Keywords

1 Tissue Engineering: Definition and Requirements

Tissue engineering employs a combination of engineering, biology, and bioactive constructs to improve function by repairing, replacing, or regenerating tissue. The expanded concept of regenerative medicine includes tissue engineering but also incorporates research on the regeneration of tissue directly in vivo, where the body uses its own systems to repair, replace, or regenerate function in damaged or diseased tissue with the help of exogenous cells, scaffolds, or biological factors (Dzobo et al. 2018). The regenerative medicine relies on harnessing the body’s natural ability to self-heal. However, tissue engineering evolved from the field of biomaterials development and refers to the practice of combining scaffolds, cells, and biologically active molecules into functional tissues (Brien 2011).

Actually, there are six basic requirements widely accepted for designing polymer scaffolds: high porosity and proper pore size; high surface area; biodegradability and a proper degradation rate to match the rate of neotissue formation; mechanical integrity; not be toxic to cells (i.e., biocompatible); and finally, bioactivity which means interaction with cells, including enhanced cell adhesion, growth, migration, and differentiated function (Ma 2004).

The success of the development of tissue engineering is closely related to the conditions for tissue culture that involves the in vitro maintenance and propagation of cells in optimal conditions. The comprehension of interaction between the ligands present in the extracellular matrix and the receptors of the cell allows multiple intracellular signaling processes that can result in the alteration of cellular behaviors, such as growth, migration, and differentiation and several natural biomaterials have been explored recently. Three-dimensional biomaterials with large pore size (greater than 100 μm) carry a high number of functional units essential for the regeneration of various tissues. Pore size greater than 100 μm is essential for the cell adhesion and proliferation. However, to design functional units of tissue, not only are the subcellular and cellular scales required, but also nanostructures, 1–100 nm in size. This type of structural arrangement is essential to control cell behavior, in particular cell–cell interactions, cell–molecular interactions, and the cellular environment (Bhatia and Goli 2018).

Cell adhesion, mitosis, and growth are often caused by proteins that have been attached to scaffold material, so bioactive molecules like growth factors or proteins of the extracellular matrix (ECM) have been included in the polymers to support these functions. Bifunctional groups can also be added to the polymer materials for improved spreading of cells. These include glycolipids, oligopeptides, and oligosaccharides. Fibronectin, collagen, laminin, tenascin, vitronectin, and thrombospondin, a glycoprotein that mediates cell-to-cell and cell-to-matrix interactions, are some examples that increase the spreading of cells (Ogueri et al. 2019).

Particularly the “RGD” arginine-glycine-aspartate sequence in fibronectin is responsible to stimulate the cellular response, in this way several researcher groups are testing only this tripeptide RGD anchored in the polymer surface, instead of the whole molecule (Ruoslahti 1996). Scaffolds coated with polylysine, polyornithine, or lactose and N-acetlyglucosamine and micropaths have been created and a positive effect in the cell attachment and spreading have been observed (Sigma-Aldrich 2008; Lam and Longaker 2012).

Actually, the researcher’s centers are looking for materials that mimic natural ECMs in terms of their composition, structural characteristics, and mechanical properties. To find scaffolds capable to transmit a signal to actively construct and degrade their microenvironment, providing cellular adhesion, proteolytic degradation, and growth factor (GF)-binding, as well as space-filing mechanical support capable to behavior as bioactive and dynamic environment to mediate cellular functions is a challenge nowadays and 3D scaffolding materials can represent a new choice to get these properties (Dutta et al. 2019).

Cellulose has been widely applied in engineering of blood vessels, reconstruction of urethra and dura mater, liver and adipose tissue, neural tissue, bone, cartilage, repairing connective tissue and congenital heart defects, and constructing protective barriers and contact lenses.

This chapter reviewed some of the newest researches in the last 5 years, reporting the development of scaffolds based in cellulose for tissue engineering (bone, cartilage, and skin). The intention is to highlight cellulose structural characteristics that make their application more attractive while tailoring them to tissue regeneration demands improving the methods of repair, replacement, or regeneration of damaged tissues and organs .

2 Cellulose: Function, Structure, and Properties

Structure–property and structure–function relationships have long been considered important explanatory concepts at hierarchical levels and provide insight to design new mimetic tissues able to be employed into tissue engineering.

Cellulose is a semicrystalline polymer formed by (1-4)-linked betha-D-glucosyl residues that are alternately rotated by 180° along the polymer axis to form flat ribbon-like chains. Each glucosyl unit bears three hydroxyl groups, one on hydroxymethyl group. It has been long recognized that these hydroxyl groups and their ability to bond via hydrogen bonding not only play a major role in directing how the crystal structure of cellulose forms but also in governing important physical properties of cellulose materials (Brown and Saxena 2000).

Cellulose is a polycrystalline material and the crystals are aligned along the microfibrils and present a polymorphism (Delmer and Amor 1995; Atalla and VanderHart 1984). As a result, cellulose has several polymorphs, namely cellulose I, II, III, and IV and their varieties Iα, Ibetha, IIII, IVI, IIIII, and IVII. Most of these polymorphs result from chemical treatments of polymorph (Šturcova et al. 2004; VanderHart and Atalla 1984). The degree of crystallinity, i.e., the quality of the cellulose crystal, is another important factor that varies extensively from one cellulosic material to another (Revol 1985).

The interaction between water and cellulose is of utmost importance in order to understand and control the properties of cellulosic materials (Sugiyama 1984). Indeed, the unusual physical and chemical properties of cellulose such as highly hydrophilic nature, good mechanical properties, low density and thermal conductivity, and good thermal stability arise from its structural architecture (Chami Khazraji and Robert 2013). Therefore, it is possible to get more assertive manipulation process of cellulose when we have knowledge of its structural crystalline organization.

In nature cellulose occurs as a slender rodlike or threadlike entity, called microfibril (collection of cellulose chains); this entity forms the basic structural unit of any “cellulose” independent of its origin (Fig. 1). Each microfibril can be considered as a string of cellulose crystals linked along the chain axis by amorphous domains. The outer regions of wood microfibrils are strongly disordered, mostly due to the direct contact with hemicellulose, which is largely amorphous in nature. These regions can show the form of paracrystalline or fully amorphous cellulose (Fig. 2) (Dufresne 2019; Nishiyama 2009).

Fig. 1
figure 1

Layers of cellulose microfibril composed by crystalline and amorphous regions surrounded by hemicellulose

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Fig. 2
figure 2

Crystalline and paracrystalline regions

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Not only the microfibril diameter (nm) is different but also the DP (degree of polymerization). Values of DP ranging from hundreds and several tens of thousands have been reported and the DP is heavily dependent on the source of the original cellulose (Habibi et al. 2010).

Different protocols focusing in chemical and physical modifications have been applied to cellulose with the purpose to obtain particular morphologies and structures seeking to meet specific properties capable to stimulate specific tissue regeneration .

3 Cellulose: Derivatives and Cellulose Nanostructures

As mentioned above, our body is formed by different tissues and they possess distinct characteristics raised from its structural organization. In this way cellulose usually is obtained and modified in order to provide the specific characteristics necessary to distinct functions. Cellulose can be tailored to exhibit particular physical and chemical properties using different methodologies, one of them is by varying the pattern and degrees of substitution within the cellulose backbone.

There is a large distribution in the worldwide market of cellulose ethers and esters, in biomedicine cellulose ethers derivatives are more used (e.g., methylcellulose [MC], ethyl cellulose [EC], hydroxyethylcellulose [HEC], hydroxypropyl cellulose [HPC], hydroxypropyl methyl cellulose [HPMC], and carboxymethyl cellulose [CMC]). Recently, hydroxypropyl methyl cellulose (HPMC) have been evaluated in association with other polymers for tissue engineering, e.g., for cell-based cartilage engineering (Rederstorff et al. 2017), corneal regeneration (Long et al. 2018), and filler material for use in oral and craniofacial fields (Huh et al. 2015), and methylcellulose (MC) was crosslinked to form hydrogels (Niemczyk-Soczynska et al. 2019) for bone regeneration (Kim et al. 2018), thermally reversible hydrogels (Kummala et al. 2020), and more recently aroused interest as a versatile printing material for bio-fabrication of tissues (Law et al. 2018; Ahlfeld et al. 2020; Roushangar Zineh et al. 2018). Carboxymethyl cellulose has been intensively researched for bone regeneration (Singh and Pramanik 2018; Hasan et al. 2018; Matinfar et al. 2019).

Due to the reduced structure of the cellulose chain arrangement in the disordered region, it has a lower density and thus exhibits more free volume than the crystalline region (De Souza Lima and Borsali 2004). Therefore, several researches have been focusing to obtain nanostructures of cellulose through preferentially acid hydrolyses which remove the disordered amorphous region leaving the crystalline region largely intact due to their tight packing. The correct timing and hydrolysis conditions enabling the obtention of CNC (cellulose nanocrystals) produced as individualized particles (De Souza Lima and Borsali 2004; Anglès and Dufresne 2001). These nanoscale crystalline structures are isolated from native cellulose mostly via an acid hydrolysis, but enzymatic hydrolysis with cellulases (Siqueira et al. 2010; Henriksson et al. 2007) TEMPO ((2,2,6,6-tetramethylpiperidin-1-oxyl)-mediated oxidation (Saito et al. 2007), and ionic liquids has also been reported to prepare cellulosic nanoparticles (Man et al. 2011).

Typically, nanocellulose can be categorized into two major classes: (1) nanostructured materials (cellulose microcrystals and cellulose microfibrils) and (2) nanofibers (cellulose nanofibrils, cellulose nanocrystals, and bacterial cellulose) (Trache et al. 2017).

Nanocellulose market increased a lot in the last years and the employment of new applications has driven the researchers and the industry to exploit even more its employment. Therefore, International Organization for Standardization (ISO), Technical Association of the Pulp and Paper Industry (TAPPI), and Canadian Standards Association (CSA) standards on cellulose nanocrystals (CNCSs) are being developed and published. In 2011, TAPPI proposed international standards for cellulose-based nanomaterials that could remove the trade barriers and harmonize research and development in nanocellulose-based products (Reid et al. 2017).

These standards help to categorize nanocellulose avoiding overcoming the problems related to multiple definitions and ambiguity. The physicochemical and structural properties of nanocellulose are dependent of initial biomass type or microbial source selected (Trache et al. 2017), cellulose polymorphs (Gong et al. 2018), pretreatment process of cellulose extraction and acid hydrolysis, or enzymatic treatment (Mondal 2017), followed for nanocellulose fabrication.

Among the standards are ISO/TC 229 – TS 20477:2017: Standard terms and their definition for cellulose nanomaterial and some terms and abbreviations of cellulose nanomaterials were established, e.g., nanocrystalline cellulose (NCC), nanofibrillar cellulose (NFC), cellulose nanocrystals (CNC), cellulose nanowhiskers (CNW), cellulose nanofibrils (CNF), bacterial cellulose (BC), and tempo-oxidized cellulose nanofibrils (TOCN) (Reid et al. 2017). The good choice of biomaterial is the first step to design of scaffold. Scheme 1 shows some important points related to selection and characterization of cellulose materials.

Scheme 1
scheme 1

Steps for selection and characterization of cellulose

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4 Cellulose Applied to Bone Tissue Engineering

Bone acts as the supportive structure of the body, functions as a mineral reservoir, guards vital organs, is the site of blood cell production, and helps maintain acid–base balance in the body (De Witte et al. 2018). The aim of bone tissue engineering is to develop 3D scaffolds that mimic the extracellular matrix (ECM) and provide mechanical support, thereby aiding in the formation of new bone.

Scaffolds provide a template for cell attachment and stimulate functional bone tissue formation in vivo through tailored biophysical cues to direct the organization and behavior of cell (Bose et al. 2013). Bone tissue scaffolds present some specific biological requirements between them which are as follows (De Witte et al. 2018):

  1. (a)

    Biodegradable, nontoxic, osteogenic, presence of GFs (growth factors)

  2. (b)

    Mechanical requirements: mechanical properties, compressive strength ~2–12 MPa, and Young’s modulus ~0.1–5 GPa

  3. (c)

    Structural requirements: interconnected porosity, average pores size 300 nm, and nanotopography

Bone tissue engineering requires the presence of a bioactive component like hydroxyapatite Ca5(PO4)3OH (HAp) and tricalcium phosphate (TCP) Ca3(PO4)2. Several researches report different methodologies to achieve the formation of these biocomposites (cellulose and Hap or TCP). Scaffolds were fabricated with silk fibroin (SF) and carboxymethyl chitosan (CMCS) incorporated with strontium substituted hydroxyapatite (Sr-HAp) and there were enhanced protein adsorption and ALP (alkaline phosphatase) activity.

The expression of osteogenic gene markers such as RUNX2 (Runt-related factor-2), ALP, OCN (osteocalcin), OPN (Osteopotin), BSP (Bone Sialoprotein), and COL-1 was also stimulated (Zhang et al. 2019a).

A smoother scaffold with better distribution of HA with good interconnectivity, hardness range of scaffolds of 550–640 MPa, compression strength range of 110–180 MPa, an elastic modulus of ~5 GPa, and a fracture toughness value of ~6 MPa1/2 in the range of cortical bone was obtained using the association of (TEMPO)-oxidized cellulose nanofibrils (TCNF) and cellulose nanocrystals (CNC) with hydroxyapatite (HA) (Ingole et al. 2020).

The alignment of cellulose nanofibers hydrogels appeared to be a key structural feature in the successful and thorough infiltration of minerals as observed in a study where mineralized hydrogels were fabricated with TEMPO-oxidized cellulose and well-aligned nanofibers using a biomimetic method. The scaffold presented roughly 70 wt.% mineral content in the mineralized cellulose scaffold comparable with the mineral content in natural hard tissues (ranging ∼70–85 wt.%) (Qi et al. 2019).

Biomimetic growth of biphasic ceramics (HA/β-TCP) was used also to produce mineralized tissue with nanocellulose obtained from açaí integument (Euterpe Oleracea Mart.) (HA/β-TCP) (Valentim et al. 2018).

Bacterial cellulose (BC) has been an important choice to fabricate mineralized tissues proving to be a template for the ordered formation of calcium-deficient hydroxyapatite (CDHAP) (Hutchens et al. 2006). BC associated to hydroxyapatite (HA) and anti-bone morphogenetic protein antibody (anti-BMP-2) produced a noncytotoxic, genotoxic, and mutagenic biomaterial in MC3T3-E1 cells with increased mineralization nodules and the levels of ALP activity (Coelho et al. 2019).

Aligned scaffolds using cellulose nanocrystals loaded with bone morphogenic protein-2 (BMP-2) were produced by electrospinning and cellulose seems aligned human mesenchymal stem cells (BMSCs) growth and mineralized nodules formation in vitro (Zhang et al. 2019b). Cryogels formed upon contact with body fluids, with high porosity and high specific surface area, a rough hydroxyapatite layers and release of ions (Si, Ca, P, and Na) that were produced because there is a synergy between cellulose nanofibrils/bioactive (organic and inorganic) materials. So the cell differentiation is directly affected by the combination of high porosity, hydroxy apatite formation, and ion release and these set of events and conditions increases the release of BMP-2 greatly improving bone formation (Ferreira et al. 2019).

Nanocellulose (NC) containing BMP2-VEGF (BNBV) was loaded in porous sponge biphasic calcium phosphate (BCP) scaffold produced by replica method and bone marrow mesenchymal stem cells (RBMSCs) were seeded in these scaffolds. Bone formation increased because BMP2 stimulated the differentiation of stem cells to osteoblasts and the angiogenesis was facilitated by VEGF drawing the attention to the important role of nanocellulose as carrier for growth factors (Sukul et al. 2015).

The cellulose modification followed by the incorporation of bioactive molecules expand the possibilities to change its properties and reactivity; it can be achieved using ex situ chemical (e.g., periodate oxidation and grafting (Leguy et al. 2018) functionalization through linker (Ribeiro-Viana et al. 2016), or crosslinking reactions (Kirdponpattara et al. 2015)) or physical modifications (physical absorption from solutions or particle suspensions, the homogenization, or dissolving BC mixing with additive material or yet to add the additive material in the culture medium) (Lopes et al. 2014).

Recently through controlled nucleation, HA nanocrystals were produced in CNCs functionalized with sulphonic groups and aerogels obtained from nanocellulose with incorporated sulfate and phosphate groups crosslinked with hydrazine increased the cell metabolism of Saos-2 cells on the porous scaffolds. Twelve weeks after implantation in rats the osteoconductivity and bone volume were increased (Osorio et al. 2019).

Considering the importance of biodegradability and functionality to regulate the bone regeneration process, injectable bone composed of bisphosphonate-modified nanocellulose (pNC) was prepared with bisphosphonate groups on nanocellulose. The results demonstrated that pNC released under osteoclast microenvironment can control the osteoclast activity and, moreover, pNC-α/β-TCP composites promoted osteoblast differentiation (Nishiguchi and Taguchi 2019).

3D printing has been a recent trend to get the production of hydrogels with biomimetic structures for tissue regeneration and organ reconstruction. For this purpose, the development of bioinks capable to mimetic the properties and morphologies of tissues is fundamental to achieve the success. Bacterial cellulose nanofibers demonstrated a benefic effect improving the shape fidelity and mechanical properties of the 3D printed scaffolds composing silk fibroin and gelatin (Huang et al. 2019). Scaffolds hydrogels were produced using 3D printing through partial crosslinking of TEMPO-oxidized cellulose with alginate and biomimetic mineralization using simulated body fluid was the method used to get hydroxyapatite nucleation using calcium ions (Abouzeid et al. 2018).

Other approaches have demonstrated that scaffolds derived from apple hypanthium tissue can act as a bioactive biomaterial (Modulevsky et al. 2014, 2016; Hickey et al. 2018). After removing the native cellular components, the reminiscent structure presented pore size (100 and 200 μm) which was shown to be the optimal pore size for biomaterials used for bone tissue engineering. Additionally, pre-osteoblasts were seeded (MC3T3-E1) and the localized mineralization was proved by the presence of calcium deposits after 4 weeks, specifically on the edge of the pores (Karageorgiou and Kaplan 2005).

Table 1 summarizes some representative researches realized in the last 5 years focusing cellulose modification to use in bone tissue engineering.

Table 1 Examples of researches with cellulose applied for bone tissue engineering
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5 Cellulose Applied to Cartilage Tissue Engineering

Cartilage is a connective tissue which does not spontaneously heal, it is a tough, semitransparent, elastic, flexible connective tissue consisting of cartilage cells scattered through a glucoprotein material that is strengthened by collagen fibers. The main purpose of cartilage is to provide a framework on which bone deposition may begin. Another important purpose of cartilage is to cover the surfaces of joints, allowing bones to slide over one another, thus reducing friction and preventing damage; it also acts as a shock absorber (Zhang et al. 2009). Engineered cartilage regeneration in gels necessitate the control of mechanical properties (strength, rigidity, and elongation) and easy processing into complex shapes (Fu et al. 2017). Hydrogels with nanocellulose also have been extensively explored for cartilage scaffolds, however, to match these requirements. Cellulose-based gels are often combined to other materials and are produced by different methodologies in order to achieve efficient and bioactive scaffolds.

As already mentioned, 3D bioprinting is a powerful tool emerged in the last years for the production of highly structured tissue engineering scaffolds, allowing to dispense hydrogels in three dimensions with precision and high resolution (Kang et al. 2016; Mandrycky et al. 2016). The printed material gel is prepared with cells encapsulated with homogeneous density, which permits homogeneous cell distribution and the scaffold can be fully colonized and the combination of crosslinked sodium alginate and NC has been recently explored for cartilage tissue engineering, for articular and nasal reconstruction (Puelacher et al. 1994; Nguyen et al. 2017; Engineering and Wood 2017).

Not only for bone tissue but also for human cartilage nanocellulose–alginate hydrogels is a promising combination to obtain scaffolds with 3D printed (Table 2). Recently one of the bionks developed is composed by the induction of pluripotent stem cells (iPSCs) and human chondrocytes printed together with the hydrogel matrix, e.g., nanofibrillar cellulose/alginate (NCF/A) bionk was embedded with human bone marrow–derived stem cells (hBMSCs) and human nasal chondrocytes (hNC) (Möller et al. 2017). The coculture enhanced chondrogenesis and after 60 days chondrocyte cell clusters indicated the ability of embedded cells to proliferate and the formed tissue presented all qualitative features of proper cartilage. Furthermore, cell clusters contained human chromosomes proving their human origin (de Windt et al. 2014). Additionally was observed high cell viabilities of 73% and 86% after 1 and 7 days of 3D culture, respectively, in coculture NFC/A bioinks for bioprinting iPSCs to support cartilage production and formation of cartilaginous tissue by expression of collagen II was observed after 5 weeks (Nguyen et al. 2017). Similarly, human auricular cartilage was obtained from cells cultured for 28 days inside a 3D printed scaffold with 75% of cells were still viable and increased by 20% the expression of ECM proteins (Martínez Ávila et al. 2016). In another study the viability of human chondrocytes within an ear-like structure was 86% after 7 days (Markstedt et al. 2015).

Table 2 Examples of researches with cellulose applied for cartilage tissue engineering
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Bacterial NFC or named BCN (bacterial nanocellulose) was also applied to design layered scaffolds and the cellulose solvent system “ionic liquid EMIMAc,” was used to bond tightly the two layers. Particularly the scaffolds were able to produce and accumulate cartilage-specific ECM components and the chondrogenesis shown by upregulation of the expression of chondrogenic markers. Bone marrow mononuclear cells (MNC) was also loaded and 8 weeks post-implantation had a macroscopically cartilage-like appearance (Martínez Ávila et al. 2015). Alginate acts as a crosslinker to increase gel viscosity. It improves the scaffold structure and guarantees shape to be maintained during the process (Engineering and Wood 2017).

Taking into account the importance and potential application of sodium alginate with nanocellulose as cartilage tissue scaffold (nasal and articular cartilage), one of new challenges is to understand how crosslinking and sterilization methods can affect structural and mechanical properties of nanocellulose-based hydrogels, contemplating cellulose nanofibrils, cellulose nanocrystals, or a blend of the two. So, recently the effect of crosslinking – using calcium chloride (CaCl2) – on the structural and mechanical properties of AVAP® produced was evaluated (Al-Sabah et al. 2019).

American Process Inc.’s patented AVAP® technology produces cellulose nanocrystals (CNC); cellulose nanofibrils (CNF); and hydrophobic, lignin-coated varieties of CNC and CNF directly from woody and nonwoody biomass (Nelson 2014). The mechanical properties of the hydrogels were mildly affected by the sterilization method, apart from the chemical sterilization using ethanol that yielded significantly stronger hydrogels, possibly due to the dehydration. Whereas UV and ethanol sterilization have shown roughly similar pore sizes in all NC-based hydrogels not affecting the porosity. All sterilization methods did not significantly affect the stiffness of NCB hydrogels, but in contrast the stiffness of CNC was affected independent of the sterilization method used (Sulaeva et al. 2015).

In 2001 bacterial cellulose (BC) was molded into tubular form with diameter <6 mm and tubes having 1 mm diameter and 5 mm length with a wall thickness of 0.7 mm was obtained. The normal blood presents a tensile strength (800 mN) and the tubular BC showed comparable value demonstrating its potential to be employed as blood vessel to replace part of the carotid artery (Klemm et al. 2001). A clinical product named BActerial SYnthesized Cellulose (BASYC®) was used in microsurgery and presented favorable mechanical properties, including shape retention and tear resistance and a better mechanical strength than organic sheets, like polypropylene, polyethylene terephtalate or cellophane, and polyester (Dacron). The BASYC® tubes resisted to the blood pressure of the test animal (white rat) of 0.02 MPa (150 mmHg). After 4 weeks treated blood vessels showed that BASYC®-prosthesis was wrapped up with connective tissue, pervaded with small vessels like vasa vasorum. The BASYC®-interposition was completely incorporated in the body without any rejection. The regeneration nerve was improved after 10 weeks, compared to an uncovered anastomosed nerve (Belgacem and Gandini 2008). Recently, various properties and biology evaluation of BC tubes as blood vessel replacement have been investigated (Fink et al. 2010; Andrade et al. 2010; Esguerra et al. 2010).

At the beginning of this era there were two main specifications for tissue engineering scaffolds, the first was that the material had to be degradable, while the second was that this degradable material shall to have prior approval by the FDA for use in medical devices. For this reason the first scaffold biomaterials were the bioabsorbable materials approved to be used to produce surgical sutures, plates, and drug delivery systems (Williams 2019). The second specification is highly questionable, in fact the regulatory approval for medical devices is predicated on the ability to show that the material does no harm, which means the material needs to be “biologically safe.” Thus, depending on the precise application, the materials are subjected to the biological safety tests of ISO 10993 (International Standards Organization 2018) to show that they pass the tests that demonstrate a lack of cytotoxicity, acute systemic toxicity, reproductive toxicity, thrombogenicity, complement activation, and so on. Scheme 2 demonstrates some biological, mechanical, and morphological requirements for effectiveness of scaffolds. This scheme do not have relationship with ISO 10993.

Scheme 2
scheme 2

Biological requirements for development of scaffolds

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The first requirement for tissue engineering development is the choice of “best” material to be changed and adaptable to implanted in the body. Of course, this material needs to be noncytotoxic, non immunogenic, and minimally pro-inflammatory. As already mentioned, the body needs to recognize the material and interact with it, which means the biomaterial should be capable of orchestrating molecular signaling to the target cells, either by directing endogenous molecules or delivering exogenous molecules. Scheme 3 demonstrates some modifications that can be made to improve the bioactivity of the biomaterials and cellular factors that affect cellular behavior.

Scheme 3
scheme 3

Modifications to improve cell-scaffold interactions and intracellular and extracellular factors that affect the response of cells

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6 Cellulose for Skin Tissue Engineering and Wound Healing

The largest organ in the human body is the skin with several vital functions mainly as barrier against adverse effects (chemical damage, radiation, e.g., by ultraviolet light, and microbial infection). Three layers epidermis, dermis, and the fat layer, hypodermis, compose the skin (Kanitakis 2002).

Bacterial cellulose is the most widely cellulose applied for reconstruction of skin layers due to its resemblance to natural soft tissues. The use of bacterial cellulose in skin wound therapy proposed as “temporary skin substitutes” for treating burns, ulcers, abrasions, and other skin injuries was first reported in 1990 (Fontana et al. 1990).

Joining mechanical and morphological characteristics to biological properties, nontoxic, biocompatible, and biodegradable, different biocomposites were produced and different properties were improved as reported by Portela et al. (2019).

Like bacterial nanocellulose, plant-derived nanocellulose has repeatedly been shown to be promising for skin tissue engineering, especially after its physical and chemical properties have been modified (e.g., cellulose nanofibrils (CNFs) modified by TEMPO-mediated oxidation) (or introduction of electrical charge functionalized with biomolecules, e.g., cell adhesion peptides (Trovatti et al. 2018) and silk fibroin (Shefa et al. 2017)).These modifications improved the capacity of nanocellulose for wound healing.

There are many reviews focusing the cellulose application in skin tissue and wound dressing (Sulaeva et al. 2015; Portela et al. 2019; Ullah et al. 2016; Czaja et al. 2007; Wu et al. 2017; Naseri-Nosar and Ziora 2018; Pinho and Soares 2018; Rasouli et al. 2019) between others. Then, the commercial products available for wound dressing and hemostatic agents based on cellulose will be highlighted.

Oxidized cellulose is an excellent biodegradable and biocompatible derivate of cellulose, which has become one of the most important hemostatic agents used in surgical procedures. Oxidized cellulose-based hemostatic materials have proven local hemostatic efficacy and antibacterial activity and appear to be a longstanding, frequently used, safe, and effective hemostat for hemorrhage in the surgical setting (WO2016171633A1 2016).

Recently a biodegradable antibacterial nanocomposite based on oxidized bacterial nanocellulose was prepared and exhibited greater procoagulant properties and blood-clotting capability and higher adhesion of erythrocytes and platelets with concomitant lower blood loss, in addition to ultrafast cessation of bleeding, superior to the commercial hemostatic ORC product Surgicel™ gauze (Yuan et al. 2020).

Surgical products are procoagulant materials composed of a scaffold of cellulose polymers that have been in clinical use for more than 60 years and are manufactured in fibrillar consistency as well as woven sheets. The major benefit of these agents is that they are totally absorbable, which means that can be left on or within the wound; the wound can even be sutured closed over them.

Several strategies to adsorb, covalently bind, or physically entrap antimicrobial compounds in BC, including antibiotics, silver nanoparticles, chitosan, and cationic antiseptics, have been developed (Sulaeva et al. 2015). The efficacy of BC membranes and improved properties after inclusion of antimicrobial agents has been evidenced in the literature (Hosseini et al. 2020; Liu et al. 2018). A group of antimicrobial compounds that has been less extensively investigated in this context are antimicrobial peptides (AMPs) (Lei et al. 2019). Recently BC was functionalized with ε-poly-L-Lysine (ε-PLL) using carbodiimide chemistry and the growth of S. epidermidis on the membranes was inhibited without significant effects on the morphological structure and mechanical properties of BC. The cytocompatibility of BC functionalized to cultured human fibroblasts was the same of native BC (Fürsatz et al. 2018). At present, nanocellulose is produced on an industrial scale for commercial application. The manufacturers are mainly located in the USA, Brazil, and Poland. A summary of commercial products from bacterial cellulose and oxidized regenerate cellulose is shown in Table 3.

Table 3 Some commercially available cellulose-based products as skin dressing and hemostatic agent
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7 Conclusions, Future Directions, and Challenges

The aim of this chapter is to demonstrate the main aspects of cellulose and its applications in bone as well as cartilage, and skin tissue engineering focusing products already on the market. The remarkable versatility of cellulose permits scientists to explore its unique mechanical and morphological properties associated with biocompatibility, nontoxicity, and biodegradability. The levels of structural organization and extraordinary supramolecular nanofiber 3D network permits the potential application in tissue engineering, an emerging and challenged research field. This chapter brings an overview of researches focusing in distinct steps of research development initially in the choice of material, chemical, and/or physical modification, physicochemical characterization, in vitro biological characterization, clinical evaluation, and the bioactive potential affecting the cellular response. Tissue engineering is a radically different approach to reconstruction of the body demanding interdisciplinary research, regulatory requirements for products and processes, and high cost of productive investments. Despite these challenges, the success of biopolymers application, variety of methods, and the results obtained ensure the terrific opportunity for the development of responsive structures focusing on the production of fully functional tissue.