2.2.6. Manufacturing Technologies

**Figure 3.** The necessary ideal scaffold requirements include biocompatibility, biodegradability, mechanical properties, scaffold architecture, and manufacturing technology. As stated by Place et al., TE products must be both productive and cost-effective, introducing a potential dichotomy between the need for sophistication and ease of production [39]. While ensuring scaffold efficiency, it is also essential to consider the cost and availability, ensuring scale-up production of the scaffolds is feasible when required. Another key factor to consider is delivering and packaging the scaffolds to the clinicians. Even though clinicians usually prefer off-the-shelf availability to lessen waiting time before implantations, it may not be possible for some tissue types [40]. Therefore, this should be considered while implementing a TE strategy.

#### *2.3. Materials Used for Developing Scaffold*

The material source for scaffolds should depend on the patient's status. For instance, patients with cancer or osteoporosis generally experience low bone metabolism; hence, the scaffold material should be non-resorbable. Nevertheless, the material source would come under biomaterials. According to the European Society for Biomaterials (ESB), a biomaterial is a material meant to interface with biological systems to treat, evaluate, augment or replace any tissue, organ or function of the body [18,40]. The four major

biomaterials typically used in the fabrication of scaffolds are polymers, bio-ceramics, metals, and carbon-based nanomaterials. As each group has specific advantages and disadvantages, scaffolds may comprise more than one of these biomaterial types [40,41]. Natural polymers, synthetic polymers, bio-ceramics, biodegradable metals, and carbon-based nanomaterials are currently used in scaffold development [1].

#### 2.3.1. Polymer

A polymer is a long-chained macromolecule built up by repeated monomers, and polymer-based biomaterials are considered a good choice for fabricating a scaffold [42]. Polymers are a good candidate in TE applications for their great versatility and flexibility in providing a wide range of mechanical, chemical, and physical properties. They show good biocompatibility, are light in weight, and are resistant to biochemical attack. Moreover, polymers are highly available at a reasonable cost and quickly processed into desired shapes. In addition, the inertness of polymers towards host tissues makes them an eligible candidate for a drug delivery system. Some biomedical applications involving polymers include artificial organs and blood vessels, breast implants, contact lenses, coatings for pharmaceutical tablets and capsules, external and internal ear repairs, cardiac assist devices, and joint replacements [43]. Polymeric biomaterials have been obtained from natural and synthetic polymers, each having pros and cons [44,45]. Carbohydrates such as chitin, cellulose, starch, alginate, and hyaluronic acid and proteins such as collagen, elastin, keratin, gelatin, and fibrin fall under natural polymers, where polyesters such as poly εcaprolactone (PCL), polylactic acid (PLA), and polyglycolic acid (PGA) and Polyurethanes come under synthetic polymers [44].


#### 2.3.2. Bio-Ceramics

Ceramic materials obtained from natural products, termed bio-ceramics, have been widely used in dental and bone tissue engineering. Bio-ceramics are organic, non-metallic solids with good compatibility, bio-inertness, bioactivity, osteoconductivity, and mechanical strength [18,43]. In addition, bio-ceramics can promote new bone generation and the osteo-potential of scaffolds. However, bio-ceramics are low in elasticity with a brittle surface, limiting their use in implants. Thus, they are usually blended or coated with other materials to improve their elasticity and strength. Among the biomaterials, bio-ceramic scaffolds have been proven to be more successful in treating minor bone defects, such as orthopaedic implants and bone-filling applications. There are three main types of ceramics: bioinert, bioactive, and bioresorbable. Bioinert ceramics include alumina (Al2O3), Zirconia (ZrO2), and pyrolytic carbon; bioactive ceramics include bioglasses (BG) and glass ceramics, while bioresorbable ceramic contains calcium phosphates. Of all three types, the most commonly used ceramics in BTE applications are HAp, tricalcium phosphates (TCP), and their composites [3]. Human bone and teeth are composed of an inorganic compound known as hydroxyapatite, which constitutes calcium, phosphate, and OH radicals with high tensile strength and quickly adheres to host tissues. Many studies revealed that HAp is non-toxic and lacks inflammatory and pyrogenetic response [47].

HAp is a naturally occurring calcium phosphate-based mineral with the chemical formula Ca10(PO4)6(OH)2. It is structurally similar to a biological apatite in the human body, known as bone mineral, which makes up approximately 60–70% of human bone tissues on a dry weight basis. It shows similar chemical and physical properties to human bone and dental tissues [48,49]. Hence, some hydroxyapatite-rich natural products such as shells, corals, algae, fish scales, and animal bones are used to develop scaffolds [50,51]. HAp exhibits excellent biocompatibility and bioactivity, high osteoinductivity and osteoconductivity, non-toxicity, and non-inflammatory characteristics. Moreover, it vitalizes growth factors and promotes cell growth and proliferation. HAp is therefore considered a highly potential implant material and bone substitute. Nevertheless, HAp also shows poor mechanical properties, slow resorption and remodeling rates, and slow degradation rates in vivo, making it unsuitable for all BTE applications. Thus, HAp is usually synthesized with other natural or synthetic polymers to create more effective composite scaffolds [19,52].

HAp can be obtained through extractions from natural sources or chemical syntheses, divided into three categories: high-temperature methods, wet methods, and dry methods. Dry methods include mechanochemical methods and solid-state reactions. Here, dry precursors of calcium and phosphate are mixed without any precisely controlled conditions to synthesize HAp. According to Sadat-Shojai et al., this is to ease the mass production of HAp powders. Wet methods include sol-gel, chemical/wet/co-precipitation, hydrothermal, hydrolysis, sonochemical method, and emulsion method. With this method, the morphology and average powder size can be controlled. However, HAp yielded usually exhibits low crystallinity due to low operating temperatures. High-temperature processes include combustion and pyrolysis methods, where samples undergo thermal decompositions. These two methods are rarely used for HAp synthesis due to poor control over the operating conditions [53].

HAp can also be extracted from food wastes or biological sources such as aquatic or marine sources, mammalian bones shell sources, and plant sources [54]. This method is relatively safe, more sustainable, and economical to fabricate HAp, thereby contributing to the economy, environment, and general health. However, it is notable that natural HAp is non-stoichiometric, either calcium or phosphorus-deficient. Generally, calcium positions would be the vacancy, where cations such as Na<sup>+</sup> , Mg2+, and Al3+ are substituted into the vacant space. Likewise, carbonate ions would replace phosphate or hydroxyl ions, and fluoride ions would substitute in place of hydroxyl ions. These trace elements present in the natural HAp resemble the apatite in human bone, which is crucial in accelerating bone formation and regeneration. For instance, blending 3–5 mol% silicon with synthetic HAp can boost cell growth density, enhancing osteoblast growth. Another example is adding 1–10% of strontium ions in synthetic HAp, which improves osteoblast activity and material differentiation [55]. Calcium carbonate is abundantly found in the exoskeleton of most marine organisms such as corals, sea urchins, and some algae. HAp produced from these exoskeletons are highly porous, have good vascularization and blood supply, and help to form new tissue [56]. Over the past 20 years, extensive research has been done to constantly improve the synthesis methods and introduce new technologies, aiming to develop an ideal HAp composite or scaffold that fulfills all the desired specifications.

#### 2.3.3. Metals

Metals such as stainless steel, cobalt–chromium–molybdenum alloy, aluminium, lead, silver, and titanium alloys have been considered good load-bearing implants because of their excellent quality electrical and thermal conductivity, appropriate mechanical properties, corrosion resistance, biocompatibility, and reasonable cost. However, metals are non-biodegradable. Therefore, researchers introduced the use of biodegradable metals [18]. Biodegradable metals are metals having controlled corrosion properties. They can be grouped into pure biodegradable metals (Mg−- and Fe- -based), biodegradable alloys, and biodegradable metal matrix composites [43]. Pure biodegradable metal implants have similar mechanical properties to stainless steel and bone and are non-toxic. However, they show slow degradation rates and are incompatible with MRI (Magnetic Resonance Imaging). These problems can be addressed through newly-developed fabrication methods such as casting, electroforming, powder metallurgy, and inkjet 3D printing. Moreover, it is essential to note that the patients implanted with biodegradable metals should not have an iron-related disease, and the patient's intestines can absorb only Fe2+. Thus, any Fe3+ released should be first reduced to Fe2+ before being absorbed [43].

Biodegradable porous metal scaffolds have attracted researchers in scaffold development by their high compressive strength. Biodegradable metals overcome problems such as innate immune rejection and have good load-bearing capacity during bone healing. However, biodegradable metals such as Mg and their alloys have a high corrosion rate. Recently, scientists have concentrated on the Zn-based alloy system to produce biodegradable metal scaffolds [57,58].

#### 2.3.4. Carbon-Based Nanomaterials

Researchers developed carbon-based nanomaterial scaffolds by combining tissue engineering and nanotechnology to enhance the scaffold's features. Carbon nanotubes (CNTs), graphene oxide (GO), carbon dots (CDs), fullerenes, and nanodiamonds are some carbon nanomaterials used as scaffolds in tissue engineering. Biocompatibility, mechanical stability, low cytotoxicity, facilitating cell communication, and nutrition delivery are advantages of carbon-based nanomaterials that pull down to use in scaffold development. However, limited biodegradability and potential cytotoxicity are significant drawbacks [59].

Carbon-based nanomaterials, including graphene oxide (GO), carbon nanotubes (CNTs), fullerenes, carbon dots (CDs), nanodiamonds (NDs), and their derivatives, are highly potential scaffold materials for bone restoration applications. They are biocompatible, mechanically stable, and commercially available. In addition to that, they show essential qualities such as good biodegradability, efficient cell proliferation and osteogenic differentiations, significant cell growth stimulations, proper mass transfer of nutrients in the scaffold microenvironment, improved cell distributions, and appropriate cell bioactivity. Yet, further studies regarding the low cytotoxicity and the adverse environmental effects of carbon-based nanomaterials are to be conducted before they can be clinically tested and brought into application [1,60]. The materials used for developing the scaffold are summarized in Figure 4.

2.3.4. Carbon-Based Nanomaterials

the scaffold are summarized in Figure 4.

cant drawbacks [59].

**Figure 4.** Materials used for scaffold development. (Materials are divided into four broad categories such as polymers, bio-ceramics, metals, and carbon nanomaterials. Classification with a few examples is summarized.) **Figure 4.** Materials used for scaffold development. (Materials are divided into four broad categories such as polymers, bio-ceramics, metals, and carbon nanomaterials. Classification with a few examples is summarized).

Researchers developed carbon-based nanomaterial scaffolds by combining tissue engineering and nanotechnology to enhance the scaffold's features. Carbon nanotubes (CNTs), graphene oxide (GO), carbon dots (CDs), fullerenes, and nanodiamonds are some carbon nanomaterials used as scaffolds in tissue engineering. Biocompatibility, mechanical stability, low cytotoxicity, facilitating cell communication, and nutrition delivery are advantages of carbon-based nanomaterials that pull down to use in scaffold development. However, limited biodegradability and potential cytotoxicity are signifi-

Carbon-based nanomaterials, including graphene oxide (GO), carbon nanotubes (CNTs), fullerenes, carbon dots (CDs), nanodiamonds (NDs), and their derivatives, are highly potential scaffold materials for bone restoration applications. They are biocompatible, mechanically stable, and commercially available. In addition to that, they show essential qualities such as good biodegradability, efficient cell proliferation and osteogenic differentiations, significant cell growth stimulations, proper mass transfer of nutrients in the scaffold microenvironment, improved cell distributions, and appropriate cell bioactivity. Yet, further studies regarding the low cytotoxicity and the adverse environmental effects of carbon-based nanomaterials are to be conducted before they can be clinically tested and brought into application [1,60]. The materials used for developing

In recent years, some innovative synthetic scaffolds based on natural products have been developed by applying recombinant DNA technology and advanced genetic engineering. Elastin-like recombinant [ELR] and elastin-like peptides [ELP] are a few scaffolds developed by obtaining the principles of advanced genetic engineering techniques. In recent years, some innovative synthetic scaffolds based on natural products have been developed by applying recombinant DNA technology and advanced genetic engineering. Elastin-like recombinant [ELR] and elastin-like peptides [ELP] are a few scaffolds developed by obtaining the principles of advanced genetic engineering techniques. The Arginine, Glycine, and Aspartic acid (RGD) sequence is an integrin-binding sequence in the ELP scaffolds, which helps in cell adhesion and proliferation. ELR scaffolds constitute a fibronectin domain that helps cell adhesion, particularly in vascular regeneration [61,62]. B. Gurumurthy et al. developed a collagen-based scaffold by incorporating it with an elastin-like polypeptide obtained from genetically modified *Escherichia coli* bacteria and bioglass to examine the osteogenic differentiation [63]. Repeated sequences of elastin and silk blocks are recombinantly combined to form silk-elastin-like protein polymers (SELPs). The hydrogels of SELPs play a vital role in wound healing [62].

On the whole, bio-based polymers have good features such as compatibility, versatility, and adaptability, and are also abundant in nature; they can be obtained from various agricultural resources and biodegradable waste materials. Hence, the processing and synthesis cost is low and environmentally friendly [50,64]. Many researchers have tried to produce scaffolds from natural polymers by keeping this in mind by modifying and enhancing their stability using various fabrication methods [65].
