*2.4. Common Natural Polymers Used in Tissue Regeneration Applications* 2.4.1. Cellulose

Cellulose is a fundamental structural unit of the plant cell wall. It is also found in red, green, and brown algae, some fungi, and as an extracellular component in bacteria [66]. Cellulose is a homopolysaccharide composed of D-glucose units connected by β-(1→4) glycosidic bonds [67]. Cellulose is an ideal material for tissue growth. It has several features such as biocompatibility, biodegradability, and cheap cost. It is already used as a scaffolding material in wound repair, cartilage tissue regeneration, differentiating endothelial cells, and bone tissue engineering [60]. Scaffolds developed based on bacterial cellulose are widely

used in various biomedical applications [68]. Based on recent research, the performance of the material toward cell growth or biocompatibility is mentioned in Table 2.

**Table 2.** Recent research on the performance of the materials toward cell growth or biocompatibility.



#### **Table 2.** *Cont.*

*Gels* **2023**, *9*, 100


**Table 2.** *Cont.*

*Gels* **2023**, *9*, 100


#### **Table 2.** *Cont.*


**Table 2.** *Cont.*

## 2.4.2. Chitin and Chitosan

Chitin is the second most common polysaccharide globally, followed by cellulose. It exists in the exoskeleton of arthropods such as crabs, shrimps, lobsters, insects, prawns, and fungal cell walls. Chitin comprises repeated units of 2-(acetylamino)-2-deoxy-D-glucose. Chitin and chitosan are differentiated by a degree of deacetylation. Chitin has various biomedical applications in tissue engineering due to its outstanding properties such as non-toxicity, biocompatibility, biodegradability, and chelating of metal ions. It also supports cell adhesion, differentiation, and migration. Chitin also has structural similarity with N-glycosaminoglycans, essential components of connective tissues; hence, it is a good option for skin tissue regeneration. Further, it is also used in dental, bone, and cartilage implants [3,114,115]. Mokhtari et al. have developed a scaffold hydrogel by combining chitosan with collagen and aldehyde-modified nanocrystalline cellulose loaded with gold nanoparticles, showing a potential application in tissue engineering [116].

#### 2.4.3. Alginate

Alginate is a seaweed-derived polysaccharide extracted from Phaeophyceae-brown algae. Alginate comprises β-(1–4)-d-mannuronic acid and α-(1,4)-l-guluronic acid connected as repeated linear chains [66,117]. Alginate displays biocompatibility, biodegradability, a simple production process, and tunable mechanical properties, leaping to join in developing scaffolds in cartilage tissue engineering [118]. Moreover, alginate is hydrophilic, so it is used in wound dressing to absorb the pus and help it heal. It is also used in cell growth scaffolds, supporting blood vessels' formation, healing bone injuries, cartilage regeneration, and drug delivery systems [119]. Alginate-based scaffolds are widely used in various tissues or organs, including skeletal muscles, pancreas, nerve, liver, and dental tissue engineering [117]. For cardiac repair, Rosellini. E. et al. produced a scaffold using alginate, elastin, and gelatin, which successfully attained the desired cellular response [103]. The molecular structure of some polysaccharides is shown in Figure 5.

in tissue engineering [116].

2.4.3. Alginate

**Figure 5.** Molecular structure of polysaccharides: (**a**) cellulose-microcrystalline [120]; (**b**) chitosan [121]; (**c**) alginate [122]; (**d**) hyaluronic acid [123]. **Figure 5.** Molecular structure of polysaccharides: (**a**) cellulose-microcrystalline [120]; (**b**) chitosan [121]; (**c**) alginate [122]; (**d**) hyaluronic acid [123].

outstanding properties such as non-toxicity, biocompatibility, biodegradability, and chelating of metal ions. It also supports cell adhesion, differentiation, and migration. Chitin also has structural similarity with N-glycosaminoglycans, essential components of connective tissues; hence, it is a good option for skin tissue regeneration. Further, it is also used in dental, bone, and cartilage implants [3,114,115]. Mokhtari et al. have developed a scaffold hydrogel by combining chitosan with collagen and aldehyde-modified nanocrystalline cellulose loaded with gold nanoparticles, showing a potential application

Alginate is a seaweed-derived polysaccharide extracted from Phaeophyceae-brown algae. Alginate comprises β-(1–4)-d-mannuronic acid and α-(1,4)-l-guluronic acid connected as repeated linear chains [66,117]. Alginate displays biocompatibility, biodegradability, a simple production process, and tunable mechanical properties, leaping to join in developing scaffolds in cartilage tissue engineering [118]. Moreover, alginate is hydrophilic, so it is used in wound dressing to absorb the pus and help it heal. It is also used in cell growth scaffolds, supporting blood vessels' formation, healing bone injuries, cartilage regeneration, and drug delivery systems [119]. Alginate-based scaffolds are widely used in various tissues or organs, including skeletal muscles, pancreas, nerve, liver, and dental tissue engineering [117]. For cardiac repair, Rosellini. E. et al. produced a scaffold using alginate, elastin, and gelatin, which successfully attained the desired cellular response [103]. The molecular structure of some polysaccharides is shown in Figure 5.

#### 2.4.4. Starch 2.4.4. Starch

Starch is a popular polysaccharide produced by plants for energy storage. It consists of amylose and amylopectin. Amylose (a linear polymer linked by α (1–4) linkages) is connected to amylopectin (highly branched polymer) by α (1–6) linkages. Starch is highly porous and allows cells to penetrate vascularization and tissue growth. Biocompatibility, biodegradation, osteoconduction, and osteo production are some characteristics that display starch to apply in tissue engineering [124]. A study revealed that starch membrane, collagen, and chitosan enhance epithelial Starch is a popular polysaccharide produced by plants for energy storage. It consists of amylose and amylopectin. Amylose (a linear polymer linked by α (1–4) linkages) is connected to amylopectin (highly branched polymer) by α (1–6) linkages. Starch is highly porous and allows cells to penetrate vascularization and tissue growth. Biocompatibility, biodegradation, osteoconduction, and osteo production are some characteristics that display starch to apply in tissue engineering [124].

tissue regeneration during wound healing, clearly showing that starch-based scaffolds have more significance in wound healing [125]. It also helps in cell adhesion, growth, proliferation, and differentiation. Starch generally shows poor mechanical properties in aqueous media and is easily dissolved. Starch was incorporated with bio-additives to attain good mechanical properties to overcome this problem. For instance, researchers combined starch-based scaffolds with bio-additives such as citric acid, cellulose nanofibers, and hydroxyapatite to obtain the desired result. Many in vivo and in vitro assessments certified that starch-based scaffolds are better for bone regeneration [86]. The molecular structure of starch is shown in Figure 6. A study revealed that starch membrane, collagen, and chitosan enhance epithelial tissue regeneration during wound healing, clearly showing that starch-based scaffolds have more significance in wound healing [125]. It also helps in cell adhesion, growth, proliferation, and differentiation. Starch generally shows poor mechanical properties in aqueous media and is easily dissolved. Starch was incorporated with bio-additives to attain good mechanical properties to overcome this problem. For instance, researchers combined starch-based scaffolds with bio-additives such as citric acid, cellulose nanofibers, and hydroxyapatite to obtain the desired result. Many in vivo and in vitro assessments certified that starch-based scaffolds are better for bone regeneration [86]. The molecular structure of starch is shown in Figure 6.

**Figure 6.** Molecular structure of starch soluble [126]*.* (Starch is a polysaccharide mainly found in plant cells.) **Figure 6.** Molecular structure of starch soluble [126]. (Starch is a polysaccharide mainly found in plant cells.)

#### 2.4.5. Hyaluronic Acid

ChitosanMAXIAOYU[137–143]

2.4.5. Hyaluronic Acid Hyaluronic acid is a glycosaminoglycan that deficits sulfate bonds commonly secreted by chondrocytes and fibroblasts. It comprises repeated β-1,4-D-glucuronic acid and β-1,3-N-acetyl-D-glucosamine disaccharide units. It is mainly present in the synovial fluid, connective tissues of the dermis, the vitreum, and the dental pulp matrix. It maintains the viscoelasticity of ECM by acting as a lubricant [127]. It plays a vital role in the cell's structural maintenance, keeps tissue hydrated, and helps cell signaling and wound repair. It is highly biocompatible, biodegradable, and can be easily modified chemically. Therefore, it is widely used as scaffolds in various forms such as sponges, cryogels, hydrogels, and injectable hydrogels [128*–*130]. A combination of collagen and hyaluronic acid scaffold material was used in cartilage regeneration, which plays a significant role in tissue repair. Mohammadi et al. prepared a scaffold by combining hyaluronic acid and collagen loaded with prednisolone to make a proper dosage form for cartilage repair [130]. According to Sieni et al., scaffolds based on hyaluronic acid show several more valuable features than collagen scaffolds in breast cancer treatment [131]. The ad-Hyaluronic acid is a glycosaminoglycan that deficits sulfate bonds commonly secreted by chondrocytes and fibroblasts. It comprises repeated β-1,4-D-glucuronic acid and β-1,3-N-acetyl-D-glucosamine disaccharide units. It is mainly present in the synovial fluid, connective tissues of the dermis, the vitreum, and the dental pulp matrix. It maintains the viscoelasticity of ECM by acting as a lubricant [127]. It plays a vital role in the cell's structural maintenance, keeps tissue hydrated, and helps cell signaling and wound repair. It is highly biocompatible, biodegradable, and can be easily modified chemically. Therefore, it is widely used as scaffolds in various forms such as sponges, cryogels, hydrogels, and injectable hydrogels [128–130]. A combination of collagen and hyaluronic acid scaffold material was used in cartilage regeneration, which plays a significant role in tissue repair. Mohammadi et al. prepared a scaffold by combining hyaluronic acid and collagen loaded with prednisolone to make a proper dosage form for cartilage repair [130]. According to Sieni et al., scaffolds based on hyaluronic acid show several more valuable features than collagen scaffolds in breast cancer treatment [131]. The advantages, disadvantages, and applications of each polysaccharide are mentioned in Table 3.

vantages, disadvantages, and applications of each polysaccharide are mentioned in Table 3. **Table 3.** Advantages, disadvantages, and applications of polysaccharides in various tissue regeneration applications.

> cle, neural, and skin

Bone, cartilage, skin, cardiac, MAXIAOYUmus-

Low mechanical MAXIAOYUresistance, stiff and


Easy digestion, biocompatibility, biodegradability, antibacterial MAXIAOYUactivity, and hemo-


**Table 3.** *Cont.*

#### 2.4.6. Guar Gum

Guar gum is a galactomannan gum, a polysaccharide obtained from the seed of a leguminous plant, namely, guar beans, commonly known as cluster beans (*Cyamopsis teteragonolobha*). Easy accessibility, biodegradability, biocompatibility, non-toxicity, and nonimmunogenicity are attractive features that tempt many researchers to develop scaffolds from guar gum [159].

#### 2.4.7. Pullulan

Pullulan is a polysaccharide made up of repeated maltotriose units connected by alpha (1–6) linkages obtained from fungi known as *Aureobasidium*. Pullulan plays a vital role in tissue engineering due to its adjustable property, biocompatibility, biodegradability, and adhesive nature. Oxidized pullulan was cross-linked with collagen, and scaffolds were produced for various biomedical applications [160–162].

#### 2.4.8. Collagen

Collagen is the critical protein in the connective tissues of animals, mainly in mammals. It is a protein with high biocompatibility and biodegradability. Therefore, it is applied in the medical field in various forms, such as a scaffold, drug carrier, and wound dressing [163]. The latest research shows that collagen obtained from marine organisms is used in multiple biomedical applications [164]. Collagen-based scaffolds are widely used in myocardial tissue engineering [137], cartilage tissue engineering [165], neural tissue engineering [166], musculoskeletal tissue engineering [167], and bone tissue engineering [48]. Massimino et al. developed a collagen-based scaffold obtained from bovine tendon for dermal regeneration applications [49]. Pericardial bovine and porcine tissue underwent TRICOL decellularization (detergent-based treatment), and decellularized pericardial scaffold containing collagen and elastin was considered a potential biomaterial for tissue replacement [52].

#### 2.4.9. Fibroin

Fibroin is protein silk produced by some larvae such as spiders, silkworms, mites, scorpions, and flies. The silk obtained from *Bombyx mori* (silkworm) and spiders such as *Araneus diadematus* and *Nephila clavipes* are widely used commercially [50]. Due to its excellent structural integrity and mechanical properties, silk fibroin-based biomaterial is used in musculoskeletal tissue engineering [168]. Hadisi et al. developed a silk fibroinbased scaffold composed of hardystonite loaded with gentamicin as an antibiotic agent to evaluate the in vitro and in vivo studies on bone tissue engineering applications [169]. According to Zakeri-Siavashani et al., fibroin-based scaffold containing keratin and vanillin particles acts as a potential antibacterial agent in skin tissue engineering [170].

#### 2.4.10. Keratin 2.4.10. Keratin

2.4.9. Fibroin

Keratin is a fibrous protein rich in cysteine and is widely present in hair, nails, wool, feathers, and horns [171]. The flexible transverse bonds in the keratin molecular chain provide suitable mechanical properties to its fibrous protein structure [172]. Keratin is insoluble, highly durable, chemically unreactive, and has binding factors that help cell adhesion and growth [173]. Keratin-based scaffolds are widely used in skin, bone, and nerve regeneration [100]. Wan et al. developed a biocomposite mat that constitutes poly (ε-caprolactone), keratin, heparin, and vascular endothelial growth factor, which acts as a well-suited scaffold in vascular tissue engineering [174]. The molecular structure of some protein molecules is shown in Figure 7. Keratin is a fibrous protein rich in cysteine and is widely present in hair, nails, wool, feathers, and horns [171]. The flexible transverse bonds in the keratin molecular chain provide suitable mechanical properties to its fibrous protein structure [172]. Keratin is insoluble, highly durable, chemically unreactive, and has binding factors that help cell adhesion and growth [173]. Keratin-based scaffolds are widely used in skin, bone, and nerve regeneration [100]. Wan et al. developed a biocomposite mat that constitutes poly (ε-caprolactone), keratin, heparin, and vascular endothelial growth factor, which acts as a well-suited scaffold in vascular tissue engineering [174]. The molecular structure of some protein molecules is shown in Figure 7.

pericardial scaffold containing collagen and elastin was considered a potential bio-

Fibroin is protein silk produced by some larvae such as spiders, silkworms, mites, scorpions, and flies. The silk obtained from *Bombyx mori* (silkworm) and spiders such as *Araneus diadematus* and *Nephila clavipes* are widely used commercially [50]. Due to its excellent structural integrity and mechanical properties, silk fibroin-based biomaterial is used in musculoskeletal tissue engineering [168]. Hadisi et al. developed a silk fibroin-based scaffold composed of hardystonite loaded with gentamicin as an antibiotic agent to evaluate the in vitro and in vivo studies on bone tissue engineering applications [169]. According to Zakeri-Siavashani et al., fibroin-based scaffold containing keratin and vanillin particles acts as a potential antibacterial agent in skin tissue engineering [170].

*Gels* **2023**, *9*, x FOR PEER REVIEW 22 of 45

material for tissue replacement [52].

2.4.11. Elastin

lagen [60].

2.4.12. Fibrin

gineering [184].

2.4.13. Gelatin

**Figure 7.** Molecular structure of some protein molecules: (**a**) collagen I [175]; (**b**) keratin [176]; (**c**) fibrin [177] ; (**d**) elastin [178]. **Figure 7.** Molecular structure of some protein molecules: (**a**) collagen I [175]; (**b**) keratin [176]; (**c**) fibrin [177]; (**d**) elastin [178].

rial regeneration and helps to understand the macrophage-mediated immune response created after implantation [180]. Rodrigues I. C. P. et al. stated that adding elastin and collagen to his polyurethane-based scaffold improves cellular response and wettability [181]. Matriderm and glyaderm are some dermal substitutes used in wound healing made up of elastin combined with collagen, whereas matriderm constitutes bovine col-

Elastin is a structural protein with elastic properties widely found in connective tissue and other load-bearing tissues. Elastin is in the collagen network in many organs,

Fibrin is a protein molecule formed during blood clotting by polymerizing thrombin and fibrinogen in blood plasma. Easy fabrication, rapid biodegradability, and good biocompatibility are some properties that make fibrin used in tissue regeneration applications. It is mainly used in nerve tissue engineering, skin tissue engineering, musculoskeletal tissue engineering, and cardiac tissue engineering [182,183]. According to Bluteau et al., the low thrombin concentration increased the rate of osteoblastic marker expression. It brought out the increased angiogenic response of osteoblasts by vascular endothelial growth factor (VEGF) expression. Thus, fibrin also helps in bone tissue en-

Gelatin is a protein molecule obtained by the hydrolysis of collagen, and it constitutes the Arg–Gly–Asp (RGD) peptide sequence, which helps in cell adhesion, proliferation, and differentiation [185]. The primary source of gelatin production is extracted from mammals, especially bovine hides and porcine skin [186]. Scaffold coated with gelatin inhibits complement system and opsonization. Thus, it reduces their immunogenicity [187]. In vitro studies show that scaffolds based on gelatin can control cell differentiation and gene expression [188]. Dehghan M. et al. combined gelatin, polycaprolactone, and

#### 2.4.11. Elastin

Elastin is a structural protein with elastic properties widely found in connective tissue and other load-bearing tissues. Elastin is in the collagen network in many organs, including the lungs, skin, and blood vessels [179]. In vascular tissue engineering, the successful development of elastin-based vascular graft materials helps to facilitate arterial regeneration and helps to understand the macrophage-mediated immune response created after implantation [180]. Rodrigues I. C. P. et al. stated that adding elastin and collagen to his polyurethane-based scaffold improves cellular response and wettability [181]. Matriderm and glyaderm are some dermal substitutes used in wound healing made up of elastin combined with collagen, whereas matriderm constitutes bovine collagen [60].

#### 2.4.12. Fibrin

Fibrin is a protein molecule formed during blood clotting by polymerizing thrombin and fibrinogen in blood plasma. Easy fabrication, rapid biodegradability, and good biocompatibility are some properties that make fibrin used in tissue regeneration applications. It is mainly used in nerve tissue engineering, skin tissue engineering, musculoskeletal tissue engineering, and cardiac tissue engineering [182,183]. According to Bluteau et al., the low thrombin concentration increased the rate of osteoblastic marker expression. It brought out the increased angiogenic response of osteoblasts by vascular endothelial growth factor (VEGF) expression. Thus, fibrin also helps in bone tissue engineering [184].

#### 2.4.13. Gelatin

Gelatin is a protein molecule obtained by the hydrolysis of collagen, and it constitutes the Arg–Gly–Asp (RGD) peptide sequence, which helps in cell adhesion, proliferation, and differentiation [185]. The primary source of gelatin production is extracted from mammals, especially bovine hides and porcine skin [186]. Scaffold coated with gelatin inhibits complement system and opsonization. Thus, it reduces their immunogenicity [187]. In vitro studies show that scaffolds based on gelatin can control cell differentiation and gene expression [188]. Dehghan M. et al. combined gelatin, polycaprolactone, and polydimethylsiloxane to produce a scaffold, and further investigations on tests regarding biocompatibility, biodegradability, and mechanical properties gave a positive result [9].

Singh S. et al. used gelatin as a fabricating material for a cellulose-based scaffold produced from cotton to improve cell adhesion [189]. Goudarzi Z. M. et al. concluded that a poly (ε-caprolactone) and gelatin composite scaffold incorporated with acetylated cellulose nanofiber is an ideal scaffold for soft tissue engineering [190]. The list of advantages, disadvantages, and applications of each protein is mentioned in Table 4.

**Table 4.** Advantages, disadvantages, and applications of proteins in tissue regeneration applications.



**Table 4.** *Cont.*

Figures 8 and 9 depict the number of publications on polysaccharides and proteins in tissue engineering applications. It can be seen from both figures that there is an apparent increase in terms of publications in research involving the usage of polysaccharides and proteins as a natural ingredient in developing suitable scaffolds for tissue engineering applications *Gels* **2023**, *9*, x FOR PEER REVIEW 25 of 42

**Figure 8.** Publications on the usage of polysaccharides in tissue engineering. (The number of papers published on individual polysaccharides such as cellulose, chitin, alginate, starch, hyaluronic acid, and pullulan is drawn based on the year and the respective total number of papers published, using search engine: www.scopus.com.) **Figure 8.** Publications on the usage of polysaccharides in tissue engineering. (The number of papers published on individual polysaccharides such as cellulose, chitin, alginate, starch, hyaluronic acid, and pullulan is drawn based on the year and the respective total number of papers published, using search engine: www.scopus.com, accessed on 15 January 2023.)

**Figure 9.** Publications on the usage of proteins in tissue engineering. (The number of papers published on individual proteins such as collagen, fibrin, fibroin, keratin, elastin, and gelatin is drawn based on the year and the respective total number of papers published, using search engine:

**2001**

**2003**

**2005**

**2007**

**2009**

**2011**

**2013**

**2015**

**2017**

**2019**

**2021**

Usually, the tissue comprises repeated 3D units such as islets that act as a base for coordinating multicellular processes, maintaining mechanical properties, and integrating various organs through the circulation process. Hence, while designing the scaffold for

**Publications on usage of Proteins in Tissue Engineering**

www.scopus.com.)

**Number of publications**

**1965**

**1967**

**1971**

**1973**

**1975**

**1977**

**1979**

**1981**

**1983**

**1985**

**1987**

**1989**

**1991**

**1993**

**1995**

**Year**

**Collagen Fibrin Fibroin Keratin Elastin Gelatin**

**1997**

**1999**

*2.5. Scaffold Fabrication Techniques* 

**Number of publications**

**1969 1976 1978 1982 1984 1987 1989 1991 1993 1995 1997 1999 2001 2003 2005 2007 2009 2011 2013 2015 2017 2019 2021**

**Publications on usage of Polysaccharides in Tissue Engineering**

**Year**

**Cellulose Chitin Starch Hyaluronic acid Alginate Pullulan**

**Figure 9.** Publications on the usage of proteins in tissue engineering. (The number of papers published on individual proteins such as collagen, fibrin, fibroin, keratin, elastin, and gelatin is drawn based on the year and the respective total number of papers published, using search engine: www.scopus.com.) **Figure 9.** Publications on the usage of proteins in tissue engineering. (The number of papers published on individual proteins such as collagen, fibrin, fibroin, keratin, elastin, and gelatin is drawn based on the year and the respective total number of papers published, using search engine: www.scopus.com, accessed on 15 January 2023).

**Figure 8.** Publications on the usage of polysaccharides in tissue engineering. (The number of papers published on individual polysaccharides such as cellulose, chitin, alginate, starch, hyaluronic acid, and pullulan is drawn based on the year and the respective total number of papers published,

#### *2.5. Scaffold Fabrication Techniques 2.5. Scaffold Fabrication Techniques*

using search engine: www.scopus.com.)

Usually, the tissue comprises repeated 3D units such as islets that act as a base for coordinating multicellular processes, maintaining mechanical properties, and integrating various organs through the circulation process. Hence, while designing the scaffold for Usually, the tissue comprises repeated 3D units such as islets that act as a base for coordinating multicellular processes, maintaining mechanical properties, and integrating various organs through the circulation process. Hence, while designing the scaffold for tissue repair, we must remember that tissue substitutes should have desired mechanical properties and facilities for transporting nutrients and wastes [2]. Fabrication techniques are needed to create a proper scaffold with good mechanical properties, interconnected pores, 3D porous structure, and uniform distribution [220]. The scaffold architectural design is characterized into three levels (nano, micro, and macro) to maintain scaffold parameters such as anatomical features, cell–matrix interactions, and nutritional transportation. The nano-level architecture includes surface modification, including attachment of signaling molecules for cell adhesion, proliferation, and differentiation. Micro-level architecture constitutes pore size, porosity, interconnected pores, and spatial arrangements. The anatomical features and organ and patient specificity include macro-level architecture [221].

Fabrication techniques are classified into two categories: conventional and rapid prototyping. Techniques such as freeze drying, solvent casting, particle leaching, electrospinning, gas foaming, and thermal-induced phase separation come under conventional fabrication techniques. These techniques are suitable for constructing porous scaffolds, but the main limitation is the lack of tunable properties to control shapes and internal architecture. In other words, achieving complex micro- and macro-level architecture is difficult in conventional fabrication techniques. Rapid prototyping is developed to overcome the drawbacks caused by conventional fabrication techniques. Rapid prototyping is known as solid free-form fabrication (SFF) and additive manufacturing (AM). It is the fastest fabrication method for assembling the desired item by using computer generation tools such as computer-aided design (CAD), magnetic resonance imaging (MRI), and computer tomography (CT). Nearly 30 rapid prototyping technologies were applied in various fields, of which 20 were used for biomedical applications [222]. Stereolithography, bioprinting, selective laser sintering, solvent-based extrusion-free forming, and fused

deposition modeling are standard rapid prototyping methods used in tissue engineering for scaffold fabrication.

Usually, the primary protocol includes forming and slicing a virtual computer model ensured by layer-by-layer fabrication steps that are similar in all the various rapid prototyping techniques. Initially, a CAD model is captured or formulated from a physical unit by digital method, and then the obtained model is converted into a stereolithography file for virtual slicing. Further, it allows for digital slicing to gain cross-sectional layers. This process is termed pre-processing. Then, rapid prototyping starts to print the layer of the prototype. The post-processing steps, including surface treatment and hardening, are applied. It entirely depends on the purpose and manufacturing techniques. The desired complex micro- and macro-level architecture can also be achieved by using rapid prototyping [223].

#### 2.5.1. Freeze Drying

The freeze-drying technique is otherwise known as lyophilization or ice templating. This technique includes three steps: dissolution, solidification or freezing, and sublimation. At first, the chosen polymer is dissolved in a solvent. Secondly, the solution is loaded into a mold and placed in the freezer for solidification or freezing. It is then allowed to cool down using chemicals such as dry ice in aqueous methanol, liquid nitrogen, or mechanical refrigeration. Care should be taken at this step to maintain temperature, or else it will result in the formation of large crystals, which may affect the properties of the scaffold later. Thirdly, the sublimation process is carried out to remove water and other solvent molecules in the frozen component. This technique is highly suitable for producing scaffolds with high porosity, which provides vascularization and helps in cell proliferation and differentiation. The lyophilization method can be combined with salt leaching, gas foaming, gel casting, and liquid dispensing practices to improve the scaffold's properties. No involvement of heat is the primary advantage of this method, so heatsensitive molecules such as proteins or growth factors can be incorporated into it without hesitation. However, it consumes a longer time and high energy, and the cost of a freeze dryer is expensive, which are some of the drawbacks [224]. C. M. Brougham et al. developed a heart valve-shaped tissue engineering scaffold using collagen and glycosaminoglycan copolymer and fabricated it using the freeze-drying method [225]. During electro-spinning, toxic substances from organic solvents may involve scaffold preparation. Moreover, it can cause damage to the biological activity of cells. To avoid this situation, A. Izadyari Aghmiuni et al. combined freeze-drying and electro-spinning methods to develop a scaffold for tissue engineering [226].

## 2.5.2. Solvent Casting and Particle Leaching

3D specimens with thin walls or membranes were produced using solvent casting and particle leaching methods. These thin membranes are prepared by adding salt particles to the solvent polymeric solution. Then, the solvent is allowed to evaporate, and the resulting membrane is washed with distilled water to leach out the salt. The main advantages of solvent casting and particle leaching methods are high porosity, cheapness, and straightforwardness. This technique's usage of toxic solvents, poor interconnectivity, and irregularly shaped pores are limitations [227,228]. N. Thadavirul et al. developed a polycaprolactone porous scaffold using solvent casting and particle leaching techniques for bone tissue engineering [228]. To enhance the mechanical properties, researchers incorporate hydroxyapatite into blends of the biodegradable polymer [229].

#### 2.5.3. Gas Foaming

The gas foaming technique was introduced to avoid using organic cytotoxic solvents and high temperatures. However, the resultant material obtained had closed pores, which limited its usage, especially in cell transplantation. In this method, chosen polymer was mixed with salt particles and molded to form solid disks. Then, disks were exposed to inert gas foaming agents such as nitrogen gas or carbon dioxide with high pressure for saturation. Then, gas was decreased to ambient pressure to create thermodynamic instability, resulting in nucleation and facilitating carbon dioxide pores between polymer matrices. Finally, the salt was removed by leaching the polymer using distilled water [2,230].

#### 2.5.4. Electrospinning

It is a simple technique in which solutions produce fibers by applying high-voltage electricity. The main principle behind this technique is the interaction between electrostatic repulsion and surface tension of charging liquid that receives high voltage droplets. This machine consists of four major parts: a power supply unit, a syringe pump, a metallic needle, and a grounded collector [2,231], as shown in Figure 10. Usually, this technique is widely used for producing nano-fibrous scaffolds. The liquid is injected into the capillary tube of the syringe pump. The muscle power of the electric field from a high-voltage power supply increases the surface tension of liquid extruding from the nozzle of the metallic needle. *Gels* **2023**, *9*, x FOR PEER REVIEW 29 of 45

**Figure 10.** Simplified diagram of an electrospinning device, which consists of four main components such as power supply, syringe pump, metallic needle, and grounded collector. Reprinted with permission from Ref. [2]. Copyright 2019 Abdalla Eltom et al. **Figure 10.** Simplified diagram of an electrospinning device, which consists of four main components such as power supply, syringe pump, metallic needle, and grounded collector. Reprinted with permission from Ref. [2]. Copyright 2019 Abdalla Eltom et al.

2.5.5. Thermal-Induced Phase Separation Method This method is widely used to fabricate microcellular foams or microporous membranes. This technique de-mixes the homogenous polymer solution into polymer-rich and poor phases by applying variant temperatures. Further, lyophilization of phase-separated polymer solution helps produce microcellular structure [233]. Adjustment of pore size can be practically made possible in this method by allowing drugs and fillers. Moreover, these particles are also homogeneously distributed within the pore size. Further, the liquid jet is continuously whipped due to electrostatic repulsion and is collected in the form of fibers in the grounded collector. Electrospinning techniques help produce scaffolds with good porosity, patterned architecture, and aligned fibers, which further help cellular response and enhance tissue regeneration. Precise control over fiber formation, homogeneous cell distribution, and lack of cellular infiltration are drawbacks of the electrospinning method [231]. Cellulose nano fiber (CNF) scaffolds developed using potato peel waste promote the adhesion and proliferation of BALB-3T3 fibroblasts cells [232].

#### Inadequate resolution and usage of limited materials for fabrications are the main 2.5.5. Thermal-Induced Phase Separation Method

1. Freeze drying

2. Solvent casting and practical

5. Thermal-induced phase

MAXIAOYUseparation

drawbacks of this method. The phase separation technique plays a vital role in fabricating a 3D nanofibrous scaffold, and it can be highly recommended to use along with another fabricating technique such as solid free form [2]. The advantages and disadvantages of various fabrication techniques are mentioned in Table 5. **Table 5.** Advantages and disadvantages of various fabrication techniques. **S.No Techniques Advantages Disadvantages**  Capability to do away with high temperatures, applicable in a vari-Long time consumption, high This method is widely used to fabricate microcellular foams or microporous membranes. This technique de-mixes the homogenous polymer solution into polymer-rich and poor phases by applying variant temperatures. Further, lyophilization of phase-separated polymer solution helps produce microcellular structure [233]. Adjustment of pore size can be practically made possible in this method by allowing drugs and fillers. Moreover, these particles are also homogeneously distributed within the pore size. Inadequate resolution and usage of limited materials for fabrications are the main drawbacks of this method. The phase separation technique plays a vital role in fabricating a 3D nanofibrous scaffold, and it can be highly recommended to use along with another fabricating technique such as

ety of purposes, and pore size can

and usage of simple equipment

Fast, controllable, scalable, and formation of intrinsically interconnected pores

6. Stereolithography High resolution, fast processing, Expensive, high temperature,

leaching Cheap and high porosity Usage of toxic solvents

energy consumption, usage of MAXIAOYUcytotoxic solvents, and irregular pore size

Poor interconnectivity, low reproducibility, and structural uniformity

Limited control of pore structure, use of toxic solvents, and many variables involved in the process

Only used for thermoplastic

3. Gas foaming Absence of caustic solvents

4. Electrospinning Porosity, control over morphology,

solid free form [2]. The advantages and disadvantages of various fabrication techniques are mentioned in Table 5.


**Table 5.** Advantages and disadvantages of various fabrication techniques.

## 2.5.6. Stereolithography

Stereolithography is considered the first rapid prototype technique commercially available in the fabrication process—an aqueous photo-curable polymer was used as a raw material. An ultraviolet laser beam was used as a light source to irradiate the material surface for solidification where the untreated region remains liquid. Once the solidification of one layer is completed, the lifting table starts to move to the next layer. Subsequently, the solidified layer is recoated with new liquid resin. This photo-polymerization process is repeated until the remaining layer is done. This technique's scaffold material has enhanced cell growth and adhesion. High resolution and uniformity in pore interconnectivity are this method's main advantages [234]. The process involved in stereolithography is shown in Figure 11.

7. Selective laser sintering

9. Solvent-based extrusion 3D

printing method

2.5.6. Stereolithography

8. Fused deposition model No requirement for solvents and

**Figure 11.** Simplified diagram of stereolithography, which consists of a tank, lifting table, laser **Figure 11.** Simplified diagram of stereolithography, which consists of a tank, lifting table, laser scanner, and a computer.

and smoother surface and toxic uncured resin

High temperature, rough surface finish

Filament requirement and high temperature

Temperature MAXIAOYUextrusion

MAXIAOYUexistence

Fast processing, high resolution, and no support is needed during MAXIAOYUmanufacturing

good mechanical properties

Applicable to precise control of micron-level scaffold structure, suitable for ceramic and MAXIAOYUmetals too

Stereolithography is considered the first rapid prototype technique commercially available in the fabrication process—an aqueous photo-curable polymer was used as a raw material. An ultraviolet laser beam was used as a light source to irradiate the material surface for solidification where the untreated region remains liquid. Once the solidification of one layer is completed, the lifting table starts to move to the next layer. Subsequently, the solidified layer is recoated with new liquid resin. This photo-polymerization process is repeated until the remaining layer is done. This technique's scaffold material has enhanced cell growth and adhesion. High resolution and uniformity in pore interconnectivity are this method's main advantages [234]. The process

10. Bioprinting method Cheap and higher accuracy Depends on cell

involved in stereolithography is shown in Figure 11.

#### 2.5.7. Selective Laser Sintering

scanner, and a computer.

2.5.7. Selective Laser Sintering It is an additive manufacturing technique in which a high-intensity laser beam fabricates a scaffold layer-wise using computer-aided design models. Usually, materials are used in powder, and this technique can be applied to produce various materials such as ceramic, polymer, and metals. The laser beam is used to heat powder particles to glass transition temperature (near their melting point). The material was sintered to form a solid model directly without permitting the melting phase. Then, the workstation moves It is an additive manufacturing technique in which a high-intensity laser beam fabricates a scaffold layer-wise using computer-aided design models. Usually, materials are used in powder, and this technique can be applied to produce various materials such as ceramic, polymer, and metals. The laser beam is used to heat powder particles to glass transition temperature (near their melting point). The material was sintered to form a solid model directly without permitting the melting phase. Then, the workstation moves down layer by layer. At the same time, fresh powder is spread on the sintered object with the help of a roller, and the process is repeated until the completion of a 3D material. The scaffolds from this method provide excellent compressive strength, fracture toughness, osteoconduction, and osteoinduction. However, the high operating temperature limits the resolution, and additional procedures such as removing injected powder after processing the phase spin are some drawbacks of this method [2,234,235]. The process involved in selective laser sintering is shown in Figure 12. *Gels* **2023**, *9*, x FOR PEER REVIEW 31 of 45 down layer by layer. At the same time, fresh powder is spread on the sintered object with the help of a roller, and the process is repeated until the completion of a 3D material. The scaffolds from this method provide excellent compressive strength, fracture toughness, osteoconduction, and osteoinduction. However, the high operating temperature limits the resolution, and additional procedures such as removing injected powder after processing the phase spin are some drawbacks of this method [2,234,235]. The process involved in selective laser sintering is shown in Figure 12.

**Figure 12***.* Simplified diagram of selective laser sintering, which consists of a reservoir platform, moving workstation, roller, and a scanner*.*  **Figure 12.** Simplified diagram of selective laser sintering, which consists of a reservoir platform, moving workstation, roller, and a scanner.

#### 2.5.8. Fused Deposition Model 2.5.8. Fused Deposition Model

ing [2,234,236].

According to Xia et al., the fused deposition model is a filament-based additive manufacturing method. Plastic materials are used in the form of filament. They are inserted into a heating nozzle, where the filament is melted, extruded, and deposited into According to Xia et al., the fused deposition model is a filament-based additive manufacturing method. Plastic materials are used in the form of filament. They are inserted into a heating nozzle, where the filament is melted, extruded, and deposited into a plate to

a plate to produce a 3D structure, layer-by-layer manner, with the help of comput-

establishment, which results in a lack of cell growth and vascularization. The processing time is too long, and the heating process hinders the integration of biomolecules into the scaffold, resulting in a smooth surface unsuitable for cell adhesion, which needs further coating. Many experiments were carried out to overcome these problems, and some series were developed based on the fusion deposition model. Low-temperature deposition modeling is one of the series created, and it also gave positive responses such as better biocompatibility, biodegradability, and all required properties for bone tissue engineer-

The solvent-based extrusion 3D printing method keeps biomaterials in solvents to produce inks. Then, obtained inks are extruded from the nozzle in filament to create a scaffold structure in a layer-wise manner. Natural polymers, synthetic polymers, and ceramics are the biomaterials currently being used to produce ink. This technique was widely applied to fabricate scaffolds for cartilage tissue, bone tissue, blood vessel, heart valve tissue, and skin tissue. Difficulty in obtaining appropriate levels of filament uniformity, lack of ink feasibility, and poor fidelity between the structure of computer

2.5.9. Solvent-based Extrusion 3D Printing Method

models and printed scaffold structures are some disadvantages [237].

produce a 3D structure, layer-by-layer manner, with the help of computer-based devices. This technique is simple, cheap, versatile, and has wide applications. However, some significant deficiencies are there, too, such as difficulty in microporosity establishment, which results in a lack of cell growth and vascularization. The processing time is too long, and the heating process hinders the integration of biomolecules into the scaffold, resulting in a smooth surface unsuitable for cell adhesion, which needs further coating. Many experiments were carried out to overcome these problems, and some series were developed based on the fusion deposition model. Low-temperature deposition modeling is one of the series created, and it also gave positive responses such as better biocompatibility, biodegradability, and all required properties for bone tissue engineering [2,234,236].

#### 2.5.9. Solvent-based Extrusion 3D Printing Method

The solvent-based extrusion 3D printing method keeps biomaterials in solvents to produce inks. Then, obtained inks are extruded from the nozzle in filament to create a scaffold structure in a layer-wise manner. Natural polymers, synthetic polymers, and ceramics are the biomaterials currently being used to produce ink. This technique was widely applied to fabricate scaffolds for cartilage tissue, bone tissue, blood vessel, heart valve tissue, and skin tissue. Difficulty in obtaining appropriate levels of filament uniformity, lack of ink feasibility, and poor fidelity between the structure of computer models and printed scaffold structures are some disadvantages [237].

#### 2.5.10. Bioprinting Method

Bioprinting technology is a promising fabrication technique to develop highly mimicked tissue with digital control. A typical bioprinting method consists of pre-processing, processing, and post-processing phases. At first, in the pre-processing step, the tissue blueprint is created using computer-aided design (CAD). The vital information regarding histological structure and composition, anatomy, and human organ topology for the design can be extracted using imaging approaches. Moreover, parameters for biomaterials are also finalized during this stage. A suitable bioprinter prints the desired structure in the processing step. The bio-ink used for the bioprinter plays a crucial role in delivering the desired scaffold. Finally, post-processing steps are carried out to maturate the obtained scaffold before host implantation. Using an ideal bioreactor for the scale-up process is also under this category. Computer-aided design (CAD) and computer-aided manufacturing (CAM) are used in all three phases and play a crucial role.

Bio-CAD mimics the 3D internal structure, differentiates heterogeneous tissue types, and creates desired models. Bio-CAM is used to predict the feasibility of the fabrication process. The combination of Bio-CAD and Bio-CAM helps accelerate the bioprinting process and enhance the quality of printed tissues. The biomaterials used in this process should be printable, non-toxic, and biodegradable in vivo. Inkjet bioprinting, extrusion bioprinting, laser-assisted bioprinting, and stereolithography are the widely applied bioprinting approaches. Due to their advantages, low cost, accuracy, and high speed, bioprinting technologies have already marked their footprints in cartilage, skin, aortic valve, bone, vascular, and kidney tissues. Dependence on existing cells is the main drawback of this method [238].

#### 2.5.11. Aerosol Jet Printing

The focused airstream is used as ink instead of liquid droplets in aerosol jet printing. Either organic or inorganic materials can be used for this printing technique. A composite suspension is atomized into an aerosol using an ultrasonic or pneumatic atomizer. Then, it is transported to the deposition head by nitrogen gas, which acts as a carrier gas, and jets onto the substrate to form a 3D structure in a layer-wise manner. Polymer, ceramic, and metals can be used for aerosol jet printing. Scaffolds developed from aerosol jet printing show better cytocompatibility in in vitro studies, and it is a low-temperature process, so

it is suitable for biomanufacturing too [239]. Some research on natural polymers used to fabricate scaffolds for various tissue regeneration applications is explained in Table 6.

**Table 6.** Natural polymers used to fabricate scaffolds for various tissue regeneration applications.


#### **3. Conclusions**

Scaffolds based on natural products have gained more importance than synthetic products. The research in developing scaffolds from natural-based biomaterials for tissue regeneration applications is rapidly growing due to their outstanding properties such as promoting cell adhesion, proliferation, migration, biocompatibility, biodegradability, porosity, ease of production, inexpensive, and non-toxic. However, natural-based biomaterials have poor mechanical properties. They can be fabricated with suitable materials and used in various biomedical applications, including tissue engineering. The selection of suitable materials is crucial in tissue engineering. In that way, this paper provides a clear idea about the natural-based materials that are currently used in tissue engineering applications. In addition to that, the applications of fabrication techniques in scaffold development have been illustrated. Each technique has its respective benefits and drawbacks, and, as mentioned, appropriate selection to satisfy the need for the tissue to be repaired plays a vital role.

**Author Contributions:** M.K. and W.Y.S. performed the review, analysis, and editing of the manuscript; H.S. and N.S.S. performed the structure conceptualization, review, and editing of the manuscript. All authors have read and agreed to the published version of the manuscript.

**Funding:** This research and the APC were funded by Universiti Brunei Darussalam Research Grant No: UBD/RSCH/1.3/FICBF(b)/2020/005.

**Institutional Review Board Statement:** Not applicable.

**Informed Consent Statement:** Not applicable.

**Data Availability Statement:** No new data were created or analyzed in this study. Data sharing is not applicable to this article.

**Conflicts of Interest:** The authors declare no conflict of interest.

#### **References**


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