DE102009024133A1 - Bacterial nanocellulose for cartilage regeneration - Google Patents

Abstract

Bacterial nanocellulose comprises a structure consisting of a wide-meshed phase (1) of the bacterial nanocellulose and at least one close-meshed phase (2) of the bacterial nanocellulose firmly connected with wide-meshed phase. Independent claims are included for: (1) use of bacterial nanocellulose, where the bacterial nanocellulose is used without drying after the cultivation- and purification-process as initial-wet implant for cartilage formation; and (2) producing the bacterial nanocellulose, comprising generating the structure in a cultivation step of the cultivation process as a two-phase implant.

Description

The Invention relates to a special bacterial nanocellulose (BNC) for use as a scaffold for a particular In vivo colonization by cartilaginous cells for training of cartilage tissue. The invention is used for example for correction traumatic and degenerative damage of cartilage tissue, like damage to articular cartilage, by application of a initial cell-free and non-absorbable material in the cartilage defect. For this serves a network, which of the local cells of cartilage colonized and filled with newly formed cartilage matrix becomes.

cartilage has a very low potential for natural regeneration of traumatic or degenerative defects.

It are different possibilities or approaches to Regeneration of cartilage defects known, for. B. after Differentiate the type of materials used. That's one Classification in cell-containing and cell-free as well as in absorbable and non-absorbable materials possible.

A cell-containing material treatment method used in practice is autogenous chondrocyte transplantation, briefly referred to as ACT (e.g. Brittberg et al .: Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation, Engl J Med, 1994, 331, 889 or T. Minas: Autologous chondrocyte implantation in the arthritic knee, Orthopedics, 2003, 26, 945 ). In this case, the patient is a healthy cartilage tissue piece removed from the unloaded area of the joint and the cartilage cells are propagated under autologous conditions and then added as a cell suspension in the defect. The ACT has several disadvantages, such as in particular use only in certain regions of the knee, "leakage" of the cell suspension and hypertrophy of the chondrocytes or the periosteum.

To circumvent these problems, "tissue engineered" cartilage grafts based on resorbable polymers have been developed to combine the regenerative potential of autologous cartilage cells with stabilizing resorbable biomaterials to provide even distribution of cells, better initial mechanical stability, promote tissue maturation, and to achieve easier handling by the surgeon. In this matrix / biomaterial-assisted ACT, in vitro propagated autologous cartilage cells are introduced into hyaluronic acid membranes, into collagen membranes or into resorbable polymers (polyglycolic acid and co-polymers of polyglycolic acid and polylactic acid) (for example S. Nehrer et al .: Three-year clinical outcome of age chondrocyte transplantation using a hyaluronan matrix for cartilage repair, Eur J Radiol, 2006, 57, 3 ; P. Gehrens et al .: Matrix-associated autologous chondrocyte transplantation / implantation (MACT / MACI) -5-year follow-up, Knee, 2006, 13, 194 ; C. Ossendorf et al .: Treatment of posttraumatic and focal osteoarthritic cartilage defects of the knee with autologous polymer-based three-dimensional chondrocyte grafts: 2-year clinical results, Arthritis Res Ther, 2007, 9, R41 ). The resorbable materials, eg. B. on the basis of hyaluronic acid or collagen, however, have significant disadvantages due to poor fixability, especially for the positioning and fastening (sewing process) of implants, with too low dimensional stability. The more stable synthetic polylactide-co-glycolide (PLGA) and polyglycolide (PGA) materials can elicit chronic inflammatory cell reactions when degraded.

One particular problem of all resorbable materials is that the speed and type of scaffolding degradation of the implant be synchronized with the building speed of the cartilage need to perform the function of the cartilage to be regenerated to ensure. These processes are at the individual Patients highly variable and also age-dependent. In principle, however, one can assume that resorbable materials already degraded after about 4 to 6 weeks. The formation of new intact and functional cartilage tissue is complete but over a period of several months.

Cell-free hydrogels were the first biomaterials to be designed rationally for human use (Review: J. Kopecek, J. Yang: Hydrogels as Smart Biomaterials, Polymer International 2007, 56/9, 1078 ). They are usually characterized by very good biocompatibility, which is partly due to their high water content. However, hydrogels often have inadequate mechanical stability, which has limited their applications. The already known nonabsorbable biomaterials based on hydrogels of synthetic polymers such as polyvinyl alcohol are not accepted by the organism as an in vivo scaffold, ie no endogenous cells migrate into such implants. In contrast, it has been shown that non-absorbable bacterial nanocellulose (BNC) is a material that, as a result of numerous studies, shows very good tissue and blood compatibility and is rapidly and permanently colonized by cells (eg endothelial cells, fibroblasts). At the same time, the nanofibers of the BNC have strengths in the range of steel and Kevlar (Review: D. Klemm et al.: Nanocelluloses as Innovative Polymers in Research and Application, Adv. Polym. Sci. 2006, 205, 49 ).

In the EP 1953174 A1 becomes the application of oxidized bacterial nanocellulose as a tissue substitute. Oxidized BNC is, however, resorbable and offers by their degradation no permanent, but only a temporary support structure, so that this material for the present specific use for the construction of permanently dimensionally stable cartilage tissue is not suitable.

WO 2007/064881 A2 and WO 2007/146946 A2 describe the use of BNC for the repair and replacement of soft and hard tissues. In both cases, after biosynthesis, the BNC is subjected to intensive mechanical treatment to remove the components derived from the culture medium. In addition, the BNC in the case of WO 2007/064881 A2 brought into the final implant form by drying and compressing. In the described procedure, it can be assumed that the network structure of the BNC is severely damaged and is no longer suitable for cell infiltration, in particular for the formation of a cartilage tissue to be generated.

It is also known ( WO 2008/079034 A2 ), bacterial nanocellulose preferably used as a wound dressing or for the treatment of hernias. Continuous surfaces of BNC, but also surfaces with artificially generated holes are described. The diameters of these holes are in the range of 0.08-0.5 cm in each case at a distance of 0.7-2.5 cm. Our own investigations have shown that this network structure of the BNC is not sufficiently populated with cartilage cells. The artificially generated holes do not represent a three-dimensional structure because of the average diameter for the cells which are very small in relation to the hole size, but a two-dimensional surface which promotes dedifferentiation processes in chondrocytes. This network with the holes of the said size and distances generated therein therefore does not seem suitable for the formation of a corresponding cartilage replacement.

In WO 2005/018435 thermally modified BNC is described. The temperature in the BNC is lowered to below 0 ° C and the water by pouring, blotting, pressing or the application of vacuum partially or completely removed. Possible fields of application for this material are the tissue replacement and the use as a filler or "surgical" network in surgery, plastic surgery and neurosurgery. In the drying method, which is not explicitly disclosed, however, the risk of damage to the BNC network structure, for example due to thermal and / or mechanical stress, can not be ruled out, so that for use in the generation of cartilage tissue, the colonization of the BNC network with cells inhibited or at least not negligibly affected. Nonthermal drying methods for removing water, such as squeezing, can also cause damage to the BNC network structure.

The use of BNC as an in vitro / ex vivo cell carrier is described in DE 2003-10361898 and in A. Svensson et al .: Bacterial cellulose as a potential scaffold for tissue engineering of cartilage, Biomaterials, 2005, 26 (4), 419-431 described. However, this type of application is possible only in vitro. In addition, it has been shown that with this method, only a superficial (two-dimensional), but not in-depth (three-dimensional) colonization of the network is achieved. Therefore, the formation of a functional cartilage matrix is not expected. About a related use of such a carrier for in vivo cartilage formation is also known in the art nothing.

It is also known (for example CR Rambo et al .: Template assisted synthesis of porous nanofibrous cellulose membranes for tissue engineering, Materials Science & Engineering, C: Biomimetic and Supramolecular Systems, 2008, 28 (4), 549-554 ) to introduce optical fibers or fibers of polystyrene vertically into the BNC. So BNC could be produced with pores between 60-300 μm. Another disadvantage is the drying process and the associated danger of damage to the BNC network structure. Another problem is the deep-seated colonization of the network, since a colonization of cells exclusively in the surface area of the pores of the BNC network structure does not lead to the generation of complete and high-quality cartilage tissue. The danger of damage to the BNC network structure by the said drying process further restricts this field of application.

Furthermore, the imitation of cartilage-like properties by the construction of Doppelhydrogelnetzwerken, z. B. by the combination of BNC with gelatin, has been described (for example A. Nakayama et al .: High Mechanical Strength Double-Network Hydrogel with Bacterial Cellulose, Adv. Funct. Mater. 2004, 14, 1124-1128 ). However, composites, which can temporarily increase, for example, the stability of the BNC network, adversely affect the space for the colonization of the network structure, so that the generation of cartilaginous structures is at least temporarily limited. Even resorbable composites, such as gelatin, require a considerable amount of time to break down, during which time the colonization of the network structure is inhibited.

The invention is therefore based on the object, a universally usable dreidimensiona To create a network that has both a durable, scaffold-forming stability and ensures the fastest possible and homogeneous colonization with cartilage-forming cells for the formation of functional cartilage tissue in vivo.

The The task is solved by building up a bacterial nanocellulose, consisting of a wide-meshed phase of the BNC and at least one solved with this firmly connected close-meshed phase of the BNC, which can be used as an implant for cartilage regeneration. Such a construction is generated in a cultivation step, and the material is preferably as so-called initial-moist (i.e., without complete drying) scaffold for in particular in vivo colonization by cartilaginous cells in order Training of cartilage tissue used.

It has been shown to build with the proposed layered network system a said framework for the generation of cartilaginous tissue represents, on the one hand due to the at least one closely meshed Phase (low water content) a favorable dimensional stability and thus an immediate and consistent "scaffold-supporting" effect for the wide-meshed phase (high water content) and thus guaranteed for implant use. moreover This close-meshed phase ensures good surgical handling, d. H. it gives the BNC the necessary strength to do so these can be cut and also easily fixed, in particular sewable, is. On the other hand, offers the wide-meshed phase of the implant structure Excellent conditions for an immediate, uninhibited and homogeneous in vivo colonization by cartilage-own cells of the patient, without first degrading supporting elements of the implant must be before these spaces as settlement locations are usable. The invention advantageously combines the known positive properties of bacterial nanocellulose (especially very good tissue and blood compatibility, fast and permanent colonization by cells) with the characteristics a stable shape and the support function of a scaffold for Colonization with cells. These new characteristics of the bacterial Nanocellulose can also be reached in a cultivation process. The proposed construction of bacterial nanocellulose allows the use as an implant for cartilage regeneration and avoids due to its special properties the ones described above Disadvantages of known methods.

by virtue of the absence of cellulases in the human organism, is the BNC not absorbable, so that no substances are released cause the chronic inflammatory cell reactions could. Furthermore, the problem of synchronization on the type and speed of scaffolding removal with the building speed of the cartilage to warrant circumvented the function of the cartilage to be regenerated. The inventive BNC gives the implant long-term stability right from the start and at the same time acts as a stable three-dimensional framework, which is filled up by the formed regeneration tissue becomes.

By the gentle treatment of BNC in the removal of the Culture medium-derived components after biosynthesis without structurally destructive Drying and / or mechanical treatment steps remains the BNC nanowire structure, which allows the BNC to handle large volumes of fluid to bind, completely preserved. Unlike hydrogels synthetic polymers allows this BNC after implantation, especially by the presence of the wide-meshed phase, the immigration mainly located on the edge of the implant local cartilage precursor or cartilage cells in vivo. These BNC cells offers the BNC one three-dimensional framework structure, by training homogeneous and fully functioning cartilaginous tissue can.

BNC is sterilizable and biocompatible. In a Sterile packaging is the initial moist material, preferably in water, stable for at least one year, preferably at temperatures at 4 ° C and in the dark. The storage can as well after gentle drying at room temperature. By suitable Drying methods (eg freeze drying, critical point drying) the structure of the BNC according to the invention is preserved, thus ensuring complete re-usability remains.

A Such BNC is considered a cell-free, bioactive, permanently stable Implant for the regeneration of cartilage defects, for example in larger joints, proposed. Since they are not autologous cells is a previous biopsy not necessary for the patient. The implant can be either arthroscopic (minor defects) or surgery directly in place (big defect) easy and uncomplicated to be used. It forms due to its biphasic invention BNC network a dimensionally stable hydrogel structure and allowed the passing or immigration and settlement of ions, organic Molecules and cells. Its high strength allows reliable surgical handling and use different fixation techniques (eg cartilage suture, adhesive bonding, Fixation with pins / nails).

The implant may additionally contain cytokines or growth factors which enhance the immigration and proliferation of local cells into the body Promote defect-filling implant and stimulate the development of full-quality cartilage matrix. The BNC implant represents a long-term stable framework structure, which is filled up by a fully functional, newly forming cartilage tissue. This cell-free implant is long-term storable and easy to ship. It can be produced in large quantities and, if needed, used directly and quickly for surgical intervention.

to Use of the implant for cartilage regeneration in cartilage damage after diagnosis (eg by MRI, arthroscopy) the cartilage damage the patient either classic by opening the joint or arthroscopically exposed. If necessary, a Smoothing of the cartilage lesion or the arthritic Defective. Depending on the size of the cartilage defect area, the surgeon can choose from a range of prefabricated BNC implants and select a BNC implant of appropriate shape and Insert size into the defect. The fixation The BNC implant is made by sewing with the marginal Cartilage shoulder using resorbable suture material. It is the same Gluing the implant with surgical adhesives, z. Fibrin livers, and anchoring the implant in the underlying bone by means of resorbable pins / nails possible. To Suturing the surgical opening should be the patient due to the relatively gentle procedure (no injury intact Cartilage areas for the production of chondrocytes as in the ACT) and the mechanical stability of the implant after a short time Rest phase can burden the joint again.

The Invention will be described below with reference to the drawing Embodiments explained in more detail become.

In show the drawing:

1 : Schematic representation of the intended for implant use biphasic BNC

• A) Besiedlung der engmaschigen Netzwerkstruktur des BNC-Netzwerkes
• B) Besiedlung der weitmaschigen Phase des BNC-Netzwerkes

2 : Migration of chondrocytes into the BNC network

• A) Colonization of the dense network structure of the BNC network
• B) Colonization of the wide-meshed phase of the BNC network

3 : In vitro model for the endogenous regeneration of a cartilage defect with a BNC network fitted in it

4 : Early stages of cartilage matrix regeneration in the BNC network after 8 weeks of in vitro culture with bovine cartilage

Embodiment 1

Production of the two-phase BNC network

A nutrient solution, the distilled water per liter. 20.00 g of glucose anhydrous, 5.00 g of bactopeptone, 5.00 g of yeast extract, 3.40 g of disodium hydrogen phosphate dihydrate and 1.15 g of citric acid monohydrate, and a pH of between 6.0 and 6.3 (Schramm -Hestrin medium) is steam sterilized at 120 ° C for 20 minutes. After inoculation of 20 ml of nutrient medium with the bacterium Gluconacetobacter xylinus from a 7-day-old liquid stock in Schramm-Hestrin medium (1 ml), 0.3 ml of this inoculated nutrient solution in a vessel of polystyrene, with a capacity of 1.8 ml filled. To ensure sterility and a moist, aerobic environment in the vessel, this is closed during the cultivation process with a lid. The cultivation time is 14 days at a temperature between 28 ° C and 30 ° C. Surprisingly, it has been found that a bacterial nanocellulose is formed under these cultivation conditions, which in a two-phase structure to 67% of a weitmaschigen phase 1 and 33% from a close-knit phase 2 of bacterial nanocellulose. The biphasic structure of the generated bacterial nanocellulose was taken from the vessel (for reasons of clarity not shown in the drawing), treated with boiling, aqueous 0.1 N sodium hydroxide solution for 10 min and washed thoroughly with water until neutral. In this way, bacteria-free and usable as an implant for cartilage formation bacterial nanocellulose having a diameter of 11.5 mm and a height of 1.0 mm. It was found, surprisingly, that the two-phase system described remained structurally stable even after treatment with the said sodium hydroxide solution.

1 shows a schematic representation of the structure of this generated in the cultivation process two-phase bacterial nanocellulose as a three-dimensional cylindrical shaped body in perspective view (left figure) and in cross section (right figure). From these figures, the structure of the BNC network is from the lower said wide-meshed phase 1 as well as with this tightly connected upper close-meshed phase 2 seen.

Embodiment 2:

Colonization of the BNC network with isolated chondrocytes

The three-dimensional colonization of BNC was studied with isolated cartilage cells. The isolation of the human or bovine cartilage cells from the surrounding cartilage matrix was carried out by enzymatic digestion and subsequent purification by means of double passaging of the cells in monolayer cultures. A migration of chondrocytes 3 into the BNC network was studied in so-called transwell systems. The use of the transwell system allows the adjustment of a serum gradient, which should induce a targeted attraction of the chondrocytes into deeper layers of the BNC network. The previously isolated bovine or human chondrocytes were applied as a cell suspension on the located in the Transwelleinsätzen BNC and cultured. In the upper transwell chamber was serum-poor medium (1% serum), in the lower transwell chamber serum-rich medium (10% serum). The presence of the fine-pore membrane between the two chambers produced a serum gradient for attracting the superficially applied chondrocytes to deeper regions of the BNC network. Because the tight-meshed BNC forms a very dense network of nanocellulose fibers whose pores are of critical size relative to the comparatively large chondrocytes, isolated chondrocytes adhered upon colonization of tight-meshed BNC on the surface of the BNC network.

at Colonization of the wide-meshed BNC with isolated chondrocytes was histologically after 24 hours of cultivation a chondrocyte migration into deeper layers of the BNC network observed. This wide-meshed BNC has a looser network, its pore sizes in a favorable Ratio to the size of the applied Chondrocytes are standing.

2 shows schematically the colonization of the BNC network with the chondrocytes 3 , While these at the close-knit phase 2 of the BNC network ( 2a ) adhered to the surface, colonized the chondrocytes 3 the wide-meshed phase 1 of the BNC network within a short time even in depth ( 2 B ).

Embodiment 3

In vitro cultivation of an implant BNC network with bovine cartilage

An in vitro model for endogenous cartilage regeneration has been established ( 3 ) by an artificial defect 4 of a cartilage 5 (bovine cartilage slices) with a preformed, bacterially synthesized BNC network 6 filled in and a model implant system formed therefrom 7 (artificial defect 4 of cartilage 5 with inserted BNC network 6 ) in one with agarose 8th filled culture vessel 9 was given. Subsequently, the model implant system 7 with the addition of a culture medium 10 cultured in vitro under TGF-b1 stimulation (see 3 ). The shape of the BNC network 6 oriented to the dimensions of the cartilage 5 generated defect 4 , After 4- or 8-week culture, but not after 2 weeks of culture, early stages of cartilage matrix regeneration were evident in the defect-filling BNC network. The integrity of the cartilage was maintained despite the relatively long culture time for in vitro studies. The early stages of cartilage matrix regeneration in both unstimulated and TGF-b1 stimulated samples were in the form of immunohistological detection of intact aggrekans and collagen type II in the BNC network 6 detectable ( 4 ). Furthermore, chondrocytes left 3 the cartilage 5 and adhered to the surface of the BNC network 6 (see also 4 ).

4 schematically shows these early stages of cartilage matrix regeneration in the artificial defect 4 of cartilage 5 fitted BNC network 6 after 8 weeks of culture with bovine cartilage. The incorporation of proteoglycans into the BNC network 6 was by a positive safranin-O staining of the BNC network 6 seen.

This finding was confirmed by immunohistological detection of newly formed aggrecan. Another important cartilage component was collagen type II in the framework of the BNC network 6 demonstrated. In addition to a storage of cartilage matrix in the framework of the BNC network 6 became a colonization of the surface of the BNC network 6 through the chondrocytes migrated out of the cartilage 3 observed.

For the use of implants according to the invention for cartilage regeneration in the case of cartilage damage, after diagnosis (eg by means of MRT, arthroscopy) the cartilage damage of the patient is either revealed by opening the joint or arthroscopically. If necessary, a smoothing of the cartilage lesion of the arthritic defect takes place. Depending on the size of the cartilage defect area, the surgeon can select from a range of prefabricated BNC implants and insert a BNC implant of appropriate shape and size into the defect. The peculiarity of this BNC implant is that the relatively loose, wide-meshed BNC network serves to colonize cells migrating from the cartilage. These cells are able to migrate well and form new cartilage matrix due to the particular nature of the wide-meshed BNC. The stability required for attachment of the BNC web during the operation is ensured by the compacted / close-meshed part of the BNC, which is firmly connected to the wide-meshed phase of the BNC. The fixation of the BNC implant is thus carried out by suturing the compacted / closely meshed portion of the BNC implant with the marginal cartilage shoulder resorbable suture material. Gluing the implant with surgical adhesives (eg fibrin glue) as well as anchoring the implant in the underlying bone by means of resorbable pins / nails is also possible. After suturing the surgical opening, the patient should be able to reload the joint after a short period of rest due to the relatively gentle procedure (no injury to intact cartilage areas to obtain chondrocytes as in ACT) and the mechanical stability of the implant.

1

wide-mesh Phase of the BNC network

2

close-knit Phase of the BNC network

3

chondrocytes

4

artificial malfunction

5

cartilage

6

BNC Network

7

Model Implant System

8th

agarose

9

culture vessel

10

culture medium

QUOTES INCLUDE IN THE DESCRIPTION

This list The documents listed by the applicant have been automated generated and is solely for better information recorded by the reader. The list is not part of the German Patent or utility model application. The DPMA takes over no liability for any errors or omissions.

Cited patent literature

• - EP 1953174 A1 [0008]
• - WO 2007/064881 A2 [0009, 0009]
• WO 2007/146946 A2 [0009]
• - WO 2008/079034 A2 [0010]
• WO 2005/018435 [0011]
• - DE 2003-10361898 [0012]

Cited non-patent literature

• - M. Brittberg et al .: Treatment of deep cartilage defects in the knee with autologous chondrocyte transplantation, Engl J Med, 1994, 331, 889 [0004]
• - T. Minas: Autologous chondrocyte implantation in the arthritic knee, Orthopedics, 2003, 26, 945 [0004]
• - S. Nehrer et al .: Three-year clinical outcome of age chondrocyte transplantation using a hyaluronan matrix for cartilage repair, Eur J Radiol, 2006, 57, 3 [0005]
• - P. Gehrens et al .: Matrix-associated autologous chondrocyte transplantation / implantation (MACT / MACI) -5-year follow-up, Knee, 2006, 13, 194 [0005]
• - C. Ossendorf et al .: Treatment of posttraumatic and focal osteoarthritic cartilage defects of the knee with autologous polymer-based three-dimensional chondrocyte grafts: 2-year clinical results, arthritis Res Ther, 2007, 9, R41 [0005]
• J. Kopecek, J. Yang: Hydrogels as Smart Biomaterials, Polymer International 2007, 56/9, 1078 [0007]
• - D. Klemm et al .: Nanocelluloses as Innovative Polymers in Research and Application, Adv. Polym. Sci. 2006, 205, 49 [0007]
• A. Svensson et al .: Bacterial cellulose as a potential scaffold for tissue engineering of cartilage, Biomaterials, 2005, 26 (4), 419-431 [0012]
• CR Rambo et al .: Template assisted synthesis of porous nanofibrous cellulose membranes for tissue engineering, Materials Science & Engineering, C: Biomimetic and Supramolecular Systems, 2008, 28 (4), 549-554 [0013]
• - A. Nakayama et al .: High Mechanical Strength Double-Network Hydrogel with Bacterial Cellulose, Adv. Funct. Mater. 2004, 14, 1124-1128 [0014]

Claims (18)

Bacterial nanocellulose for cartilage regeneration, characterized by a structure consisting of a wide-meshed phase ( 1 ) of the bacterial nanocellulose and of at least one tightly connected phase ( 2 ) of bacterial nanocellulose. Bacterial nanocellulose according to claim 1, characterized in that the structure consisting of the wide-meshed phase ( 1 ) and at least one close-knit phase ( 2 ) of the bacterial nanocellulose, has a substantially cylindrical shape with a diameter of 3 mm to 40 mm, preferably 3 mm to 10 mm, and a height of 1 mm to 10 mm, preferably 2 mm to 4 mm. Bacterial nanocellulose according to claim 1, characterized in that the proportion of the wide-meshed phase ( 1 ) in the structure is 10% to 90%, preferably 30% to 80%. Bacterial nanocellulose according to claim 1, characterized in that the nanocellulose in the wide-meshed phase ( 1 ) and / or in the at least one close-knit phase ( 2 ) Growth and / or recruitment factors and / or other proteins and other drugs, such as drugs are added. Bacterial nanocellulose according to claim 1, characterized in that the wide-meshed phase ( 1 ) has a mean pore diameter of 10 .mu.m to 500 .mu.m, preferably 100 .mu.m to 200 .mu.m, and a cellulose content of 0.1 ‰ to 5.0 ‰ (water content 99.50% to 99.99%), preferably 0.5 ‰ to 1.5 ‰ (water content 99.85% to 99.95%). Bacterial nanocellulose according to claim 1, characterized in that the close-meshed phase ( 2 ) has an average pore diameter of 1 .mu.m to 20 .mu.m, preferably 2 .mu.m to 6 .mu.m, and a cellulose content of 5.1 to 10.0 ‰ (water content 99.00% to 99.49%), preferably 7.0 to ‰ 9.0 ‰ (water content 99.10% to 99.30%). Bacterial nanocellulose according to claim 4, characterized in that in the nanocellulose growth factors introduced, for example growth factors of the TGF superfamily, in particular TGF-b1 and TGF-b3, and / or members of the BMP (Bone morphogenetic protein) family, and / or representatives of EGF (epidermal growth factor) and / or FGF (fibroblast groth factor) and / or IGF (insulin like growth factor) and / or PDGF (Platelet derived growth factor) and / or VEGF (Vascular Endothelial Growth Factor) and / or GDF (Growth and differentiation factor) families. Bacterial nanocellulose according to claim 4, characterized in that in the nanocellulose recruiting factors introduced, mainly chemokines, for example Members of the CXC chemokine family, in particular CXCL-7, and / or members other chemokine families (CC, CX3C and XC). Bacterial nanocellulose according to claim 1, characterized in that in the nanocellulose cartilage matrix components, such as proteoglycans and collagens are introduced. Bacterial nanocellulose according to claim 1, characterized in that in the nanocellulose, for example for the purpose of increasing their initial mechanical properties biocompatible materials, such as Calcium alginate and crosslinked gelatin, are introduced. Use of the bacterial nanocellulose according to a or more of claims 1 to 10, characterized that the bacterial nanocellulose without drying after the cultivation and Cleaning process as an initially moist implant for cartilage regeneration is used. Use according to claim 11, characterized in that the initially moist implant from the bacterial nanocellulose without further modification required intended for in vivo cartilage regeneration. Use according to claim 11, characterized in that the bacterial nanocellulose first completely or partially colonized in vitro with cartilage cells and then used as an implant. Use of the bacterial nanocellulose according to a or more of claims 1 to 10, characterized that the nanocellulose for storage, for example by freeze-drying or Critical point drying, gently dried, the structure and maintainability of nanocellulose, and that the implant before surgical use or before in vitro use, for example in saline or physiological saline in patient's own or allogeneic (pharma-grade) serum becomes. Process for the preparation of the bacterial nanocellulose according to one or more of Claims 1 to 10, characterized in that the structure consisting of the wide-meshed phase and of the at least one close-meshed phase firmly connected to the wide-meshed phase bacterial nanocellulose is generated in a culture step of the cultivation process as a two-phase implant. A method according to claim 15, characterized characterized in that following the cultivation process and after purification of the cultured bacterial nanocellulose the bottom of the wide-meshed phase for the purpose of solidification preferably is compressed by partial dehydration. A method according to claim 15, characterized characterized in that following the cultivation process and after purification of the cultured bacterial nanocellulose at the bottom of the wide-meshed phase for the purpose of solidification trained there further polymer network, for example Calcium alginate, calcium carboxymethyl cellulose or cross-linked acrylates, is built. A method according to claim 15, characterized characterized in that following the cultivation process and after purification of the cultured bacterial nanocellulose the surfaces by mechanical or laser technology Treatment to be opened.