Bacterial Cellulose: A Sustainable Source for Hydrogels and 3D-Printed Scaffolds for Tissue Engineering

Bacterial cellulose is a biocompatible biomaterial with a unique macromolecular structure. Unlike plant-derived cellulose, bacterial cellulose is produced by certain bacteria, resulting in a sustainable material consisting of self-assembled nanostructured fibers with high crystallinity. Due to its purity, bacterial cellulose is appealing for biomedical applications and has raised increasing interest, particularly in the context of 3D printing for tissue engineering and regenerative medicine applications. Bacterial cellulose can serve as an excellent bioink in 3D printing, due to its biocompatibility, biodegradability, and ability to mimic the collagen fibrils from the extracellular matrix (ECM) of connective tissues. Its nanofibrillar structure provides a suitable scaffold for cell attachment, proliferation, and differentiation, crucial for tissue regeneration. Moreover, its mechanical strength and flexibility allow for the precise printing of complex tissue structures. Bacterial cellulose itself has no antimicrobial activity, but due to its ideal structure, it serves as matrix for other bioactive molecules, resulting in a hybrid product with antimicrobial properties, particularly advantageous in the management of chronic wounds healing process. Overall, this unique combination of properties makes bacterial cellulose a promising material for manufacturing hydrogels and 3D-printed scaffolds, advancing the field of tissue engineering and regenerative medicine.


Introduction
In recent years, bacterial cellulose has been a subject of interest for researchers due to its unique macromolecular structure and biological properties [1][2][3][4][5].Early studies focused on optimizing the production and purification methods of bacterial cellulose, in order to improve its quality and suitability for various biomedical applications [6,7].The material was predominantly utilized to design wound dressings, tissue engineering scaffolds, and controlled drug delivery systems [4,5].However, its utilization in 3D printing for biomedical purposes was relatively limited due to challenges in processing and scalability [8].Research efforts in the past have laid the foundation for understanding the fundamental characteristics of bacterial cellulose and exploring its potential in the realm of biomedicine [4,5,9].
Bacterial cellulose is a natural biopolymer produced by various species of bacteria, such as Rhizobium, Gluconacetobacter, Sarcina, Komagataeibacter, Agrobacterium, and Rhodobacter [10], and is known for its remarkable properties, including high purity, biocompatibility, mechanical strength, and water retention capacity [4].These unique characteristics make bacterial cellulose an attractive material for a wide range of biomedical applications, with 3D printing emerging as a promising avenue for its utilization.Traditional biomaterials used in 3D printing, such as synthetic polymers, often face limitations in terms of biocompatibility, degradation rates, and mechanical properties.In contrast, bacterial cellulose offers a sustainable and eco-friendly alternative that has the potential to address these challenges and revolutionize the field of biomedical 3D printing.The scientific community is starting to show an increased interest in harnessing the capabilities of bacterial cellulose for 3D printing applications in the biomedical field [4,5,11].Researchers are actively exploring innovative strategies to functionalize bacterial cellulose-based materials with bioactive compounds to enhance their performance in tissue regeneration, drug delivery, and medical device manufacturing [7,12].Advances in additive manufacturing technologies have facilitated the development of customized 3D printing techniques and parameters that allow the fabrication of intricate structures using bacterial cellulose-based composites with improved mechanical properties and structural integrity [5,7,9].Current research endeavors are focused on evaluating the biocompatibility, degradation kinetics, and efficacy of bacterial cellulose-based 3D printed constructs in both in vitro and in vivo settings across a wide spectrum of biomedical applications [9,13].
The number of scientific papers regarding bacterial cellulose has significantly increased over the last 10 years, but the most rapid growth was observed in the last five years, specifically between 2020 and 2024, when over half of the publications were published.When searching for the term "bacterial cellulose", 6081 publications in total were found in the online Web of Science core collection database in 2020-2024.An analysis of these publications on "bacterial cellulose", as a common denominator, and "scaffold" or "wound healing", indicated 536 and 426 articles, respectively, and revealed an uneven distribution according to the most addressed applications, as depicted in Figure 1.A high percentage was found when "regenerative medicine" was superimposed.
Gels 2024, 10, x FOR PEER REVIEW 2 of 21 to address these challenges and revolutionize the field of biomedical 3D printing.The scientific community is starting to show an increased interest in harnessing the capabilities of bacterial cellulose for 3D printing applications in the biomedical field [4,5,11].Researchers are actively exploring innovative strategies to functionalize bacterial cellulose-based materials with bioactive compounds to enhance their performance in tissue regeneration, drug delivery, and medical device manufacturing [7,12].Advances in additive manufacturing technologies have facilitated the development of customized 3D printing techniques and parameters that allow the fabrication of intricate structures using bacterial cellulose-based composites with improved mechanical properties and structural integrity [5,7,9].Current research endeavors are focused on evaluating the biocompatibility, degradation kinetics, and efficacy of bacterial cellulose-based 3D printed constructs in both in vitro and in vivo settings across a wide spectrum of biomedical applications [9,13].
The number of scientific papers regarding bacterial cellulose has significantly increased over the last 10 years, but the most rapid growth was observed in the last five years, specifically between 2020 and 2024, when over half of the publications were published.When searching for the term "bacterial cellulose", 6081 publications in total were found in the online Web of Science core collection database in 2020-2024.An analysis of these publications on "bacterial cellulose", as a common denominator, and "scaffold" or "wound healing", indicated 536 and 426 articles, respectively, and revealed an uneven distribution according to the most addressed applications, as depicted in Figure 1.A high percentage was found when "regenerative medicine" was superimposed.To refine this search, the search strategy included the keywords "bacterial cellulose", "hydrogel", and "3D printing", and the research domain of biomedical applications, inclusive of "tissue engineering", "drug delivery", and "wound healing".In total, 22 full-text articles fit all our inclusion criteria (Figure 2).To refine this search, the search strategy included the keywords "bacterial cellulose", "hydrogel", and "3D printing", and the research domain of biomedical applications, inclusive of "tissue engineering", "drug delivery", and "wound healing".In total, 22 full-text articles fit all our inclusion criteria (Figure 2).
In this context, the present paper aims to explore the current landscape of promising research on bacterial cellulose, highlighting its physicochemical and structural properties, biocompatibility, formulation as hydrogel (ink), and its customizability for applications in the field of biomedicine with a specific focus on the emerging 3D printing technology.In this context, the present paper aims to explore the current landscape of promising research on bacterial cellulose, highlighting its physicochemical and structural properties, biocompatibility, formulation as hydrogel (ink), and its customizability for applications in the field of biomedicine with a specific focus on the emerging 3D printing technology.

Bacterial Cellulose Hydrogels-Physicochemical, Structural, and Biological Properties
Hydrogels play a vital role in various biomedical applications, providing a hydrated, biocompatible matrix for cell encapsulation, tissue regeneration, and controlled drug delivery.The numerous properties of hydrogels make them indispensable tools in tissue engineering, providing a versatile platform for creating biomimetic tissue constructs, promoting cell growth and tissue regeneration, allowing for controlled drug delivery, and facilitating advanced research and therapeutic applications in regenerative medicine [7].Their biocompatibility, adjustable characteristics, and capacity to imitate the natural tissue milieu make them important in the advancement of tissue engineering and regenerative medicine.Certain hydrogels, for example, can alter tissue engineering by acting as biomimetic scaffolds that mimic the mechanical properties of genuine tissues, boosting

Bacterial Cellulose Hydrogels-Physicochemical, Structural, and Biological Properties
Hydrogels play a vital role in various biomedical applications, providing a hydrated, biocompatible matrix for cell encapsulation, tissue regeneration, and controlled drug delivery.The numerous properties of hydrogels make them indispensable tools in tissue engineering, providing a versatile platform for creating biomimetic tissue constructs, promoting cell growth and tissue regeneration, allowing for controlled drug delivery, and facilitating advanced research and therapeutic applications in regenerative medicine [7].Their biocompatibility, adjustable characteristics, and capacity to imitate the natural tissue milieu make them important in the advancement of tissue engineering and regenerative medicine.Certain hydrogels, for example, can alter tissue engineering by acting as biomimetic scaffolds that mimic the mechanical properties of genuine tissues, boosting integration with surrounding tissues and cell viability [14].Studies have shown that by modifying the mechanical properties of hydrogels, it is possible to meet specific needs of tissue engineering applications for bone, cartilage, and blood vessels.
The main components used as building blocks for hydrogels in tissue engineering include natural polymers such as alginate, elastin, chitosan, silk fibroin, fibrinogen, collagen, hyaluronic acid, and gelatin, along with synthetic polymers like poly (ethylene glycol), methacrylates, polyvinyl alcohol, polyacrylate, and polyacrylamide [9,15,16].However, most of the time, these materials provide limited mechanical properties or may not contain nano-to-mesoscale topographic features.As a result, hydrogels based on self-assembling/fibrillizing (bio)molecules or nanofibrils, such as bacterial cellulose, are appealing due to their ease of chemical modification, advanced and tunable mechanical properties, and the potential for the mesoscale alignment of colloidal scale nanofibrils to guide cell alignment [15,17].The presence of many hydroxyl groups on the surface of the cellulose matrix allows for functionalization while maintaining the surface's hydrophobicity and thermodynamic stability [9,18].

Bacterial vs. Plant-Derived Cellulose
Cellulose, a polysaccharide comprising β-1,4 connected D-glucose units, is one of the most prevalent biodegradable materials on the planet.The main source of cellulose is represented by plants and agricultural residues, including wood, cotton, hemp, bamboo, and sisal bagasse.On the other hand, cellulose can be generated by a number of microorganisms from the genera Acetobacter, Gluconobacter, Komagataeibacter, Rhizobium, Agrobacterium, and Sarcina [10].Bacterial cellulose is also manufactured by specific lactic and acetic acid bacteria in SCOBY consortia in a regulated fermentation process, yielding a very pure and consistent form of cellulose nanofibrils [19].Therefore, bacterial cellulose's purity and uniformity make it more suitable for scientific and industrial applications that require careful control over material qualities.Unlike plant-derived cellulose, which is a primary component of plant cell walls, bacteria generate cellulose as a mechanism to protect themselves and establish a stable environment for growth (Figure 3).

Bacterial vs. Plant-Derived Cellulose
Cellulose, a polysaccharide comprising β-1,4 connecte most prevalent biodegradable materials on the planet.T represented by plants and agricultural residues, including and sisal bagasse.On the other hand, cellulose can be gen organisms from the genera Acetobacter, Gluconobacter, Kom bacterium, and Sarcina [10].Bacterial cellulose is also manu acetic acid bacteria in SCOBY consortia in a regulated fer very pure and consistent form of cellulose nanofibrils [19].purity and uniformity make it more suitable for scientific a require careful control over material qualities.Unlike plan primary component of plant cell walls, bacteria generate protect themselves and establish a stable environment for  Although both types of cellulose share some similar characteristics, bacterial cellulose is often preferred over plant cellulose as a base material in hydrogel composition for several reasons: its purity and consistency, nanostructure, mechanical properties, biocompatibility, low thermal expansion, high optical transparency, scalability, and efficiency [20] (Table 1).Bacterial cellulose has a distinct nanostructure that includes a highly porous network of nanofibers.Cellulose chains form basic fibrillary units or elementary fibrils with lengths of 0.1 to 0.2 µm organized into microfibrils measuring 0.1 µm in width and 0.1 to 1 µm in length [21].This structure improves water absorption and retention, these being preferred for hydrogel production, which requires high water content (Figure 4).
In regard of mechanical properties, bacterial cellulose has a significant tensile strength (200 MPa) [22], while plant-derived cellulose has a value of tensile strength of up to 1 GPa.Still, bacterial cellulose presents higher flexibility with a Young modulus of 15 GPa vs. plantderived cellulose with a Young modulus of 130 GPa.These mechanical qualities make it ideal for applications requiring strength and durability, such as wound dressings and other biomedical devices.Due to its purity, bacterial cellulose showed excellent biocompatibility, and is widely adopted as a scaffolding biomaterial for the modeling and engineering of biomimetic tissues, showing appropriate cellular response and behavior [23].
Another aspect to consider is the environmental impact of the chosen cellulose source.While vegetal cellulose is sourced from renewable plant materials, bacterial cellulose manufacturing can be more efficient and scalable, particularly when using specific bacterial strains in controlled fermentation conditions.Because of its high efficiency, bacterial cellulose may be a more cost-effective solution for the manufacture of large-scale hydrogel and other materials [24].
work of nanofibers.Cellulose chains form basic fibrillary units or elementary fibrils with lengths of 0.1 to 0.2 μm organized into microfibrils measuring 0.1 μm in width and 0.1 to 1 μm in length [21].This structure improves water absorption and retention, these being preferred for hydrogel production, which requires high water content (Figure 4).In regard of mechanical properties, bacterial cellulose has a significant tensile strength (200 MPa) [22], while plant-derived cellulose has a value of tensile strength of up to 1 GPa.Still, bacterial cellulose presents higher flexibility with a Young modulus of 15 GPa vs. plant-derived cellulose with a Young modulus of 130 GPa.These mechanical qualities make it ideal for applications requiring strength and durability, such as wound dressings and other biomedical devices.Due to its purity, bacterial cellulose showed excellent biocompatibility, and is widely adopted as a scaffolding biomaterial for the modeling and engineering of biomimetic tissues, showing appropriate cellular response and behavior [23].
Another aspect to consider is the environmental impact of the chosen cellulose source.While vegetal cellulose is sourced from renewable plant materials, bacterial cel-
In recent years, bacterial cellulose has gained increasing attention as a base material for hydrogel formulation, particularly in the context of 3D printing.Its unique architecture imparts exceptional mechanical strength and structural integrity to hydrogels, making them well suited for 3D printing applications that demand precise control over shape, resolution, and design complexity [29].Better mechanical performance, even in wet conditions, was achieved via the ion crosslinking of bacterial cellulose nanofibers using Fe 3+ to obtain macrofibers with higher tensile strength [30].The nanofibrous network of bacterial cellulose offers a biomimetic scaffold that mimics the microstructural, physicochemical, and mechanical characteristics of the extracellular matrix, promoting cell adhesion, proliferation, and differentiation, and favoring oxygen and nutrient permeability [31].The natural constituents also provide biophysical and biological cues to stimulate and direct cell function and signaling through cell surface receptors, growth factors, or signal proteins [32].Microscopically, bacterial nanocellulose fibrils resemble collagen fibrils, presenting the same size range, i.e., a width of roughly 100 nm [33][34][35].
Bacterial cellulose hydrogels possess a remarkable capacity for water absorption and retention, creating a hydrated microenvironment conducive to cell viability, proliferation, and differentiation.The ability of bacterial cellulose to retain water molecules within its porous structure enhances nutrient transport, waste removal, and signaling molecule diffusion, essential for supporting cellular activities within the hydrogel matrix [33,36].
The thermal characteristics of hydrogels are closely related to their viscosity and therefore to their printability, and thermographic analysis provides important data to determine the parameters, compositions, and properties of hydrogel viscosity [36,37].Rheological investigations showed the flow properties of hydrogels used for the 3D printing of scaffolds, viscosity being an essential parameter in determining printability.Also, rheological experiments were conducted to evaluate the changes in the mechanical properties of bacterial cellulose hydrogel that occurred during the UV curing process [38].The linear and non-linear tests provided information on temperature, component, and water concentration that can greatly influence the thermoelastic properties and behavior of hydrogels when printed, and the subsequent mechanical properties of 3D-printed structures [36,[39][40][41][42][43][44].
A key advantage of bacterial cellulose is its biocompatibility, making it an attractive choice for biomedical applications where interaction with living tissues is paramount.Hydrogels incorporating bacterial cellulose exhibit minimal cytotoxicity and immunogenicity, enabling their safe use in tissue engineering, wound healing, and regenerative medicine [45].Recently, an improvement in cell adhesion and a growth of cells were reported after the surface modification of bacterial cellulose via drying and plasma treatment to obtain higher porosity and hydrophylicity [46].Moreover, the biodegradability of bacterial cellulose ensures that the hydrogel scaffold can gradually degrade over time, allowing for tissue ingrowth and remodeling.
Bacterial cellulose itself has no antimicrobial activity, but due to its ideal structure (including an ultrafine and highly porous network of nanofibrils), it can serve as a matrix for other biologically active molecules (Figure 5).Thus, it becomes an essential part of hybrid products with antimicrobial applications [47] that provide a large contact surface area for interactions with microorganisms [48].2024, 10, x FOR PEER REVIEW hybrid products with antimicrobial applications [47] that provide a area for interactions with microorganisms [48].Nanocellulose-based antimicrobial materials are classified into two types based on the used antimicrobial agent: inorganic (silver nanoparticles) [48][49][50][51][52][53] and organic (honey, curcumin, antibiotics) [54][55][56].Acting as a stabilizer of the antimicrobial formulation, bacterial cellulose can sustain controlled release and improve the bioactivity of poorly soluble compounds [48].Therefore, more effectiveness in the targeted biomedical field can be provided via the structural and physical modification of bacterial cellulose, on the one hand, and the process of in situ or ex situ tuning using antimicrobial compounds or polymers, on the other hand [57].
In regenerative medicine, bacterial cellulose promotes cellular adhesion, proliferation, migration, and differentiation to accelerate re-epithelization and speed up wound healing [10,27,45,58].The molecular mechanisms revealed during these processes were based on the interleukin 10 (IL 10) signaling pathway, with important immunoregulatory function and vascular endothelial growth factor (VEGF) signaling involved in cell proliferation and angiogenesis [10].Furthermore, bacterial cellulose has wide-ranging uses in biomedical device manufacturing, including dental implants, artificial blood vessels, vascular grafts, implants for the urethra and nerve, artificial corneas, and retinas.Its main characteristics are low tissue adhesion, low toxicity, thermal insulation, and preservation of a moist environment for gas exchange at the wound site [10,24,45,[59][60][61].For bacterial cellulose to function in vivo, cell-cell contact is essential [4,5,[9][10][11][12][13][14][15][16]27].More importantly, because of its topography, wettability, and surface charge, bacterial cellulose demonstrates remarkable physicochemical and pharmacological properties [1,4,27,59,60].In tissue engineering applications, bacterial cellulose is used in two main ways: as grown directly on application-specific substrates, or mechanically and chemically processed to serve as a reinforcing or crosslinking agent.The crystallinity of bacterial cellulose is the main property correlated to its reinforcing ability [61].

Customizability and Tunability for 3D Printing
The mechanical properties, porosity, degradation rate, and bioactive functionalities of bacterial cellulose-based hydrogels can be tailored through various processing techniques, such as blending with polymers, the incorporation of bioactive molecules, or physical crosslinking methods.This customizability allows researchers to fine-tune the properties of the hydrogel to meet the specific requirements of diverse applications, ranging from soft tissue engineering to cartilage regeneration and drug delivery systems.Various investigation techniques can be used to assess and optimize hydrogel properties (Figure 6).serve as a reinforcing or crosslinking agent.The crystallinity of bacterial cellulose is the main property correlated to its reinforcing ability [61].

Customizability and Tunability for 3D Printing
The mechanical properties, porosity, degradation rate, and bioactive functionalities of bacterial cellulose-based hydrogels can be tailored through various processing techniques, such as blending with polymers, the incorporation of bioactive molecules, or physical crosslinking methods.This customizability allows researchers to fine-tune the properties of the hydrogel to meet the specific requirements of diverse applications, ranging from soft tissue engineering to cartilage regeneration and drug delivery systems.Various investigation techniques can be used to assess and optimize hydrogel properties (Figure 6).Thus, the micromorphology, fibril length, and thickness of unmodified or modified bacterial cellulose diluted solutions were investigated via transmission electron microscopy (TEM) [45,61], this being essential for determining the tunability of hydrogels in view of 3D printing.Also, a modification on the surface of bacterial-generated cellulose nanofibers with 3-(trimethoxysilyl)propyl methacrylate (TMSPM) was revealed via TEM Thus, the micromorphology, fibril length, and thickness of unmodified or modified bacterial cellulose diluted solutions were investigated via transmission electron microscopy (TEM) [45,61], this being essential for determining the tunability of hydrogels in view of 3D printing.Also, a modification on the surface of bacterial-generated cellulose nanofibers with 3-(trimethoxysilyl)propyl methacrylate (TMSPM) was revealed via TEM at an accelerating voltage of 200 kV [61].
An atomic force microscopy (AFM) study demonstrated a thickening of bacterial cellulose nanofibers from 55-95 nm to 85-140 nm via poly(acrylic acid) grafting on their surface during UV-induced polymerization, which reinforced the mixtures for the 3D printing of stable structures (Figure 7) [62].
SEM investigations showed modifications to surface morphology and pore size variation between 2 and 190 µm in bacterial cellulose hydrogels, according to the increasing content of acrylic acid in their composition, presenting denser inter-polymeric networks after electron beam irradiation and improved thermal stability (Figure 7) [63].An investigation of the in situ arrangement of bacterial cellulose filaments in spheroids and of their size and shape was conducted via optical, SEM, and TEM analyses to allow for a tunable strategy for biomedical applications [64].
Changes in the functional groups of the hydrogels components were highlighted via Fourier transform infrared spectrometry (FT-IR) to determine the optimal chemical structure of bacterial cellulose hydrogels used as inks for 3D printing [10].Improvements in mechanical properties and the thermal stability of bacterial cellulose-gelatin composite hydrogels at varying mixing ratios between 25:1 and 400:1 were observed via FT-IR, highlighting hydrogen bond formation between amine and hydroxyl groups (Figure 7) [65].Alongside this, the structural characteristics and crystallinity of bacterial celluloses investigated via Raman spectroscopy and X-ray diffraction varied according to static or dynamic regimes of cultivation with Gluconacetobacter sucrofermentans, allowing the selection of parameters to strengthen the derived inks [66].Crystalline regions alternating with amorphous ones were revealed after the addition of silver sulfadiazine to bacterial cellulose-chitosan hydrogels, via X-ray diffraction, simultaneously improving the mechanical and antibacterial properties required for tissue engineering applications (Figure 7) [67].
Changes to surface wettability observed via contact angle measurements in nanofibrous porous microstructures of bacterial cellulose revealed enhanced biocompatibility and the potential to interact with mouse embryonic stem cells [68].In addition, optimal values of bacterial nanocellulose concentration and mixing ratios with gelatin methacryloyl influenced the mechanical properties of composite hydrogels intended for 3D printing [69].
The intrinsic properties of bacterial cellulose, including its structural integrity, biocompatibility, and water retention capabilities, make it highly compatible with 3D printing technologies.By integrating bacterial cellulose as a base element in hydrogel formulations for 3D printing, researchers can achieve precise control over the spatial distribution of cells, growth factors, and biomaterials, enabling the fabrication of complex, multi-material constructs with tailored mechanical and biological properties [9,12,70].The versatility of 3D printing techniques, such as extrusion-based printing, stereolithography, and inkjet bioprinting, further enhances the potential of bacterial cellulose hydrogels for creating patient-specific implants, organ-on-a-chip models, and personalized medical devices.
By applying living cells, also known as bioink, to a non-living surface, living items can be 3D printed.The structural integrity of the entire 3D design and the survival and functionality of the cells in the bioprinted structures depend heavily on the characteristics of the printing substrate and the makeup of the bio-ink [71].On the other hand, cell behavior, beyond the physical and chemical properties of the hydrogel, is also influenced by its 3D geometrical structure, since a better resemblance to the extracellular matrix leads to better cell proliferation.Therefore, for biomedical applications targeting the preparation and conditioning of nanocellulose-based hydrogels, a number of methods have been developed to generate a 3D structure that mimics the extracellular matrix, including homogenization, freeze-thawing cycling, freeze-drying, free radical polymerization, photocrosslinking and 3D bioprinting [13,72].
site hydrogels at varying mixing ratios between 25:1 and 400:1 were observed via FT-IR, highlighting hydrogen bond formation between amine and hydroxyl groups (Figure 7) [65].Alongside this, the structural characteristics and crystallinity of bacterial celluloses investigated via Raman spectroscopy and X-ray diffraction varied according to static or dynamic regimes of cultivation with Gluconacetobacter sucrofermentans, allowing the selection of parameters to strengthen the derived inks [66].Crystalline regions alternating with amorphous ones were revealed after the addition of silver sulfadiazine to bacterial cellulose-chitosan hydrogels, via X-ray diffraction, simultaneously improving the mechanical and antibacterial properties required for tissue engineering applications (Figure 7) [67].Changes to surface wettability observed via contact angle measurements in nanofibrous porous microstructures of bacterial cellulose revealed enhanced biocompatibility and the potential to interact with mouse embryonic stem cells [68].In addition, optimal values of bacterial nanocellulose concentration and mixing ratios with gelatin methac-

Applications of Bacterial Cellulose Hydrogels
Because of their biocompatibility, mechanical robustness, and bioactive properties, bacterial cellulose hydrogel applications include wound healing, drug delivery systems, and tissue engineering [11,12,73].Synthesized data on recent advances are presented in Table 2.

Characterization of Bacterial Cellulose-Based 3D-Printed Scaffolds
For the characterization and analysis of bacterial cellulose-based 3D-printed scaffolds, several investigation techniques serve to assess their morphological, structural, and mechanical properties and their biocompatibility (Figure 6).Various microscopy techniques, such as optical, transmission and scanning electron, atomic force, and confocal microscopy were used for the morphological and structural characterization of bacterial cellulose 3D-printed structures.Each microscopy technique has its own advantages and disadvantages, such as equipment resolution, the complexity of sample preparation, the time required, and last but not at least the cost of conducting a good evaluation of certain morphological characteristics of 3D-printed bacterial cellulose scaffolds.Thus, SEM of polylactic acid 3D-printed composite scaffolds containing bacterial cellulose facilitated stability evaluation via observations of strand width, with the morphology of the surface and pore size, and the microstructure controlled via layer printing (Figure 8) [82].SEM also allowed observations for tuning of fine characteristics, like pore shapes from round to square and sizes between 301 and 314 μm in 3D-printed polycaprolactone/gelatin scaffolds, via the addition of different concentrations of bacterial cellulose and hydroxyapatite in the hydrogel composition, to better meet the required mechanical properties and to facilitate the interaction with cells for bone tissue engineering applications (Figure 9) [83].SEM also allowed observations for tuning of fine characteristics, like pore shapes from round to square and sizes between 301 and 314 µm in 3D-printed polycaprolactone/gelatin scaffolds, via the addition of different concentrations of bacterial cellulose and hydroxyapatite in the hydrogel composition, to better meet the required mechanical properties and to facilitate the interaction with cells for bone tissue engineering applications (Figure 9) [83].
Structural details of bacterial nanocellulose/alginate 3D-printed porous scaffolds before and after oxidization with (2,2,6,6-tetramethylpiperidin-1-yl)oxyl (TEMPO) and laponite nanoclay incorporation were provided with a high degree of precision via field emission SEM [76].The team also reported that this composition allowed the preparation of porous 3D-printed scaffolds with different pore sizes, which varied according to the used working parameters, such as line spacing, but were also suitable for multiple-layered materials printed as complex nose-and ear-like structures with high-fidelity shape and stability for long-term preservation (Figure 10).
High-resolution topographical images obtained via AFM allowed the calculation of the fiber-length-to-diameter ratio of bacterial cellulose nanofibers, highlighting the positive interaction with maleic acid to decrease their diameter and to further use combinations with gelatin for high-precision stable 3D-printed osteogenic constructs [81].
With regard to mechanical property characterization, various bacterial nanocellulosebased bioprinted scaffolds were tested, and the effect of the composition and crosslinking was assessed.Thus, the influence of component concentration, including bacterial nanocellulose concentration, on the mechanical properties of composite hydrogels with gelatin methacryloyl, was analyzed to determine the optimal values and mixing ratios for obtaining reinforced 3D-printed cartilage [69].With regard to mechanical property characterization, various bacterial nanocellulose-based bioprinted scaffolds were tested, and the effect of the composition and crosslinking was assessed.Thus, the influence of component concentration, including bacterial nanocellulose concentration, on the mechanical properties of composite hydrogels with gelatin methacryloyl, was analyzed to determine the optimal values and mixing ratios for obtaining reinforced 3D-printed cartilage [69].Confocal laser scanning microscopy observations of living materials with self-healing and self-regenerating capabilities manufactured via the 3D printing of xanthan gel incorporating Gluconacetobacter xylinus revealed the morphology of a heterogeneous cellulose network produced by bacteria in situ after incubation in culture medium [84].Confocal laser scanning microscopy observations of living materials with self-healing and self-regenerating capabilities manufactured via the 3D printing of xanthan gel incorporating Gluconacetobacter xylinus revealed the morphology of a heterogeneous cellulose network produced by bacteria in situ after incubation in culture medium [84].

Conclusions and Future Development
Ongoing research efforts are focused on expanding the utility of bacterial cellulose in more sophisticated biofabrication applications, including vascularized tissue constructs, organoids, and bioartificial organs.Future studies might explore novel approaches for enhancing the functionalization of bacterial cellulose hydrogels with bioactive molecules, growth factors, and cell-laden bioinks to create biomimetic tissues with improved physiological relevance and therapeutic efficacy.
Looking ahead to the future, bacterial cellulose is poised to emerge as a key player in revolutionizing the field of biomedical 3D printing.With ongoing advancements in research and technology, the potential applications of bacterial cellulose are expanding rapidly, particularly in the areas of tissue engineering, regenerative medicine, and personalized healthcare.Future developments may involve the integration of bacterial cellulose with bioactive molecules, stem cells, or growth factors to create functionalized constructs that closely mimic native tissue structures and promote enhanced tissue regeneration.The further optimization of 3D printing techniques utilizing bacterial cellulose-based materials is anticipated to lead to the fabrication of patient-specific implants, organ-on-a-chip devices, and advanced drug delivery systems tailored to individual patient needs.The continued exploration of bacterial cellulose in the realm of 3D printing holds significant promise for addressing crucial healthcare challenges and driving forward the development of cuttingedge therapies and interventions that have the potential to positively impact patients on a global scale.

Figure 1 .
Figure 1.Correlation between publications on bacterial cellulose found as a common denominator in the Web of Science databases between 2020 and April 2024, and (a) scaffold and (b) wound healing, and distribution according to the most addressed applications.

Figure 1 .
Figure 1.Correlation between publications on bacterial cellulose found as a common denominator in the Web of Science databases between 2020 and April 2024, and (a) scaffold and (b) wound healing, and distribution according to the most addressed applications.

Figure 2 .
Figure 2. Flowchart of the articles found in the Web of Science database between 2020 and April 2024.

Figure 2 .
Figure 2. Flowchart of the articles found in the Web of Science database between 2020 and April 2024.

Figure 3 .
Figure 3. Transmission electron micrograph of bacterial nanocell Equisetum arvense in Kombucha consortia of yeasts and bacteria the authors).

Figure 3 .
Figure 3. Transmission electron micrograph of bacterial nanocellulose obtained via fermentation of Equisetum arvense in Kombucha consortia of yeasts and bacteria (unpublished results obtained by the authors).

Figure 4 .
Figure 4. Main differences between structure and purity of bacterial and plant cellulose.

Figure 4 .
Figure 4. Main differences between structure and purity of bacterial and plant cellulose.

Figure 5 .
Figure 5. Bacterial cellulose-based antimicrobial biomedical device for s printed from [47] under a Creative Commons Attribution License 4.0 Deed|Attribution 4.0 International|Creative Commons (accessed on 21 Ma

Figure 6 .
Figure 6.Main techniques used for the morphological and structural characterization of bacterial cellulose-based hydrogels and 3D-printed scaffolds.BC-bacterial cellulose.

Figure 6 .
Figure 6.Main techniques used for the morphological and structural characterization of bacterial cellulose-based hydrogels and 3D-printed scaffolds.BC-bacterial cellulose.

Figure 9 .
Figure 9. Pore shape and size (a-h) and SEM images (i-l) of 3D-printed polycaprolactone/gelatin scaffolds supplemented with different concentrations of bacterial cellulose and hydroxyapatite for bone tissue engineering.Reprinted from [83] under a Creative Commons Attribution License 4.0 (CC BY), "CC BY 4.0 Deed|Attribution 4.0 International|Creative Commons (accessed on 30 May 2024)".

Figure 9 .
Figure 9. Pore shape and size (a-h) and SEM images (i-l) of 3D-printed polycaprolactone/gelatin scaffolds supplemented with different concentrations of bacterial cellulose and hydroxyapatite for bone tissue engineering.Reprinted from [83] under a Creative Commons Attribution License 4.0 (CC BY), "CC BY 4.0 Deed|Attribution 4.0 International|Creative Commons (accessed on 30 May 2024)".

Author
Contributions: Conceptualization, E.I.O. and O.C.; writing-original draft preparation, E.U. and V.S.M.; writing-review and editing, E.I.O. and O.C.All authors have read and agreed to the published version of the manuscript.

Table 1 .
Advantageous characteristics of bacterial cellulose vs. plant-derived cellulose that are useful for obtaining hydrogels and 3D-printed scaffolds for tissue engineering.

Table 2 .
Applications of bacterial-cellulose-based materials as inks and 3D-printed scaffolds for tissue engineering.BC-bacterial cellulose.