18 shows a flow chart of a standard process for producing BNC nonwoven,

Figure 19 shows force-elongation curves for BNC samples with different moisture contents,

Figure 20 shows a flow chart of a standard stabilization procedure for obtaining stable BNCs,

Figure 21 shows electron microscopy images of stabilized BNCs with different stabilizer concentrations,

Figures 22A-C show electron microscope images of BNC samples stabilized with PEG400,

FIG. 23 shows swelling factor as a function of stabilizer concentration for stabilized BNC samples.

Figure 24 shows a flow chart of a standard process for manufacturing BNC tubes,

Figure 25 shows a flow chart of a standard process for manufacturing stent grafts,

Figure 26 shows a flow chart of a standard process for manufacturing 3D BNC components,

Figure 27 shows a flow chart of a standard process for manufacturing locally swellable BNCs,

Fig. 28 shows a schematic view of a pressing apparatus.

Figure 29 shows a 3D stamper which,

Figure 30 shows a picture of a TAVI prosthesis with BNC skirt,

Figure 31 shows a graph of fiber volume and density of BNCs as a function of incubation time,

Figure 32 shows a graph of fiber volume and density of BNCs as a function of bacterial solution and nutrient solution.

Detailed Description

Fig. 1 shows in top view an electron micrograph of a prior art bacterial nanocellulose sheet obtained by a method according to example 1, wherein the bacterial nanocellulose is obtained in a silicone mold.

Fig. 2 shows in top view an electron micrograph of a bacterial nanocellulose sheet obtained by the method according to example 2, wherein the bacterial nanocellulose is produced by using PEEK rods. As compared to fig. 1, it can be seen that the bacterial nanocellulose of example 2 has a higher fiber density than the bacterial nanocellulose of example 2.

Fig. 3 shows in side view an electron micrograph of a prior art bacterial nanocellulose sheet obtained by a method according to example 1, wherein the bacterial nanocellulose is obtained in a silicone mold.

Fig. 4 shows in side view an electron micrograph of a bacterial nanocellulose sheet obtained by the method according to example 2, wherein the bacterial nanocellulose was produced by using PEEK rods. As compared to fig. 3, it can be seen that the bacterial nanocellulose of example 2 has a higher fiber density than the bacterial nanocellulose of example 1.

Fig. 5A shows a photograph of dried and rehydrated tubular bacterial nanocellulose. Fig. 5B shows a schematic diagram of the photograph of fig. 5A.

Fig. 6A shows a comparison of pictures of bacterial nanocellulose obtained according to example 1, left side being a collapsed hollow cylinder, right side being a shape stable hollow cylinder, both being dried and rehydrated. The bacterial nanocellulose according to example 1 collapsed, whereas the bacterial nanocellulose according to example 2b had a stable shape. The BNCs of example 1 were synthesized in silicone hoses and did not show any dimensional stability even after a synthesis time of 7 days, because the oxygen supply through the silicone membrane was limited. The bacterial nanocellulose obtained in example 2b (using a rotary PEEK rod) has higher internal mechanical stability and higher mechanical strength compared to the bacterial nanocellulose according to example 1. Fig. 6B shows a schematic diagram of the picture of 6A.

Fig. 7 shows a schematic view of a shaped article 9 for manufacturing a heart valve prosthesis. The shaped article comprises a holding portion 92 for mounting the shaped article in a holder of the bioreactor. The shaped article comprises a circular skirt 8 and leaflet portions 92 in the shape of heart valve leaflets. The shaped article may also include a focal point for laser cutting. On such shaped articles bacterial nanocellulose can be grown. The shaped article is preferably made of PEEK.

Fig. 8A shows a schematic diagram of a transcatheter heart valve prosthesis 1 comprising bacterial nanocellulose according to the present invention in a side view. The transcatheter heart valve prosthesis 1 comprises a stent matrix 2, the stent matrix 2 comprising metal struts. The outer skirt 3 (also called peripheral sealing shell) and/or the inner skirt (not visible) are made of bacterial nanocellulose and are fixed to the stent base 2, for example by gluing or stitching using threads (for example polytetrafluoroethylene threads). The outer and/or inner skirt 3 adjoins heart valve leaflets 5, for example three heart valve leaflets, which may be made of bacterial nanocellulose or pericardial tissue.

Fig. 8B shows a schematic top view of a heart valve prosthesis comprising the bacterial nanocellulose of fig. 8A. A heart valve 5 (e.g., three heart valve leaflets) is secured to the stent base 2. For example, the heart valves 5 may be formed from bacterial nanocellulose sheets or pericardial tissue, each valve opening or closing according to the blood flow forces acting on it.

Fig. 9 shows a schematic view of an apparatus 6 for producing bacterial nanocellulose. The apparatus comprises at least one (here three) culture vessel 7 for receiving a culture medium for bacterial nanocellulose-producing bacteria in which bacterial nanocellulose can be produced. The apparatus further comprises more than one (here three) rotary units 8 for rotatably mounting the shaped articles 9, respectively. The apparatus comprises more than one shaped article 9, each rotatably mounted by a rotary unit 8. The rotation unit 8 is based on a belt drive driven by a motor 10 to rotate each shaped article within its respective incubation container.

Fig. 10 shows a detailed schematic of an apparatus for producing bacterial nanocellulose, which shows a culture vessel 7 for receiving a culture medium of bacterial nanocellulose-producing bacteria and for receiving a shaped article 9. The shaped article 9 is rotatably mounted by a rotary unit 7. The rotation unit 7 may be driven by a motor (not shown) to rotate the molded article in the culture container. The shaped article is rod-shaped and comprises a support 11 on its surface. When the shaped article is rotated together with the scaffold structure, a covered scaffold can be obtained, wherein the scaffold structure is covered with the obtained bacterial nanocellulose.

Fig. 11 shows a schematic view of a scaffold 11 covered with bacterial nanocellulose 12. The strut portions of the stent are embedded within the cellulose. The stent may be made of nitinol struts. The stent may have a diameter of 7.6mm and a length of 87mm

Fig. 12 shows SEM images comparing the fiber size ranges of (a) plant cellulose and (b) bacterial nanocellulose of bacterial strain k.hansenii. Bacterial nanocellulose fibres of bacterial strain k.hansenii are in the range of 30nm to 60 nm.

Fig. 13 shows the molecular structure of bacterial nanocellulose consisting of adjacent Anhydroglucose (AGU) units. These AGU units are covalently linked to each other by a-1, 4-glycosidic bond. The cyclic glucose monomers are covalently bonded to each other by a polycondensation reaction between the hydroxyl group on the carbon atom C-1 of one glucose unit and the C-4 of the adjacent glucose unit. Each glucose unit, also called AGU, is rotated alternately about 180 °. Two adjacent AGU units form a disaccharide cellobiose, which is considered to be a repeating cellulose unit. At the end of the C1-carbon atom, cellulose exhibits a reducing function due to rearrangement of the hydroxyl groups to aldehyde groups. At the other end (C-4), a non-reduced alcoholic hydroxyl group was found. Depending on the source, the chain length or degree of polymerization varies between 300 and 10000AGU, all properties of cellulose being present between 20 and 30 units. Each AGU has hydroxyl groups on the carbon atoms C-2 (secondary), C-3 (equatorial) and C-6 (primary). Due to the strong electronegativity of oxygen, their partially positively charged hydrogen atoms form intermolecular and intramolecular hydrogen bonds (fig. 2.4). Hydroxyl groups and bridging oxygens produce a fibrous composite material having crystalline and amorphous regions.

Fig. 14 shows the natural BNC-fluff obtained after 7 days of synthesis according to example 2 a.

FIG. 15 shows the sample geometry for a uniaxial tensile test according to the modified German ISO 527-2 standard (type 1 BA). The total length of the BNC was 50mm and the width of the handle portion was 10mm. The distance between the shoulders of the standard gauge section was 30mm and the width was 5mm.

Fig. 16 shows a schematic of different process variants for bacterial nanocellulose post-treatment. The individual process steps and the parameters investigated in each case are listed.

Fig. 17 shows force-elongation diagrams of different drying methods of BNCs obtained according to example 2 a. GT represents BNCs freeze-dried for 72 hours and KS represents BNCs dried in a climatic chamber with a relative humidity of 10% at 23 ℃. A significant increase in initial modulus was observed at higher drying temperatures (in an oven at 100 ℃). The elongation at break and the force modulus do not show any significant difference, whereas the force at break is significantly increased due to the drying process compared to the natural sample.

Fig. 18 shows a flow chart of a standard process for producing nonwoven from BNCs. r.f. stands for relative humidity. The process sequentially comprises the following steps:

BNC-growth (culture)

2. Drying BNC

3. Compression dried BNC

4. Clean dried and pressed BNC

5. Rehydrating the dried, pressed and cleaned BNC

The cultivation was performed under static conditions using the growth medium and nutrient solution described in example 2 a.

Figure 19 shows force-elongation curves for BNC samples with different relative humidity/moisture content. For dry BNCs, the relative humidity is 20%. For rehydrated boron nitride, the relative humidity was 70%. The relative humidity of natural BNCs is 98%. The aggregation of the fibers and the additional hydrogen bonding formed during drying results in significantly higher breaking strength (breaking force) and higher F-modulus (F-Modul) compared to the natural or rehydrated sample.

Figure 20 shows a flow chart of a standard stabilization procedure for BNC post-treatment to obtain stabilized BNCs. r.f. stands for relative humidity. The process comprises the following steps (in sequential order):

BNC-growth (culture)

Stabilization of BNC

3. Drying of stabilized BNC

4. Pressing the stabilized and dried BNC optionally cleans the stabilized, dried and pressed BNC (flow chart not shown)

5. Rehydrating (clean) stabilized, dried and pressed BNC

After incubation (static synthesis in incubator (7 days, 28 ℃) and subsequent rinsing with ultrapure water, the biological material was stabilized with glycerol and PEG 400. Thus, the addition of the stabilizer occurs prior to the drying process to prevent hydrogen bonding due to dehydration.

Figure 21 shows electron microscopy images of coating BNC fiber networks with glycerol at different glycerol stabilizer solution concentrations.

Fig. 22A, B, C shows electron microscopy images of BNC samples stabilized with PEG400 after cobalt thiocyanate staining. FIGS. 22 (a) - (c) show cross-sections of samples, with the bottom two photographs showing the surface of the samples as a function of stabilizer concentration.

Figure 23 shows a plot of swelling factor of stabilized BNC samples as a function of stabilizer concentration (3 wt%, 5wt%, 10 wt%, 20 wt% glycerol or PEG 400) after rehydration, as a function of material thickness in the compressed state. The control group represented an unstable sample.

Figure 24 shows a flow chart of a standard process for manufacturing BNC tubes. The process sequentially comprises the following steps:

1. BNC synthesis in bioreactor (molded article Using rotating rod)

2. Drying BNC in bioreactor (molded article Using rotating rod)

3. Rehydration of dry BNC

4. Laser cutting rehydrated BNC

5. Clean laser cut BNC

6. Sterilizing the cleaned BNC

Such BNCs can be used as vascular grafts.

Figure 25 shows a flow chart of a standard process for manufacturing a stent graft with BNC membranes. The method comprises the steps of:

1. BNC synthesis in bioreactor (using a rotating rod-like article covered with a scaffold)

2. Drying BNC

3. Rehydration of dry BNC

4. Laser cutting rehydrated BNC

5. Clean laser cut BNC

6. Sterilizing the cleaned BNC

In the case of BNCs synthesized only outside the stent, the post-treatment steps of drying, rehydration and laser cutting of the stent graft can be performed on the shaped article (rotating in the bioreactor). In the case of a stent coated with BNCs on both sides, the shaped article is manually removed prior to drying.

Fig. 26 shows a flow chart of a standard procedure for manufacturing three-dimensional tissue elements made of BNCs (preferably using the shaped article of fig. 7 for percutaneous aortic valve replacement). The process sequentially comprises the following steps:

1. BNC synthesis in bioreactor (use of shaped articles)

2. Drying BNC in bioreactor

3. Laser cutting of dry BNC

4. Rehydration of dry BNC

5. Clean rehydration BNC

6. Sterilizing the cleaned BNC

The drying of the natural three-dimensional shaped BNCs occurs on the shaped article which is rotated in the bioreactor. After complete drying, the skirt and leaflet edges were three-dimensionally laser cut. The advantage of performing the laser cutting under dry conditions is that the fabric component on the shaped article is prevented from slipping. After rehydration, and after final processing steps such as cleaning and sterilization, the three-dimensional BNC component is removed from the molded article.

Figure 27 shows a flow chart of a standard process for manufacturing locally swellable BNCs. The process sequentially comprises the following steps:

BNC Synthesis

2. Stabilization using glycerol and/or polyethylene glycol

3. Pressing stable BNC in 3D die

2. Dry pressed and stabilized BNC

3. Rehydration of dried, pressed and stabilized BNC

5. Clean rehydrated BNC

It should be noted that in alternative processes, the process steps of stabilization and pressing may also be performed in reverse order (thus pressing first and then stabilization).

Fig. 28 shows a schematic view of a pressing apparatus for producing locally swellable BNCs. The pressing apparatus comprises two platens (upper platen 100 and lower platen 700), a 3D press 400 (optional solid body 300), a pressure compensation layer 200 and optionally a foil 600 on the lower platen 700. The BNC 500 is located between the two platens 100, 700 on the foil 600. The 3D stamper 400 (e.g., made of PLA) is placed on the surface of the BNC 500. For uniform pressure distribution, a solid 300 (e.g. made of PLA) and a pressure compensation plate 200 (shore hardness 50, e.g. silicone pad) are also placed on the stamp 400. The stabilization zone using the stabilizer solution can be created locally in the BNC due to the rigid platen in combination with the die having grooves or openings.

Fig. 29 shows a 3D stamper. The 3D stamper comprises grooves or openings for creating locally swellable regions. Such a die may be used to make a TAVI skirt.

Fig. 30 shows a picture of a TAVI prosthesis 20 with a BNC skirt 21, the skirt 21 having locally swellable regions 22 (here ridges between the struts of the prosthesis). These locally swellable regions may provide a sealing function upon rehydration (the picture shows the rehydrated state).

Figure 31 shows a graph showing fiber volume and density of BNCs as a function of incubation time. Bacterial nanocellulose obtained from K.hansenii (incubation time 3 to 10 days) consists of nanocellulose fibres with a density in the range of 1.100g/cm ³ to 1.500g/cm ³, preferably 1.30.+ -. 0.10g/cm ³.

Figure 32 shows a graph of fiber volume and density of BNCs as a function of bacterial solution and nutrient solution. Bacterial nanocellulose obtained from k.hansenii (bacterial solution: nutrient solution 1:6 to 1:96) consisted of nanocellulose fibres with a density in the range of 1.0g/cm ³ to 1.35g/cm ³. A density of 1.30.+ -. 0.10g/cm ³ was obtained using a 1:18 bacterial solution/nutrient solution (see example 2 a).

Claims

1. Method for producing a shaped element made of bacterial nanocellulose, comprising the following steps

-Providing a shaped article of manufacture,

Providing a growth medium for bacterial nanocellulose and a nutrient solution for said bacteria, said medium comprising Komagataeibacter of Komagataeibacter hansenii bacteria of the family Acetobacter, preferably in the form of a bacterial suspension,

2. The method of claim 1, wherein the Komagataeibacter Komagataeibacter hansenii bacteria of the family aceraceae are Komagataeibacter hansenii, american Type Culture Collection (ATCC) code 53582.

3. The method according to claim 1 or 2, wherein rotating the shaped article is performed at a rotational speed of at most 60 rpm.

4. The method of any of the preceding claims, wherein rotating the shaped article is performed at a speed of 10 to 60 rpm.

5. The method according to any one of the preceding claims, wherein the nutrient solution comprises at least one monosaccharide and/or one disaccharide, at least one peptone and a yeast extract, and wherein the growth medium has an acidic pH value.

6. The method according to any one of the preceding claims, wherein the nutrient solution comprises or consists of glucose, peptone, yeast extract, disodium hydrogen phosphate and citric acid.

7. The method of claim 5 or 6, wherein the peptone is soybean peptone.

8. The method of any one of the preceding claims, wherein the ratio of the bacterial suspension to the nutrient solution is 1:18.

9. The method of any of the preceding claims, wherein the shaped article is made of a polymer.

10. The method of claim 9, wherein the polymer does not contain Si-O groups.

11. The method of claim 9 or 10, wherein the polymer has a polymer backbone containing alternating ketone and ether groups.

12. The method of any one of claims 9 to 11, wherein the polymer comprises polyetheretherketone.

13. The method according to any one of the preceding claims, wherein 40% to 60%, preferably 50% of the surface of the shaped article is in contact with the growth medium.

14. The method according to any of the preceding claims, wherein the method is performed in an oxygen-containing environment, preferably in air.

15. The method of any of the preceding claims, wherein rotating the shaped article is performed at a temperature of 23 ℃ to 30 ℃ for at least 30 hours.

16. The method of any of the preceding claims, wherein rotating the shaped article is performed at a temperature of 26 ℃ to 30 ℃ for 48 hours to 114 hours, preferably at a temperature of 26 ℃ to 28 ℃.

17. The method according to any one of the preceding claims, wherein the method further comprises the step of drying the obtained shaped element made of bacterial nanocellulose to obtain a dried shaped element made of bacterial nanocellulose.

18. The method of claim 17, wherein the drying step is performed in air.

19. The method of claim 17 or 18, wherein the drying step is performed in air during rotation of the shaped article.

20. The method according to claim 19, wherein the drying step is performed in air during rotation of the shaped article at a rotational speed of less than 10rpm, preferably less than 5 rpm.

21. The method according to any one of claims 17 to 20, wherein an additional step of treating the obtained bacterial nanocellulose with at least one structural stabilizer is performed before the drying step, to obtain a stable shaped element made of bacterial nanocellulose.

22. The method according to claim 21, wherein the at least one structural stabilizer comprises or consists of glycerol and/or polyethylene glycol, preferably comprises 5 to 50% by weight of glycerol and/or polyethylene glycol.

23. The method according to any one of claims 17 to 22, wherein the method further comprises the step of treating the obtained bacterial nanocellulose with a hydroxide solution before or after the drying step.

24. The method of any of the preceding claims, wherein the shaped article is a rod, a rotationally symmetrical body, or a medical implant.

25. The method of any of the preceding claims, wherein the shaped article is covered by a stent, a prosthetic heart valve, a polymer frame, a metal frame, or a metal alloy frame.

26. The method of claim 25, wherein after obtaining bacterial nanocellulose, the shaped article is removed from the stent, heart valve prosthesis, polymer frame, metal frame, or metal alloy frame.

27. A shaped element made of bacterial nanocellulose, preferably produced by the method of any one of claims 1 to 21, consisting of bacterial nanocellulose fibers having a diameter of 30nm to 60nm and a density of 1.0g/cm ³ to 1.5g/cm ³, preferably 1.29g/cm ³ to 1.31g/cm ³.

28. A shaped element made of stabilized and dried bacterial nanocellulose, preferably produced by the method of claim 27, having a refractive index of 1.30 to 1.40 and/or a density of 1.29g/cm ³ to 1.31g/cm ³.

29. Profiled element according to claim 28, having a breaking strength in the range 40N to 63N and/or a tensile strength of more than 30MPa and/or an elongation at break in the range 30% to 45% and/or an F-modulus in the range 130N to 200N.

30. A profiled element obtained by the method of any one of claims 1to 26.

31. Use of a shaped element of bacterial nanocellulose produced by the method according to any one of claims 1 to 23 in biomedical applications, preferably for vascular grafts, antibacterial films, medical implants, medical stents, cardiac pacemakers or leadless pacemaker covers, prosthetic valves, prosthetic heart valves, prosthetic venous valves, transcatheter heart valve prostheses, stents, stent grafts, tissue patches, drug coatings or biosensors.

32. An apparatus for producing bacterial nanocellulose comprising

At least one rotating profile and/or at least one shaped body, rotatably mounted on the rotating unit,

At least one drive unit with a gearmotor,

At least one rotary unit driven by a gearmotor,

Optionally at least one evaluation unit and/or control unit for evaluating and/or detecting the rotational speed of the at least one rotating profile and/or the at least one molded body.

33. The device of claim 32, wherein the gear unit comprises at least one shaft, at least one toothed belt, and at least one gear.

34. Device according to claim 32 or 33, wherein the device comprises at least one detection unit for detecting the rotational speed of at least one rotating profile and/or at least one shaped body.

35. The apparatus of claim 34, wherein the at least one detection unit comprises at least one hall sensor.

36. A method for producing bacterial nanocellulose, comprising the steps of:

-Culturing the growth medium to obtain bacterial nanocellulose.

37. The method of claim 36, wherein Komagataeibacter Komagataeibacter hansenii bacteria of the family aceraceae are Komagataeibacter hansenii, american Type Culture Collection (ATCC) code 53582.

38. The method of claim 36 or 37, wherein the nutrient solution comprises or consists of glucose, peptone, yeast extract, disodium hydrogen phosphate and citric acid.

39. The method of any one of claims 36 to 38, wherein the ratio of the bacterial suspension to the nutrient solution is 1:18.

40. The method according to any one of claims 36 to 39, wherein the method is performed in an oxygen-containing environment, preferably in air.

41. The method of any one of claims 36 to 40, wherein the culturing of the growth medium is performed at a temperature between 23 ℃ and 30 ℃ for at least 30 hours to obtain bacterial nanocellulose.

42. The method according to any one of claims 36 to 41, wherein the culturing of the growth medium is performed at a temperature of 26 ℃ to 30 ℃ for 48 hours to 114 hours, preferably at a temperature of 26 ℃ to 28 ℃.

43. The method of any one of claims 36 to 42, wherein the culturing is performed in the dark.

44. The method of any one of claims 36 to 43, wherein the growth medium is contacted with at least a portion of the shaped article.

45. The method of any of claims 36 to 44, wherein the shaped article comprises or consists of a polymer.

46. The method of claim 45, wherein the polymer does not contain Si-O groups.

47. The method of claim 45 or 46, wherein the polymer comprises a polymer backbone comprising alternating ketone and ether groups.

48. The method of any one of claims 45-47, wherein the polymer comprises polyetheretherketone.

49. The method according to any one of claims 45 to 48, wherein the shaped article is a rod, a rotationally symmetrical body or a medical implant, such as a stent or a heart valve prosthesis.

50. The method of any one of claims 45 to 49, wherein the method further comprises the step of drying the obtained bacterial nanocellulose to obtain dried bacterial nanocellulose.

51. The method of claim 50, wherein the drying step is performed in air.

52. The method according to claim 50 or 51, wherein an additional step of treating the obtained bacterial nanocellulose with at least one structural stabilizer is performed to obtain a stabilized bacterial nanocellulose before the drying step.

53. The method of claim 52, wherein the at least one structural stabilizer comprises or consists of glycerol and/or polyethylene glycol, preferably comprises 5% to 50% by weight of glycerol and/or polyethylene glycol.

54. The method of any one of claims 36 to 53, wherein the method further comprises the step of treating the bacterial nanocellulose with a hydroxide solution before or after the drying step.

55. The method of any one of claims 36 to 54, wherein the method further comprises the step of compacting the bacterial nanocellulose before, during or after the drying step to obtain compacted bacterial nanocellulose.

56. The method of claim 55, wherein a pressure of 2N/mm ² to 40N/mm ², preferably 10N/mm ², is applied.

57. The method according to claim 55 or 56, wherein the pressing step is performed for more than 5 minutes, preferably 15 minutes.

58. The method according to any one of claims 55 to 57, wherein the pressing step is performed at a temperature between 20 ℃ and 90 ℃, preferably 50 ℃.

59. The method of any one of claims 50 to 58, wherein the method further comprises the step of rehydrating after drying and/or pressing.

60. Bacterial nanocellulose, preferably prepared by the method according to any one of claims 36 to 49, consisting of nanocellulose fibres having a diameter of 30nm to 60nm and/or a fibre density of 1.30±0.10g/cm ³.

61. A medical implant comprising the bacterial nanocellulose of claim 60.

62. A stable and dry bacterial nanocellulose, preferably produced by the method of claim 53, having a refractive index in the range of 1.30 and 1.40 and/or a density between 1.0g/cm ³ and 1.5g/cm ³, preferably 1.29g/cm ³ and 1.31g/cm ³.

63. The stable and dried bacterial nanocellulose of claim 62 having a breaking strength in the range of 40N to 63N and/or a tensile strength exceeding 30MPa and/or an elongation at break in the range of 30% to 45% and/or an F modulus in the range of 130N to 200N.

64. A medical implant comprising the stable and dried bacterial nanocellulose of claim 62 or 63.

65. A dried bacterial nanocellulose obtained by the method of any one of claims 51 to 54.

66. A medical implant comprising the dried bacterial nanocellulose of claim 65.

67. A stabilized bacterial nanocellulose obtained by the method of claim 54.

68. A medical implant comprising the stabilized bacterial nanocellulose of claim 65.

69. A pressed bacterial nanocellulose obtained by the method of claim 55.

70. A medical implant comprising the pressed bacterial nanocellulose of claim 69.

71. A rehydrated bacterial nanocellulose obtained by the method according to claim 59.

72. A medical implant comprising the rehydrated bacterial nanocellulose of claim 65.