Hydroxyapatite Composites in Tissue Engineering

Abstract

In the last few decades, material sciences, particularly tissue engineering, have advanced significantly. Biomaterials, including bioceramics, such as hydroxyapatite and bioglass, have shown to be quite useful in a variety of biomedical applications. Naturally produced polymers of protein or carbohydrate origin have also been employed as scaffolds in tissue engineering for many years. Collagen has been the most widely researched natural polymer for scaffold creation. Besides, aliphatic synthetic polymers such as polylactic acid, polyglycolic acid, and polycaprolactone are effective for scaffold fabrication. The improvements in material science have led to the procurement of biomaterials from natural sources, then processed using a variety of techniques, including porogen leaching, gas foaming, phase separation, fiber meshing, and three-dimensional printing. This generates a variety of three-dimensional scaffolds with various porosities and surface characteristics. When compared to the original components, hydroxyapatite composites have been proven to have superior characteristics. In the field of bone tissue repair and engineering, the biological performance of composites containing hydroxyapatite and other abundant natural biopolymers such as chitosan, collagen, gelatin, and cellulose is thoroughly investigated. This chapter discusses the various hydroxyapatite composite scaffolds utilized in in vitro and in vivo bone tissue engineering investigations, including their fabrication techniques.

1. Introduction

Bone deformities frequently demand surgical therapy along with bone grafts. Autografts provide considerable osteogenic properties and are considered as “gold standard.” However, donor site morbidity and hematoma have been linked to this procedure. Furthermore, with the limited supply of autografts and the risks associated with allografts, surgeons, and engineers are looking at novel ways to treat bone deformities. Among all the reported methods, tissue engineering is a multidisciplinary approach that integrates biological science and engineering concepts to augment or substitute biological tissues, and it has provided a novel treatment option for bone deformities. Several factors have critical effects on the process of tissue engineering; the scaffolds are one of the key factors, which act as substrate and also provide structural and mechanical support for cell growth. Hydroxyapatite (HA)—one of the main components of natural bone, has widely been applied as scaffolds in tissue engineering owing to its bioactivity and osteoconductivity. Based on these features, HA is an excellent choice for orthopedic and dental implants [1]. Up to now, various HA-based materials (natural and synthetic) have been developed and studied. Nowadays, a variety of materials and manufacturing methods, including 3D printing, is widely regarded as a novel alternative to traditional bone grafts for fabricating sophisticated biological products, such as biological scaffolds, tissues, organs, and customized medical devices, utilizing biological materials, living cells, and signaling molecules, and the use of computer-aided design (CAD) modeling to integrate 3D printing into tissue engineering has substantially improved scaffold production accuracy and repeatability.

In this review, we aim to provide an overview of HA-based materials, their types, and preparation as well as their applications in bone tissue engineering will also be introduced. This review may be useful for researchers interested in bone tissue engineering to receive an insight into HA-based materials and then choose the appropriate material depending on their preferences.

2. Natural sources of hydroxyapatite

Chemically formed HA has long been employed for bone tissue engineering, but its low durability and stability have limited its utility in the biomedical field. Because of the disadvantages of chemically and physically manufactured HA, natural biowastes have been used. Researchers have been attempting to find a way to synthesize HA using natural products. Several studies have shown that natural sources, such as corals, fish scales, eggshells, fish bones, seaweed, and animal bones, can be used for the successful preparation of HA [2]. Organic food waste, such as bovine/fish bones, seashells, and eggshells, may have ideal potential for generating HA, with extremely high availability, which could improve orthopedic applications. Scaffolds can also be made by successfully integrating biodegradable polymers (for adaptive degradation and biocompatibility) and bioceramics (for strength and bioactivity) to improve the bioactivity of the produced materials. With simple and efficient methods, numerous researchers have demonstrated the potential of transforming food waste into immensely useful bioceramics. Animal bones, eggshells, fish bones, oyster shells, and corals have all been used in various synthesis procedures. Additionally, Rocha et al. demonstrated the hydrothermal conversion of natural aragonite from cuttlefish bone in HA. HA derived from biological sources retains many of the attributes of the precursor materials, including chemical composition and pore structure. In this regard, it contains high calcium content as well as important trace minerals for bone formation, such as Mg and Na. The use of biowaste is cost-effective and environmentally friendly. Biowaste, such as eggshells, animal bones, and sea shells, has also shown considerable promise in this direction. The ability to make HA from fish bones has been established in recent years by simple calcination. This method produces HA with a structure and shape that is highly comparable to human bone. Furthermore, all microorganisms and organic components from the source are eliminated by heating it to a high temperature. Furthermore, HA derived from natural sources commonly contains ions such as Mg2+, Na+, Zn2+, and K+. These ions improve the effectiveness of natural HA by encouraging bone growth and regeneration [3].

3. Fabrication techniques for hydroxyapatite-based composite scaffolds

To create scaffolds with the desired properties, HA-based composites have been used in combination with other biomaterials, such as polymers or other inorganic materials. In addition to the “biological” advantages of using such biomaterials, these decrease the requirement for synthetics (materials derived from fossil fuels) in the biomedical business, improving ecological impacts all around.

According to the literature, a variety of techniques can be used to manufacture HA-based composites. Biomimetic mineralization, electrochemical deposition, lyophilization, electrospinning, self-assembling, and chemical vapor deposition are a few of these methods [4].

3.1 3D printing technologies for HA-based nanocomposites

With or without encapsulating cells, the 3D printing process involves the precise layering of biomaterials. The preparation phase, printing phase, and post-handling phase make up the majority of the entire process. In the preliminary stage, computer graphics tools, such as CAD/CAM and biomaterials, are used. In vitro transplantation, animal implantation, and tissue maturation in bioreactors all include the post-handling procedure. The most popular 3D printing techniques are inkjet printing, stereolithography (SLA), extrusion printing, and laser printing (Figure 1) [5].

Figure 1.

Most popular 3D printing techniques.

3.1.1 Inkjet-based 3D printing

Inkjet-based 3D printing with additional names such as drop-on-demand inkjet printing and continuous inkjet uses a nozzle driven by thermal or acoustic forces to eject liquid droplets onto the substrate. Different inkjet printers generate different droplets.

Two distinct methods of inkjet-based 3D printing exist continuous inkjet printing which creates an ongoing stream of liquid drops and drop-on-demand inkjet printing where individual drops are generated. Thermal and piezoelectric drop-on-demand inkjet printing technologies are employed in the drop-on-demand inkjet printing process to create pressure pulses and enhance droplet production and ejection. A small thin-film heater is used in the fluid chamber of the thermal drop-on-demand inkjet printing technique so that the fluid in direct contact with the heater can be heated more easily by applying a voltage gradient across the heater. Small vapor pockets or bubbles can form more easily when the fluid is heated continuously above its boiling point. Since there is no longer any heat transmission from the heater to the fluid, these bubbles quickly deflate when there is no voltage gradient. Using mechanical actuation, the piezoelectric drop-on-demand inkjet printing technique creates a pressure pulse. The liquid phase is used during the inkjet-based 3D printing process in both of the approaches [6].

Strobel et al. [7] generated the porous biphasic calcium phosphate (BCP) scaffolds via indirect 3D printing of a powder composed of homogenized 35 wt.% HA, 35 wt.% TCP, and 30 wt.% of a modified potato starch powder. Starch consolidation led to considerable porosity. Additionally, growth factor (BMP-2) and osteogenic cells (primary osteoblasts) were seeded and cultured for a few weeks in a flow bioreactor. Warnke et al. also printed BCP scaffolds by 3D printing, and the BCP scaffold was seeded with human osteoblasts [7].

3.1.2 Stereolithography (SLA)-based 3D printing

The “father of 3D printing,” Chuck Hull, invented SLA, which is typically used to create polymeric constructs. SLA, where a platform moves the scaffold after each new layer is built while a photoreactive resin is selectively cured. An ultraviolet (UV) laser beam is employed in the SLA-based 3D printing method to selectively cure the photopolymer resin. SLA-based 3D printing provides many benefits over inkjet-based printing, including fast speed, high resolution, and consistency. This technique employs a digital mirror array, usually, either UV light or near-UV blue light (405 nm) is employed.

Woesz et al., utilizing visible light, showed the use of printing systems. They fabricated microporous HA scaffolds using the SLA approach with visible light; the scaffold had a strut size of 450 m, with designed, fully interconnected macro-porosity. Although the SLA approach has been used for 3D printing, Le Guéhennec et al. claimed that the use of SLA for 3D printing of HA composites is constrained by several issues. For instance, the entrapment of unreacted monomers and residuals and the use of photo-initiators and radicals may compromise the integrity of the bone matrix synthesis in addition to elevating the risk of cytotoxicity. Despite these challenges, the incorporation of HA via SLA has the overall effect of promoting bone regeneration due to the increase in osteoblast activity on the HA surface [8, 9].

3.1.3 Extrusion-based 3D printing

The principle of extrusion-based 3D printing relies on extruding a viscous material using an extruder that is steered through a mechanical or electromagnetic actuator to create 3D objects According to Derakhshanfar et al., the extrusion-based 3D printing technique is characterized by different extrusion systems that can be cataloged as pneumatic pressure, piston, and screw-driven systems. Numerous benefits are available for extrusion-based 3D printing, including high cell seeding density, rapid printing, and scalability. The printed structures may then be cross-linked utilizing ionic, photo, and thermal crosslinking methods. This printing technology can also be utilized to manufacture continuous cylindrical filaments using various types of inks.

Direct ink writing (DIW, also known as robocasting) and fused deposition modeling (FDM), in which the raw material is expelled by a nozzle, are the two extrusion-based methods. The process of FDM is based on heating the material (polymer and polymer-ceramic composites) before squeezing it out of a nozzle, and by moving the nozzle, the material is deposited on a substrate, layer-by-layer.

The resulting printed constructs are subsequently heat treated to eliminate the binder and densify the ceramic. Sun et al. also utilized the DIW technique in applying silk fibroin ink, filled with HA nanoparticles, to print 3D scaffolds characterized by gradient pore spacings, ranging from 200 to 750 nm through the DIW technique [10, 11].

3.1.4 Laser-assisted 3D printing

The working principle relies on a pulsed laser beam for deposition of bio-ink, including cells, onto a substrate to fabricate 3D objects. The component of the printing system includes a pulsed laser source, a target coated with the substance to be printed (also known as ribbon), and a receiving substrate. Deckard and Beaman created printing in 1986 at the University of Texas in Austin in the United States. A powerful laser beam is focused onto the powder bed during SLS printing to selectively and continuously irradiate the surface of the powders, fusing them to produce the 3D construct. Xia et al. fabricated nano-HA/poly-caprolactone (PCL), using the SLS technology, such that the porosity (78.54–70.31%) and mechanical strength (1.38–3.17 MPa) of the printed scaffold could be regulated by variation of the printing parameters. The printed nano-HA/PCL scaffolds were more bioactive than the PCL scaffolds, according to the in vitro data. Compared to HA, BCP is usually challenging to fabricate as a porous scaffold by SLS printing because of the short sintering time. The sintering ability of BCP ceramics can be significantly improved via compositing with polymers [12].

3.2 Hydroxyapatite (HA) and HA-based nanocomposites via 3D printing

3.2.1 Hydroxyapatite

The hexagonal crystalline structure of hydroxyapatite (HA), also known as Ca₁₀(PO₄)₆(OH)₂, is what distinguishes it from other minerals and is what gives bone its mineralized components. Additionally, HA possesses good physicochemical qualities, such as osteoconductivity, bioactivity, resorbability, and delayed decomposition characteristics. Furthermore, nanometer-sized HA can increase intracellular absorption and lower cell survival in vitro.

Due to the lack of bonding and flowability for the printing process, considerable study on pure-HA printed materials has not been done, despite the fact that HA is widely considered for hard tissue regeneration due to its presence in the native extracellular matrix (ECM) of bone tissue. To print precise HA structures, various kinds of sacrificial materials and polymers are used as binders during the 3D printing process [13].

3.2.2 Hydroxyapatite (HA)/polymer-based nanocomposites

The printability of HA constructions can be improved by combining a polymer with HA nanoparticles. Due to the suitability and compatibility with cellular environments, various polymers could be used to fabricate (no matter how complex) constructs in ambient or relatively mild chemical and environmental conditions (Table 1) [14].

Hydroxyapatite-based nanocomposites

HA/collagen nanocomposites

Hydroxyapatite (HA)/gelatin nanocomposites

Hydroxyapatite (HA)/silk nanocomposites

Hydroxyapatite (HA)/alginate nanocomposites

Hydroxyapatite (HA)/cellulose nanocomposites

Hydroxyapatite (HA)/chitosan nanocomposites

Hydroxyapatite (HA)/poly (lactic acid)-based nanocomposites

Hydroxyapatite (HA)/poly-caprolactone nanocomposites

Hydroxyapatite (HA)/polymethyl methacrylate nanocomposites

Hydroxyapatite (HA)/polyvinyl alcohol nanocomposites

Hydroxyapatite (HA)/poly (propylene fumarate) nanocomposites

Table 1.

Hydroxyapatite-based nanocomposites.

3.2.2.1 HA/collagen nanocomposites

Collagen, fibrin, gelatin, silk, and other high-weight biomacromolecules found in nature can be employed as bio-ink network precursors. The amino acid sequences in natural polymers, such as collagen and gelatin, specifically the adhesion ligand arginine-glycine-aspartic acid (RGD), make them ideal for cell attachment. Collagen refers to a family of fibrillary proteins with a triple-helix structure of polyproline-II (PP-II) type. Significantly, collagen type I represents 90% of the collagen present in the human body, mainly in the skin, bones, tendons, and organs. The main structural element of the ECM is type I collagen, which is also frequently employed as a 3D hydrogel. Collagen/HA (collagen type I) was created by Lin et al. using a low-temperature robocasting technique. The 3D structure of the printed scaffolds was remarkable. In vivo data show that the printed scaffolds with interconnected pores might facilitate cell penetration and mineralization and further enhance bone regeneration after being implanted in a rabbit femoral condyle lesion model. A homogenous collagen/HA suspension was generated by Montalbano et al. using rod-like HA nanoparticles combined with type I collagen and an ammonium-based dispersion agent (Darvan 821-A). This suspension was used as bio-ink for extrusion 3D printing. The obtained collagen/HA bio-ink showed that the shear thinning and sol-gel transition upon stimulus-physiological conditions and the mesh-like constructs could be printed [15].

3.2.2.2 Hydroxyapatite (HA)/gelatin nanocomposites

Compared to collagen, gelatine presents no cytotoxicity, good cell adhesion, faster biodegradability, easier preparation, and low cost, thus it can be considered as a sufficient candidate for printing.

Animal tissues from diverse species, including those from pigs, cows, and fish, can be used to create gelatin polymers with a range of molecular weights and isoelectric points. The combined use of gelatine and loaded HA presents an ideal microenvironment for cell adhesion, proliferation, and differentiation toward an osteogenic phenotype, due to the presence of intrinsically cell adhesive motifs of gelatin. The combined use of gelatin and HA was demonstrated in the study by Samadikuchaksaraei et al. [15] where an HA/gelatin scaffold was fabricated using the layer solvent casting in combination with lamination techniques. The prepared HA/gelatin scaffold could support osteoblasts’ adhesion and growth, and in vivo results confirm that the scaffold could accelerate collagen content during bone healing. Nosrati et al. fabricated a HA/gelatin scaffold using a 3D printing method, with reduced graphene oxide (rGO) nanosheets used to reinforce the printed scaffold. The addition of rGO/HA could result in smaller pores and higher 3D accuracy of scaffolds. Notably, the gelatin may also be applied in drug release, due to its induction of degradation and deposition on the apatite layer [16, 17].

3.2.2.3 Hydroxyapatite (HA)/silk nanocomposites

Silk fibroin (SF), a protein fiber generated from Bombyx mori cocoons, has traditionally been employed as a natural polymer in the production of surgical sutures. Due to its unique structure, which consists of hydrophobic sheet crystalline blocks staggered by hydrophilic amorphous acidic spacers, SF possesses outstanding mechanical properties and good biocompatibility both in vitro and in vivo [18].

SF has also established a good reputation for bone TE applications due to its many unique properties, including impressive biocompatibility, strong mechanical behavior, minimal/non-immunogenicity, biodegradability, and ease of processability. Furthermore, the silk fibroin scaffolds showed improved anticoagulant activity. According to Lee JW et al. [19], HA/SF composites can encourage bone repair by activating signaling pathways linked to cell-biomaterial interactions. The obtained 3D printed scaffolds showed good porosity of 70% with interconnected pores with a diameter of ~400 m and relatively high compressive strength of over 6 MPa. While retaining cell adhesion and penetration, the printed scaffolds also showed well in vitro biomineralization activity in SBF.

3.2.2.4 Hydroxyapatite (HA)/alginate nanocomposites

The alginate structure is composed of a linear repetition of (1,4)-linked-D-mannuronic acid (M) and L-guluronic acid (G) units, with 4C1 ring conformation. Alginate has a strong affinity for di- and tri-valent cations and rapidly forms a gel in the presence of low concentrations of such ions (Mg2+ being an exception) at a range of pH values and temperatures. Alginate is also a polysaccharide that is negatively charged and works well as a scaffold for cell growth. Alginate can be modified by adding functional groups (such as heparin) that can bind to and immobilize various growth factors. These changes allow the growth factors to be micropatterned in three dimensions. Several studies have also indicated that HA/alginate nanocomposites are suitable for TE, with enhanced bioactivity [20].

Pre-crosslinking the HA/alginate nanocomposite with D-gluconic acid lactone improved the mechanical characteristics of the printed HA/alginate scaffold (GDL). To ensure continuous drug release, curcumin, an anti-inflammatory medicine, might be added to the printed scaffold during printing. According to in vitro experiments, the mouse bone mesenchymal stem cells (mBMSCs) may have adhered to the porous HA/alginate scaffolds. Curcumin, an anti-inflammatory medication, could be placed onto the printed scaffold during printing to achieve sustained drug release. The mouse bone mesenchymal stem cells (mBMSCs) may have attached to the porous HA/alginate scaffolds, according to in vitro studies [21].

3.2.2.5 Hydroxyapatite (HA)/cellulose nanocomposites

The most prevalent naturally occurring polymeric material in nature is cellulose, which is a well-known fact. Cellulose is used in TE because of its great biocompatibility, particular protein-binding locations, and remarkable mechanical strength. The high density of reactive hydroxyl groups on cellulose fiber can also aid in the immobilization of cell adhesion proteins like fibronectin on the surface of cellulose. Biocompatible cellulose fiber can be used to create a variety of scaffolds. Liu H et al. [22] fabricated HA/bacterial cellulose nanocomposites by inkjet 3D printing, which could be applied in bone engineering. Favi et al. [23] prepared HA/bacterial cellulose scaffold with well-defined honeycomb pore arrays using a laser patterning technique. The fabricated scaffold was shown to have a honeycomb pore array with a diameter of 300 m, which was suitable for bone TE applications. The nanocomposite scaffold can have good mechanical strength because of the addition of HA to cellulose. The nanocomposite scaffold can have good mechanical strength because of the addition of HA to cellulose. However, more research is required to determine whether HA/cellulose bio-ink can be printed in 3D.

3.2.2.6 Hydroxyapatite (HA)/chitosan nanocomposites

Chitin is converted into the polysaccharide complex chitosan, which has strong biocompatibility, degradability, and solubility in weak acids, and is nontoxic. Chitosan is a linear copolymer of -(1–4) linked 2-acetamido-2-deoxy-Dglucopyranose and 2-amino-2-deoxy-D-glucopyranose. Being a positively charged polysaccharide, chitosan still needs to be chemically altered and/or combined with other biomaterials to achieve the best mechanical and physiological qualities for TE. There are several reports on HA-reinforced chitosan scaffolds fabricated using different methods, including 3D printing. The high uniformity of the structure enhanced the mechanical strength of the printed HA/chitosan scaffold, thus improving its capacity to maintain its shape during the shrinkage phase of the dispensing medium.

Venkatesan J et al. [24] discovered that the mechanical property of HA/chitosan composites could be regulated by adjusting the weight ratio of HA/chitosan, such that the maximum value of the compressive strength attains 120 MPa at a mass ratio of HA/chitosan of 70/30. The printed HA/chitosan nanocomposites scaffold offers a wide range of possible applications in bone TE because of these great features.

3.2.2.7 Hydroxyapatite (HA)/poly-(lactic acid) based nanocomposites

Poly (lactic acid), sometimes known as PLA, is a nontoxic, biodegradable thermoplastic polymer created by the ring-open polymerization of lactide. Sugar feedstock fermentation is a source of PLA. Because of its linear aliphatic structure, PLA has appealing biodegradability, outstanding biocompatibility, and great mechanical qualities. For the above reasons, PLA was widely used as a matrix material in constructing biodegradable composites for bone repair, and bone fixation devices used in orthopedics and oral surgery applications. However, due to its unpredictable hydrolysis and weak hydrophilicity, PLA still has a narrow range of applications. However, these issues might be resolved by fusing PLA and bioactive ceramics like HA.

In the beginning, the HA/PLA composite was thought of as a viable biomaterial for bone replacement and repair. The distribution of HA nanoparticles may delay the rate at which PLA degrades, while increasing the distribution of HA nanoparticles may enhance mechanical properties. By using finite element modeling and simulation, the compression strength of printed structures could be changed. The authors claim that printed HA/PLA scaffold exhibited a greater rate of cell adhesion and proliferation than PLA scaffold based on in vitro studies. Using FDM, Domenech M et al. [25] created HA microspheres/PLA scaffold. HA/PLA scaffolds displayed increased porosity and a rougher surface compared to printed PLA scaffolds. Poly-L-lactic acid (PLLA) degrades more slowly than PLA, which is thought to lead to a slower inflammatory tissue reaction. Due to the bioresorbable characteristic, HA/PLLA has bone-bonding potential for bone regeneration [25].

3.2.2.8 Hydroxyapatite (HA)/poly-caprolactone nanocomposites

Poly-caprolactone (PCL) is commonly used as a synthetic biomaterial for bone tissue and periodontal applications due to its biocompatibility, suitability for various scaffold fabrication techniques, prolonged degradation rate, and mechanical stability. However, due to PCL’s slow rate of disintegration and prolonged durations of intactness, scaffolds may have a negative impact on bone repair. Because PCL and PCL-based scaffolds are easily printable and quickly solidify following extrusion, they can be produced using 3D printing. PCL and PCL-based scaffolds could be easily fabricated via 3D printing because of their good printability, and quick solidification after extrusion. Yang Hu et al. [26] fabricated HA/PCL scaffolds with hierarchical porous structures and tunable multifunctional performance via 3D printing. Peter SJ et al. [27] also fabricated HA/PCL scaffolds using the SLS technique. The printed scaffolds had porosity ranging from 78.54 to 70.31%, and the corresponding compressive strength ranged from 1.38 to 3.17 MPa. The in vivo results confirm that the printed HA/PCL scaffolds not only enhanced the formation of new bone but also orthopedic and reconstructive surgery. Xia et al. also fabricated HA/PCL scaffolds using the SLS technique. The printed scaffolds had porosity ranging from 78.54 to 70.31%, and the corresponding compressive strength ranged from 1.38 to 3.17 MPa. The in vivo outcomes demonstrate that the printed HA/PCL scaffolds not only improved new bone development but also satiated all the fundamental criteria for bone TE scaffolds, indicating a sizable potential for usage in orthopedic and reconstructive surgery.

3.2.2.9 Hydroxyapatite (HA)/polymethyl methacrylate nanocomposites

Polymethyl methacrylate (PMMA) is an FDA-approved synthetic polymer widely employed in ophthalmic, orthopedic, and dental applications. PMMA is also used as bone cement to fill defects of any shape or size, As a result, it can be used to treat osseous malignancies, trauma, illnesses, and birth defects in the skeletal structure. Lal B et al. [28] developed HA/PMMA using solvent casting particulate leaching technique; computational fluid dynamics (CFD) analysis concluded that HA/PMMA scaffold with 60 wt.% HA content tended to be the most potential option for bone TE applications due to the finest compromise between porosity, permeability, and compressive strength.

As filaments for 3D printing, Esmi et al. combined HA/PMMA with carbon nanotubes (CNTs). The modulus and hardness of HA/PMMA/CNTs were found to be greater than those of HA/PMMA using nano-indentation, and the generated nanocomposites accelerated cell adhesion, growth, and proliferation, according to the findings of a biocompatibility test [29].

3.2.2.10 Hydroxyapatite (HA)/polyvinyl alcohol nanocomposites

PVA, a thermoplastic that dissolves in water, is frequently utilized as a support material in 3D printing. Due to its excellent biocompatibility, high water solubility, and chemical resistance, it is often used in medical equipment. PVA is mostly used in cartilage TE because it has a tensile strength that is comparable to that of human articular cartilage. Notably, compositing PVA with calcium phosphate nanoparticles, such as HA, TCP, and BCP, showed promising applications for the fabrication of scaffolds in bone TE. The osteoconductive HA/PVA scaffold could be created for bone replacement, according to several research findings. For instance, Nie L et al. [30] fabricated HA/PVA scaffolds by powder-based 3D printing. The results show that the printed scaffold with 1.0 wt.% of PVA showed the best compressive strength. The HA/PVA scaffolds’ performances were superior and considerably more appropriate as bone scaffolds than those of the HA/polyvinylpyrrolidone (PVP) and HA/polyacrylamide (PAM) scaffolds made using the same method. In addition, the printed HA/PVA produced had good cytocompatibility.

3.2.2.11 Hydroxyapatite (HA)/poly (propylene fumarate) nanocomposites

Unsaturated linear polyester poly (propylene fumarate, or PPF), which has carbon double bonds throughout its backbone, can be crosslinked. PLA, PCL, and PPF are regarded as bioresorbable polymers that can break down in vivo either through hydrolysis or through enzymatic cleavage. PPF may be degraded into nontoxic products of propylene glycol, poly (acrylic acid-coumaric acid), and fumaric acid. Numerous medical applications, including vascular stents, cartilage, blood vessel engineering, bone TE, have made significant use of PRF. This may be printed into a variety of 3D shapes using extrusion-based printing and SLA3D printing. Besides linear PPF oligomers, Fer et al. [31] developed PPF bio-ink for continuous DLP, with the printing speed improved. PPF is one of the promising candidate materials for load-bearing applications due to its suitable mechanical properties. But PPF still needs to have its biomechanical and osteoconductive qualities improved, perhaps by adding ceramic components. Lee et al. [32] asserted that the osteoconductive ability of HA/PPF nanocomposites was increased compared with pure PPF. Lee et al. fabricated HA/PPF scaffolds with micro-SLA (MSTL) technology. During the preparation of the HA/PPF bio-ink, diethyl fumarate (DEF) was incorporated to reduce the viscosity, and the photo-initiator bis-acyl phosphine oxide (BAPO) and 7 wt.% of HA were designed. Interconnected pores could be seen in the printed scaffolds. Additionally, in vitro research indicates that printed HA/PPF scaffolds promoted MC3T3-E1 cells’ proliferation and cell adhesion more effectively than PPF scaffolds did.

3.3 Hydroxyapatite (HA)-based ceramics

3.3.1 Hydroxyapatite (HA)/beta-tricalcium phosphate (BCP)-based ceramics

Beta-tricalcium phosphate is of low mechanical strength and degrades too quickly in a physiological environment which can be improved via its combination with HA. For 30 years, BCP has been utilized to create bone graft materials; BCP-based ceramics have demonstrated clinical success. Asran AS [33] fabricated porous BCP ceramics using extrusion-based 3D printing with a motor-assisted micro-syringe (MAM) system; the morphology, pore size, and porosity of printed BCP scaffolds could be precisely controlled to optimize their mechanical properties.

3.3.2 Hydroxyapatite (HA)/bioglass-based ceramics

The field of bioactive inorganic materials, which may bind with bone tissues, was introduced by the discovery of bioglass. Due to its osteoconductivity and osteo-productivity, bioglass has demonstrated significant potential in bone regeneration. According to numerous investigations, the remarkable bioactivity of HA/bioglass composites makes them suitable for use in bone regeneration. However, bioglass scaffolds may deteriorate before the formation of new bone due to the rapid rate of bioglass dissolution in bodily fluids. Ferraz MP et al. [34] indicated that HA/bioglass could stimulate early osteogenesis and osteointegration at the interface in the biological environment. For instance, Zebarjad SM et al. [35] fabricated the calcium sulfate hydrate (CSH)/mesoporous bioactive glass (MBG) scaffolds using the inkjet 3D printing approach (4th 3D Bioplotter™, EnvisionTEC GmbH, Germany). The printed scaffolds were well used for apatite mineralization because of their regular and consistent structure. Seyedmajidi et al. additionally acquired HA/bioactive glass for use as cell scaffolds in the restoration of the rat tibia. The radiographic, histological, and histomorphometric evaluations showed that the trabecular thickness and rate of new bone formation were elevated.

3.3.3 HA-based composites of titanium ceramics

Besides calcium phosphate ceramics, titanium and its alloys, such as titanium dioxide (TiO₂) and titanium alloy (Ti-6Al-4 V, Ti64), can be used to fabricate scaffolds for TE. TiO₂ is also capable of enhancing the growth of bone and vascular tissues and osteoconductivity. For instance, Kim et al. created HA/TiO₂ nanocomposites utilizing HA-doped TiO₂ particles and concluded that these materials had greater strength and bioactivity than TiO₂ compounds. Additionally, Ti64, known for its excellent strength-to-weight ratio, is an alloy that can be employed in fabricating porous scaffolds. SLM, or selective laser melting, can be used to create such porous Ti64 scaffolds.

However, since Ti64 lacks some functionality, such as blood compatibility and bone conductivity, the surface of Ti64 may be coated using HA to improve its physicochemical properties as demonstrated in the literature. For instance, Cai X et al. [36] used a nanorod-structured HA as a coating on the surface of Ti64 via atmospheric plasma spraying in combination with hydrothermal treatment and subsequently demonstrated how the created nano-structured surface will improve osseointegration and cell responses.

4. Different types of hydroxyapatite polymer composite for bone tissue engineering

4.1 Hydroxyapatite as tissue scaffolding material

Hydroxyapatite (HA) (Ca10(PO4)6(OH)2) is a biocompatible ceramic material that belongs to the calcium phosphate cement family (CPCs). Natural bones are mainly made up of HA, collagen, and water. Biological apatite, the main mineral form present in mammals is a carbonate-rich, hydroxyl-deficient apatite with a Ca/P ratio of less than 1.67. Synthetic HA has a comparable composition and characteristics to natural HA found in bones and teeth. Synthetic HA is also a biocompatible, bioactive, nontoxic, and osteoinductive substance. In two alternative scaffold material combinations, HA can be utilized. Hydroxyapatite has significant remodeling and cell adhesive qualities when combined with natural materials, but it can also trigger an immunological response. Gelatin, fibrinogen, and collagen are examples of natural materials (Figure 2). Synthetic materials, on the other hand, are less likely to elicit an immune response, although they can still be hazardous [37].

Figure 2.

Natural and synthetic materials used in combination with hydroxyapatite.

4.2 Hydroxyapatite in combination with natural materials

4.2.1 Collagen

Collagen is a natural substance that is biodegradable, porous, and biocompatible. It is a natural component of many soft and hard tissues and provides raw protein for the ECM. They are made up of 28 different proteins that fall into six different categories: fibrillar, fibril-associated collagens with interrupted triple helices (FACIT), beaded filament, basement membrane, short-chain, and transmembrane collagens. Because type I collagen is plentiful in the bones, it is the molecule of interest when it comes to bone regeneration. Collagen, on the other hand, lacks stiffness, making its usage in load-bearing regions challenging [37].

The idea of creating collagen (Col)-HA biocomposites is supported since they make up the microarchitecture of the bones. Collagen has long been known for its high biocompatibility, low degradation, and positive interactions with cells and other organic macromolecules in our bodies. To stimulate osteogenic differentiation—HA biocomposites combine collagen’s cell migration and binding capabilities with HA’s inherent bioactivity. Collagen improves the mechanical strength of porous HA scaffolds by reducing porosity. The increased mechanical strength is due to the creation of intermolecular H-bonds between collagen and HA, which raises fracture energy and hence improves failure resistance. Because of all these factors, tissue engineering can also be done with collagen-HA composites. When implanted in the rat tibia, these scaffolds exhibited osteoconductivity and begin to disintegrate after 12 weeks. In animal tests, these collagen-HA composite materials have also been employed to cover titanium implants with encouraging results. Collagen-HA scaffolds containing recombinant growth factors and antibiotics are also being developed which helps to improve regeneration [37]. Hoyer et al. created resorbable collagen/hydroxyapatite porous scaffolds with appropriate connectivity and mechanical characteristics (2012) [38]. Sotome et al. further verified the advantages of porous HAp-collagen composite for bone regeneration. Collagen-Hap was also successfully used for cheekbone augmentation, resulting in significant ossification with little inflammation and limiting bone development over time.

4.2.2 Gelatin

Gelatin is a natural polymer made from collagen that has been denatured. It’s been employed in adipose tissue, blood vessels, bone, intervertebral disc, muscle, tendon, heart, liver, and skin tissue engineering. Bone tissue engineering can also be facilitated by gelatin/HA scaffolds. In terms of osteogenic development of periodontal ligament cells, these scaffolds have demonstrated encouraging outcomes. Due to the immunogenic reaction induced by collagen by-products, an alternative organic matrix component source was necessary as a backup. In this case, the organic component in the composite was preferable to gelatin. The role of gelatin as a matrix was investigated after it was degraded in lysozyme under physiological circumstances. In thermal solution, gelatin is a linear, amorphous, hydrophilic, and moldable biomaterial and also biocompatible, and has a slower rate of degradation, and the by-products are not immunogenic. The addition of gum to the HA/gelatin composite can improve the mechanical characteristics of the scaffold. The scaffolds are seeded with Mesenchymal Stem Cells (MSCs) extracted from Wharton jelly that protects the umbilical cord. According to White et al., the preparation of gelatin-HAp composite can be done through the casting method as it is a simple and economical approach in the preparation of porous scaffold materials [39].

Because of their biodegradability and biocompatibility, gelatin and hydroxyapatite films in various ratios appear to have promise in bone tissue engineering, according to the findings of multiple research. However, in order to accurately assess the mechanical characteristics of this composite, additional detailed experiments were necessary.

4.2.3 Chitosan

Chitosan (CS) is a D-glucosamine/N-acetyl-D-glucosamine copolymer, which is a versatile natural biopolymer made by partial deacetylation of chitin under alkaline environments. Chitin and its derivatives have received a lot of interest as a scaffold material in bone tissue engineering. Pure chitosan scaffolds, on the other hand, have poor mechanical characteristics and lack osteoconductivity. HA can be employed to improve the ability of CS to differentiate into osteoblasts [40]. By including HA in the CS scaffolds, the bioactivity and biocompatibility of the CS can be improved. CS/HA systems have also been employed as carriers to transport drugs, stem cells, and other growth factors into hosts. The very first attempt to create a CS/HA biocomposite material was the creation of a bone-filling cement made of powdered HA/ZnO/CaO combined in a chitosan sol. The final paste had a quick setting time and a high compressive strength. The HA nanoparticles are evenly distributed throughout the CS matrix, and the HA crystallites increase during nucleation due to interactions generated between calcium ions and CS amino acids [40]. The aggregation of HA nanoparticles is a disadvantage of this type of material; however, Hu et al. devised a simple in situ hybridization approach to make HA/CS nanocomposites that can overcome this issue. Furthermore, the biocompatibility of the HA/CS scaffold is quite excellent.

The HA/CS composite improved cell adherence, spreading, and proliferation of human mesenchymal stem cells as compared to a pure chitosan scaffold. HA/CS biocomposites have been shown to stimulate bone growth in a variety of bone defects by inducing osteoinduction and osseointegration. To repair the femoral condyle defect in 43 adults New Zealand white rabbits, HA/CS and pure chitosan were implanted into the left femoral condyle. The results demonstrated that after 12 weeks of surgery, rabbits implanted with the HA/CS scaffold had completely healed their bone abnormalities, whereas the defects remained in the pure chitosan group. The HA/CS composite can also be employed as a functional layer on other implants to create outstanding osteoinduction characteristics. Wang et al., for example, coated HA/CS on a titanium surface(cTi) and employed it in diabetic patients. After 12 weeks, the cTi implant had greater bone contact and a higher volume of new bone formed into it than the Ti implants [41].

4.2.4 Alginate

Alginate (Alg) is a marine-sourced biopolymer produced from brown algae, similar to chitosan. Alginates, which produce partially soluble hydrogels in water after partial interaction with divalent cations, have a variety of uses in bone regeneration, wound healing, and drug administration. According to Becker et al., who also researched their mechanical characteristics, alginates are biodegradable and biocompatible materials [42]. This study discovered a link between biocompatibility and alginates purity, with pure versions causing less unfavorable reactions in tissues than their less purified equivalents. In comparison with hydrogels with high mannuronic acid concentration, hydrogels with high glucuronic acid content had improved tensile strength and ductility qualities.

Ceramics, such as HA, can be used to strengthen the mechanical qualities of alginate. Some studies also discovered that a slight increase in Sodium Alginate (SA) content resulted in the production of biocomposites with enhanced density and hardness due to decreased porosity and the establishment of strong linkages within the SA, allowing SA/HA composites to outperform pure SA scaffolds mechanically. In vitro and in vivo tests were performed on biocomposites including carbonated nano-HA with strontium and sodium alginate (SrCHA) spheres and without strontium (CHA). Vancomycin-loaded Alg/Sr-HA microspheres had improved sustained drug release capabilities. Although the mechanical characteristics of Alg/HA composites are sufficient to fill critical-sized defects (tissue defects which are the smallest one and that do not heal on their own over the course of an animal’s lifespan), their usefulness for treating defects in long bones requires additional investigation.

4.2.5 Hyaluronic acid

Hyaluronic acid (HylA) is a hydrophilic linear unbranched glycosaminoglycan composed of repeated N-acetyl glucosamine and glucuronic acid disaccharide units. This was first obtained from the vitreous humor of cows. Because of its elasticity, biocompatibility, antibacterial, and osteoinductive qualities, HylA got a variety of biological uses, including bone regeneration. HylA is engaged in cell signaling pathways and contributes to cell proliferation and differentiation. Bakos et al. investigated the effect of HylA on HA. HylA-conjugated HA/Col composites had a more compact structure and higher bend strength than non-conjugated HA/Col composites, indicating that HylA has a positive impact on the mechanical properties of HA/Col scaffolds [43]. Hyaluronic acid (HylA) is a hydrophilic linear carbohydrate that is found in the body. When the ability of biocomposite scaffolds made of calcium sulfate/HA/HylA and encapsulated with collagenase to repair alveolar bone defects in rats was investigated, it was shown that these scaffolds displayed excellent biocompatibility, compressive strength, and a sustained release of the collagenase enzyme for up to 4 days. Histological analysis of the rat models revealed that defects filled with calcium sulfate HA/HylA-collagenase scaffolds showed significant and uniform regeneration of the alveolar bones 8 weeks post-implantation verified by the increased number of osteocytes observed on the defect site. However, in in vivo circumstances, fast enzymatic degradation of HylA must be considered, as this might deplete the mechanical characteristics of HylA-based composites [44].

4.2.6 Silk

Silk may be utilized as a scaffold for MSCs since it is biocompatible and strong. To make the silk/HA scaffold, NaCl can be employed as a porogen. Scaffolds with different HA percentages are made and employed with MSCs cells. According to Bhumiratana et al., since HA stimulates mineralization and trabeculae development in its scaffold, silk/HA scaffolds can play an important role in osteogenic cell differentiation [45].

4.3 Hydroxyapatite in combination with synthetic materials

Natural materials are undeniably accessible and biocompatible, but synthetic materials, under their regulated nature, have many distinct features. Polylactic acid (PLA), poly (lactic-co-glycolic acid) (PLGA), b-tricalcium phosphate (b-TCP), and polycaprolactone (PCL) are examples of synthetic materials. It’s also possible to employ HA in conjunction with synthetic materials (Figure 2).

4.3.1 Polycaprolactone (PCL)

PCL is a biocompatible, synthetic polymer with easily adjustable mechanical characteristics. PCL (in its high molecular weight form) is recognized for its typical slow breakdown rate of up to 3 years to entirely eradicate itself from the host body, despite the fact that it is biodegradable. The volume of HA in the composite affects the mechanical characteristics of PCL/HA scaffolds. Researchers used laser sintering to create nano-HA (nHA)/PCL scaffolds. Increased nHA concentration leads to osteoblast development and mineralization, based on Alizarin Red and ALP staining. The greatest amount of nHA-containing scaffolds exhibits a slow-release profile of recombinant bone morphogenic protein; as a result, HA/PCL scaffolds are crucial in bone tissue regeneration. The utilization of a biphasic calcium phosphate (BCP) scaffold covered with nHA and PCL for the differentiation of primary human osteoblasts (HOBs) and ASCs cells has been described. The addition of HA to porous PCL has an effect on the latter’s behavior under in vitro circumstances. Shor et al. in an 11–21 days observational study revealed that porous PCL/HA scaffolds cause an increase in cell survival and proliferation of fetal bovine osteoblasts over conventional PCL scaffolds. Throughout the observation period, cell differentiation as evaluated by alkaline phosphatase activity revealed that PCL-HA scaffolds clearly outperformed conventional PCL scaffolds in terms of alkaline phosphatase activity. In comparison with the negative controls, PCL-HA matrices implanted subcutaneously in white rats demonstrated the development of connective tissues after 7 days and vascularization after 14 days, provoking a very low immune response. After 21 days, the matrices commenced to biodegrade, demonstrating their effectiveness and cytocompatibility in in vivo animals. PCL, on the other hand, is very hydrophobic, has minimal antibacterial action, and is known to degrade more slowly than poly (L-lactic acid) (PLLA) and poly(glycolic) acid (PGA) [46].

4.3.2 Polyhydroxybutyrate (PHB)

Poly 3-hydroxybutyrate (P3HB) is a crystalline polyester that belongs to the polyhydroxyalkanoates family of polyhydroxyalkanoates produced by bacteria through enzymatic synthesis. Because brittleness is a key disadvantage of P3HB, it is frequently copolymerized with polyhydroxy valerate (PHV) to improve processability. PHB and gelatin can be coupled with nHA to create a bone scaffold that is both osteoconductive and osteoinductive. The nanofibrous scaffold can be employed in conjunction with MSCs to improve bone regeneration potential. In comparison with P3HB control scaffolds, P3HB/nano-HA scaffolds enhance cell proliferation and differentiation of osteoblasts better. Both scaffolds boosted cell survival and proliferation over time, however, the P3HB/nano-HA scaffold had the best outcomes. When tested subcutaneously on rat models, PHB/HA porous scaffolds filled with bone marrow cells showed promise in in vivo environments. Forty-five days after implantation, the implants were covered with a thin connective tissue layer. In the pores of the scaffolds, a healthy connective tissue in-growth comprised of matured osteoblasts, macrophages, and capillary was seen, indicating the site of the active bone formation near the implant site, indicating their capacity to support bone regeneration. However, because both P3HB and HA are brittle materials, the mechanical strength of the P3HB/HA biocomposites is a problem. As a result, because of concerns about their long-term mechanical stability, the composite may not be the best option for implant [47].

4.3.3 Polyvinyl alcohol (PVA)

Tissue engineering can be done with polyvinyl alcohol (PVA)/BCP scaffold. Nie et al. created PVA/BCP scaffolds and seeded BMSC cells on them. The biocompatibility of this scaffold was good. The porosity and mechanical characteristics of the scaffolds are similar to those of bone. It improves MSC adherence, making PVA/BCP scaffolds useful in bone tissue engineering [48].

4.3.4 Poly (lactic acid)

Poly (lactic acid) (PLA) is formed when lactic acid undergoes a polyesterification reaction. It exhibits four different forms: poly (L-lactic acid) (PLLA), poly (D-lactic acid) (PDLA), poly (D,L-lactic acid) (PDLLA), and meso-poly(lactic acid). PLA has high tensile strength and Young’s modulus in general. These properties, however, range greatly among the various types of PLA, which defines their applicability. PLA/HA biocomposites are employed as scaffolds as well as carriers for delivering medicines and other proteins into the body. The mechanical properties of PLA/HA biocomposites are influenced by the proportion of HA in the composite and the temperature at which it is produced. Calcined PLA/HA composites with around 80% HA had Young’s modulus of 10 GPa, which was comparable to the lower limit of a cortical bone, as well as comparable flexural strength and fracture toughness. Increased cell numbers, cell adhesion, expression of bone-specific markers (osteocalcin), and promotion of osteoblast differentiation by alkaline phosphatase activity demonstrate that PLA/HA biocomposites are biocompatible with a wide range of cell lines, including MG-63 osteosarcoma cells, L929 fibroblastic cells, and MC3T3-E1 osteoblastic precursors. In rabbits with bone defects, PLA/nano-HA/collagen scaffolds seeded with rh-BMP2 (recombinant human bone morphogenic protein) exhibited promising signals of bone remodeling. After 8 weeks, the implant showed cellular infiltration into its pores, and after 12 weeks, it had fully integrated into the defect site, demonstrating the formation of new bony (trabecular) tissues and the replacement of the composite, showing the composite’s biodegradability and bone regeneration capacity [49].

4.3.5 Poly (glycolic acid)

The semi-crystalline polymer poly (glycolic acid) (PGA) has a high tensile modulus of 12.5 GPa and is insoluble in most organic solvents. PLA and PGA were the first synthetic biodegradable polymers to be approved for use in the fabrication of resorbable sutures by the US Food and Drug Administration. PGA has better mechanical qualities than PLA and degrades faster. In a 1986 study, PGA implants were found to be biocompatible in rabbits with cortical and cancellous bone abnormalities. The implants in the cancellous locations showed the greatest degradation after 12 weeks of implantation, compared to partial degradation in the cortical bones that did not result in an inflammatory response. In vitro, PGA/HA composite scaffolds showed excellent resorption and the appropriate porosity for cellular penetration and adhesion. However, due to the stiffness of PGA and the risk of inflammation caused by its breakdown by-products, their biological applications have been limited [50].

4.3.6 Poly (lactide-co-glycolide)

The biocompatibility and mechanical characteristics of PLGA are well known. PLGA is seen as a solution to both PLA and PGA’s drawbacks. Researchers were able to solve the problem of premature degradation by copolymerizing the two and altering the homopolymer ratio, which gave them some control over their degradation rates. PLGA having 75% PGA is known to be amorphous and hydrolytically unstable, causing it to degrade more quickly.

PLGA systems are used in areas such as bone, cartilage, and nerve regeneration, in addition to drug delivery. The mechanical characteristics of PLGA are strengthened by HA. Fisher et al. discovered that adding 30 percent nano-HA to PLGA matrices resulted in composites with three times the strength of the polymer alone, as well as a sixfold increase in compressive modulus. The scaffolds were designed to be injectable, and when injected into swine femoral heads, they increased the trabecular bone strength and compressive modulus from 3.5 to 5.9 MPa and 81 to 180 MPa, respectively [51]. In comparison with PLGA control scaffolds, PLGA/nano-HA scaffolds cultivated with mesenchymal stem cells demonstrated larger cell counts, improved cell adherence, increased cell proliferation, and better alkaline phosphatase activity. Twenty-one days after implantation, PLGA/HA scaffolds implanted into rabbit mandibular defects showed the presence of bone trabeculae without any ongoing osteoblast activity, whereas PLGA control scaffolds had a high number of osteoblasts on their surface, indicating increased cellular activity to generate more bone trabeculae. Also, at the end of 6 weeks, the PLGA/HA scaffolds tested positive for the bone markers osteonectin and osteopontin, indicating increasing bone deposition in vivo [52].

5. Application in tissue engineering

Tissue engineering with hydroxyapatite (HA) holds a lot of promise. Furthermore, according to current research, 3D printed HA-based nanocomposites serve as 3D templates for cells to attach, proliferate, and maintain their differentiated function in tissue regeneration. Bone, cartilage, applications in dentistry, skin, and drug delivery are just a few of the possibilities for HA-based structures. These applications are briefly covered below (Figure 3).

Figure 3.

Applications in tissue engineering.

5.1 Bone

Due to the chemical resemblance of calcium phosphates to bone minerals, notably HA (Ca10(PO4)6(OH)2), bioceramic materials are used in bone replacement applications. In comparison with autografts and allografts, a complex hierarchical artificially engineered bone scaffold with a complex structure can reduce the risk of infection, immune response, and transmission of the disease. The optimally designed bond scaffold should meet a number of requirements, including biocompatibility, osteoconduction, osteoinduction, and mechanical properties. In conjunction with natural and synthetic polymers, pure HA or other ceramics such as tetra calcium phosphate can be utilized to build scaffolds for hard tissue regeneration. Polymers, by their very nature, are flexible and lack stiffness. When inorganic materials and polymers are mixed, composite materials display both inorganic features like mechanical strength and flexibility, as well as polymer properties such as porosity and osteoconductivity. PLGA/HA is a biocompatible and osteoconductive composite scaffold that promotes uniform cell seeding, cell development, and tissue creation. When coupled with collagen, PLLA, CS, and gelatin, HA and Tri calcium Phosphate (TCP), the inorganic component of bone, are employed as scaffolding [53].

A 3D gel-printing method can be used to create an interconnected porous pure HA scaffold. According to Shao et al., the inclusion of HA-based nanocomposites constructions in bone TE can be accomplished by combining 3D printing and microwave sintering to create a hierarchical porous HA scaffold with micropores and macropores. Song et al. revealed that these hierarchical HA scaffolds can be easily incorporated with native bone [54]. Bone regeneration is being attempted in all parts of the body, and the important ones are discussed below:

5.1.1 Maxilla

Trauma, defects, and cosmetic issues in the maxillofacial region necessitate bone tissue engineering. For bone regeneration, having an adequate scaffold is critical. Osteoinductive growth factors, such as BMP-2 and BMP-7, which are present in extracts of demineralized freeze-dried bone, can also be used to induce osteogenic cartilage periodontal attachment bone cementum hydroxyapatite in tissue engineering. After a trauma or tumor removal, HA and associated materials are utilized to repair a damaged maxilla. To fill the deficiencies, granular versions of HA and TCP are utilized, which shows improvement in bone regeneration in that area. Polymer scaffolds loaded with HA and TCP are also employed to promote tissue regeneration in the craniofacial region [55].

5.1.2 Mandible

Hydroxyapatite (HA) can also be utilized to regenerate mandibular bone. A lesion in the rabbit mandibular bone was created in an in vivo investigation and filled with porous Poly Methyl Methacrylate (PMMA) with or without HA. A new bone formed 12 weeks after implantation with no other surrounding tissue infection. The findings indicated that injectable porous PMMA-HA could be a reasonable solution for craniofacial bone regeneration. Nanocrystalline HA (HA-SiO) was employed to regenerate a mandibular bone defect in another animal investigation. The study found that HA accelerated the production of new bone tissue and had a high osteogenic potential. In clinical research, HA has also been utilized to treat a mandibular bone deformity. The magnesium-enriched HA was employed as a substitute in a mandibular bone defect caused by ameloblastoma excision in the study, and it was found to be effective in bone regeneration [56].

5.1.3 Alveolar bone

Periodontitis can cause damage to the alveolar bone that supports the teeth. The defective bone could be treated with tissue engineering or regenerative medicine. By providing a proper structural foundation, materials for a bone graft can induce bone healing. Rats had their alveolar bone defects repaired using a composite material made of HA. The new alveolar bone regenerated with good mechanical strength, biocompatibility, and a faster bone remodeling process, according to the researchers [57].

5.1.4 Calvarium

For the restoration of calvarial bone deformities, various materials have been proposed. In the seventeenth century, a gold plate was used to repair a calvarial defect for the first time. Other materials used in the previous century included tantalum, silver, titanium, and stainless steel. HA has been proposed as the most suitable biomaterial for the construction of biocomposite scaffolds for bone regeneration because of its superior osteoconductive and osteoinductive capabilities [58].

5.2 Periodontal tissue regeneration

Restoring periodontal tissues to their former shape and function is a difficult task. In a canine model with a labial alveolar bone defect, Liu et al. investigated the periodontal regeneration potential of collagen-HA scaffolds combined with bone marrow mesenchymal stem cells. The authors demonstrated that collagen-HA scaffolds supported periodontal tissue regeneration with no abnormal events occurring throughout the regeneration process, implying that collagen-HA scaffolds provide a biocompatible environment for periodontal regeneration. In another work, the nanoparticle HA was employed to promote the proliferation and differentiation of primary periodontal ligament cells (PDLC). When compared to the control group, HA brings extensive PDLC proliferation and alkaline phosphatase activity, suggesting that HA could be employed as a bioresorbable agent in osseous reconstruction [59].

5.3 Temporomandibular joint

The TMJ is a joint that connects the temporal bone to the mandibular condyle and is commonly damaged. TMJ traumatic abnormalities are divided into three categories: “fracture, mandibular dislocation, and subluxation,” which cause pain during necessary oral functions. Because there are few therapies for severe TMJ illnesses, there has been a surge in interest in regenerative techniques. TMJ regeneration has been studied by Mehrotra et al. Their research found that HA-enhanced collagen scaffolds facilitated the regeneration of entire TMJ condyles in adolescents and children with TMJ ankyloses. For the replacement of the mandibular condyle, a customized 3D polyamide implant-coated nano-HA can be planned and produced. The use of such materials in a patient has been shown to have favorable clinical consequences [60].

5.4 Cartilage

Proteoglycans, glycosaminoglycans, collagen fibers, and elastin make up the matrix of cartilage. Articular cartilage injuries are prevalent, and traumatic cartilage has limited healing and regeneration capability. Unfortunately, due to its limited ability to heal, articular cartilage lesions or injuries are difficult to heal, and artificial cartilage is necessary in the clinic. Articular cartilage grafting has been established in the literature to be a promising therapy option for these injuries [61]. The researchers discovered that HA stimulates chondrocytes to secrete the calcified cartilage matrix both in vitro and in vivo [62]. For cartilage repair, 3D printing may be utilized to build constructs with high structural complexity and flexibility, such as hydrogels, which offer the benefit of personalized precise customization, allowing the construct to match perfectly with the faulty surface. It has been established that HA-based nanocomposite structures can be used in cartilage TE.

5.5 Dentin

Dentin regeneration demands a proper scaffold system and an inductive microenvironment. In orthopedics and dentistry, HA was the most commonly used substance. HA is utilized not just in dental fillings and cement, but also in a variety of toothpaste that works as a polisher to reduce biofilm build-up on teeth. Dentin remineralization can be stimulated by advancements in dental materials that lead to the production of nHA particles [63]. nHA is a great source of free calcium and is an important factor in the prevention of dental caries and erosion. As a result, nHA is regarded to be a promising hard tissue engineering candidate [61]. The effect of HA particles of various sizes on the proliferation of pre-odontoblast cells (MDPC-23) has also been studied. The researchers also discovered that the size of HA particles is inversely related to cell proliferation. According to these findings, nHA could be a beneficial substitution for odontoblast cell proliferation [63].

5.6 Cementum

Cementum is a mineralized, avascular tissue that covers the surface of the root and creates the interface between the dentin and the periodontal ligament. Varying cementum has different proportionate compositions of chemical components. Cementum has a biological component that is identical to the bone, according to previous research. In the cementum, HA is a key inorganic component that is abundant (approximately 50%). The predominant components of the remaining organic matrix are proteoglycans, glycoproteins, and collagens [62, 64]. Periodontal infections or accidental damage can cause periodontal tissue degeneration, which can lead to teeth loosening and affect oral function. Cementum regeneration has been accomplished with HA based on a few studies. Mao et al. investigated the effect of HA bioceramics with a micro-nano-hybrid surface (mnHA) on human periodontal ligament stem cell adhesion, growth, and cementoblast differentiation. The findings showed that mnHA bioceramics stimulated cell proliferation, cell adhesion, alkaline phosphatase activity, and the expression of cementoblast differentiation markers such as cementum attachment protein and cementum protein [64].

5.7 3D printing of HA nanocomposites for dental applications

3D printing for dental applications has developed significantly in recent years, particularly in the fields of oral and maxillofacial surgery, endodontics, orthodontics, prosthodontics, and periodontics. The potential for personalized dental solutions promotes the use of 3D printing in this field. Dental prostheses and crowns are frequently made of metal, ceramic, and polymer-based materials. 3D printing is indeed being used to restore lost teeth. To assure a denser and more compact construction, the mechanical qualities of prosthodontic constructs must be addressed, and porosity difficulties must be avoided. Ink-jet printing, rather than SLS or SLA printing, can produce a denser and more compact structure. Controlling infection is said to be critical to the efficacy of apical surgery of root canal-treated teeth. However, because HA lacks bactericidal characteristics, antibacterial HA-based nanocomposites can prevent the growth of microorganisms in the root canal [65].

5.8 Drug delivery applications

For more precise and long-lasting drug release, HA has been combined with biopolymer (e.g., alginate) matrices. Venkata Subbu et al. [66], for example, placed the antibiotic ciprofloxacin onto a nano-HA composite using alginate. The medication was pre-adsorbed onto the ceramic particle before the composite was formed in their investigation. They discovered that integrating HA-based nanocomposites into ciprofloxacin-loaded HA increased the duration of ciprofloxacin release when compared to ciprofloxacin-loaded HA alone. In conclusion, the importance of HA-composites cannot be underestimated. Furthermore, recent studies from 2021 have shown that they can be used in human investigations. Kim and Kim, for example, used 3D strontium-substituted HA (Sr-HA) ceramic scaffolds in human cells to stimulate fast cell proliferation, osteogenic differentiation, and cellular mineralization. They used Sr-HA scaffolds as new bone graft alternatives in people and confirmed their effectiveness. Based on the reported success, it is expected that future research will focus on the use of HA-composites in people. The next section discusses what the future holds for HA composite applications in TE [66].

5.9 Future horizons

The use of nanotechnology in the manufacturing of HA is very beneficial. Since natural raw materials are used, HA production will be cost-effective in the near future. Cell culture, drug delivery, antibody purification, catheters, and engineered artificial organs constructed of HA composites are all interesting advancements for HA. Given the utility of HA-based nanocomposites in drug delivery, the scientists speculate that future applications for HA-based nanocomposites in the delivery of gene modifiers and targeted nutrition may be possible. Clearly, in the future, such technologies will allow for advanced and targeted cell and tissue transformations. In addition, various features of HA-based composites, such as printability, appropriate mechanical strength, biodegradation, and biocompatibility, will make it easier to use 3D printing to build on-demand, highly individualized complicated designs at low costs in the future. Recognizing that vascularization is critical for tissue regeneration and that functional vascularization of the biological scaffold is challenging to achieve with current 3D printing technologies, extrusion-based printing can be employed to provide the required structural integrity of the final result. The development of HA-based nanocomposites employing 3D printing in the future will address the vascularization issues stated above. However, a better understanding of the complex biological system is still required, and customized scaffolds created with 3D printing technology must be further developed with an increase in processing speed while avoiding mistakes and errors, as the printing process is not automated. In summary, we will be able to build better composites in the future if we can completely grasp the process of HA response to various cells and the signals they trigger. Cells respond differently depending on their microenvironment. Furthermore, the advancement of materials and 3D printing techniques is expected to lead to the development of HA-based nanocomposites for future clinical applications [67].

6. Conclusion

Despite extensive and new research into the treatment, critical-sized bone lesions are becoming more common and remain a significant barrier for tissue engineers. Desirable mechanical qualities merged into single tissue-engineered constructions as scaffold fabrication has increasingly aimed to include composite materials with greater bioactivity. As a result, several effective bone and cartilage structures with therapeutic application have been produced, with ceramic and polymer composites having excellent success. In the future, it will be essential to achieve even closer replication of natural mechanical and biochemical stimuli that cells are exposed to, as well as increased construct vascularization, to maximize osteogenesis and chondrogenesis. 3D bio-fabrication and bioprinting technologies provide ever-increasing precision in constructing microarchitecture. When combined with the growing number of bioactive materials, growth factors, functionalization processes, and biomimetic scaffold designs available, the future potential for constructing sophisticated BTE scaffolds suited to patient-specific applications is enormous. This gives hope for the treatment of several challenging illnesses, such as osteonecrosis, osteoporosis, and severe bone abnormalities. As manufacturing methods advance, it is believed that in the future, treatment personalized to the individual patient can be produced in a more cost-effective and efficient manner.