Fabrication of three-dimensional components from bacterial cellulose

Abstract

The present invention relates to a method for generating a three-dimensional component from bacterial cellulose, comprising the steps of: providing a support structure (2) comprising an outer surface (2a), the support structure (2) allowing passage of oxygen therethrough as well as through the outer surface (2a), providing a layer (3) arranged on said outer surface (2a) of the support structure (2), the layer (3) being permeable to oxygen, immersing the support structure (2) and the layer (3) arranged thereon at least partially in a nutrient solution (4) comprising bacteria capable of forming bacterial cellulose in the presence of oxygen, and passing oxygen into the support structure (2) so that oxygen permeates through the layer (3) allowing bacteria in the nutrient solution to form a component that is comprised of bacterial cellulose and is arranged on an outer surface (3a) of the layer (3).

Description

[0001] Fabrication of three-dimensional components from bacterial cellulose

[0002] The present invention relates to a method and a device for producing three-dimensional components from bacterial cellulose.

[0003] For the production of a three-dimensional cellulose fleece, rotationally symmetrical matrices are usually rotated on the surface of a nutrient solution, since cellulose can only be synthesized by the bacteria there due to the oxygen present. The structure is complicated and the cultivation of non-rotationally symmetrical or complicatedly shaped components is made difficult.

[0004] Based on the above, the problem to be solved by the present invention is to provide an improved method and device for generating a component, particularly a nonwoven, formed out of cellulose. Particularly, it is an objective of the present invention to allow producing a three-dimensional, rotationally symmetrical or non-rotationally symmetrical cellulose nonwoven, particularly for use in a vascular implant.

[0005] This problem is solved by a method having the features of claim 1 and a device having the features of claim 14. Preferred embodiments of these aspects of the present invention are stated in the corresponding dependent claims and are described below.

[0006] According to claim 1 a method for generating a three-dimensional component from bacterial cellulose is disclosed, the method comprising the steps of:

[0007] - providing a support structure comprising an outer surface the support structure allowing passage of oxygen therethrough as well as through the outer surface, - providing a layer (directly) arranged on said outer surface of the support structure, the layer being permeable to oxygen or an oxygen containing gas and optionally to an inert gas (e.g. nitrogen),

[0008] - immersing the support structure and the layer arranged thereon at least partially in a nutrient solution comprising bacteria capable of forming bacterial cellulose in the presence of oxygen, and

[0009] - passing oxygen or an oxygen containing gas into the support structure so that the oxygen or the oxygen containing gas permeates through the layer allowing bacteria in the nutrient solution to form a component comprised of bacterial cellulose and arranged on an outer surface of the layer, and optionally passing the inert gas (e.g. nitrogen), into the support structure (2) so that the inert gas permeates through the layer (3) allowing a stop of the growth of bacterial cellulose.

[0010] In order to generate the three-dimensional component with varying material properties (e.g. varying thickness and / or density) of bacterial cellulose either the layer has varying thicknesses or areas of the layer are passed through with oxygen or the oxygen containing gas and other areas of the layer are passed through with the inert gas or a mixture of the inert gas and oxygen or a mixture of the inert gas and the oxygen containing gas, and / or the oxygen or oxygen containing gas or inert gas pressure is varied.

[0011] The oxygen containing gas may be air. The inert gas may be nitrogen and / or at least one noble gas (e.g. helium, neon, argon, krypton, xenon).

[0012] Optionally a cyclical variation (pulsation) of the oxygen or oxygen containing gas pressure can lead to an increased material exchange (nutrition solution, oxygen) in the growth layer and thus to a faster layer growth or to a denser layer.

[0013] By producing matrices, e.g. a layer formed from an oxygen-permeable material, which are immersed in a nutrient solution and exposed to oxygen, intricately shaped components of bacterial cellulose can be produced on the surface of such matrices. The matrices do not have to be moved along the surface of the nutrient solution but can be completely immersed. Thus, in a preferred embodiment, the support structure is stationary (i.e. is not moved) after having been immersed in the nutrient solution and is in particular only moved once the component has been generated in order to remove the component from the support structure.

[0014] According to a preferred embodiment, the component is a hollow component, particularly hollow nonwoven, enclosing the support structure and the layer arranged thereon before removal of said component / nonwoven from the support structure.

[0015] According to a preferred embodiment of the method, the layer is completely submerged in the nutrient solution. Thus, the outer surface of the layer determines the shape of the cellulose component. Preferably, the component is a nonwoven.

[0016] The growth medium for bacterial (nano)cellulose comprises (nano)cellulose producing bacteria, preferably a bacterial suspension of the (nano)cellulose producing bacteria, and the nutrient solution. The selected bacterial strain, the composition of the nutrient medium used, and the ratio of bacteria to nutrient medium during inoculation have a significant influence on the properties of the bacterial (nano)cellulose.

[0017] The (nano)cellulose producing bacteria may be gram-negative microorganisms (Gluconacetobacter, Azotobacter, Rhizobium, Pseudomonas, Salmonella, Alcaligenes) or gram-positive microorganisms (Sarcina ventriculi). Preferably Gluconacetobacter like Gluconacetobacter xylinus (G. xylinus, also called Acetobacter xylinum), Gluconacetobacter pasteurianus (G. pasteurianus) or Acetobacteraceae bacteria of the genus Komagataeibacter and the species Komagataeibacter hansenii or K. hansenii may be used. The Acetobacteraceae bacteria of the genus Komagataeibacter and the species Komagataeibacter hansenii may be Komagataeibacter hansenii with the American Type Culture Collection (ATCC) code 53582.

[0018] The nutrient solution comprises a carbon source as well as nitrogen providing source. Peptone and yeast extract may be provided as nitrogen source, which ensures good cell growth. The peptone may be a soybean peptone. The choice of carbon source significantly determines the yield as well as characteristic properties of the cellulose pellicle. The monosaccharide and / or disaccharide acts as carbon source. The monosaccharide and / or disaccharide can be glucose, fructose, sucrose or mannitol.

[0019] The acidic pH value can be obtained by using citric acid. Aerobic, gram-negative bacteria are efficiently fermented at a pH of 3 to 7 and in a temperature range of 25°C to 30°C. The metabolism of some carbohydrates leads to the side production of gluconic acid, which lowers the pH of the medium and thus has a negative effect on cellulose yield. However, the presence of antioxidants and polyphenolic compounds inhibits the formation of gluconic acid and is realized by adding disodium hydrogen phosphate and citric acid as buffers in the culture medium. The solvent of the bacterial suspension and / or the nutrient solution may be (purified) water.

[0020] The nutrient solution may comprise at least one monosaccharide and / or one disaccharide, at least one peptone and yeast extract, and wherein the growth medium has an acidic pH value. More preferably, the nutrient solution may comprise glucose, peptone, yeast extract, disodium hydrogen phosphate, and citric acid or consists of these. Most preferably, the growth medium for bacterial (nano)cellulose comprises a bacterial suspension, preferably comprising Acetob acteraceae bacteria of the genus Komagataeibacter and the species Komagataeibacter hansenii or K. hansenii, and a nutrient solution comprising 20 g / 1 glucose, 5 g / 1 peptone, 5 g / 1 yeast extract, 2.7 g / 1 disodium hydrogen phosphate, and 1.5 g citric acid. The ratio of the bacterial suspension to the nutrient solution may be between 1 : 16 and 1 :20, preferably 1 : 18.

[0021] The cultivation of the growth medium may be carried out at a temperature between 23°C and 30°C, for at least 3 Oh, to obtain bacterial (nano)cellulose. The cultivation of the growth medium may be carried out for 48 hours to 114 hours at a temperature between 26°C and 30°C, preferably at a temperature between 26°C and 28°C. The cultivation may be carried out in the dark.

[0022] In a further method step, the obtained bacterial (nano)cellulose can be dried and / or pressed.

[0023] A (partial) dehydration by drying or pressing changes the morphology of the biomaterial. The drying is preferably carried out at air, optionally with 3 rpm for 24h. The bacterial (nano)cellulose can be removed from the shaped article before or after drying.

[0024] In order to obtain a swellable bacterial (nano)cellulose, the obtained bacterial (nano)cellulose can be conserved by at least one structure-stabilizing substance before drying. Thus, the method can for example comprise a further step of treating the obtained bacterial cellulose with a solution comprising glycerol and / or polyethylene glycol before drying the bacterial (nano)cellulose. Bacterial (nano)cellulose is formed at the interface between the oxygen or the oxygen containing gas and the nutrient medium. Therefore, the method has to be carried out by using oxygen or an oxygen containing gas, preferably air.

[0025] According to yet another preferred embodiment of the method, the support structure may be flexible for easy removal of the component from the support structure and outer layer arranged thereon. Particularly, the support structure can be elastically deformable.

[0026] Furthermore, according to a preferred embodiment of the method according to the present invention, the support structure is a monolithic support structure.

[0027] According to a further preferred embodiment, the support structure is porous, wherein particularly the support structure comprises open porosity for the passage of oxygen through the support structure.

[0028] Furthermore, according to a preferred embodiment of the method, the support structure is connected to a conduit and the oxygen is passed through the conduit (e.g. from an oxygen source) into an internal space defined by the support structure, particularly said internal space is formed by interconnected pores of the support structure.

[0029] Preferably, in an embodiment, the nutrient solution is arranged in a container sealed by a lid. This allows one to prevent contamination of the nutrient solution with foreign organisms or impurities more easily. Furthermore, in a preferred embodiment of the method, the conduit extends through said lid in a sealed fashion, thus allowing to transport oxygen into the support structure when the latter is residing in the nutrient accommodated in said container. The layer being permeable to oxygen may be a polymeric or ceramic layer. In a preferred embodiment of the method, the layer is a formed out of a polysiloxane which is permeable to oxygen and allows oxygen to diffuse therethrough.

[0030] In a preferred alternative embodiment of the method, the layer is formed out of a hydrophobic fabric (e.g. an expanded polytetrafluoroethylene (ePTFE) like Goretex) or a superhydrophobic fabric. In the framework of the present invention, a hydrophobic fabric is understood to be a fabric that repels water such that a water droplet sitting on a surface of the hydrophobic fabric forms a contact angle with the surface of the hydrophobic fabric being larger than 90°. In case of a superhydrophobic fabric this contact angle is larger than 150°.

[0031] In a preferred embodiment, preferably in case the hydrophobic or superhydrophobic fabric is used as a layer arranged on the support structure, a pressure of the oxygen residing in the support structure does not exceed a hydrostatic pressure of the nutrient solution, particularly so as to avoid that the oxygen bubbles through the layer. Rather, by preferably keeping the pressure of the oxygen so as to not exceed said hydrostatic pressure, forming of an undisturbed oxygen interface on the outer surface of the fabric is promoted that allows the growth of a more homogenous component / nonwoven formed of bacterial cellulose.

[0032] Furthermore, the layer may comprise a thickness in the range from 1 pm to 1000 pm, preferably 10 pm to 300 pm, more preferably 10 pm to 80 pm. When a silicone mold or hose having no support structure is used, the silicone mold or hose has to have a certain thickness (e.g. 1 mm to 3 mm) in order to provide sufficient mechanical stability for depositing the cellulose on the silicone mold. Using a support structure enables using a thinner layer (e.g. of silicone) being permeable to oxygen. Thus, using thinner oxygen permeable layers the production time for the bacterial (nano)cellulose may be reduced.

[0033] Furthermore, in a preferred embodiment of the method according to the present invention, the three-dimensional component is one of a tube, a vascular graft, a medical implant, a vascular implant, a cover or a part of a cover for a cardiac pacemaker, a cardiac valve, a venous valve, a medical occluder or a tissue patch. According to yet another aspect of the present invention, a device for generating a three- dimensional component (particularly nonwoven) from bacterial cellulose is disclosed, wherein said device comprises:

[0034] - a support structure comprising an outer surface the support structure allowing passage of oxygen therethrough as well as through the outer surface,

[0035] - a layer arranged on said outer surface of the support structure, the layer being permeable to oxygen or an oxygen containing gas and optionally to an inert gas (e.g. nitrogen),

[0036] - a container filled with a nutrient solution comprising bacteria capable of forming bacterial cellulose in the presence of oxygen, wherein the support structure and the layer thereon are configured to be at least partially immersed in said nutrient solution residing in the container, and

[0037] - an oxygen source and a conduit connected to the oxygen source and in flow connection with the support structure so that oxygen or the oxygen containing gas can be passed from the oxygen source via the conduit into the support structure immersed in the nutrient solution to allow oxygen or the oxygen containing gas to permeate through the layer, thus allowing bacteria in the nutrient solution to form a component that is comprised of bacterial cellulose and is arranged on an outer surface of the layer , and optionally an inert gas (e.g. nitrogen) source connected to the conduit (7) and in flow connection with the support structure (2) so that inert gas (e.g. nitrogen) can be passed from the inert gas (e.g. nitrogen) source via the conduit (7) into the support structure (2) immersed at least partially in the nutrient solution (4) to allow a stop of the growth of bacterial cellulose.

[0038] According to a preferred embodiment of the device, the layer is made of a polysiloxane. Alternatively, the layer is preferably formed out of a hydrophobic (e.g. an expanded polytetrafluoroethylene (ePTFE) like Goretex) or a superhydrophobic fabric (see also above).

[0039] The device according to the present invention can be further characterized by the features and embodiments described herein in conjunction with the method according to the present invention. In the following embodiments of the present invention as well as further features and advantages of the present invention shall be described with reference to the Figure, wherein

[0040] Fig. 1 shows a schematic cross-sectional illustration of an embodiment of a device and method according to the present invention for producing a component, particularly a nonwoven, out of bacterial cellulose.

[0041] Fig. 2 shows a schematic cross-sectional illustration of another embodiment of a device and method according to the present invention for producing a component, particularly a nonwoven, out of bacterial cellulose.

[0042] Fig. 1 shows a preferred embodiment of a device 1 for generating a three-dimensional component from bacterial cellulose. The device 1 comprises a support structure 2 with an outer surface 2a. The support structure 2 as well as the outer surface 2a are configured to allow passage of oxygen therethrough. The support structure may be a porous structure. A layer 3 being permeable to oxygen is arranged on said outer surface 2a of the support structure 2. The support structure 2 is connected with an oxygen and nitrogen source 8 preferably via a conduit 7, so that oxygen and nitrogen can be passed from the oxygen source and nitrogen 8 via the conduit 7 into the support structure 2 and can permeate through the layer 3 being permeable to oxygen and nitrogen. The device further comprises a container 5 suitable for being filled with a nutrient solution 4 comprising bacteria capable of forming bacterial cellulose in the presence of oxygen. The support structure 2 and the layer 3 being permeable to oxygen thereon are configured to be at least partially immersed in said nutrient solution 4 residing in the container 5. The container 5 may comprise a lid 6 through which the conduit 7 extends.

[0043] Fig. 2 shows a preferred embodiment of a device 1 and method according to the present invention for producing a component, particularly nonwoven, out of bacterial cellulose.

[0044] The device 1 comprises a support structure 2 consisting preferably of an open-celled porous, but preferably mechanically stable (elastic or inelastic), material that is provided with a (thin) layer 3 of a (viscous) resin (e.g. a silicone / polysiloxane) arranged on an outer surface 2a of the support structure 2. For this, the support structure 2 can e.g. be dipped into a (viscous) resin (e.g. a silicone / polysiloxane resin) and pulled out again. Alternatively, the (viscous) resin (e.g. a silicone / polysiloxane resin) can be sprayed onto the support structure. The (viscous) resin (e.g. a silicone / polysiloxane resin) may be a 2-component (viscous) resin. The (viscous) resin (e.g. a silicone / polysiloxane resin) may be diluted by a (suitable) organic solvent in order to adjust the viscosity of the resin. After the (viscous) resin (e.g. silicone / polysiloxane) has cured, the resulting coated support structure 2 is preferably evacuated and then filled with pure oxygen via a conduit 7 being in flow connection to an oxygen source 8. The connection to the oxygen source 8 is then maintained so that the amount of oxygen required for synthesis can flow freely.

[0045] The prepared support structure 2 with said gas permeable layer 3 (e.g. a silicone / polysiloxane layer) thereon is immersed in a nutrient solution 4 residing in a container 5 that is sealed by a lid 6 through which the conduit 7 extends. Diffusion of the oxygen into the nutrient solution 4 produces a cellulose nonwoven on the outer surface 3a of said silicone layer 3. Preferably, the conduit 7 comprises an end section 70 into which an end portion of the support structure 2 is inserted to form the flow connection between the conduit 7 and the support structure 2. The conduit 7 and the end section 70 may be a modular construction, e.g. the end section 70 may be interchangeable with another end section having a different geometrical shape. Thus, geometrically different shapes of cellulose can be produced within one device. It is to be understood that the connection between the conduit 7 and the interchangeable end section 70 needs to be sealed in a gas tight manner.

[0046] The generated nonwoven / component formed out of bacterial cellulose can then be removed from the outer surface 3 a of the layer 3. When using a sufficiently flexible base material for the support structure 2, removing the cellulose component from the support structure 2 and layer 3 arranged thereon is ideally possible by evacuating the support structure 2 thereby compressing it to a smaller size which promotes easy removal of the cellulose component / nonwoven from the support structure 2 and layer 3.

[0047] In a preferred alternative embodiment of the present invention, instead of a gas diffusion membrane / layer 3 formed out of polysiloxane, an e.g. close-meshed fabric 3 made of a hydrophobic (e.g. an expanded polytetrafluoroethylene (ePTFE) like Goretex) or superhydrophobic material can also be used. In this case, the oxygen pressure in the internal space of the support structure 2 is preferably adjusted so as to not exceed the hydrostatic pressure of the nutrient solution 4, so that the oxygen does not directly bubble through the fabric 3 into the nutrient solution 4, but forms an interface on the outer surface of the fabric 3 that promotes forming a component / nonwoven out of bacterial cellulose. The remaining steps can be conducted as described above with respect to the first embodiment that uses the polysiloxane layer 3.

[0048] The present invention achieves in an advantageous fashion that the support structure 2 and layer 3 thereon do not have to be moved with respect to the nutrient solution but can be completely immersed and kept stationary. Another advantage is that the container containing the nutrient solution can be sealed. Contamination with foreign organisms / impurities can thus be more easily avoided. Furthermore, the invention allows to generate cellulose components / nonwovens having a complex three-dimensional shape that do not need to be rotationally symmetric.

[0049] Further, by applying an element such as a stent or another structure to the layer 3, this element can be directly encased by the generated cellulose nonwoven.

Claims

Claims1. A method for generating a three-dimensional component from bacterial cellulose, comprising the steps of:- providing a support structure (2) comprising an outer surface (2a), the support structure (2) allowing passage of oxygen therethrough as well as through the outer surface (2a),- providing a layer (3) arranged on said outer surface (2a) of the support structure (2), the layer (3) being permeable to oxygen or an oxygen containing gas and optionally to an inert gas,- immersing the support structure (2) and the layer (3) arranged thereon at least partially in a nutrient solution (4) comprising bacteria capable of forming bacterial cellulose in the presence of oxygen, and- passing oxygen or an oxygen containing gas into the support structure (2) so that the gas permeates through the layer (3) allowing bacteria in the nutrient solution to form a three-dimensional component that is comprised of bacterial cellulose and is arranged on an outer surface (3a) of the layer (3), and optionally passing the inert gas, into the support structure (2) so that the inert gas permeates through the layer (3) allowing a stop of the growth of bacterial cellulose characterized in that- the layer (3) has a varying thickness, or- that areas of the layer are passed through with oxygen or the oxygen containing gas and other areas of the layer are passed through with the inert gas or a mixture of inert gas and oxygen or a mixture of inert gas and the oxygen containing gas, and / or- that the oxygen or oxygen containing gas or inert gas pressure is varied.

2. The method according to claim 1, wherein a pressure of the oxygen or the oxygen containing gas residing in the support structure (2) does not exceed a hydrostatic pressure of the nutrient solution (4).

3. The method according to claim 1 or 2, wherein the support structure (2) is a monolithic support structure (2) and / or porous.

4. The method according to one of the preceding claims, wherein a gas pressure of the oxygen or the oxygen containing gas is cyclically varied.

5. The method according to one of the preceding claims, wherein the support structure (2) is connected to a conduit (7) and the oxygen or oxygen containing gas is passed through the conduit (7) into the support structure (2).

6. The method according to one of the preceding claims, wherein the nutrient solution (4) is arranged in a container (5) sealable by a lid (6).

7. The method according to claim 5 and 6, wherein the conduit (7) extends through said lid (6).

8. The method according to one of the preceding claims, wherein the layer (3) is formed out of a polysiloxane.

9. The method according to one of the claims 1 to 7, wherein the layer (3) is formed out of a hydrophobic fabric or a superhydrophobic fabric.

10. The method according to one of the preceding claims, wherein the support structure (2) is flexible for easy removal of the three-dimensional component from the support structure (2) and the layer (3) arranged thereon.

11. The method according to one of the preceding claims, wherein the oxygen containing gas is air and / or the inert gas is nitrogen and / or at least one noble gas.

12. The method according to one of the preceding claims, wherein the layer (3) comprises a thickness in the range from range from 1 pm to 1000 pm, preferably 10 pm to 300 pm, more preferably 10 pm to 80 pm.

13. The method according to one of the preceding claims, wherein the three-dimensional component is one of a tube, a vascular graft, a medical implant, a vascular implant, a cover or a part of a cover for a cardiac pacemaker, a cardiac valve, a venous valve, a medical occluder or a tissue patch.

14. A device (1) for generating a three-dimensional component from bacterial cellulose, the device (1) comprising:- a support structure (2) comprising an outer surface (2a), the support structure (2) allowing passage of oxygen therethrough as well as through the outer surface (2a),- a layer (3) arranged on said outer surface (2a) of the support structure (2), the layer (3) being permeable to oxygen or an oxygen containing gas and optionally to an inert gas,- a container (5) filled with a nutrient solution (4) comprising bacteria capable of forming bacterial cellulose in the presence of oxygen, wherein the support structure (2) and the layer (3) thereon are configured to be at least partially immersed in said nutrient solution (4) residing in the container (5), and- an oxygen source (8) and a conduit (7) connected to the oxygen source (8) and in flow connection with the support structure (2) so that oxygen or the oxygen containing gas can be passed from the oxygen source (8) via the conduit (7) into the support structure (2) immersed at least partially in the nutrient solution (4) to allow oxygen or the oxygen containing gas to permeate through the layer (3), thus allowing bacteria in the nutrient solution (4) to form a component that is comprised of bacterial cellulose and is arranged on an outer surface (3a) of the layer (3), and optionally an inert gas source connected to the conduit (7) and in flow connection with the support structure (2) so that inert gas can be passed from the inert gas source via the conduit (7) into the support structure (2) immersed at least partially in the nutrient solution (4) to allow a stop of the growth of bacterial cellulose.

15. The device according to claim 14, wherein the layer (3) is a formed out of a polysiloxane, or wherein the layer (3) is formed out of a hydrophobic or a superhydrophobic fabric.

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