Bacterial nanocellulose material, methods of production, and uses thereof

Abstract

The present disclosure relates to a bacterial nanocellulose material obtained by short pulse laser etching, comprising pores, wherein the largest diameter of the pore ranges from 5 µm to 10 mm, measured by micro-CT analysis. The disclosure also describes an article comprising the disclosed porous bacterial nanocellulose material and the use of said porous bacterial nanocellulose material or a bacterial nanocellulose composition in tissue engineering.

Description

BACTERIAL NANOCELLULOSE MATERIAL, METHODS OF PRODUCTION, AND USES TH EREOFTECHN ICAL FIELD

[0001] The present disclosure relates to a bacterial nanocellulose material, obtained by short pulse laser etching. The disclosed material displays customized 3D micro- and macro-porosity, recapitulating complex structures that mimic the natural porosity of biological tissues and other structures (such as hollow bacterial nanocellulose cylinders, meshes, among others, of any size and shape), while preserving the naturally crosslinked 3D fibrous network and therefore, the natural mechanical properties of bacterial nanocellulose.BACKGROUND

[0002] Bacterial nanocellulose (BNC) biosynthesized by microorganisms, most effectively by Acetobacter species, is produced in its pure form, originating a gel-like structure that contains up to 99% of water [1], BNC is composed of glucan chains self-assembled through intra and inter-hydrogen bonds, into superior hierarchical structures, from protofibrils to ribbons. When produced by static culture, the obtained BNC membranes exhibit a fascinating ultrafine 3D porous structure composed of an interwoven network of highly intricate nanofibers [2] with a high aspect ratio. These characteristics, combined with the 3D spatial organization, the existence of branching points and mechanical interlocking unifying BNC in one piecematerial, confer this biopolymer exceptional mechanical properties, namely high tensile strength and elastic modulus [2], Besides the remarkable mechanical performance, the network morphology resembling collagen fibres of the Extracellular Matrix (ECM) provides a mimetic environment for cell growth and proliferation [1], Most importantly, the biocompatibility of BNC has been demonstrated in vivo with no appreciable signs of inflammation [3], All these features have prompted BNC for several Tissue Engineering (TE) applications, particularly cartilage [4-6], bone [7-9] and vascular grafts [10-12], The application of BNC as a dressing material for wound healing has already resulted in several marketed products such as BioFill®

[0013] , Bioprocess®, XCell®, Gengiflex® and Dermafill™

[0014] , However, its nano- and sub-micron scale porosities (0.02-10 pm)

[0015] , unsuitable for cellular ingrowth, represent a major limitation for its application in the TE field

[0016] ,

[0003] As a highly versatile biopolymer, several strategies have been adopted to endow BNC with adequate porosity for its use as a tissue construct. Among these, porogen templating, i.e., the use of porogens [4, 17] on the fermentation medium

[0018] has been a popular strategy. However, these methods don't allow for a proper control of the spatial distribution of pores, a good control of the size distribution,or the production of complex microporous architectures. Moreover, the application of BNC postprocessing methods, such as grinding and blending with other polymers, reinforcing compounds or decellularized ECM [6] for the development of composites [8, 19], obtained through crosslinking [5, 6, 20, 21] or freeze drying [6, 9], have been some of the conventional approaches for the development of BNC- based TE constructs. However, by disassembling the crosslinked BNC structure, these methods result in the irremediable loss of the mechanical properties. Also, the development of bioinks reinforced with BNC has been explored [22, 23], since the fibrous nature of BNC, its insolubility in water and limited solubility in common solvents have constrained the direct printing

[0024] , Despite this panoply of processing methods to tune the porosity and functional properties of BNC, some drawbacks have been identified. For example, the inefficient and time-consuming steps for porogen removal and the introduction of toxic organic compounds. Also, BNC grinding approaches impair the mechanical properties of native BNC, since they destroy the natural 3D hierarchical organization of the BNC and its crosslinks points, resulting in the lack of control over porosity, pore size and morphology [20, 24], Although 3D printing allows some control over the definition of complex patterns, it is necessary to destroy the structure of the BNC and mix it with other compounds, to make it flowable. Also, these methods don't allow for the definition of complex microarchitectures.

[0004] Laser drilling has also been proposed to create unidirectional channels in BNC for cell colonization and growth. Several authors have applied a CO2 laser to create arrays of unidirectional vertical microchannels on BNC, with diameters comprehended between 200-300 pm, with post-modifications with functional polymers or compounds, such as gelatine and / or hydroxyapatite [25, 26] or collagen

[0027] for bone and cartilage TE applications, respectively. Using a laser technique, Arhem et al.

[0028] have developed 3D BNC scaffolds for cartilage regeneration, trough unidirectional laser perforation on different sides of a cuboid BNC sample. No chemical modifications arose from the laser perforation and the resulting BNC scaffolds supported chondrocytes migration, differentiation and matrix production

[0028] , More recently, laser drilling has also been proposed for the development of a BNC-based prosthetic mesh for pelvic organ prolapse treatment

[0029] , In this study, BNC-collagen films were perforated in a zipper-like arrangement design, using a CO2 laser cutting system. The developed BNC-based meshes have shown superior mechanical properties compared to clinical synthetic ones, and their long-term in vivo biocompatibility was assured in ewe vaginal environment, with a favoured tissue integration and a very low acute inflammatory response

[0029] , In another study, also using a CO2 laser, microchannels with varying diameters (100, 200, and 400 pm) were produced in nano-submicro-fibrous BNC scaffolds, by stepwise spraying culture medium into the electrospun cellulose acetate submicro-fibrous scaffold, followed by in situ BNC biosynthesis

[0030] , It was demonstrated that microchannel size had a significant effect on cell survival and migration. Also, a synergistic effect of the size of the microchannels and their nano-submicro- fibrous wall morphology on cell migration was reported

[0030] , Following the same methodology, an integrated bilayer small-diameter vascular graft, consisting of a dense inner layer of BNC and saponifiedcellulose acetate microfibers and a macroporous outer layer of BNC with an array of pores (225 pm in diameter) was developed by CO2 laser ablation

[0031] , The proposed bilayer vascular graft exhibited good hemocompatibility and in vitro biocompatibility, with endothelialisation of the inner layer, while the outer layer was shown to be conducive for smooth muscle cells' adhesion and proliferation

[0031] ,

[0005] Several patents report the use of a simple laser drilling technology to create channels / holes in BNC. Document CN108126248A reports a preparation method for a porous BNC membrane using UV laser drilling to create uniformly distributed holes of 1-2 mm in diameter

[0032] ,

[0006] Similarly, document CN102600507B reports the development of a BNC-collagen scaffold, where holes with a diameter of 100-300 pm (1-2 mm apart) were made by laser ablation

[0033] ,

[0007] Likewise, document W02010052584A2 reports the use of laser drilling for the development of a BNC sheet with perforations for the reinforcement and / or replacement of soft tissues, such as the abdominal wall and pelvic floor

[0034] ,

[0008] Also, document CN103272265A claims the invention of a three-dimensional BNC microporous stent. Here, a CO2 or an yttrium aluminium garnet (YAG) laser was used for laser drilling of a freeze-dried BNC stent, to obtain a 3D microporous scaffold with 100-500 pm diameter pores, with a pitch (distance between each laser drill) of 0.8-2.5 mm. The micro-hole machining is carried out along the three- dimensional coordinate axis direction of the bacterial cellulose stent (XYZ axes). The claimed material is used for TE applications, particularly in the vascular field

[0035] ,

[0009] Similarly, document CN103301505A also claims the use of CCh-laser punching for the development of a 3D microporous BNC scaffold, in which an array of micropores (100-300 pm in diameter) is punched in the processing planes (XY and YZ planes) of the freeze-dried BNC

[0036] ,

[0010] In document CN110639065A, the inventors claimed the use of femtosecond or millisecond laser perforation technology for the development of a BNC-based asymmetric double-sided anisotropic biological patch, comprising anti-adhesion and prosthetic properties. The prosthetic side consists of a microporous bacterial cellulose semi-dry film, where micropores self-assembled with a certain density and size (100-300 pm) were made by using the femtosecond or millisecond lasers

[0037] , One side of this microporous BNC surface is hydrophobized by chemical vapor deposition of a long-chain hydrophobic modifier, to confer anti-adhesion property. The use of semi-dried BC films is disadvantageous because drying BC changes its properties. On the other hand, pores are created only on the surface of BC and not across the entire depth, creating gradients of pores that allow cell infiltration.

[0011] In fact, laser technology has been highly explored for the development of microporous BNC materials with several applications in the TE field. Indeed, laser is a non-contact and contaminant-free technology that has shown great potential to modify the BNC microstructure without changing its bulk properties. It has enabled the generation of vertical micro-holes / channels, crossing the entire section ofthe material, which can be interconnected by performing laser drilling in orthogonal planes of the BNC sample. However, the application of laser technology as a simple drilling technique for the fabrication of porous BNC scaffolds still has severe limitations in terms of precision, spatial control over the drilled micropores and design options, which impair the mimicking of the natural structure of biological tissues in terms of porosity gradients, pore morphology and spatial organization. Although this simple drilling technique allows cell infiltration, and therefore represents a progress towards the use of BNC in TE, it does not allow the reproduction of the natural ultrastructure of tissues, namely the reproduction of ultrastructural anisotropic features. The recognition of gradients of pores, stiffness, density, etc, in different tissues (e.g. on cartilage) has triggered the search for scaffolds capable of mimicking these features, a recent and important trend in TE

[0038] ,

[0012] These facts are disclosed in order to illustrate the technical problem addressed by the present disclosure.GEN ERAL DESCRIPTION

[0013] The present disclosure relates to a bacterial nanocellulose (BNC) (or porous bacterial nanocellulose BCN ) material obtained by short pulse laser etching, wherein the material comprises pores, and wherein the largest diameter of the pore ranges from 5 pm to 10 mm (10000 pm), as measured by X- ray microtomography (micro-CT) analysis. The customized porous BNC can be applied in tissue engineering (TE) as a biomimetic scaffold for cell culture and growth.

[0014] An aspect of the present disclosure relates to a BNC material comprising a plurality of pores (namely micropores and macropores), wherein said pores were obtained short pulse laser etching and wherein the largest diameter of the pore ranges from 5 to 10000 pm in order to allow cell infiltration and proliferation while simultaneously facilitating the passage of nutrients.

[0015] The porous BNC of the present disclosure can be used for the repair and regeneration of human or animal tissue, such as articular cartilage, skin, and other tissues damaged by trauma or disease. The material described in the present disclosure is rupture-resistant, elastic, and biocompatible, and can be integrated with surgical procedures, facilitating rapid tissue formation.

[0016] The porous BNC of the present disclosure enables the confinement of cells within a specific area, aiding tissue repair and regeneration. This material supports directed cell development, ensuring that the newly formed tissue exhibits characteristics similar to the pre-existing one.

[0017] The porous BNC of the present disclosure allows the design of a scaffold that replicates the essential characteristics of the native extracellular matrix of the tissue to be repaired, thereby creating optimal conditions for tissue regeneration.

[0018] The common laser drilling technique only allows for the production of unidirectional holes that pass through the material from one end to the other. These holes can be made in various directions (usually perpendicular), creating a porous mesh. This way, the porous architecture of biological tissues cannot be reproduced.

[0019] To overcome the limitations of the state of the art, the BNC material (or porous BNC material) of the present disclosure was obtained by the use of short pulse laser technology, for the development of customized 3D microporosity on BNC bulk, particularly for biomedical applications. The use of short pulse lasers to etch pores (cavities, holes) with a tight control of the pore size, morphology, interconnectivity and 3D distribution, allows to closely mimic the porosity gradient (and consequently gradients of stiffness) of native tissues, emulating its 3D microenvironment, enabling cell infiltration, guiding cell ingrowth and the deposition of newly cell-derived oriented extracellular matrix (ECM)). This approach is critical to determine the regenerative outcome.

[0020] In an embodiment, in addition to the development of microporosity, the short pulse laser technology also allows for the design of complex structures on BNC membranes, through the creation of large pores within the membrane, generating structures such as, but not limited to, cylinders or meshes, which can be used in various biomedical applications.

[0021] In an embodiment, the spatially modulated short pulse laser (in particular, femtosecond laser pulses) is used to etch individual pores (cavities, holes) that are linked together (overlap) to obtain channels inside BNC.

[0022] Surprisingly, the disclosed method allowed for the creation of biomimetic gradient porous scaffolds able to reproduce the native anisotropy of different tissues. Although it has been shown by others that it is possible to cause changes in the interior of transparent materials [39-42], such as hydrogels, by using short pulse laser technology, it was not possible to anticipate whether the lasermaterial interaction allows to preserve the intrinsic properties of the BNC material near the interaction region. Surprisingly, the physical integrity of the BNC is not altered upon short pulse laser etching processing. Moreover, the works related to the application of short pulse laser technology in other biomaterials are based on transparent synthetic polymers or on soluble organic hydrogels [39-42], whereas BNC is an insoluble fibrillar structure. Surprisingly, structures with customized porosity and architecture were obtained with great spatial accuracy and dimensional control of the generated pores, by employing the disclosed method, while maintaining the intrinsic properties of BNC, in particular its 3D crosslinked structure and its native mechanical properties.

[0023] In an embodiment, by focusing the laser beam on the desired spatial location, within the BNC bulk, a variety of etching configurations, such as isolated micropores or microchannels, with unlimited options in terms of microarchitectures' design, can be precisely performed in its XYZ planes, without drilling the entire BNC section or changing the BNC processing plane. The size and morphology of theetched micropores / channels can be customized by adjusting the laser parameters, while preserving the bulk properties (3D BNC network with native mechanical properties). In this way, the short pulse laser offers the possibility to closely mimic the porosity of native biologic tissues, approaching its natural 3D microenvironment, which is critical for regenerative purposes.

[0024] In an embodiment, by employing short pulse lasers with high spatial resolution, complex microporosity are achieved in a reliable manner, even for extremely sensitive materials, such as BNC. The reduced laser pulse duration associated with the possibility of focusing on a very small spot, strongly diminishes the thermal collateral damage, normally seen when employing a pulsed CO2 laser.

[0025] In an embodiment, the present disclosure reports a porous bacterial nanocellulose material, with customized porosity / microstructures obtained by short pulse laser technology, with a tight control over its spatial location and design, paving the way to accurately mimic the porosity gradients and anisotropic features exhibited by several biological tissues such as the osteochondral, meniscus or intervertebral discs. Considering the toughness and elasticity of BNC, depending on the cellulose content, it can exhibit mechanical properties comparable to or even exceeding those of native menisci from pigs, sheep and humans. As such, its application in replacing load bearing tissues such as the meniscus is very appealing. Moreover, its hydrophilicity and high overall porosity (>99%) provide continuous nourishment of the cells and its abundant free OH groups, amenable to functionalization, combined with short pulse laser technology can be used for the development of porosity with suitable size, morphology, interconnectivity and biofunctional gradient BNC scaffolds.

[0026] In an embodiment, the process begins with the purification of the BNC membranes. In a further embodiment, the washing process comprises 3 steps, first with deionized water, next with sodium hydroxide (NaOH) and finally with deionized water until the wash waters reach a neutral pH. Optionally, endotoxins may be removed, for in vivo applications. The purified BNC membranes are then sliced longitudinally and sterilized by autoclave. If needed, they are then stored at 4°C until laser processing.

[0027] In a further embodiment, BNC slices are cut into discs using CO2 laser etching and computer-aided graphical design. BNC discs are then placed on a 3D translational stage and etched by short pulse laser, from the top surface to the bottom, according to the desired design; finally, new BNC 3D scaffolds are obtained with customized pore / microchannel diameter, morphology and spatial distribution on the XYZ axis.

[0028] The customized and flexible character of short pulse laser etching process represents a great advantage over conventional methods that have been described to confer adequate porosity for cell ingrowth in BNC scaffolds. The virtually unlimited options of microarchitectures that can be etched on BNC, with control over its spatial distribution, size and morphology, offers the possibility to better approach the organization of native tissues by mimicking their gradient porosities and other anisotropic features that naturally occur in human tissues.

[0029] The present disclosure relates to a porous BNC material obtained by short pulse laser etching, preferably short pulse laser surface and bulk etching, wherein the composition comprises pores (cavities or holes; linear or non linear pores) , and wherein the largest diameter of the pore ranges from 5 pm to 10 mm (10000 pm), preferably from 15 pm to 5 mm, as measured by X-ray microtomography (micro-CT) analysis.

[0030] An aspect of the present disclosure relates to BNC material (this is a porous BCN material) comprising pores (cavities or holes) obtainable by short pulse laser etching, wherein a largest diameter of each pore obtained by short pulse laser etching is comprised in a range from 5 to 10000 pm, as measured by X-ray microtomography (micro-CT) analysis.

[0031] In an embodiment the largest diameter of each pore is comprised in a range from 5 pm to 5000 pm; preferably 30 pm to 2000 pm; more preferably 50 pm to 500 pm.

[0032] In an embodiment, the each pore is individually obtained by short pulse laser etching and the pores are linked together to obtain complex micro or macropore geometries, wherein the complex micro or macroporore geometries comprise at least a non-linear segment.

[0033] In an embodiment, each pore is individually obtained by short pulse laser etching and the pores are linked together (this is, etched next to one another), to obtain a channel or channels inside the bacterial nanocellulose material with a predefined format, making possible the creation of tortuous channels.

[0034] In the present disclosure, a non-linear channel refers to a pathway that does not follow a straight or uniform trajectory. Instead, it may exhibit curves, branches, irregularities, or variations in width, shape, or direction.

[0035] In an embodiment, each pore is individually created through short-pulse laser etching, these pores are interconnected to form a channel, where the diameter is not uniform and can either decrease or increase, forming a gradient. This is achieved by applying multiple laser shots and / or varying intensities.

[0036] In an embodiment, the each pores obtained by short pulse laser etching form a array of parallel microchannels; and / or the pores obtained by short pulse laser etching form a orthogonal grid structures.

[0037] In an embodiment, the pores obtained by short pulse laser etching form non-linear, tortuous channels, generating anisotropic architectures.

[0038] In an embodiment, wherein the pores comprise linear pores, non-linear pores or combinations thereof.

[0039] For the scope and interpretation of the present disclosure it is defined that "non-linear pores" and "non-linear channels" are pores / channels that do not follow a straight or uniform path, thus can have irregular shapes, different sizes, or complex geometries.

[0040] In an embodiment, the largest diameter of the pore ranges from 5 to 5000 pm; preferably 30 pm to 5000 pm measured by micro-CT analysis, more preferably 30 pm to 2000 pm; even more preferably 50 pm to 500 pm; even more preferably 50 pm to 200 pm.

[0041] In an embodiment, the pores comprise a dead-end, i.e., the pore terminates without connecting to any other pore or channel.

[0042] In an embodiment, the distribution of the pores in the bacterial nanocellulose material is anisotropic.

[0043] In another embodiment, the pore distribution in the bacterial nanocellulose material is isotropic.

[0044] In an embodiment, the pores have a tubular morphology, branched morphology, oval morphology, spherical morphology, or combinations thereof.

[0045] In an embodiment, 25% of the pores (channels forming the top and bottom macroporous structures) has a width ranging from 50-80 pm; preferably 25% of the pores has a width below 60-70 pm.

[0046] In an embodiment, 50% of the pores (channels forming the top and bottom macroporous structures) has a width ranging from 80-110 pm; preferably 50% of the pores has a width below 90-100 pm.

[0047] In an embodiment, 75% of the pores (channels forming the top and bottom macroporous structures) has a width ranging from 130-160 pm; preferably 75% of the pores has a width below 140-150 pm.

[0048] In an embodiment, the pores have an aspect ratio (spot diameter / spot height) ranging from 0.1 to 0.6; preferably 0.15 to 0.4.

[0049] In an embodiment, the porous BNC material further comprises an anti-inflammatory agent, an antiseptic agent, an antipyretic agent, an anaesthetic agent, a therapeutic agent, a cell, or combinations thereof.

[0050] In an embodiment, the cell is a non-human animal cell, or a human cell, or a stem cell, or combinations thereof.

[0051] In an embodiment, the material is a hydrogel.

[0052] The present disclosure also relates to an article or product comprising the disclosed porous bacterial nanocellulose material.

[0053] In an embodiment, the article is a multicompartment medical-device, preferably a mesh, a hollow cylinder, a scaffold, a cell carrier, a therapeutic hydrogel, a drug, a delivery depot, a cardiac valve, a vascular prosthesis, a surgical mesh for hernia repair, a stent, an artificial intervertebral disc, or an artificial meniscus or combinations thereof.

[0054] An aspect of the present disclosure relates to the use of the porous BNC material for use in tissue engineering or tissue regeneration.

[0055] An aspect of the present disclosure relates to a bacterial nanocellulose device or composition for use in tissue engineering wherein the bacterial nanocellulose composition is administered in the form of a bacterial nanocellulose material comprising a plurality of pores, wherein said pores obtainable by short pulse laser etching as described in the present disclosure. Preferably, a largest diameter of each pore obtained by short pulse laser etching is comprised in a range from 5 to 10000 pm, as measured by X-ray microtomography (micro-CT) analysis.

[0056] In an embodiment, the composition or the porous BNC material is for use in the treatment of cartilage defects, bone defects, osteochondral defects, cardiac defects, or vascular defects.

[0057] The present disclosure also relates to the use of the disclosed porous bacterial nanocellulose material, or disclosed article, as a cell scaffold.

[0058] An aspect of the present disclosure relates to a method for treating cartilage defects, bone defects, or vascular defects in a subject, the method comprising administering a porous BNC material or a bacterial nanocellulose composition / device to the subject, wherein the bacterial nanocellulose composition is administered in the form of a porous bacterial nanocellulose material as defined in any of the previous embodiments.

[0059] An aspect of the present disclosure relates to a method for producing a porous bacterial nanocellulose hydrated material, which comprises the following steps: providing a bulk sample of bacterial nanocellulose; preferably bacterial nanocellulose biosynthesized by Komagataeibacter spp, Gluconocetobacter spp., Acetobacter spp., Novacetimonas spp., among others, or mixtures thereof (preferably Acetobacter hansenii, Acetobacter xylinum, A. pasteurianus, or mixtures thereof); washing the bulk sample of bacterial nanocellulose to obtain a high purity (i.e., with no bacterial cells visible by Scanning Electron Microscopy BNC (preferably medical grade BNC, without endotoxins); slicing the washed sample of BNC to obtain BNC pellicles, preferably BNC pellicles with a thickness of up to 5 mm; more preferably from 1 mm to 5 cm; etching the BNC pellicles with a short pulse laser to obtain the porous BNC material, wherein the short pulse laser has a pulse duration that ranges preferably from 1 fs to 100 ps and a pulse energy that ranges from 1 nJ to 2 J; preferably 0.025 mJ to 2 mJ; more preferably 0.1 mJ to 2 mJ.

[0060] This method provides a novel innovative approach to achieve high purity, medical-grade BNC with enhanced porosity, tailored specifically for advanced applications, such as medical devices or biotechnology with application in medicine. The integration of precise short pulse laser etching parameters— specifically, the defined ranges of pulse duration and pulse energy, allow the controlled formation of non-linear / linear and of dead-end pores with a defined range of diameter withoutcompromising their structural integrity or functional properties. Such parameters allow to obtain porous BNC material with improved biocompatibility, controlled porosity, and high mechanical strength.

[0061] In an embodiment, the short pulse has a pulse duration that ranges from 35 to 150 fs, preferably from 100 to 130fs, more preferably is 130 fs.

[0062] In an embodiment, the centre wavelength of the short pulse laser ranges from 157 nm to 27000 nm, preferably from 266 to 10600 nm, more preferably 780 to 800 nm, even more preferably is 790 nm.

[0063] In an embodiment, the pulse repetition frequency of the short pulse laser ranges from 1 Hz to 10 GHz, preferably from 1 Hz to 5 MHz, more preferably from 1 Hz to 3 kHz, even more preferably is 1 kHz.

[0064] In an embodiment, the pulse energy of the short pulse laser ranges from 1 nJ to 2 J, preferably from 0.1 pJ to 0.6 mJ, more preferably is 0.26 mJ.

[0065] In an embodiment, the pulse energy of the short pulse laser ranges from 0.1 pJ to 1 mJ, preferably from 0.26 to 0.6 mJ, more preferably is 0.26 mJ

[0066] In an embodiment, the etch volume diameter is proportional to the pulse power. In another embodiment, the diameter can also be modulated by an iris placed before the focus lens of the laser. The spot shape can also be modified using slits in the laser path, previously to the focus lens (elipsoid).

[0067] In an embodiment, the short pulse laser operates with a magnification lens ranging from 5 to 100 x and a numerical aperture (NA) ranging from 0.14 to 1.45, preferably 0.14 to 0.75 . In another embodiment, the magnification lens of the short pulse laser ranges from 20x to 50x, preferably 50x.

[0068] In an embodiment, the numerical aperture (NA) of the short pulse laser ranges from 0.42 to 0.67, preferably is 0.67.

[0069] In an embodiment, the centre wavelength of the short pulse laser is of 790 nm, the pulse frequency is 1 kHz, the pulse duration is 130 fs and the maximum pulse energy is 1 mJ.

[0070] In an embodiment, the magnification lens is 50x and the numerical aperture is 0.67.

[0071] In an embodiment, the processing speed of the short pulse laser ranges from 0.01 to 20000 mm.s'1, preferably from 1 to 6000 mm.s1, more preferably from 1 to 1000 mm.s1; even more preferably 0.01 mm.s1to 2 mm.s1; even mor preferably from 1 mm.s1to 2 mm.s1.

[0072] In an embodiment, at least 50% of the number of pores have a volume inferior to 0.4 mm3.

[0073] In an embodiment, the method further comprises a step of sterilization of the bacterial nanocellulose pellicles, preferably before the etching step.

[0074] In an embodiment, the step of sterilization is performed by autoclave, preferably for 20 min, at 130°C and 1 bar.

[0075] In an embodiment, the step of sterilization is performed by gamma irradiation.

[0076] In an embodiment, the thickness of the bacterial nanocellulose pellicle ranges from 1 to 50 mm.

[0077] In an embodiment, the method further comprises a step of cutting the bacterial nanocellulose pellicles by CO2 laser etching, to obtain discs or other regular or non-regular shapes.

[0078] In an embodiment, the CO2 laser operates in pulsed mode with an excitation power ranging from 5 to 100 W, preferably 24.5 W, a processing speed ranging from 1 to 20000 mm. s’1, preferably of 100 mm.s-1and a frequency ranging from CW to 1 MHz, preferably of 100 kHz.

[0079] In an embodiment, the diameter of the bacterial nanocellulose discs ranges from 5 to 50 mm, preferably is 10mm.BRI EF DESCRI PTION OF TH E DRAWI NGS

[0080] The following figures provide the preferred embodiments for illustrating the disclosure and should not be seen as limiting the scope of the invention.

[0081] Figure 1: Embodiment of the etching path of the femtosecond laser (grey lines) for the 3D macroporous design from different perspectives: transversal view (a), top view (b), and 3D view.

[0082] Figure 2: Embodiment of a Micro-CT 3D reconstructed tomograms with different perspectives of the femtosecond laser-etched macroporous design, comprising two layers with three distinct structures: a square pyramid (colored in red and yellow), a rhombic prism (colored in light blue and green), and a sphere (colored in dark blue and pink) (a). Pore width distribution (pm) of channels forming the top and bottom macroporous structures: square pyramid, rhombic prism, and sphere (b).

[0083] Figure 3: Embodiment of the etching path of the femtosecond laser (blue lines) for the non-linear 3D spiral channel from different perspectives: top view and 3D view (a). Micro-CT 3D-reconstructed tomogram showing top- view and a 3D perspective (b), of a femtosecond laser-etched non-linear porous structure comprising a surface circular cavity connected to a continuous 3D spiral channel. Pore width distribution (pm) of the channel forming the spiral structure (c).

[0084] Figure 4: Schematic representation of an embodiment of a femtosecond laser etching process for the assessment of the variation of spot and channel diameters as a function of laser power and etch depth. Top view schematic showing the spatial arrangement of the channel (marked 'x') and isolated pore (marked 'dot') pairs (a). The channels and pores are grouped and identified according to the applied laser pulse energy (mJ), as follows 1-0.025, 2-0.05, 3-0.1, 4-0.2, 5-0.4 and 6-0.6 (b). Transverse schematic view of the femtosecond laser etching performed from bottom to top, showing the continuous channels and the adjacent isolated pores for pairs 2, 5, and 6 (c). Transverse view of the frequently undetectable or missing pores and channels in the lower and upper parts of the sample (highlighted in shadows) observedafter laser processing, due to sample height variations and deformations caused by sample handling and freeze-drying (d).

[0085] Figure 5: Embodiment of micro-CT 3D reconstructed tomograms with top view and 3D perspectives of femtosecond laser etched channel and pore pairs, marked with different colors according to the power used (a). Individual tomograms of each channel and pore pair (b) with the sections used for the micro-CT diameter measurements.

[0086] Figure 6: Diameter (pm) of the channel (a) and pores (b) as a function of depth and power and aspect ratio (spot diameter / spot height) of individual pores etched at different powers. For the calculation of average diameter (pm) and aspect ratio, only sections from completed spots and channels were included.

[0087] Figure 7: Simulation of the pulse overlap per unit area normalised to the theoretical laser spot size for continuous pulsed laser etching at 10 mm / s and 1 kHz (a) and the total energy deposited per unit area (J) as a function of scanning speed for different power (b).

[0088] Figure 8: X-ray photoelectron survey (a) and deconvolution of the Cis and Ols photoelectron spectra (b) of a femtosecond pulsed laser etched BNC.

[0089] Figure 9: Schematic representation of an embodiment of a femtosecond laser's etched patterns from different perspectives, corresponding to circuit-shaped (a and b), Al orthogonal grid (c and d) and A2 orthogonal grid with z interconnectivity (e and f) microarchitectures.

[0090] Figure 10: Embodiment of a Micro-CT 3D cross-sectional view of the microarchitectures: circuitshaped (a, b), Al (orthogonal grid) (c) and A2 (orthogonal grid with z interconnectivity) (d), where each microchannel's layer was identified with an L according to the laser etching order (LI to LX, from the top surface to the bottom of the BNC sample.

[0091] Figure 11: Embodiment of a Micro-CT 3D reconstructed tomograms with different perspectives of the microarchitectures: circuit-shaped (a to d), Al (e to h) and A2 (I to m), where the femtosecond laser etched microchannels are coloured in blue, green and red, respectively.

[0092] Figure 12: Embodiment of a fluorescence microscopy confocal images and respective 3D projections (scale bar: 200 pm), SEM images of cross-section views (scale bars: 250 pm and 50 pm) and representation of cell viability assays of ATDC5 cells cultured on freeze-dried Al and hydrated A2 porous BNC scaffolds after 8 days, where DAPI (blue), Pha-red (red) and GFP-CBM (green) were used for nuclei, actin cytoskeleton and BNC visualization, respectively.DETAI LED D ESCRI PTION

[0093] The present disclosure relates to a porous bacterial nanocellulose material, obtained by short pulse laser etching; comprising pores and dead-end pores obtained by short pulse laser etching, preferably wherein the largest diameter of the pore ranges from 15 pm to 10 mm; the pore may be measured by micro-CT analysis. The disclosure also describes an article comprising the disclosed porous bacterial nanocellulose material and the use of a bacterial nanocellulose composition in tissue engineering.

[0094] Since the material is obtained by the use of short pulse (nano to femtosecond) laser etching, it can display 3D micro and macro-porosity on bulk BNC, with a tight control over its size and spatial distribution. This technology has several advantages over the conventional approaches used for the development of BNC porous constructs, which lack precise control over pore morphology, size and distribution and often compromise the mechanical properties of BNC. In addition, some of these methods involve the use of solvents, in particular organic solvents (for example for the removal of porogen agents) or additives (to e.g. to make BNC processable by 3D printing), which is not required in the current case. It is thus reported the development of BNC materials comprising customized 3D micro and macro porosity in bulky bacterial nanocellulose, through the application of short pulse (nano to femtosecond) laser technology, while maintaining the structural integrity of the bulk BNC. The customized porous BNC can be applied in the TE field as biomimetic scaffolds for cell culture and growth.

[0095] Surprisingly, the disclosed method enables the fabrication of biomimetic gradient porous scaffolds that accurately replicate the native anisotropy of various tissues. Contrary to expectations, our findings reveal that short-pulse laser processing does not compromise the physical integrity of BNC.

[0096] Unexpectedly, the disclosed method enables the precise generation of structures with customized porosity and architecture, achieving high spatial resolution and dimensional control over the generated pores. Importantly, this is accomplished while fully preserving the intrinsic properties of BNC, particularly its native 3D crosslinked network and mechanical robustness. This breakthrough provides a novel approach to structuring BNC, expanding its applicability in tissue engineering and biomaterial design.

[0097] In an embodiment, by using short pulse laser etching it is possible to produce customized 3D micro and macro-porosity in membranes of BNC obtained by static culture (with any size or shape) that allows to i) create complex structures, for example hollow BNC cylinders, meshes, etc, of any size and shape and ii) to create bacterial cellulose scaffolds that mimic the natural porosity of biological tissues, enabling cell infiltration, guiding cell ingrowth and the deposition of newly cell-derived oriented ECM. The same technique - short pulse laser - is used to produce pores in a multidimensional (micro and macro) top- down approach.

[0098] In an embodiment to obtain the disclosed material, the process begins with the purification of BC samples. Static cultured, food-grade BNC membranes are firstly purified by washing in deionized water at 80 °C, for 3h, followed by a washing step in 0.1M NaOH solution at 90-95 °C, for 90 min. Afterwards, the BNC membranes are continuously washed with abundant deionized water until reaching neutral pH in the wash water. Optionally, endotoxins may be removed, for in vivo applications. The resulting 15 mm thick purified BNC membranes are longitudinally sliced, preferably into 4-5 mm thick pellicles, and sterilized by autoclaving for 20 min, at 120 °C and 1 bar, and stored at 4 °C until laser processing.

[0099] In an embodiment, the preparation of the BNC samples is performed with computer-aided graphical design. A BNC pellicle, featuring preferably 4-5 mm of thickness, is cut by using CO2 laser etching, preferably into discs with 5 to 50 mm of diameter, more preferably 10 mm of diameter.

[0100] In an embodiment, the BNC pellicles are cut into discs by using CO2 laser etching. In an embodiment for better results, the BNC pellicles are cut into discs by using CO2 laser etching, operating in pulsed mode with an excitation power of 24.5W, a processing speed of lOOm / s, a frequency of 100 kHz, and the process is carried out at the laser focal point.

[0101] In an embodiment, for the etching step with the short pulse laser, the BNC samples are placed on a 3D translational stage with a resolution of 0.1 pm followed by a manual rotational and tilting assembly, for fine alignment with the focus lens optical centre. The BNC samples are etched by a short pulse laser with a centre wavelength between 780 and 800 nm, a pulse repetition frequency that ranges from 1 Hz to 3 kHz, preferably of 1 kHz, and a pulse duration that ranges from 35 to 150 fs, preferably from 100 to 130 fs. The etching process is carried out at a pulse energy that ranges from 0.26 to 0.6 mJ, preferably 0.26 mJ. In a further embodiment, the laser system is equipped with a magnification lens of 5 to 100 x, preferably 20 to 50 x, and a numerical aperture between 0.14 and 0.75, preferably 0.42 and 0.67.

[0102] In an embodiment, the combination and variation of the described parameters allows the production of customized micro and macro porosity in the BNC samples. Specifically, it is possible to define the desired diameter of the pores, between 5 pm to 10 mm; preferably 50 pm to 10 mm; the pore morphology, between oval, cylindrical or spherical; the pores' spatial distribution in the XYZ axis; and the possibility of interconnectivity between pores.

[0103] The present disclosure is more particularly described in the following examples that are intended as illustrative only, since numerous modifications and variations are possible and will be apparent to those skilled in the art.

[0104] In an embodiment, the BNC samples were etched by a short pulse laser with a center wavelength of 790nm ± 10 nm, a pulse frequency of 1 kHz, and a pulse duration of 130 fs. The etching process was carried out with BNC samples (10 mm in diameter and 8 ± 1 mm in height) securely mounted in wells and submerged in water at a pulse energy of 0.1 mJ (adjusted by a neutral density filter stage) and a scanningspeed of 25 mm / s for the two-layered macroporous design comprising a square pyramid, rhombic prism, and sphere, as depicted in Figure 1. The laser system was equipped with a magnification lens of 50x and a numerical aperture (NA) of 0.67. The laser etching process within the bulk of BNC was performed from the bottom to the top layer, with a spatial resolution of ±0.01 pm and a distance of 100 pm between laser scans within the macroporous structures and 500 pm spacing in the vertical direction between macroporous layers. The lines in Figure lc represent the laser course in XYZ with a spacing of 100 pm between laser scans.

[0105] Figure 2 shows the micro-CT 3D reconstructed tomograms from different perspectives of the two- layer macroporous design (a). The pore width distribution (pm) of the macroporous objects etched in each layer, as measured by X-ray microtomography based on the dimensions of the inner walls of the porous structure, is shown in Figure 2b. In this macroporous design, as shown in the tomograms and histograms, the macroporous structures are formed by adjacent channel lines with a range of pore widths from 8 to 264 pm and average pores of 110 ± 55, 105 ± 48, and 90 ± 42 pm for the square pyramid, rhombic prism, and sphere views, respectively. The two layers of macroporous structures are separated from each other by a vertical distance of about 473 pm.

[0106] Theoretical volumes of macroporous structures, calculated during the design process, based on computer-aided design (CAD) were calculated as 2.4 mm3for the square pyramid, 1.8 mm3for the rhombic prism, and 3.05 mm3for the sphere. Experimentally measured average volumes using micro-CT were 4.0 ± 0.4 mm3, 3.2 ± 0.7 mm3and 3.6 ± 0.7 mm3, respectively. The discrepancies observed between the theoretical and measured volumes are attributed to the laser spot size, which can introduce slight variations from the machining path simulation to post-processing deformations caused by lyophilization and sample handling. These spatial differences can be adjusted to match the desired dimensions of the final macropore.

[0107] Overall, it can be stated that the intended 3D macroporous etching designs, represented in Figure 1, were achieved. To create fully voided macroporous structures (completely etching away the cellulose fibres within the defined macropore), the distance between successive laser scans can be precisely adjusted to allow for a controlled overlap, while the laser power and the numerical aperture (NA) can be optimized to control the laser spot size and the degree of interaction with the BNC bulk. This data proves the potential of short pulse lasers to create customized macroporous structures inside the BNC bulk with a controlled spatial distribution and without piercing, which would be impossible to obtain by simple laser drilling.

[0108] In another embodiment, a non-linear and non-planar structure with a spatially modulated profile is formed by etching a three-dimensional spiral microchannel into the XYZ volume of the BNC. As shown in Figure 3a, this 3D spiral structure consists of a volumetric cavity and a continuous microchannel with a spiral shape, which follows the laser path marked by the red line. To perform this etching path, the laserwas equipped with a 20x magnifying lens having a NA of 0.45. The etching process was carried out with BNC samples (10 mm ± 1 in diameter and 8 ± 1 mm in height) securely mounted in wells and submerged in water at a pulse energy of 0.2 mJ and a scan speed of 10 mm / s.

[0109] Figure 3b shows the micro-CT reconstructed tomograms from different perspectives of the volumetric cavity defined by the three-dimensional spiral microchannel. Its pore width distribution is shown in the histogram (Fig.3c), with a range of channel diameter from 8 to 136 pm and an average width of 59 pm, as measured by X-ray microtomography.

[0110] This data supports the potential of short pulse lasers to create customized non-linear 3D porous microarchitectures inside the BNC bulk with a controlled spatial distribution which would be impossible to achieve by using simple laser drilling.

[0111] In another embodiment, the average dimensions of structures produced by continuous scanning and stationary pulsed femtosecond laser etching were evaluated as a function of depth and power. BNC samples were etched by a short pulse laser with a center wavelength of 785 nm, a pulse frequency of 1 kHz, and a pulse duration of 130 fs. The laser system was equipped with a magnification lens of 50x and a NA of 0.67. The etching process was carried out with BNC samples (10 ± 1 mm in diameter and 8 ± 1 mm in height) securely mounted in wells and submerged in water. This setup was designed to minimize the effects of laser scattering by smoothing the diffraction index variance in the laser travel path between the BNC bulk and the focusing lens, thereby reducing the laser beam refraction, improving the precision of the laser spot positioning and uniformity during etching at different depths.

[0112] Six pairs of one channel (marked 'x') and six adjacent pores (marked 'dot') with a vertical spacing of 1 mm were etched at different pulse energies: 0.025, 0.05, 0.1, 0.2, 0.4, 0.6 mJ (Fig.4a-c), with continuous scanning pulsed femtosecond laser etching at a scan rate of 1 kHz and a velocity of 10 mm / s for channels and 30 ms stationary pulsed femtosecond laser etch time for pores. Laser etching was performed from the bottom to the top surface, as shown in the scheme of Figures 4a-c.

[0113] Figure 5 presents the micro-CT 3D reconstructed tomograms of the six pairs of channels and pores from the top view and different 3D perspectives (a). The micro-CT 3D reconstructed tomograms of individual pairs of channels and adjacent pores etched at six distinct pulse energies are depicted in Figure 5b. The stationary pulsed femtosecond laser etching of the BNC bulk produces pores with a symmetric biconical geometry, which is coherent with the femtosecond laser self-focus and subsequent filamentation properties at high enough power, introduced by the ionization of the media and the consequent nonlinear reduction of the local refractive index. Some pores (deeper pores etched at 0.025 and 0.05 mJ and the third vertical pore etched at 0.1 mJ) exhibit bifurcation due to their biconical geometry, in which the region of maximum diameter has inherently lower mechanical deformation resistance, making it particularly prone to collapse under freeze-drying stress. The shape of the pores canbe modulated using different setups, optical systems, and immersion media, according to the desired requirements.

[0114] Three of six pores etched at 0.025 and 0.05 mJ were successfully detected. At 0.1 mJ, four pores were observed, of which three were complete, and one was incomplete at the bottom layer. At 0.2 mJ, five out of six pores were identified, of which three were complete and homogeneous, while the pores in the upper and lower layers appeared to be incomplete (Figure 5b). Discrepancies between the etched and detected pores are attributed to variations in sample height and structural deformations induced during post-processing, which include freeze-drying and sample handling, often resulting in undetected or collapsed pores located in the upper and lower layers of the sample, as shown in Fig. 4d. An average vertical distance of 1.2 ± 0.1 mm was calculated between the central position of the spot regions, which is consistent with the CAD, taking into account slight deviations that may occur due to post-processing sample deformation.

[0115] The diameter (pm) of channels decreased slightly with depth at 0.025 mJ, while it increased with depth at 0.05 mJ and 0.100 mJ and remained constant at other pulse energies (0.2, 0.4, and 0.6 mJ) (Figure 6a). These variations, which lack a consistent trend, probably result from local deviations in the slice measurements, indicating a low contribution of the cumulative beam nonlinear refraction during the laser path through the BNC sample. Such deviations may also result from one-off structural collapses or slight deformations after laser processing. Therefore, the results indicate that the reduction in laser spot size with depth was effectively mitigated by etching the BNC samples submerged in water. The average channel diameter (pm) increases almost linearly up to 0.2 mJ and then remains constant up to 0.6 mJ with an average diameter around 140 (pm). This indicates that in continuous scanning pulsed femtosecond laser etching, the increase in pulse energy above a certain threshold (0.2 mJ) does not increase the channel diameter (Fig.6a) for the BNC material etching.

[0116] No clear trends in mean pore diameter (pm) versus depth were observed, with slight decreases at 0.025 mJ and 0.05 mJ and increases at higher pulse energies. These variations likely result from inherent deviations in the localized slice measurements. Nevertheless, using BNC samples submerged in water allowed for a uniform laser spot size with depth (Fig.6b). The average pore diameter increased until 0.4 mJ and then tended to remain constant up to 0.6 mJ with a maximum average diameter around 200 pm. The aspect ratio exhibits a decreasing trend with increasing laser pulse energy up to 0.2 mJ, stabilizing at higher pulse energies up to 0.6 mJ with an average aspect ratio of approximately 0.2. This trend is attributed to the biconical geometry of the etched spot, where the increased laser fluence at higher pulse energies causes expansion of the local plasma spot, resulting in a vertical spread of the energy distribution, leading to a reduction in the aspect ratio until the stabilization threshold is reached.

[0117] When comparing stationary pulsed femtosecond laser etching with continuous scanning pulsed femtosecond laser etching, a slight increase in the average diameter was observed for stationary etchingat pulse energies exceeding 0.1 mJ. These results can be attributed to the total laser energy deposited per unit volume during the continuous pulse etching at 10 mm / s and stationary pulsed etching at a fixed position for 30 ms. Since the frequency is fixed at 1 kHz for the used laser system, in the first case we have an energy of 100 pulses, spread over 1 mm, while in the second we have 30 pulses per fixed position. That means that the total energy delivered per unit volume in pulsed laser etching for 30 ms for a fixed position is 3 times higher than that applied per unit volume in continuous scanning pulsed femtosecond laser etching at 10 mm / s.

[0118] For a theoretical minimum laser spot size, we can point out 0.059 microns before the diffraction limit, based on the optical properties (wavelength and numerical aperture) of the focusing optics and laser wavelength, for a fixed position. Nonetheless, the effective etching spot volume is much greater due to nonlinear interaction with the medium. At sufficiently high laser energy, the electric field causes ionization of the medium, leading to non-linear phenomena and effectively creating a plasma spot surrounding the spot, normalized to the laser spot size. In the case of continuous scanning pulsed femtosecond laser etching at 10 mm / s, we have 5 overlapping pulses per unit area (Fig. 7a). In addition to the power and numerical aperture influencing the laser spot size, the scanning speed can be adjusted to modulate the total energy deposited per unit volume, thereby influencing the resulting channel ablation, as desired. To illustrate this, Figure 7b shows the total energy deposited per unit area as a function of the scan speed for each of the power settings.

[0119] The data demonstrate the ability of pulsed femtosecond laser etching to create customized, spatially controlled isolated pores or continuous porous structures, whose dimensions and morphology can be modulated by adjusting key parameters such as pulse energy, numerical aperture, and scanning speed. The data collected enables an informed choice of settings to meet specific requirements.

[0120] In another embodiment, the potential chemical modifications arising from femtosecond laser etching of BNC were assessed by X-ray-photo electron spectroscopy (XPS). BNC samples were etched by a short pulse laser with a center wavelength of 785 nm, a pulse frequency of 1 kHz, and a pulse duration of 130 fs. The etching process was carried out at a pulse energy of 0.1 mJ (adjusted by a neutral density filter stage) and a scanning speed of 25 mm / s.

[0121] XPS analysis was performed using a Kratos AXIS Supra spectrometer (Kratos Analytical, UK). ESCApa software was used for data acquisition and CasaXPS software for data processing. Monochromatic Al Ka radiation was used at 225W. Data was acquired with a base pressure below 10'6Pa. The spectrometer was operated in 10 eV pass energy mode for detail regions and 160 eV for the survey. Spectra were analyzed using Gaussian-Lorentzian peak shape fitting and Shirley background subtraction. Binding energy scales were referenced to hydrocarbon (C-C) in Cis spectra at 285 eV. Empirical relative sensitivity factors provided for the instrument were used to determine the atomic concentration percentages of the constituent elements of the surfaces.

[0122] XPS survey spectra (Fig.8a) indicate that only carbon (C) and oxygen (O) are detected on the surface of femtosecond pulsed laser-etched BNC. The relative atomic concentrations of O and C in the femtosecond pulsed laser-etched BNC are presented in Table 1. The obtained O / C ratio was 0.76. This ratio is slightly lower than the reported theoretical value for pure cellulose (0.83), which can be attributed to the appearance of Cl (carbon linked to hydrogen or carbon (-C-H, -C-C) commonly reported by several authors as a result of ubiquitous contamination of the cellulose surface by carbon and oxygen in airexposed surfaces (Pertile et al. 2010; Luz et al. 2020).

[0123] Table 1. Atomic elemental composition (%) and peak area percentage (%) of carbon functional groups on femtosecond pulsed laser-etched BNC surfaces, as determined from XPS measurements.

[0124] Fig.8b presents the deconvolution of the high-resolution scans of the Cis and Ols regions. Theoretically, the Cis spectrum of pure cellulose should consist of only two resolved peaks, C2 and C3, where the former is assigned to a carbon bonded to a single oxygen in alcohol or ether groups (C-O) and the latter to a carbon bonded to two oxygen atoms (O-C-O) or a double bond to oxygen (C=O). The C3 / C2 ratio obtained for the femtosecond pulsed laser-etched BNC was similar to the theoretical value (0.2). The C4, corresponding to a carbon bonded to a carbonyl and a non-carbonyl oxygen atom (O-C=O), which may result from the oxidation of hydroxyl cellulose groups, is present in negligible amounts, and no differences in peak (%) were observed in comparison to pristine BNC reported in other studies. The Ols region was fitted with 2 peaks, 02 corresponding to oxygen linked to carbon in a double bond (O=C) has a vestigial amount, and 01, attributed to oxygen in hydroxyl groups of BNC, is the most intense peak. All these observations support the conclusion that no changes have occurred in the surface chemistry of BNC after femtosecond pulsed laser etching.

[0125] In an embodiment, the BNC samples were etched by a short pulse laser with a centre wavelength of 790±10 nm, a pulse frequency of 1 kHZ, and a pulse duration of 130 fs. The etching process was carried out at a pulse energy of 0.26 and 0.6 mJ (adjusted by a neutral density filter stage) for the multilayer patterns' designs identified as circuit-shaped, Al and A2 microarchitectures, respectively, depicted inFigure 9. The laser system was equipped with a magnification lens of 50x, and a numerical aperture (NA of 0.67. The laser beam focused at 10 mm generated a spot size of the expected size of X / 2NA = 0.59 pm.

[0126] In an embodiment, the micropore density and pore size are be regulated by controlling laser parameters.

[0127] In an embodiment, the laser etching process was performed from the top to the bottom surface of the BNC samples. The x mark in Figure 9a) (top view) represents the initial position of the laser beam and the lines and spots depicted in Figure 9a) (top view) and Figure 9b) (cross-sectional view), refer to the laser course in X, Y and Z axis.

[0128] In an embodiment, a BNC bulk was laser etched to produce a continuous circuit of a microchannels' array, featuring an average diameter of 70 pm, interspaced by 300 pm and 1 mm in the Y and Z planes, respectively. In this circuit-shaped microarchitecture, as depicted in the top view scheme of the laser etching course (Figure 9a), a continuous array of parallel microchannels (n=16; interspaced 300 pm apart) are etched at different Z planes (1 mm apart), as shown in its cross-section representation (Figure 9b).

[0129] In another embodiment, orthogonal grid structures can also be etched on BNC, with and without interconnectivity along the Z axis. This orthogonal grid microarchitecture consists of an array of parallel microchannels (80 pm of average diameter), interspaced by 550 and 500 pm in the X, Y and Z planes, respectively. For the orthogonal grid microarchitecture Al (Figure 9c), intercalated X and Y microchannels (interspaced 550 pm apart in the same Z plane) are etched (Figure 9d), while in the orthogonal grid with Z interconnectivity microarchitecture A2, besides the XY orthogonal grid (Figure 9f), Z planemicrochannels are made at the intersection points of the XY microchannels (Figure 9e).

[0130] In an embodiment, the different microarchitectures, i.e. circuit-shaped, orthogonal grid (Al) and orthogonal grid with Z interconnectivity (A2), etched into the BNC samples (10 mm diameter and 4-5 mm thick), were investigated using a high-resolution X-ray microtomography to confirm that the proposed designs were achieved. In this way, it was possible to visualize the internal structure of the BNC samples and quantitatively express the size of the etched channels, the channel surface area (pm2) and the channel volume (pm3). Scans were performed without a filter, employing a 110 kV source voltage, 90 pA current intensity and 250 ms exposure time / each 2D projection. Samples were rotated 360° using a 0.2° rotation step. The radiographies were acquired by averaging 3 frames per slice. Image pixel size (scanning resolution) was set at 4 pm. The 2D projections were 2452 x 1640 pixels. Tomograms were reconstructed from 2D radiography using Bruker NRecon 1.7.1.6. To view the reconstructed tomograms, CTVox 3.3.0 rl403 was used, while CTAn 1.17.7.2 was applied for sample analysis. In CTAn, tomograms were thresholded to differentiate specimen walls from pores, despeckled to remove scanning artifacts and analysed in 3D to estimate pores and walls features. After thresholding, the tomogram pixels were binarized (solid sample pixels white, pores black). After binarizing the dataset, the images were invertedto display and measure pores as solid objects and separate them by thresholded boundaries. CTVox displayed the pore tomograms inserted into the 3D BNC scaffold to better understand the microchannels network interface and their in-volume distribution.

[0131] Figure 10 depicts an embodiment of cross-section views of 3D reconstructed images of the microarchitectures circuit-shaped (a, b), orthogonal grid Al (c) and orthogonal grid with Z interconnectivity A2 (d). In these images the different electronic densities have a lighter colour for the femtosecond laser etched microchannels, while the bulk BNC is darker and, therefore, the etched microchannels have been coloured for an easier visualization. Each microchannels' layer was identified with an L according to the laser etching order - LI to LX (wherein X is a natural number higher than 1) - from the top surface, or 550 pm below it in the case of Al, to the bottom of the BNC sample: L4 for circuitshaped, L6 for Al microarchitecture, or 7, for A2 microarchitectures. In the different cross-section views of the circuit-shaped microarchitecture (Figure 10a and b) it is possible to notice the array of X-parallel microchannels (n=16) (Figure 10a) and the Z-microchannels connecting the different XY layers (Figure 10b), forming non-linear pores, similarly to the previously defined etching pattern (Figure 9a and b). The fourth layer L4 in the cross-sectional view of the circuit-shaped microarchitecture (Figure 10b) is almost undetectable. Despite the smaller diameter of the microchannels etched on the fourth layer they can be clearly noticed on Figure 2a. In the case of cross-sectional views of the Al and A2 microarchitectures, the intercalated X- and Y-oriented channels were also observed (Figure 10c and d, respectively), in a similar fashion to what has been designed (Figure 9d and f, respectively). Highlighted in the white box (figure lOd), Z-channels connecting the intersection points of A2's layers 1 and 2 can be observed. The microchannels from the different microarchitectures, etched on the same XY plane were identified according to their laser etching order.

[0132] In another embodiment, the internal top views of the 3D reconstructed images are shown in Figure lib, f, j. The measurements of the microchannels etched in each layer, including the average diameter (pm), the surface area (pm2), the volume (pm3) are shown in Table 2, as measured by X-ray microtomography. The bulk BC / microchannel ratio are also presented in Table 2.

[0133] Table 2. Measurements of average size, surface and volume of the laser etched microchannels made at different layers of the BNC samples, each one corresponding to a specific depth of the samples, from the more superficial layers (Ll:0-550 pm) to the bottom (L6 / 7: 3000-3300 pm) of the BNC sample.

[0134] Figure 11 presents the micro-CT 3D reconstructed tomograms, from different perspectives, of the microarchitectures: circuit-shaped (a to d), Al (e to h) and A2 (I to m). The microchannels' tomogram reconstructed slices of the circuit-shaped, Al and A2 microarchitectures are shown in Figure 11 a, e and i, respectively. The internal top-view micro-CT tomograms of the circuit-shaped (b), Al (f) and A2 (j) microarchitectures evidence the continuous xy microchannels' array, forming the circuit-shaped etched pattern and the X, Y and Z microchannels of Al and A2 microarchitectures. The layer-by-layer construction process of the 3D microchannels can be seen in figures c, g and i and the top-view images of the entire 3D constructed microarchitectures circuit-shaped, Al and A2 in figures d), h) and m), respectively.

[0135] Therefore, it can be stated that the intended 3D etching designs, represented on Figure 9, were overall achieved. The variation in microchannel dimensions with depth can be easily overcome by adjusting the laser parameters, such as power and numerical aperture, which will influence the laser spot size and determining the magnitude of the laser interaction with BNC. This way, a uniform size distribution of the microchannels all over the sample can be achieved. This data, particularly the etched circuit-shaped microarchitecture, proves the potential of short pulse lasers to create customized microarchitectures on BNC bulk with a controlled spatial distribution, in a different way to what has been achieved by a simple laser drilling.

[0136] To assess the potential of porous BNC scaffolds as constructs for tissue engineering applications, an in vitro assay was performed to evaluate the viability and ingrowth capacity of ATDC chondrocytes cultured on Al and A2 microarchitecture scaffolds. The chondrogenic cell line ATDC5 was maintained in Dulbecco's modified essential medium (DMEM), supplemented with 10% (v / v) inactivated fetal bovine serum (iFBS) and 1% (v / v) Penicillin (10 000 U / mL)-Streptomycin (10 000 pg / mL) at 37 °C and 5% CO2.

[0137] In an embodiment, the cytotoxicity of the etched BNC samples (sterilized by autoclaving for 20 min, at 120 °C and 1 bar) was determined by performing cell culture assays, assessing the cell viability using the alamar blue assay, based on resazurin reduction

[0043] , Two different experiments were performed. In the first assay, the freeze-dried Al BNC scaffold was seeded with a ATDC5 cell suspensionwith 2.5 x 106cells / mL on 48-well plates (400 pL / well), making up 1 x 106cells / well, corresponding to its full swelling volume, i.e., the volume needed to reach a total swelling of the sample and upon which the wet weight will not change. After 2h, the cell-suspension was fully absorbed and the 3D shape of the scaffold was fully recovered. Afterwards, 400 pL of complete medium were added to the well. In the second assay, the A2 BNC scaffold was swollen in DMEM, and then seeded with 50 pL of a cell suspension (1 x io6cells / well). Cells were allowed to attach and migrate for 2h and then complete medium was added to complete 400 pL per well. In both assays, the experiment was conducted for 8 days with medium changed every 2 days. At the end of the assay, 10% (v / v) of a 2.5 mM resazurin solution in phosphate- buffered saline (PBS) was added to each well and incubated at 37 °C, in a 5% CO2 atmosphere, for 4h. The cell metabolic activity was determined by measuring the fluorescence of resorufin (Xex 560 / em 590 nm) in a microplate reader.

[0138] To visualize cell distribution, the freeze-dried seeded BNC scaffolds (1 x 106cells / well), after 8 days of culture, were fixed with 4% paraformaldehyde (PFA) for 30 min, followed by a washing step with PBS buffer at room temperature. Cells' permeabilization was carried out with 0.5% Triton X-100 in PBS at room temperature.

[0139] For the scope and interpretation of the present disclosure it is defined that "room temperature" should be regarded as a temperature between 15-30 °C, preferably between 18-25 °C, more preferably between 20-22 °C.

[0140] In an embodiment, for BNC staining, samples were incubated with a Cellulose Binding Module fused to a Green Fluorescent Protein (GFP-CBM, 0.05 mg / mL) for lh at room temperature. The samples were washed with PBS and incubated with phalloidin-red (1 pg / mL) for 30 min for actin-cytoskeleton staining. After washing with PBS, the samples were incubated with DAPI (1 pg / mL) for 10 min for nucleus visualization. Finally, the samples were washed with PBS and image acquisition was performed with software FV10-Ver4.1.1.5 (Olympus) using a Confocal Scanning Laser Microscope, following combination of excitation / emission detection range wavelengths: 405 / 430-470nm, 488 / 505-540nm, and 559 / 575- 675nm.

[0141] In an embodiment to investigate cell ingrowth, the cell-seeded BNC scaffolds (Al and A2) were fixed with 2.5% glutaraldehyde in phosphate buffered saline solution, instantly frozen in liquid nitrogen and fractured, with the aid of a scalpel, to expose cross-sectional views and then lyophilized. Prior to scanning electron microscope (SEM) image acquisition, the samples were mounted on aluminium pin stubs and sputter-coated with a thin layer of gold (50 A). SEM images were acquired with an acceleration voltage of 5-20 kV and a working distance of 15 mm, operated in high vacuum conditions.

[0142] Figure 12 depicts an embodiment of the results of cell viability. By analysing these results, it is possible to notice, by the optical density (OD) values, that cells were viable after 8 days of culture on the porous BNC scaffolds.

[0143] In an embodiment, the cellular distribution on cell-seeded freeze-dried porous Al and A2 BNC scaffolds were assessed by confocal microscopy and SEM imaging of cross-sectional views (Figure 12). It is possible to notice that cells preferentially adhere and penetrate on the first microchannels' layer of Al scaffolds, filling these channels and being detected up to a depth of 300 pm. It is possible to see also in the SEM cross section views that cells are able to penetrate and distribute inside the xy microchannels, with no interconnectivity (Figure 12, white arrows). In the case of A2 porous BNC scaffolds, cells are preferentially organized in aggregates randomly distributed on the BNC surface and filling the microchannels and z-pores of this microarchitecture. In SEM images of these scaffolds, it is also possible to detect cell agglomerates grown inside the microchannels' network (Figure 12, white arrows).

[0144] The customized and flexible character of short pulse laser etching process represents a great advantage over conventional methods that have been described to confer adequate porosity for cell ingrowth in BNC scaffolds. The virtually unlimited options of microarchitectures that can be etched on BNC, with the control over its spatial distribution, size and morphology, offers the possibility to better approach the organization of native tissues by mimicking their gradient porosities and other anisotropic features that naturally occur in human tissues.

[0145] The term "comprising" whenever used in this document is intended to indicate the presence of stated features, integers, steps, components, but not to preclude the presence or addition of one or more other features, integers, steps, components or groups thereof.

[0146] The disclosure should not be seen in any way restricted to the embodiments described and a person with ordinary skill in the art will foresee many possibilities for modifications thereof. The abovedescribed embodiments are combinable.

[0147] The following dependent claims further set out the particular embodiments of the disclosure.References:1. Halib N, Ahmad I, Grassi M, Grassi G (2019) The remarkable three-dimensional network structure of bacterial cellulose for tissue engineering applications. Int J Pharm 566:631-640. https: / / doi.Org / 10.1016 / j.ijpharm.2019.06.0172. Wu Z, Chen S, Li J, et al (2023) Insights into Hierarchical Structure-Property-Application Relationships of Advanced Bacterial Cellulose Materials. Adv Funct Mater 33, 2214327:. https: / / doi.org / 10.1002 / adfm.2022143273. Helenius G, Backdahl H, Bodin A, et al (2006) In vivo biocompatibility of bacterial cellulose. J Biomed Mater Res Part A 76A:431-438. https: / / doi.Org / 10.1002 / jbm.a.305704. Wu J, Yin N, Chen S, et al (2019) Simultaneous 3D cell distribution and bioactivity enhancement of bacterial cellulose (BC) scaffold for articular cartilage tissue engineering. 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Claims

C L A I M S1. A bacterial nanocellulose material comprising pores obtainable by short pulse laser etching, wherein a largest diameter of each pore obtained by short pulse laser etching is comprised in a range from 5 to 10000 pm, as measured by X-ray microtomography (micro-CT) analysis.

2. The bacterial nanocellulose material according to the previous claim wherein the largest diameter of the each pore is comprised in a range from 5 pm to 5000 pm; preferably 30 pm to 2000 pm; more preferably 50 pm to 500 pm.

3. The bacterial nanocellulose material according to any of the previous claims, wherein the each pore is individually obtained by short pulse laser etching and the pores are linked together to obtain complex micro or macroporore geometries, wherein the complex micro or macroporore geometries comprise at least a non-linear segment.

4. The bacterial nanocellulose material according to any of the previous claims, wherein the each pore is individually obtained by short pulse laser etching and the pores are linked together to obtain a channel or channels inside the bacterial nanocellulose material.

5. The bacterial nanocellulose material according to the previous claim wherein the channels comprise at least a non-linear channels segment, preferably a curve.

6. The bacterial nanocellulose material according to any of the previous claims, wherein the each pores obtained by short pulse laser etching form an array of microchannels; preferably an array of parallel microchannels.

7. The bacterial nanocellulose material according to any of the previous claims, wherein the each pores obtained by short pulse laser etching form orthogonal grid structures.

8. The bacterial nanocellulose material according to any of the previous claims, wherein the each pore comprise linear pores, non-linear pores or combinations thereof.

9. The bacterial nanocellulose material according to any of the previous claims, wherein the chanel comprise linear chanels, non-linear chanels or combinations thereof.

10. The bacterial nanocellulose material according to any of the previous claims, wherein the pores obtained by short pulse laser etching form non-linear, tortuous channels, generating anisotropic architectures.

11. The bacterial nanocellulose material according to any of the previous claims wherein the pores have a tubular morphology, branched morphology, oval morphology or spherical morphology, or combinations thereof.

12. The bacterial nanocellulose material according to any of the previous claims wherein the each pores / channel obtainabed by short pulse laser etching comprise a dead-end.

13. The bacterial nanocellulose material according to any of the previous claims, wherein each pore / channel obtained by short-pulse laser etching is inside the material, preserving the structure of the material surface.

14. The bacterial nanocellulose material according to any of the previous claims wherein the pores distribution in the bacterial nanocellulose material is anisotropic.

15. The bacterial nanocellulose material according to any of the previous claims 1-7 wherein the pores distribution in the bacterial nanocellulose material is isotropic.

16. The bacterial nanocellulose material according to any of the previous claims further comprising an anti-inflammatory agent, an antiseptic agent, an antipyretic agent, an anaesthetic agent, a therapeutic agent, a cell, or combinations thereof.

17. The bacterial nanocellulose material according to the previous claim wherein the cell is a non-human animal cell, or a human cell, or a stem cell, or combinations thereof.

18. The bacterial nanocellulose material according to any of the previous claims wherein the material is a hydrogel.

19. An article comprising the bacterial nanocellulose material of any of the previous claims.

20. The article according to the previous claim wherein the article is a multicompartment medical-device, preferably a mesh, a hollow cylinder, a scaffold, a cell carrier, a therapeutic hydrogel, a drug, a delivery depot, a cardiac valve, a vascular prosthesis, a surgical implant, a surgical mesh, a stent, an artificial intervertebral disc, or an artificial meniscus or combinations thereof.

21. A bacterial nanocellulose composition for use in tissue engineering and / or tissue regeneration wherein the bacterial nanocellulose composition is administered in the form of a bacterial nanocellulose material comprising a plurality of pores, wherein said pores are obtainable by short pulse laser etching , preferably as described in claims 1-18.

22. The bacterial nanocellulose for use according to the previous claim wherein a largest diameter of each pore obtained by short pulse laser etching is comprised in a range from 5 to 10000 pm, as measured by X-ray microtomography (micro-CT) analysis.

23. The bacterial nanocellulose material or bacterial nanocellulose device or composition according to any of the previous claimsfor use in the treatment of cartilage defects, bone defects, cardiac defects, or vascular defects.

24. Use of a bacterial nanocellulose material as defined in any of the claims 1-18 or an article as defined in any of the claims 19-20 as a cell scaffold, or an implantable device.

25. A method for treating cartilage defects, bone defects, or vascular defects in a subject, the method comprising administering a bacterial nanocellulose material or a bacterial nanocellulose device or composition to the subject, wherein the bacterial nanocellulose composition is administered in the form of a bacterial nanocellulose material as defined in any of the claims 1-18.

26. A method for obtaining a bacterial nanocellulose material according to any of the previous claims comprising the following steps: providing a bulk sample of bacterial nanocellulose; preferably bacterial nanocellulose biosynthesized by Komagataeibacter spp, Gluconocetobacter spp., Acetobacter spp., Novacetimonas spp., or mixtures thereof; washing the bulk sample of bacterial nanocellulose to obtain a high purity BNC; slicing the washed sample of BNC to obtain BNC pellicles, preferably BNC pellicles with a thickness of up to 5 mm; etching the BNC pellicles with a short pulse laser to obtain the porous BNC material; wherein the short pulse laser has a pulse duration ranging from 1 fs to 100 ps; wherein the short pulse laser has a pulse energy ranging from 1 nJ to 2 J.

27. A use of the bacterial nanocellulose material / composition material according to any of the previous claims for the manufacture of a medicament for tissue engineering and / or tissue regeneration.

28. A method for tissue engineering and / or tissue regeneration in a subject, wherein the method comprise administering a bacterial nanocellulose material / composition of any of the previous claims to the subject.

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