Electrospun Nanocomposites Containing Cellulose and Its Derivatives Modified with Specialized Biomolecules for an Enhanced Wound Healing

Wound healing requires careful, directed, and effective therapies to prevent infections and accelerate tissue regeneration. In light of these demands, active biomolecules with antibacterial properties and/or healing capacities have been functionalized onto nanostructured polymeric dressings and their synergistic effect examined. In this work, various antibiotics, nanoparticles, and natural extract-derived products that were used in association with electrospun nanocomposites containing cellulose, cellulose acetate and different types of nanocellulose (cellulose nanocrystals, cellulose nanofibrils, and bacterial cellulose) have been reviewed. Renewable, natural-origin compounds are gaining more relevance each day as potential alternatives to synthetic materials, since the former undesirable footprints in biomedicine, the environment, and the ecosystems are reaching concerning levels. Therefore, cellulose and its derivatives have been the object of numerous biomedical studies, in which their biocompatibility, biodegradability, and, most importantly, sustainability and abundance, have been determinant. A complete overview of the recently produced cellulose-containing nanofibrous meshes for wound healing applications was provided. Moreover, the current challenges that are faced by cellulose acetate- and nanocellulose-containing wound dressing formulations, processed by electrospinning, were also enumerated.


Introduction
Skin is the largest and outermost organ that covers the entire body, forming 8% of the body weight [1]. It is responsible for the body physical protection and sensitivity, serves as barrier to microbial and UV radiation, and regulates biochemical, metabolic, and immune functions, such as temperature, water loss (preventing dehydration) and synthesis of vitamin D3 [2,3].
When the skin barrier is disrupted through wounds, a series of complex physiochemical processes take place in an attempt to repair and regenerate the damaged tissue [4]. Wound healing is based on a complex series of cellular and biochemical processes, starting with inflammatory reactions (immune response to prevent infection), followed by proliferation (regeneration of tissues), and finalizing with tissue remodeling [5]. Based on the time that is required for wound healing, two types of wounds can be established: acute and chronic. Acute wounds usually heal within eight to 12 weeks after injury, while chronic wounds that include diabetic, pressure, and venous stasis ulcers, are unable to follow the normal healing steps, taking more than three months to heal. This inability to heal in a The most widely used dressings in chronic wounds are the interactive/bioactive dressings, such as films, foams, hydrogels, hydrocolloids, and alginates ( Figure 1) [48,49]. The films are flexible semipermeable dressings, impermeable to fluids and bacteria, and permeable to air and water vapor [50]. Hydrogels stand out by their insoluble, highly absorbent three-dimensional (3D) polymeric network, capable of maintaining a moist microenvironment at the wound bed. Hydrogels can be formulated as particles, sponges, films, and other 3D structures, and their porosity can be controlled by embedding particles of various sizes. These are particularly effective in wounds with minimal to moderate exudates [51][52][53][54]. Hydrocolloids typically consist in carboxymethylcellulose, pectin, or gelatin. In their intact state, hydrocolloids are impermeable to water vapors, but as the gelling process takes place the dressing becomes progressively more permeable. The loss of water enhances the ability of the dressing to cope with the exudates production and lower the pH, this way hindering the bacterial growth and contributing to an optimal, stable temperature, and moisture level that stimulates all phases of healing [46]. Alginates are usually classified as bioactive dressings, being available in the form of non-woven sheets and ropes or as calcium-enriched fibrous structures that are capable of absorbing fluid up to 20 times their weight [55,56]. Upon contact with the wound, calcium is exchanged with the sodium from the exudates turning the dressing into a gel. Because of this exchange, alginates act as a hemostat and are, therefore, useful in managing bleeding wounds. They also activate human macrophages to produce tumor necrosis factor-α (TNFα), which initiates the inflammatory signals. However, these dressings might not be the most suitable to fight infections, as they generate an environment that is conducive to bacteria proliferation [50,55,56].
As seen in the alginates, bioactive dressings can directly deliver active compounds to the wound. They may also be composed of materials with endogenous activity which play an active role in the healing process, by activating or driving cellular responses [57,58]. Various antibiotics, vitamins, proteins, minerals, enzymes, insulin, growth factors, cells, and antimicrobial agents have been used in this class of dressings to accelerate healing [26,29,59].  [11], with permission from Elsevier, 2020).  [11], with permission from Elsevier, 2020).
As seen in the alginates, bioactive dressings can directly deliver active compounds to the wound. They may also be composed of materials with endogenous activity which play an active role in the healing process, by activating or driving cellular responses [57,58]. Various antibiotics, vitamins, a complete review and discussion of the most successful alternatives, containing cellulose and its derivatives, to the conventionally used dressings was provided. Special attention will be given to clean strategies and to the issues still faced in this line of research, as the environment conservation remains a challenge and a major focus of this work.

Cellulose
Cellulose is the most abundant organic, eco-friendly polymer on Earth. It is a polysaccharide that consists of D-glucopyranose units (commonly composed by 10,000-15,000 units, depending on the source) that are linked by a covalent β-1,4-glycosidic bond through acetal functions, between -OH groups of C4 and C1 carbon atoms, that forms a linear and high molecular weight homopolymer [74,75]. For each anhydroglucose unit, the reactivity of the -OH groups on different positions is heterogeneous. The -OH at the 6th position acts as a primary alcohol, whereas the -OH in the second and third positions behave as secondary alcohols. It has been reported that, on the structure of cellulose, the -OH group at the sixth position can react ten times faster than other -OH groups, while the reactivity of the -OH on the second position was found twice as high of that of the third position [71]. The many -OH groups that are present in the cellulose backbone establish numerous intra-and intermolecular bonds that result in its semicrystalline structure. However, this molecular structure may undergo modifications, depending on the source of the material, method of extraction, and treatment, giving rise to different polymorphs. There are four types of polymorphs of crystalline cellulose (I, II, III, IV). Cellulose I, which is also known as "natural" cellulose, is sourced in its vast majority from plants, tunicates, algae and bacteria, being a structural component in cell walls. Because its structure is thermodynamically metastable, cellulose I can be converted into cellulose II or III. Cellulose II is the most stable structure and it can be produced either by regeneration (solubilization and recrystallization) or by mercerization (aqueous sodium hydroxide treatments). This type of cellulose has a monoclinic structure and it has been used in the production of cellophane, Rayon, and Tencel. Cellulose III results from subjecting cellulose I and II to alkaline treatments, while cellulose IV originates from the thermal treatment of cellulose III [76,77]. Another important parameter that is influenced by the source and processing of cellulose is the degree of polymerization (DP), which is the number of monomer units in the polymer backbone and affects the material viscosity and mechanical properties. For instance, while cellulose from wood pulp has only 300-1,700 units, BC has a DP of 800-10,000 repeat units [78].
The primary natural source of cellulose is the lignocellulosic material that is present in wood (40-50 wt.%). It can also be extracted from vegetable fibers like cotton (87-90 wt.%), jute (60-65 wt.%), flax (70-80 wt.%), ramie (70-75 wt.%), sisal, and hemp. However, most of these sources require large arable spaces and considerable amounts of fresh water, fertilizers, and pesticides. Additionally, cellulose can also be produced from bacteria, algae, fungi, and some animals (e.g., tunicate) [74]. To reduce the environment impact associated with its production, strategies are being developed to effectively reuse wood pulp and agricultural and food wastes, or even take advantage of wastes from the textile industry (e.g., used garments) to recover cellulose and produce new fibers with similar properties to those regenerated from conventional wood pulp [74,78,79]. Table 1 introduces some of the alternative sources of cellulose and their inherent pretreatments to obtain an efficient extraction and new unconventional solvents systems that are applied in their solubilization. For instance, the solvent N-methylmorpholine-N-oxide (NMMO), which is used in Lyocell production (rayon fiber obtained from wood pulp), has been successful in dissolving cellulose, however it entails high costs and high temperatures; the NaOH/thiourea solvent system has been used in green processes for the production of regenerated cellulose textile fibers, yet it also entails limitations, namely the inability to effectively dissolve high degree polymerized cellulose or high concentrated cellulose solutions. The ionic liquids (IL) are another alternative that has attracted lots of attention for their effectiveness. However, even Nanomaterials 2020, 10, 557 7 of 64 though they are compatible with cellulose, their elevated cost, toxicity and incapacity to be reused has hindered large-scale, multi-filament productions [80].
Cellulose has attracted considerable interest in the last years because of its potential for generating several high-value products with low impact on the environment and low cost [81]. Among its important characteristics for large-scale production, the cellulose intrinsic mechanical, chemical, and biological properties make this polymer extremely suitable for applications in composite engineering, food science, filtration processes, paper engineering, and medical engineering [82]. The clinical application of cellulose-containing 3D scaffolds includes repair, reconstruction, and regeneration of almost all types of tissues in the mammalian organism; this polymer endows scaffolds with the ability to support cell adhesion and growth. In wound dressings, it is no accident that these cellulose-based materials have been used since the mid-1970s, in the form of cotton gauze or non-woven mixtures of rayon and polyester or cotton fibers, since they are capable of absorbing excess exudates through their bulk polar groups (-OH) and allow for the production of highly porous structures, permeable to air, steam, and heat, ensuring the patients comfort [83].
Nowadays, with the rise of nanotechnology approaches, cellulose nanofibers have been engineered by electrospinning in the form of nanocomposite wound dressings that not only protect the wounds, but they are capable of releasing drugs that inhibit post-operative adhesions, stimulate hemodialysis and hemostasis, and repair tissue defects [20]. Cellulose electrospun nanofibers have been studied due to their ultrafine and highly porous structure, biocompatibility, biodegradability, hydrophilicity, low density, thermostability, low thermal expansion, and easy chemical modification [20,84]. Even though research continues in this field, the disappointing mechanical properties and the difficulties in processing cellulose in the form of nanofibers via electrospinning remain very important challenges. Its strong inter-and intra-molecular interactions originated from hydrogen bonding, and its rigid backbone structure is responsible for its insolubility in most conventional solvent systems and its inability to melt [85]. Several strategies have been proposed to address these limitations. For instance, cellulose nanofibers have been obtained via direct electrospinning by using NMMO (one of the most popular cellulose solvents), lithium chloride/dimethylacetamide (LiCl/DMAc), or ionic solvents,nsuch as 1-ethyl-3 methylimidazolium acetate 1-ethyl-3 methylimidazolium acetate (EmimAc) [86]; however, these processes are cumbersome and very expensive. This has limited the use of cellulose in nanofibrous constructs for wound healing and has increased the use of cellulose derivatives, particularly CA [87]. Uniform regenerated fibers were obtained with diameters ranging from 23.9 to 33.0 µm; A structural shift from cellulose I in the original/hydrolyzed cotton fibers to cellulose II in the regenerated fibers was observed; A small amount of dye was lost during hydrolysis but no dye leaching was observed during spinning; The intrinsic color of the regenerated fibers eliminates the need for dyeing processes.
[90] The surface of all studied fibers appeared to be smooth; Fibers spun from CWG had higher molecular weight than standard lyocell fibers; ReCell-2 exhibited superior mechanical and molecular properties in relation to the typical fibers regenerated from wood pulp. [93]

Cellulose Acetate (CA)
Many cellulose derivatives have arisen in order to overcome the limited solubility of cellulose in general organic solvents [28]. CA is one of the most important cellulose derivatives, with applications in textile, plastics, cigarette filters, diapers, sensors, LCD screens, catalysts, coatings, semi-permeable membranes for separation processes, nano and macro composites, and fibers and films for biomedical devices [97,98]. This polymer is under great consideration in the biomedical industry due to its biodegradability, biocompatibility, mechanical performance, non-toxicity, high affinity, good hydrolytic stability, relative low cost, and excellent chemical resistance [99]. These exceptional properties have driven the processing of CA-containing polymeric blends in the form of electrospun nanofibrous composites, in this way generating a new smart option for biotechnology and tissue engineering, drug delivery systems and wound dressing applications [25][26][27]. Electrospun CA has also been used to immobilize bioactive substances as vitamins and enzymes, biosensors, bio-separation, and affinity purification membranes, while non-porous CA have been used for stent coatings or skin protection after burns or wounds. Interestingly, CA has also been proven as an effective material for tissue scaffold engineering, providing good mechanical stability, and ability to mimic the extracellular matrix for cell attachment, growth, and advanced formation of targeted tissues (e.g., bones and skin) [100]. Chainoglou et al. has even demonstrated the possibilities of CA for heart valve tissue engineering, through a successful promotion of cardiac cell growth and proliferation [101]. Each one of these applications is dependent on their overall properties, which, in turn, are dependent on the polymer chemical characteristics, such as molar mass, molar mass distribution, DP, and degree of substitution (DS) [102].
DS can be easily understood as the average number of acetyl groups replacing hydroxyl groups per glucose unit. The maximum degree of acetylation is obtained when all of the -OH groups are replaced by acetyl groups, which leads to a DS that is equal to three [103]. DS is a parameter that demands a detailed understanding as it affects the chemical, physical, mechanical and morphological properties of the polymer, altering its polarity, aggregation behavior, biodegradability, and solubility [103,104].
The acetylation process reduces CA crystallinity and insolubility in water [100]. Industrially, CA is produced by the reaction of cellulose with an excess of acetic anhydride in the presence of sulfuric acid or perchloric acid as catalysts, in a two-step process of acetylation, followed by hydrolysis [105,106]. These steps are taken due to the heterogeneous nature of the reaction, since the structure of cellulose is made up of amorphous parts, which react first, and crystalline parts, which then react second; hence, being impossible to synthesize directly partially substituted CAs. An extra hydroxylation step is required for producing CA with the desired DS [21]. CA production entails very high-quality cellulose as raw material, with a high alpha cellulose content [21,[105][106][107][108][109][110]. This high-quality cellulose is generally obtained from cotton or wood dissolving pulp, where the cellulose rate is generally more than 95%. However, this is considered to be an expensive material. Alternative sources of cellulose have been researched, finding lignocellulosic biomass as an attractive alternative due to its renewability and large availability worldwide. Preliminary work has uncovered some important sources of CA that are based on biomass that include microcrystalline cellulose (MCC), cotton linter pulp, wheat straw pulp, bamboo pulp, bleached softwood sulfite dissolving pulp, bleached hardwood kraft pulp (HP) [21], oil palm empty fruit bunches [105], sugarcane straw [106], waste cotton fabrics [107], sugarcane bagasse [108], sorghum straw [109], babassu coconut shells [110] and waste polyester/cotton blended fabrics (WBFs) [111]. Still, there is a long way until process optimization occurs, since there are major barriers to the production of cellulose-containing products from agricultural residues, including the heterogeneity of the raw material, the processing conditions reproducibility, the heterogeneous phase of the synthesis reaction, the difficulty of purification, the effluent disposal, and the control of product quality [112].
Many researchers apply alkali or acid pretreatment to remove lignin and hemicellulose of material resources to increase the yield of CA production from wastes, affecting the cellulose crystalline structure, which then becomes more amorphous [110,112,113]. For instance, L. Cao et al. used diluted phosphoric acid at different temperatures and B. Ass et al. used NaOH to disrupt the crystalline structure of cellulose, which increases the amorphous region and renders cellulose more accessible to acetic anhydride, resulting in an acetylation process more effective for CA production [113,114]. Additionally, H.R. Amaral et al. resorted to acid pretreatment of babassu coconut shells to increase the yield of the acetylation of cellulose to obtain CA [110]. These works have shown the importance of pretreating lignocellulosic biomass to increase CA synthesis yield. However, bio-residues from these treatments are a serious environmental challenge due to their aggregation with household wastes, causing disturbances in the ecological cycle of the soil, and resulting in soil infertility and environmental pollution; thus, attention should be urgently paid [110,113,115]. A great opportunity has arisen to explore more of these bio-based polymers and their alternative production methods, given the current environmental and energy policies. Table 2 offers a general overview of this topic, covering some of the most effective alternative solutions for CA processing. The process was optimized by varying the temperature and the amounts of (CH 3 CO) 2 O and iodine; The best yields obtained were of 15-24%, which corresponded to a conversion of 50-80% of the starting cellulose.
[116]    (2) the mixture was poured into ethanol; (3) the solid, which consisted of CA and PET was filtered, washed and dried; (4) to extract acetone-soluble CA, part of the sample was refluxed using acetone; (5) the filtrate was dried and refluxed using DMF.
[Hmim]HSO 4 at 0.4 molar equivalents of IL was the most acetone-soluble formulation; The extraction yield of acetone-soluble CA was 49.3%, which corresponded to a conversion of 84.5% of WBFs original cellulose; 96.2% of the original PET were recovered.

Nanocellulose
The increased demand for high-performance materials with tailored mechanical and physical properties has elevated the nanocellulose status to one of the most attractive renewable materials for advanced medical applications. Nanocellulose is a considered to be a new generation of nanomaterials that combines important cellulose properties, including high specific strength, hydrophilicity, low density, flexibility and chemical inertness, with the ability to be chemically modified to incorporate specific features at the nanoscale [120,121]. In biomedicine, its exceptional water-retention capacity and large surface area that are associated with enhanced cell attachment, proliferation, and migration with no reports of toxic responses, has increased its desirability for a variety of uses that include packages, membranes for hemodialysis, vascular grafts, drug delivery systems, wound dressings, and tissue engineering strategies [122][123][124].
Nanocelluloses can be classified in three main categories: (1) cellulose nanofibers (CNFs), also known as microfibrillated cellulose (MFC), and nanofibrillated cellulose (NFC); (2) cellulose nanocrystals (CNCs), also designated by nanocrystalline cellulose (NCC) or cellulose nanowhiskers (CNWs); and, (3) BC or also named bacterial nanocellulose (BNC) [121]. The major difference between the CNFs and CNCs lies in their dimensions and crystalline structure. While CNFs have lengths in the microscale and diameters in the nanoscale, CNCs have both length and diameter that are in the nanoscale [125]; more precisely, CNFs are fibrils with lengths of a few micrometers and with diameters that range between 3 and 50 nm, whereas CNCs have a rod-like nanocrystal configuration with lengths ranging from 10 to 500 nm and diameters of few nanometers ( Figure 2) [120]. Differences in the CNFs and CNCs crystalline structure result from their extraction process. CNF contains either crystalline regions or amorphous regions, in which the amorphous domains provide a certain flexibility to NFC. In turn, CNC are mostly nanoparticles that are made predominately of pure crystalline cellulose [126,127].

Nanocellulose
The increased demand for high-performance materials with tailored mechanical and physical properties has elevated the nanocellulose status to one of the most attractive renewable materials for advanced medical applications. Nanocellulose is a considered to be a new generation of nanomaterials that combines important cellulose properties, including high specific strength, hydrophilicity, low density, flexibility and chemical inertness, with the ability to be chemically modified to incorporate specific features at the nanoscale [120,121]. In biomedicine, its exceptional water-retention capacity and large surface area that are associated with enhanced cell attachment, proliferation, and migration with no reports of toxic responses, has increased its desirability for a variety of uses that include packages, membranes for hemodialysis, vascular grafts, drug delivery systems, wound dressings, and tissue engineering strategies [122][123][124].
Nanocelluloses can be classified in three main categories: (1) cellulose nanofibers (CNFs), also known as microfibrillated cellulose (MFC), and nanofibrillated cellulose (NFC); (2) cellulose nanocrystals (CNCs), also designated by nanocrystalline cellulose (NCC) or cellulose nanowhiskers (CNWs); and, (3) BC or also named bacterial nanocellulose (BNC) [121]. The major difference between the CNFs and CNCs lies in their dimensions and crystalline structure. While CNFs have lengths in the microscale and diameters in the nanoscale, CNCs have both length and diameter that are in the nanoscale [125]; more precisely, CNFs are fibrils with lengths of a few micrometers and with diameters that range between 3 and 50 nm, whereas CNCs have a rod-like nanocrystal configuration with lengths ranging from 10 to 500 nm and diameters of few nanometers ( Figure 2) [120]. Differences in the CNFs and CNCs crystalline structure result from their extraction process. CNF contains either crystalline regions or amorphous regions, in which the amorphous domains provide a certain flexibility to NFC. In turn, CNC are mostly nanoparticles that are made predominately of pure crystalline cellulose [126,127]. The isolation of CNFs from different cellulosic origins is accomplished by means of mechanical treatments, often in combination with some chemical or enzymatic pretreatment followed by a disintegration step. The most common chemical pretreatments are perhaps those that render the pulp fibers (used when the source is wood) charged, e.g. anionic or cationic. This modification increases the electrostatic repulsion between the fibers, which is beneficial in the subsequent mechanical treatment steps, as it further promotes the fibers disintegration into nanofibers. The most used mechanical processes are the high-pressure homogenization, microfluidization, refining, and grinding, while the least used are the electrospinning, ultrasonication, cryocrushing, and steam explosion. However, all of these mechanical processes demand high energy consumption. Hence, chemical and enzymatic pretreatments, such as cationization, hydrolysis, (2,2,6,6-Tetramethylpiperidin-1-yl) oxyl(TEMPO)-mediated oxidation, acetylation, and silylation, have been used to ease the mechanical treatment and, thus, reduce the energy consumption, while attaining a desirable surface chemistry. Still, caution should be taken during mechanical processing, since the The isolation of CNFs from different cellulosic origins is accomplished by means of mechanical treatments, often in combination with some chemical or enzymatic pretreatment followed by a disintegration step. The most common chemical pretreatments are perhaps those that render the pulp fibers (used when the source is wood) charged, e.g., anionic or cationic. This modification increases the electrostatic repulsion between the fibers, which is beneficial in the subsequent mechanical treatment steps, as it further promotes the fibers disintegration into nanofibers. The most used mechanical processes are the high-pressure homogenization, microfluidization, refining, and grinding, while the least used are the electrospinning, ultrasonication, cryocrushing, and steam explosion. However, all of these mechanical processes demand high energy consumption. Hence, chemical and enzymatic pretreatments, such as cationization, hydrolysis, (2,2,6,6-Tetramethylpiperidin-1-yl) oxyl(TEMPO)-mediated oxidation, acetylation, and silylation, have been used to ease the mechanical treatment and, thus, reduce the energy consumption, while attaining a desirable surface chemistry. Still, caution should be taken during mechanical processing, since the nanofibers length depends on the degree to which the material has been exposed to this processing step. In addition, the cellulose source will also play a major role in the final product, as it determines the pretreatments that are to be carried out [127,128].
Two steps are also required to process CNC from raw cellulose: (1) homogenization pretreatment/purification, and (2) the separation of the purified cellulose into nanocrystals. To obtain cellulose nanocrystals, cellulose can be directly hydrolyzed. Acid hydrolysis has been the method of choice for many years to produce CNC. Generally, it requires sulfuric and hydrochloric acids, which starts by dissolving the disordered or para-crystalline regions, leaving behind the crystalline domains or the CNC that possess a higher acidic resistance. The temperature and time of hydrolysis, nature, and concentration of the acids and the fiber-to-acid ratio play an important role in the CNCs particle size, morphology, crystallinity, thermal stability, and mechanical properties. It is worth noting that surface sulfate esters are introduced to the CNCs during sulfuric acid hydrolysis, conferring the surface with a highly negative charge and making it accessible, for instance, to enzymes or proteins, a desirable outcome in biomedical applications [71]. Aside from hydrolysis, other methods have been reported to isolate CNCs, such as enzymatic hydrolysis, mechanical refining, ionic liquid treatment, subcritical water hydrolysis, and oxidation processes. Different sources, like plant cell walls, cotton, microcrystalline cellulose, algae, animals, and bacteria, have been used to obtain CNCs [81,129]. Like CNFs, the geometric dimensions and the final properties of the CNCs are directly dependent on the cellulosic source, the post-or pretreatments, and the subsequent preparation and processing conditions [130]. CNCs are characterized by their biocompatibility, biodegradability, high level of crystallinity (54-88%), excellent stability and mechanical performance (high strength as well as modulus), exceptional optical properties, and flexible surface chemistry [123,131].
BC is another class of nanocellulose materials that has been engineered with the goal of surpassing the limitations of cellulose and other natural or synthetic materials [132]. BC is chemically similar to the cellulose obtained from plants; however, it is free from lignin, pectin, and hemicelluloses, and it has a very low amount of carbonyl and carboxyl in its structure. BC is a biocompatible, highly porous, and highly crystalline (84-89%) polymer, with a high degree of polymerization (up to 8000), a finer web like network, and an extraordinary mechanical strength, particularly in the wet state, which was comparable to other nanofibers from plants. It is characterized by a superior water-retention capacity, and the ability to accelerate granulation tissue formation, making it very attractive for wound healing ( Figure 2) [75,126]. Another, relevant property of BC is its in situ moldability (e.g., shaping during biosynthesis) [133]. Many bacteria from the genus Acetobacter, Agrobacterium, Pseudomonas, Rhizobium, Achromobacter, Bacillus, Azotobacter, Enterobacter, Klebsiella, Salmonella, and Sarcina have been reported to secret BC as a protection mechanism against ultraviolet light or other microorganisms, like fungi and yeasts [126,129]. BC properties are highly influenced by the origin organism and culture conditions [128]. The most common BC producer is the gram-negative bacteria Gluconacetobacter xylinus, previously known as Acetobacter xylinum and secretes cellulose during metabolism of carbohydrates [132]. The fermentation method that is used more to produce BC has been the static culture, which increases the yields of BC, by producing BC layers of several centimeters of thickness under the surface of the culture medium. Here, however, it is necessary to monitor the media pH, since the accumulation of acids, such as gluconic, acetic or lactic, decreases the pH far below the optimum for bacteria growth and cellulose production. Alternatively, agitated cultures, airlift bioreactors, rotating disk bioreactors, stirred tank reactors with a spin filter, biofilm reactors with plastic composite supports, and trickling bed reactors may also be employed in the production of this cellulose type, preventing the conversion of cellulose-producing strains into cellulose-negative mutants [128]. BC can be produced in various forms, depending on the fermentation method; pellicles arise under static culture condition, while fibrils and sphere-like particles emerge under motion conditions [134]. The wastes from several industries (often rich in sugars), e.g., domestic, agricultural, cotton-based textiles, among others, are also gaining significance as carbon sources for BC production, as evidenced in Table 3 [132]. Celluloses with different degrees of crystallinity can be produced, depending on the source and culture production [133]. This is one of the most important properties in BC, since the crystalline microfibrils in its structure are Nanomaterials 2020, 10, 557 23 of 64 responsible for its high tensile strength  and thermal stability. Its poor solubility in physiological media, as well as the absence of cellulases and beta-glucanases, which increase the stability and functionality of the polymer, has increased the interest of BC as additive or base for potential new biomaterials. To date, BC has been employed in the development of biomaterials for wound dressings, blood vessels, dental implants, scaffolds for tissue engineering of cornea, heart valve, bone and cartilage, and drug delivery applications [134]. Alkaline peroxide post-treatment was further conducted to obtain purified lignocellulosic nanofibrils (P-LCNF) with low lignin content and thin diameters; The low-temperature fractionation process on WS and WWS fibers could yield cellulose nanomaterials with potential value-added for a variety of applications and uncover a new efficient processing tool for agricultural wastes. [135] Arecanut husk (AH) HCl, NaOH Isolation of cellulose nanofibrils: (1) the dried AH fibers were dewaxed with a mixture of toluene and ethanol for 48 h at 50 • C, followed by washing with boiling water and dried in air; (2) the dried fibers were then cut; (3) to remove lignin and hemicelluloses, a treatment with NaOH was applied at 50 • C for 4 h; (4) samples were washed to remove the alkali compounds and treated with HCl to break the cell walls and separate the microfibrils; (5) fibers were washed with dH 2 O to eliminate any acid traces; (6) fibers were grinded into a pulp form and treated again with alkali to remove the remaining non-cellulosic components, followed by acid hydrolysis; (7) the delignification was further carried out by bleaching with NaClO 2 and glacial acetic acid for 2 h at 60 • C.
Highly crystalline and thermally stable cellulose nanofibrils, with very high aspect ratio, were prepared from AH fibers by HCl hydrolysis followed by mechanical fibrillation.
[136] It was possible to isolate cellulose nanofibrils and extract lignin by discarding the hemicellulose using a soda cooking pretreatment followed by fiber defibrillation by acidic hydrolysis.
[138] (2) in the end the samples were washed.
CNCs were successfully isolated from groundnut shells, after purification and acid hydrolysis treatment, reaching a yield of 12%.
[142] (2) the pH of each mixture was neutralized to 6 with 1 M NaOH; PPW as alternative media for BC production: five factors were tested to optimize BC production, initial pH (7-11), media volume (mL), inoculum size (4-12%), temperature (25-45 • C), and incubation time (2-6 days); BC purification: (1) the produced BC was collected, rinsed in dH 2 O, and immersed in 1 N NaOH at 60 • C for 90 min to remove attached cells and impurities; (2) pellicles were rinsed with methanol, washed with the dH 2 O and dried at 60 • C for 24 h.
Maximum BC yield was achieved using PPW-nitric acid hydrolysate at 2.61 g/L followed by PPW-sulfuric acid hydrolysate at 2.18 g/L; Optimal BC production conditions were determined as pH 9 with 8% inoculum size and volume of 55 mL, at 35 • C and incubation of 6 days.
[144] [145] Kitchen waste (KW) α-amylase; amylglucosidase Pretreatment: (1) samples were subjected to a washing process using tap water to separate the KW into solid fraction (starch-rich solid) and liquid fraction (oil/water mixture); (2) the solid fraction was sterilized at 121 • C for 15 min; Enzymatic saccharification of the solid fraction: (1) samples were hydrolyzed using α-amylase and amylglucosidase at 55 • C for 24 h, at 150 rpm; BC production: (1) the glucose concentration of the resultant hydrolysate was diluted to 50 g/L; (2) then 5 g/L peptone, 5 g/L yeast extract, 1.15 g/L citric acid and 2.7 g/L disodium hydrogen phosphate were added to prepare the BC production media; (3) the seed culture was incubated at 30 • C and 150 rpm for 2 days; (4) 10 mL of the cultured seed were inoculated in 100 mL of production media (pH of 5.0), which was cultivated at 30 • C under static conditions for 15 days; (5)

Application in Wound Healing: Synergistic Effect with Specialized Biomolecules
In wound care, infections are a major concern, since they delay the healing process, leading to tissue disfigurement or even patient death. S. aureus and P. aeruginosa are the most common bacteria that are isolated from chronic wounds, being S. aureus usually detected on top of the wound and P. aeruginosa in the deepest regions. They can express virulence factors and surface proteins that affect wound healing. The co-infection of S. aureus and P. aeruginosa is even more problematic, since the virulence is increased; both bacteria have intrinsic and acquired antibiotic resistance, making the clinical management of these infections a real challenge [147]. In fact, the World Health Organization considers P. aeruginosa as one of the organisms in urgent need for novel, highly effective antibacterial strategies that combat its prevalence. Multiple strains of S. aureus, including methicillin-resistant and vancomycin-resistant strains, have been identified as high priority microbes in the fight against antimicrobial resistance build up [15]. In addition to the above, other microorganisms, such as beta-hemolytic streptococci, and mixtures of Gram-negative species, such as E. coli and Klebsiella strains, are also present in wounds. Bacterium native to human skin such as Staphylococcus epidermidis (Gram-positive), may also turn pathogenic when exposed to systemic circulation in the wound bed [148]. Therefore, immediate care of open wounds is pivotal in preventing infection [149]. To treat this problem, new alternatives of wound dressings have emerged with incorporated bio actives that are capable of fighting these infections and accelerating the healing process.
The performance of bioactive dressings processed via electrospinning is dependent on the polymer or polymer blends properties (i.e. hydrophilicity and hydrophobicity), drug solubility, drug-polymer synergy, and mat structure. Antimicrobial agent-loaded electrospun mats have shown superior performance to films produced by other techniques, in regard to water uptake (four to five times superior), water permeability, drug release rate, and antibacterial activity [9].
Drugs, nanoparticles, and natural extracts ( Table 4) are some of the antimicrobial agents that have been incorporated in nanofibrous dressings, in order to reduce the risk of infection [61]. These compounds have been used for their anti-inflammatory, pain-relieving, vasodilation, and antimicrobial features [11].
Several researchers claim that producing cellulose-based electrospun mats is a big challenge due to its highly crystalline structure, long chain length, increased rigidity, and strong inter-and intramolecular hydrogen bonding [150]. Selecting a proper solvent, adding other complementary polymers, or converting cellulose into its derivatives can facilitate this task. As seen in Section 3.1, the solvents or solvent systems most used for cellulose are the ILs, aqueous alkali/solvents (NaOH/urea), and polar aprotic solvents in combination with electrolytes (DMAc/LiCl); however, these are not very volatile, not being completely removed during electrospinning and, thus, limiting the use of cellulose in large scale productions. A proper solvent system is also very important in attaining appropriate viscosity levels, required for a successful electrospinning process. In fact, this is such an important processing parameter that to guarantee proper polymer solubilization, heaters have been placed within the electrospinning apparatus generating a new system, the melt-electrospinning (minimize the viscosity of spinning dopes) [151]. The option of transforming cellulose into its derivatives, such as CA, cellulose acetate phthalate (CAP), ethyl cellulose (EC), carboxymethyl cellulose (CMC), hydroxypropylcellulose (HPC), among others, is by far the most recurrent alternative to reduce the complexity of processing cellulose via electrospinning. Besides, most of these derivatives require different pHs for solubilization, which is a great advantage in biomedical applications [152].
Modifications have been proposed to increase the effectiveness of immobilized drugs, natural compounds, peptides, or other biomolecules within a cellulose-based nanostructured surface. For example, Nada et al. activated CA by introducing azide functional groups on the residual -OH groups of the polymeric chains, enhancing the release kinetics of capsaicin and sodium diclofenac from the electrospun mat and, thus, promoting patient relief [153]. To confer biocidal properties to CA nanofibers, Jiang et al. modified their surface with 4,4'-diphenylmethane diisocyanate (MDI). This resulted in a 100% inactivation of S. aureus and a 95% of E. coli within 10 min of exposure, and complete death after a 30 min contact [154]. Nano complexes with CNCs were developed with cationic b-cyclodextrin (CD) containing curcumin by ionic association and used in the treatment of colon and prostate cancers [155]. Nanocellulose has also contributed to the development of new and more efficient strategies for these biomolecules' delivery. The three -OH groups that were present in each individual glucose unit originate a highly reactive structure, which allows interaction with other molecules or with enzymes and/or proteins, contributing to overcome the low solubility of most drugs in aqueous medium [127]. Besides, the -OH groups can also be tailored by physical adsorption, surface graft polymerization, and covalent bonding to further improve the performance of the biomolecules. As a consequence of the bonds established, strong polymer-filler interactions are generated, significantly increasing the mechanical properties of material [156]. Nonetheless, the in vivo behavior of nanocelluloses is still little explored. Studies have reported that its toxicity depends on the solution concentration and its surface charges. In recent literature, nanocelluloses have not shown any toxicity at concentrations lower than 1 mg/mL; however, there are studies that reveal a concentration-dependent apoptotic toxicity of CNFs at 2-5 mg/mL. Additionally, anionic nanocelluloses, e.g., carboxymethylated CNF, have been reported to be more cytotoxic than cationic nanocelluloses, e.g., trimethylammonium-CNF [34]. Toxicity effects might arise from the diversity of chemical structures and properties between cellulose types and sources. Among nanocelluloses, BC is considered to be the most biocompatible and has already been applied in wound dressings [71]. Still, its electrospinnability is very challenging for the same structural reasons of cellulose [150].
The incorporation of BC into synthetic and natural polymers has been carried out to enhance their morphological features as well as physicochemical and biological performances. A wide variety of polymers, such as chitosan, polycaprolactone (PCL), polyethylene oxide (PEO), ethylene vinyl alcohol (EVOH), polyvinyl alcohol (PVA), polylactic acid (PLA), polyacrylonitrile (PAN), polyester, silk, and zein, have been blended with BC and processed by electrospinning. Functionalization with 3-aminopropyl triethoxysilane (APS) has been attempted to further enhance cell attachment and antibacterial properties of BC-containing electrospun membranes for wound healing. BC membranes grafted with two organosilanes and acetyled have also shown an improved moisture resistance and hydrophobicity [134]. Naeem et. al even synthetized in situ BC on CA-based electrospun mats in a process known by self-assembly to produce a new generation of wound dressings [157].
Even though CNF has already been applied as a reinforcing agent in many polymeric composites via electrospinning, no reports have been found regarding the incorporation of biomolecules along its fibers [158]. As such, in the following sections BC and CNCs will be explored in more detail.  [190][191][192]

Drug Loading
Numerous hydrophilic and hydrophobic drugs have been incorporated into electrospun polymeric nanofibers. In general, the polymer is dissolved in an organic solvent and the drug is slowly added to the polymer solution under stirring in order to guarantee a homogeneous distribution. This strategy allows for a large amount of drugs to be loaded into the nanofibers by simply adjusting the final concentration of the solution. However, adding drugs directly to a polymer solution alters its conductivity, viscosity, and surface tension, affecting the electrospinnability of the polymer and the morphology of the obtained nanofibers. Besides, in this scenario, drugs tend to very rapidly leach in an aqueous environment [193], since they are preferentially located at or near the fibers' surface [166]. The conventional electrospinning technique allows a somewhat control of drug release by modulating the pores size and density, and the polymers degradation rate; still, bursts of drug followed by cytotoxic effects remain [63]. Several research teams have focused on developing new drug delivery systems with a so-called effective controlled release to overcome this weakness. Multiple-fluid, coaxial and triaxial electrospinning approaches, capable of generating complex nanostructures, may allow a more effective control of this initial burst release by confining part of the drug to the fiber core and another to the surface. This way, during dissolution, the molecules at the core need to diffuse through an insoluble shell until reaching the bulk solution [169]. However, this is not always as straightforward. Yu et al. compared nanofibers that were produced from coaxial electrospinning at varying feeding rates. They realized that by varying just this one parameter the morphologies of the fibers obtained were completely different and that to condition drug release. They proved that the production of high quality ketoprofen-loaded CA nanofibers is not simply a result of dilution of the core solution by the sheath solvent. The most uniform fibers, with the smallest diameters, and extended drug release time were those that were produced with the lowest feed rate being applied to the sheath of CA [171].
Many attempts have been made to optimize the release kinetics of drugs over time resorting to different immobilization methods (Figure 3). Table 5 compiles some of the most successful formulations of drug and electrospun nanocomposites containing cellulose, CA, or any variation of nanocellulose. As explained earlier, CA is the oldest, most researched derivative of cellulose, and, as such, the drug loading of CA-containing electrospun wound dressings are more recurrent.  [194], with permission from Elsevier 2020).

Figure 3.
Three modes of physical drug loading on the surface of electrospun nanofibers (reproduced from [194], with permission from Elsevier 2020). Single nozzle and core-shell electrospinning; Graft copolymerization: NaCMC was grafted with MA originating NaCMC-co-MA copolymer (TCMC); Single nozzle: 5% w/w TH (in relation to methanol concentration) was added to TCMC/PEO and processed at 15 kV, distance of 20 cm and feed rate of 3 mL/h; Core-shell: TCMC was used at the shell and 5% w/w TH/PEO was used at the core, fibers were produced using potential of 15 kV, distance of 18 cm and feed rate of 0.4 mL/h.
Fibers produced from polymer blend were more uniform and bead free than those generated from core-shell; The TH release profile in core-shell nanofibers was more efficient, with an initial burst release of only 26% (first 30 min), and a 92% released within 72 h; TH-loaded TCMC/PEO core-shell nanofibers revealed excellent antibacterial effects against Gram-positive bacteria. [159] CIF 13% w/v of EC or PVP in HFIP Single nozzle electrospinning; 5% and 15% w/w of CIF (with respect to the polymer concentration) was added to PVP and to EC; Fibers were produced using potential of 20 kV, distance of 16 cm and a feed rate of 0.8 mL/h. Fibers were collected from an aluminum foil and from a gauze covering the foil.
As the feed rate at the sheath increased the diameters decreased and the fibers became smoother and uniform; Fiber produced with a 0.2 mL/h feeding rate averaged 240 nm and were capable of sustaining a more controlled release profile of KET.
[171]  Addition of 3 to 6% w/w CNCs to the PHBV nanofibers (1025 ± 96 nm) decreased the fibers from 748 ± 62 to 620 ± 33 nm, respectively; The tensile strength and Young's modulus increased with the increased CNCs content, and reached a maximum with 6% w/w CNCs; The higher CNCs content improved the hydrophilicity of PHBV nanocomposite; The percentage of drug loaded and the loading efficiency were 25.0 and 98.8%, respectively (≈ 86% HF was delivered within 540 h for nanofibrous containing 6% w/w CNCs). [183] 16% w/w PCL in acetic acid/dH2O 90/10 v/v Single nozzle electrospinning; Synthesis of CNC: (1) high molecular weight cellulose was extracted from cotton waste; (2) cellulose was hydrolyzed in H2SO4; 1% w/w TH was dissolved in 90% acetic acid; 0, 0.5, 1.0, 1.5, 2.5, 4% CNCs were added to the TH solution and then mixed with PCL; Fibers were produced using potential of 17 kV with a distance of 16 cm and a feed rate of 0.9 mL/h.
The lowest fiber diameters were obtained with 4% CNCs; The highest tensile stress was obtained was with 1.5% CNCs; During biodegradation studies the weight loss of CNCs-incorporated samples was much higher than for pure PCL nanofibers; Drug release was slower with increasing amounts of CNCs in the PCL nanofibers. [184]

Nanoparticles (NPs)
Nanotechnology tools, particularly NPs, have been recognized as occupying a fundamental role in promoting wound healing, with reports on their exceptional antimicrobial, angiogenesis, immunomodulation, and cell and drug delivery, leading the way to new strategies for improving the response to antimicrobial and tissue regeneration therapies [8].
NPs are classified in light of their impact in cellular uptake, dimension (1-100 nm), shape, role, and nature (inorganic and organic). Carbon-based, metal and metal oxide, semiconducting and ceramic NPs are classified as inorganic, while organic NPs integrate those that are produced from polymers and derived from biomolecules [195]. NPs can act as delivery vehicles, protecting and releasing active compounds locally, or by intervening in specific functions via their intrinsic properties [6]. Their antibacterial potential results from their production of reactive oxygen species and their capability to bind and disrupt DNA or RNA functions that obstruct microbial reproduction [130]. By associating NPs with a textile or polymeric matrix synergistic actions can be revealed, generating a new formulation of active dressings [6,148]. The NPs with antimicrobial activity that have been explored in combination with dressings are the bioactive glass, gold, copper, cerium, zinc oxide, carbon-based, titanium dioxide, gallium, nitric oxide, and AgNPs [130]. These display bacteriostatic and bactericidal capacity, reduced in vivo toxicity (at low concentrations), are low cost, and possess physical, chemical, and biological features that trigger complex biological responses [196]. AgNPs can be highlighted from the group for their proved potential against multidrug resistant (MDR) bacteria [188]; they are capable of blocking the respiratory pathways of specific enzymes and damage the bacteria DNA, or even block the action of selected proteins involved in key metabolic processes [189,197]. In addition, AgNPs have been associated with decreased levels of pro-inflammatory cytokines TNF-α and IL-8 and increased levels of anti-inflammatory cytokine IL-4, EGF, KGF, and KGF-2, with enhanced fibroblast migration and differentiation into myofibroblasts, macrophage activation, and improved proliferation and relocation of keratinocytes, all being very important phenomena in wound healing [198,199]. AgNPs are already clinically used, being found in dressings, gels or ointments for topical treatment of infected burns and open wounds, including chronic ulcers [6]. However, there are still some adverse effects arising from the excess use of AgNPs. At high concentrations, AgNPs may be toxic to the human cells, by inhibiting the recruitment of immune cells, the regrowth of epidermal cells, and, ultimately, hindering wound healing. Besides, like antibiotics, prolonged treatment with metal ions may result in the emergence of resistant bacterial strains. A balance between cell exposure and action against microorganisms is, therefore, required to prevent such events. Table 6 summarizes some of the most recent systems for wound healing that combine NPs with electrospun mats containing cellulose or its derivatives, in the most successful way.
Nowadays, the growing awareness of the NPs impact in the environment has led to the development of more eco-friendly approaches for inorganic NPs synthesis, which justifies new choices of solvents and reductive and stabilizing agents [200]. In fact, there are now approaches that resort to microbes, fungi, and vegetable, fruit, and plant extracts to produce metal and metal oxide NPs. There is still a long way until the optimization of such alternatives; however, it is already clear their economic and environmental potential over the current physical and chemical technologies [201,202]. Table 6. Processing of cellulose-, CA-and nanocellulose-containing electrospun mats incorporated with nanoparticles.

Nanoparticles
Polymer(s) and solvent(s) Processing conditions Results Ref.

Cellulose
AgNPs 4% w/v CMC and 4% PEO w/v in water Single nozzle electrospinning; Fibers were produced using potential of 22 kV, with distance of 15 cm and feed rate of 2 mL/h; After, electrospinning CMC/PEO mats were carefully immersed in AgNO 3 solution (0.1 mol/L, to substitute Na+ with Ag+) and irradiated with UV-light.
The average diameter of CMC/AgNPs fibers (89 ± 23 nm) was smaller than that of CMC/PEO fibers (103 ± 30 nm); CMC/AgNPs nanofiber mats were 100% effective against S. aureus and E. coli. [162] 17% w/w CA in DMF/acetone at 1/2 v/v Single nozzle electrospinning; Cellulose nanofibers were prepared from CA nanofibrous mats by a simple alkaline treatment with NaOH and coated with silver by immersion in AgNO 3 , forming CEAgNP; Fibers were produced using potential of 15 kV, with distance of 15 cm and feed rate of 0.06 mL/h. CA nanofibers showed a smooth and regular morphology with an average diameter of 291 nm, and cellulose displayed diameters averaging 289 nm; All CEAgNP samples were 100% bactericidal, being effective in preventing growth of E. coli and S. aureus strains. [163] ZnO NPs 2% w/v CMC and 10% w/v PVA/dH 2 O Single nozzle electrospinning; 1/1 w/w PVA/CMC was combined with 3% w/w of ZnO NPs (relative to PVA/CMC blend) and then with EM at 5% w/w (relative to PVA/CMC blend) and mixed until a homogenous mixture was obtained; Fibers were produced using potential of 16 kV, with distance of 20 cm and feed rate of 0.3 mL/h; -Crosslinking was performed with 2% glutaraldehyde vapor in a desiccator for 48 h and then dipped in 3% AlCl 3 in ethanol.
PVA/CMC nanofibers ranged 214.5 ± 26.0 nm, while PVA/CMC/EM averaged 238.9 ± 18.0 nm; The average size of the fibers was determined in 193.5 ± 20.0 nm and 234.9 ± 28.0 nm for PVA/CMC/ZnO and EM-loaded PVA/CMC/ZnO nanocomposites, respectively; The PVA/CMC/EM nanofibrous mat showed a high initial burst release of EM (58%) Incorporation of 3% w/w ZnO NPs decreased the initial burst release of EM; EM-loaded PVA/CMC/ZnO nanocomposites were effective against S. aureus and E. coli.
CA, CA/ZnO and CA/ZnO/AgNP nanofibers were regular and bead free; Addition of AgNPs to CA/ZnO reduced the fibers diameters; CA/ZnO/AgNPs nanofibers were effective against E. coli and S. aureus bacteria; Nanocomposites containing 10% w/w ZnO/AgNPs yielded 0% viable bacteria cells in relative cell viability experiments.
[172] NPs became entrapped within the fibers during production; NPs were found well dispersed with occasional aggregates randomly distributed along the fibers; Diameters varied between 400 and 500 nm; All metal-loaded CA nanocomposites impaired significantly the growth of Aspergillus niger; The amount of metal NPs released daily by the nanocomposite represented ≈ 1% of the total amount of Ag or Cu. [174] Ag ions/AgNPs 10% w/w CA in acetone/water at 80/20 w/w Single nozzle electrospinning; 0.0, 0.05, 0.30 and 0.50% w/w AgNO 3 were added to CA; Fibers were produced using potential of 17 kV, distance of 10 cm and feed rate of 3 mL/h; Silver ions on the electrospun CA fibers were submitted to UV irradiation (photoreduction).
Fiber diameters decreased with AgNO 3 increased content; Silver ions in ultrafine CA fibers were successfully photoreduced into AgNPs; The average diameters of the AgNPs were in the range of 3-16 nm; Both AgNO 3 (non-reduced) and AgNPs (photoreduced) ultrafine CA fibers showed very strong antimicrobial activity.
[203] Films were smooth, highly flexible and displayed a highly homogeneous appearance; AgNPs coupled to the CNC were more effective against P. aeruginosa.
[204] Nanofibers had a multi-size distribution with diameters ranging from 80 to 360 nm; After soy protein surface modification, nanofibers became more stretchable, increasing the elongation at break; Nanofibrous with soy protein NPs showed superior biocompatibility compared to pure BC electrospun nanofibers. [190]  Mats with uniform morphologies were attained with 1.5% GO, however with 2% GO smaller diameters were generated; High amounts of GO increased the scaffold mechanical strength; A reduction in the hydrophilicity of the electrospun nanofibers and their water vapor permeability with the addition of GO was also reported. [191] Abbreviations

Natural Extracts
Biomolecules that are derived from natural extracts are gaining more interest in biomedicine as alternatives to overcome the concerns associated with the resistance and toxicity of antibiotics and the overuse of silver-based compounds [206,207]. The use of plant extracts for the treatment of wounds and wound-related diseases is a very common practice. Thymol, asiaticoside, curcumin, zein, acid gallic, and gingerol are some examples of bioactive molecules used in combination with cellulose derivatives-containing electrospun nanocomposites. Their bioactive properties arise from alkaloids, phenolic, flavonoids, and terpenoids compounds, which are also endowed with immunomodulatory activities, which make these biomolecules capable of controlling the inflammatory response. Besides, these compounds are also responsible for these biomolecules antibacterial, insecticidal, antiviral, antifungal, and antioxidant properties.
The use of plant extracts in medicine dates back hundreds of years. For instance, natural extracts derived from Aloe vera such as emodin (3,8-trihydroxy-6-methyl-anthraquinone), an antioxidant compound, have been frequently used in the treatment of burns. Neem (Azadiracta indica) extracts containing omega fatty acids also have numerous medical and cosmetic applications. Ginsenosides found in the plant genus Panax (Ginseng) are often used in traditional Chinese medicine and exhibit anticancer activity. Indeed, various plant extracts and active components, formulated as nanofibers or nanoparticles, are regaining interest for therapeutic purposes [208], because of their low cost, bioavailability, and superior efficacy, with limited side effects, over the more current and synthetic alternatives.
Essential oils (EOs), which are extracted from aromatic plants, have intrinsic antibacterial, antifungal and insecticidal properties. Moreover, EOs are widely available natural compounds with a low degree of toxicity. They can be easily and efficiently combined with polymeric matrices to generate nanocomposites with improved antimicrobial features [67,168]; these are mainly conferred by active molecules present in their composition, namely terpenes, terpenoids, and other aromatic and aliphatic compounds. The EOs, and their respective components, hydrophobic character promote the partition of the lipids that are present in the bacteria cell membrane, increasing their permeability and, consequently, leading to the membrane rupture and leakage of intracellular content, ultimately inducing cell death. Therefore, EOs loaded dressings may act as powerful tools to circumvent bacteria multi-drug resistance in infected wounds [209]. In fact, studies have already shown the improved synergistic effect of the oregano EO with CA-based nanofibers against S. aureus, E. coli and the yeast Candida albicans (C. albicans), as a result of the potent antimicrobial character of the oregano oil molecular components carvacol and thymol [67]. Cinnamon, lemongrass, and peppermint EOs that are loaded onto CA electrospun mats have shown similar outcomes. However, it was also seen that the morphology of the mat is a determinant factor in the EOs antimicrobial assessment as the nanostructure fibrous network developed might impair direct contact with large sized microorganism, such as C. albicans. Regarding the modified dressings cytotoxicity, even though fibroblasts and human keratinocytes could attach and spread on the fibers surface, cell viability seemed to decrease with exposure time. The anti-proliferative effect of EOs against eukaryotic cells has already been reported [168]. This is the greatest limitation to a large-scale use of EOs as antimicrobial and regenerative biomolecules in wound healing. Still, the capacity to design and engineer systems that allow a gradual and continuous release of EOs at concentrations below the cytotoxic, while using the electrospinning technique, has been improving and has already revealed very promising results. In fact, studies have shown that CA-based electrospun nanostructures loaded with EOs to display a higher capacity to retain water and aromatic compounds, thus reducing the initial drug burst and extending release over time, this way increasing the effectiveness of the therapy above other non-reticulated systems [179,180]. Table 7 presents some of the most recent EOs loaded electrospun systems containing cellulose, CA or nanocellulose formulations, in which the above-mentioned properties and outcomes are the most noticeable. Several of those works also report on the modifications introduced by the EOs to the fiber diameters and the relative porosity of the engineered mats, which intimately affect the cell proliferation, migration, and capacity of EOs release without an adverse biological response.    The release rate of gingerol increased drastically in the first 4 h (≈ 92%) and remained constant after that period; 2,2-diphenyl-1-picrylhydrazyl (DPPH) scavenging assays and in vitro cytotoxicity tests showed the antioxidant activity of the prepared fibers and a viability above 60% for L-929 mouse fibroblast-like cells.
[181] CA solution was mixed with PVP at 8:5, which made the ratio of the dry weight of PVP to CA of 3/2; For every 13 g of PVP/CA 1 g of glycerine was added (PVP/CA/glycerine) or 1 g of garlic extract (PVP/CA/garlic); combinations of the two were also made; Fibers were produced using potential of 15 kV and distance of 12 cm.
The composite nanofibrous mats were uniform, bead-free with a size ranging from 350 nm to 900 nm; Release of garlic extract from PVP/CA/glycerine/garlic was the most important due to the large diameter of the fibers; The antibacterial activity of the PVP/CA/garlic nanofibrous mat was effective against both S. aureus and P. aeruginosa; PVP/CA/glycerine/garlic fibers were the most antimicrobial. [182]

Cellulose Nanocrystalline
Thymol 9% w/v PVA in dH2O Single nozzle electrospinning; 30% w/w CNCs (in regard to PVA concentration) were prepared in dH2O/H2SO4 and added to PVA; Fibers were produced using potential of 10 kV, distance of 10 cm and feed rate of 0.25 mL/h. Electrospun PVA/CNCs was mixed with PLA in CHCl3 to obtain blends with a final concentration of 1 % w/w; nanocomposite films were impregnated with thymol dissolved in supercritical carbon dioxide (scCO2).
PVA/CNCs nanofibers impregnated with thymol registered a yield of 20%, while the PLA films obtained 24%; The release rate of thymol was significantly slower when PVA/CNCs were incorporated within a PLA matrix. [187]

Bacterial Cellulose
Tragacanth gum (TG) 7.7% w/w of keratin/PEO at 70/30 in dH2O; 0, 1, 3 and 5% w/w of BC were added to the keratin/PEO solution; Fibers were produced using potential of 22 kV, with distance of 10 cm and feeding rate of 0.1 mL/h; TG was incorporated by electrospraying as the nanofibers were being electrospun.
The mean fiber diameter of the mats composed by keratin/PEO was 243 ± 57 nm and reduced to 150 ± 43 nm with the addition of 1% or higher % of BC; BC (1%) significantly reduced the hydrophobicity of the mat; TG and BC modified mats promoted cell attachment and proliferation on the surface of the nanofibers. [192] Abbreviations

Wound Healing Alternative Methods Containing Cellulose-Based Compounds
Wound healing is a highly complex process of tissue repair that relies on the synergistic effect of a number of different cells, cytokines, enzymes, and growth factors. A deregulation in this process can lead to the formation of a non-healing chronic wounds. Current treatment options are unable to meet the demand set by the environment surrounding these wounds. Therefore, multifaceted bioactive dressings have been developed to more efficiently respond to these wounds demands [206].
Surfaces have been physically and chemically modified, by changing the dressing topography or by introducing functional groups, like cell-recognizable ligands and bioactive molecules at the outermost layer, in order to improve the performance of electrospun polymeric nanofibers for skin regeneration. To accomplish such task, surface functionalization techniques, like the wet-chemical method, plasma treatment and graft polymerization have been applied. Pre-and post-electrospinning surface modifications are also very common; in pre-electrospinning bioactive molecules can be dissolved or dispersed in the polymeric solution, while in pos-eletrospinning physical adsorption, layer-by-layer (LbL) assembly and chemical immobilization are the most common strategies [61].
Alternatively to the earlier mentioned additives, drugs, nanoparticles, or natural extracts, other molecules, like growth factors, hormones, or enzymes, have also been incorporated onto nanofibrous dressings to promote wound healing [211]. Huang et al. produced CA nanofibrous mats that were used as a substrate to deposit LbL films, alternating between positively charged lysozyme-N-[(2-hydroxy-3-trimethyl-ammonium)propyl] chitosan chloride (LY-HTCC) compositions and negatively charged sodium alginate. The average fiber diameter increased with the increased number of bilayers, but only the samples that contained lysozymes were effective against bacteria [212]. Similar observations were made by Li et al. Here, lysozyme was combined with rectorite and electrosprayed onto negatively charged electrospun CA nanofibrous mats. The release profiles of lysozyme and its activity over time both demonstrated this formulation suitability for long-term applications [213]. Bio-based electrospun nanocomposites containing a pain reducing local anesthetic, the benzocaine (BZC), and the in situ pH-detecting dye bromocresol green (BCG) have been engineered to serve as a dual nano-carrier system for the treatment of infected wounds. BZC and BCG were introduced to CA-based nanofibers while using a single-step needleless electrospinning process. In vitro release studies demonstrated a pH dependent, controllable release of BZC, and confirmed the expected maximum drug release rate at pH 9.0, the average pH of an infected wound [214]. B. Ghorani et al. designed a β-Cyclodextrins (β-CD)/CA electrospun nanocomposite to efficiently trap and adsorb volatile molecules that are responsible for the unpleasant odors in chronic wounds. The data demonstrated an enhanced direct adsorption of a model odor compound, the hexanal (up to 80%), indicating the feasibility and potential of this formulation [24]. Vitamin A or retinol and Vitamin E or α-tocopherol have also been combined with CA solutions and electrospun in the form of cross-sectionally round, smooth fibers, with the average diameters ranging between 247 and 265 nm. The contents of Vitamin E and Vitamin A within the as-spun fiber mats were of ≈ 83% and ≈ 45%, respectively. Vitamin E was found to be more stable over time, with a maximum release of ≈ 95% of its loaded content after a 24 h period, against an ≈ 96% release of Vitamin A in only 6 h [215]. Cui et al., to improve the interaction between cells and scaffolds, modified the surface of thermoplastic polyurethane (TPU) nanofibers with CNF particles by ultrasonic-assisted technique and used polydopamine as binding agent. These composites increased cell attachment and viability, revealing excellent biological and mechanical properties [216]. In another approach Kolakovic et al. produced drug loaded CNF microparticles via the spray drying method and revealed a sustained drug release by means of a tight network that limited the drug diffusion from the system [217]. These studies offer new structures for the delivery of effective treatments in wound healing, in which the sustainability of the materials and the preservation of the environment are decisive factors during processing.

Conclusions and Future Perspectives
Wound healing is a complex process that is regulated by three essential and distinct phases. Dysregulation or disruption of this process results in non-healing, very difficult to treat chronic wounds. In the last decades, remarkable progress has been achieved in the development of therapeutic approaches for these wounds. Electrospinning is regarded as one of the most effective tools for the production of dressings with a 3D structure that is similar to the skin extracellular matrix. These electrospun dressings display a large surface area-to-volume ratio and a porous structure that enhances homeostasis, exudates absorption, gas permeability, cell adhesion, migration, and proliferation and prevents the development of complicated infections. Herein, insights on the recent advances attained in the production of electrospun nanofiber meshes containing cellulose and its derivatives and modified with specialized biomolecules were provided. New, different, and more effective approaches have been developed for overcoming the concerns that are associated with the resistance generated by antibiotics and the overuse of silver compounds. Natural extracts from plants, alternative drugs, and organic and inorganic nanoparticles have been combined with selected nanofibrous systems based on cellulose components for an accelerated wound healing.
Even though many studies have reported on the availability of cellulose, its processing remains very challenging, with researchers turning to CA for facilitating dressing production via electrospinning. Indeed, CA is the most recurrent derivative of cellulose applied in wound dressings production, with many drug-loaded systems already engineered. Yet, nowadays, nanocellulose is gaining more ground by promoting binding with various biomolecules, including proteins and enzymes, via its highly available -OH groups that are also responsible for overcoming the low solubility of other forms of cellulose. These nanofillers have also contributed to significantly increasing the mechanical properties of wound dressings. Despite these advantages, more in vivo studies are required, since there is no consensus regarding its toxicity to human cells. From the collected data, it is clear that the different forms of cellulose presented are very attractive as renewable materials for wound dressings applications due to their high specific strength, high water-retention capacity, enhanced cell attachment, proliferation, and migration with no reports of toxic responses, and ability to be chemically modified to incorporate specific biomolecules. However, the incorporation of these agents is not always simple, with it being necessary to overcome the limitations that are associated with their electrospinnability. The correct selection of appropriated solvents, combination of polymers, pre-treatments to increase the solubility of these natural resources, and introduction of new chemical functional groups at the surface for biomolecule binding, are essential to obtain reproducible and effective wound dressings. The studies analyzed in this review reflect well the hard work around this subject and the increasing concern with the development of sustainable solutions that are still capable of accelerating the healing of wounds and preventing possible infections. There is still a long way for these formulations to reach large scale production with little environment impact. Even though, there are already greener alternatives resorting to clean solvents, low energy demand technologies, and biodegradable complementary polymers, there is still much work to be done to obtain a "green" and effective wound dressing to treat of infected wounds.