# Advances in Synthesis and Functional Modification of Nanohydroxyapatite

Authors: Linbo Dou; Yancong Zhang; Hanwen Sun
Journal: Journal of Nanomaterials
 (2018)

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## Abstract

Nanohydroxyapatite material has been used to substitute for bone repair materials in clinical therapy in recent years. However, its osteogenesis effects are different due to its morphology, size, calcium phosphate ratio, crystallinity, and other differences. Thus, synthesis methods are continuously being improved to obtain synthetic materials similar to the nanohydroxyapatite in natural bone tissues in terms of biocompatibility and biological activity. Many synthesis methods are available for nanohydroxyapatite, and, among them, biological template biomimetic synthesis is the optimal method for obtaining highly bioactive and biocompatible nanohydroxyapatite, achieved by manipulating the morphology and physical and chemical properties, such as size, calcium phosphorus ratio, and degree of crystallinity. This article reviews the synthesis and functional modification of nanohydroxyapatite.


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## Body

## 1. Introduction

Tumors, trauma, congenital developmental deformities, and severe inflammatory diseases often have varying degrees of bone tissue defects [1–4]. Bone allograft immunogenicity cannot prevent trauma and pain of patients undergoing bone transplantation; thus, using apatite as a bone repair replacement material has become one of the important methods of bone repair replacement [5, 6]. With the rapid development of nanotechnology in recent years, nanomaterials are being increasingly used in biomedical science. Some natural or synthetic polymers and inorganic nonmetallic materials have been developed into nanomaterials. The use of passive targeting or the modification of specific molecules on the surfaces of active targets and drug carriers on the target cells provides effective solutions for treating diseases, such as tumors. Hydroxyapatite (HA) is a main inorganic component that is similar to those of natural bone and teeth. The eosinophilic degradation of the nano-HA ensures that it is stable under normal physiological conditions, while it is degraded in low pH environments such as vacuumization, lysosomes, or tumors. Nano-HA has relatively good biocompatibility and biodegradability and thus can be used as an ideal drug carrier material [7, 8]. Given that HA crystals exhibit hexagonal system, their a and c crystals have different properties; thus, the HA particles with a variety of molecular (acidic and alkaline protein) bonding, nano-HA, are used as growth factors, antibodies, and carriers of anticancer drugs in some studies [9, 10]. In addition, Ca2+ plays an important role in the inclusion body escape, stable cytoplasm, and nuclei of DNA through nuclear pores, making the nano-HA particle a new generation of nonviral gene-delivery carriers in gene therapy [11, 12]. At present, the application of HA is limited by its low initial fracture and can only be applied to non-load-bearing bone defects or as coatings of high-strength materials, such as stainless steel. Nano-HA has a high specific surface area, which makes it highly bioactive and improves its sintering performance (densification), thereby enhancing its mechanical properties.


## 2. Physical and Chemical Properties of Nanohydroxyapatite in Natural Bone Tissue

Humans have been using biological materials for a long time. Jade was used as a restorative material in yama until the 1960s. With the rapid developments in materials science and biotechnology, studies on biomedical materials have advanced. Nanoapatite bone repair material is an active research direction in biological materials. Hydroxyapatite (HA) has bone mineral composition. Its theoretical composition is Ca10(PO4)6(OH)2 with a Ca/P ratio of 1.67. Its crystals have a hexagonal system, and its structure is a hexagonal cylinder. Each unit cell contains 10 Ca2+, 6 PO43-, and 2 OH−. Its theoretical density is high at 3.156 g/cm3, and its refractive index is 1.64–1.65. Its Mohs hardness is 5; it is slightly soluble in water and weakly alkaline (pH = 7–9), soluble in acid, and difficult to dissolve in alkali. Moreover, HA performance depends on the surface of the structure [13]. Two surface adsorption positions exist. When OH− is located in the crystal surface, the position is at the connection of the two Ca2+ ions in aqueous solution. The surface of the OH− becomes vacant at one instance because two Ca2+ cations are positively charged, thereby forming an adsorption position. Similarly, when the surface Ca2+ is vacant at a certain moment, the surface forms another adsorption position, which is negatively charged and can adsorb the group on the cationic and protein molecules, such as Sr2+ [14]. Nano-HA essentially lacks Ca, and its calcium and phosphorus ratio is only 1.5 instead of 1.67, which is more closely related to tricalcium phosphate Ca3(PO4)2 than to hydroxyapatite Ca10(PO4) 6(OH)2. The apatite in human bone tissue is not composed of stoichiometric apatite crystals but contains small amounts of CO32-, Na+, and F− to replace the apatite lattice. As one of the main substitutes, the mass fraction of CO32- in the bone mineral is approximately 5% to 8%. CO32- can replace the OH− or PO43- in the HA lattice and form type A or B substitution. Moreover, it can occupy two positions at the same time to form type AB substitutions [15, 16]. In fact, in natural bone tissue, a type B substitution occurs, namely, -PO43– substitution. The shapes of apatite crystals are irregular, with a thickness of 2–5 nm, width of approximately 20 nm, and length of usually 40–60 nm. Its crystallinity is lower than that of the chemical synthesis of nano-HA [17].


## 3. Synthesis of Hydroxyapatite

### 3.1. Chemosynthesis

Many methods exist for preparing HA powder, such as wet and dry methods. Wet methods include precipitation, hydrothermal synthesis, sol-gel, ultrasonic synthesis, and microemulsion [18, 19].


#### 3.1.1. Chemical Precipitation Method

Chemical precipitation is mixed aqueous solution of certain concentration of calcium salt and phosphate salt, by controlling the pH value, produces chemical reaction, generates colloidal hydroxyapatite sediment, and gets its crystalline powder by calcining grinding. This method is simple and easy to prepare, and the powder has high purity, fine particle, and low cost [20]. It is a widely used method for preparing medical HA powder [21, 22].


#### 3.1.2. Hydrothermal Synthesis

The hydrothermal synthesis method is in a closed pressure vessel, using aqueous solution as the reaction medium, heating the reaction vessel, so that the dissolved or insoluble substance is dissolved and recrystallized in normal conditions [23, 24]. The main advantage of this method is that the product has high crystallinity and no need for secondary crystallization treatment, thus reducing the chance of HA particle agglomeration during recrystallization [25]. However, the method also has a complex process, and it is difficult to obtain the HA powder of quantitative Ca/P, and the quality is unstable.


#### 3.1.3. Sol-Gel Method

Sol-gel method is to dissolve alcohol salt in organic solvents and to dissolve, polymerize, and form sol by adding distilled water. Then, with the addition of water, the sol turns to gel, and the gel is processed by low temperature drying and high temperature calcining through vacuum state, which can obtain nanometer powder ceramics. The advantage of this method is that the purity is high, which is beneficial to the preparation of high purity biological ceramics, and the particle size distribution is narrow. However, the application of this method is restricted by the high raw material price, the toxicity of organic solvents, and the easy and rapid aggregation of particles during heat treatment. Sodium- (Na-) doped hydroxyapatite HA powder with crystallite sizes varying from 35 nm to 65 nm and pore sizes that vary between 100 and 300μm can be prepared by sol-gel method [26].


#### 3.1.4. Dry Method

The dry method is the solid-state reaction method. The process involved mixing solid calcium phosphate and other compounds; when the temperature is higher than 1000°C, reaction will occur with the presence of water vapor and resulted in hydroxyapatite powders. Though dry method prepared hydroxyapatite powder exhibited no lattice contraction and good crystallization performance, impurity phase often exists. Phase pure highly crystalline HA derived from eggshell was reported recently. A flower-like morphology HA (Figure1) can be obtained via a direct solid-state sintering method after preheat treatment of calcined eggshell and dicalcium phosphate dihydrate at 800°C [27].Figure 1
SEM images of the flower-like morphology HA powder derived from eggshell [27].


### 3.2. Template Synthesis

Nanometer apatite with crystal structures, orientations, and morphology that are similar to that of human bone tissue is difficult to prepare by traditional chemical methods because crystal morphology and orientation are affected by numerous factors, such as solution pH, temperature, ionic strength, solvents, anionic, ratio of reactants, and crystallization behavior of the material [30–33]. In human bones, apatite is formed with nanometer needle or columnar structure, and theC axis along the long axis of the collagen fibers is arranged (Table 1), thus gaining better mechanical properties than single crystals or the chemical synthesis of nano-HA. In the biological activity, the chemical synthesis of nano-HA has certain bone conduction performance, but its bone induction performance is low, out of the standard requirement to replace the function of the bone tissue [34, 35]. Therefore, measures must be taken for improvement. At present, differences exist among the morphology, size, calcium and phosphorus ratio, and crystallinity of chemically synthesized nanoapatite granules and apatite crystals in natural bone tissue. Therefore, reduction of the calcium and phosphorus ratio is important. Template synthesis occurs under conditions similar to the physical environment, thereby avoiding cumbersome steps and the use of special equipment during chemical synthesis methods. In recent years, the principle and method of synthesis have been introduced into the synthesis of nanohydroxyapatite. An important method of biomimetic synthesis is using a template to control the growth direction of the crystal and to obtain nanoapatite that has a similar structure and components as that in the natural bone. The current popular research template has two kinds: organic polymer and biological templates. Template synthesis yields nanoapatite with relatively low degree of crystallinity and low calcium phosphate ratio [36–38].Table 1
Difference between natural bone and synthetic nanoapatite.
Morphology Dimension Calcium phosphate ratio Crystallinity Representative method Natural bone Needle 5–10 nm 1.50 low Flaky 20–50 nm  Chemical synthesis Needle 130–170 nm 1.640–1.643 high Hydrothermal 15–25 nm Coprecipitation  Organic polymer template synthesis Needle 70–90 nm 1.35 low Polyamide 10–20 nm Polyacrylic acid 2–5 nm Polyethylene glycol Spherical 30–50 nm Polyethyleneimine 1–5µm  Biological template synthesis Flaky 7–8 nm 1.5–1.66 low Collagen Fusiform 30–40 nm Chitosan Flaky 6 nm Chondroitin sulfate 12 nm


#### 3.2.1. Organic Polymer Templates

Polyacrylic acid (PAA) is added to the nanohydroxyapatite synthesis to prepare a HA/PAA composite material; and through molecular thermodynamic simulation of the performance of nano-HA-PA interface, the nHA grows along the c axis of nano-HA under the guidance of polyacrylic acid [39, 40]. Polyethylene glycol (PEG) is an organic matter that is avirulent, without excitants, and has good water solubility. A spherical nanohydroxyapatite with a size of 30 to 50 nm can be obtained by adding 2%–6% PEG to spherical nanometer hydroxyapatite. The nano-HA crystallinity increases with the increase of PEG concentration. The different concentrations of PEG and the interaction between calcium greatly affect the nanometer apatite morphology and growth (Figure 2) [28, 41]. Cross-linked polyethylene imine (PEI) is a template to preparing nHA/PEI in composite materials. The unlinked PEI cannot guide the nucleation and growth of calcium phosphate, and the cross-linked PEI can form a spongy ball with a diameter of 1–5 μm. The inorganic membrane on the ball is made up of tiny, slightly spherical particles that further form larger spheres. The main contents include calcium phosphate and nanoscale hydroxyapatite, and the inorganic phase content accounts for only 10% (Ca/P ratio is 1.35) [42, 43].Figure 2
TEM pictures of nanometer apatite morphology using PEG as template. (a) Control, (b) 8% PEG6000, and (c) 16% PEG6000 [28].
(a) (b) (c)


#### 3.2.2. Biological Templates

Inspired by oysters, which have strong adsorption ability, dopamine was investigated by polymerization. Polydopamine easily adheres to almost any surface in alkaline solutions without requiring advanced treatment processes for the other surface. The amino and sulfonium groups of polydopamine can further take Schiffer’s alkali or Michael addition reactions with other substances to graft other biological molecules with functional activity, thus modifying the surface of biomaterials. Polydopamine can promote the adhesion of cells to multiple surfaces. Given its structure, polydopamine-o-phenol diphenol can chelate calcium ions, adsorb phosphate ions, and activate nanoapatite crystal nucleation as a template for nanoapatite synthesis. On the other hand, hydroxyl of phthalates can react to functional groups of polypeptides or proteins, as the intermediate means of grafting biological molecules. Recently, scholars reported the use of polydopamine-coated nanoapatite, titanium materials, and polyglycolic acid materials for enhancing bone implant material surfaces that improved bone tissue regeneration. Moreover, polydopamine promotes the adhesion function of cells on multiple surfaces. The structure of polydopamine catechol can chelate calcium; thus, further adsorption of phosphate ions is used for the template synthesis of nanoapatite to start the nanoapatite crystal. Catechol hydroxyl can react with peptides or protein amino functional groups, such as in grafting biologically functional molecules [44–46].Gelatin has protein-rich functional groups, such as carboxyl and hydroxyl groups. It is the local hydrolysis product of collagen, which is the main organic composition of the tendon and bone tissue and has a typical three-dimensional spiral structure. It contains numerous specific spiral biometric sites in the molecular chain. Control experimental conditions can cause the nano-HA to nucleate directly on the gelatin fibers, and the c axis is along the direction of the collagen helix. Self-assembly method confirms that the gelatin can regulate nano-HA so that it can be directed to grow along a certain direction [47, 48].Chitosan (CS) is a rare and positively charged alkaline polysaccharide. It is a product of deethylation (1–4), and its chemical name is 2-amino-2-deoxy-beta-d-glucose. By using chitosan as the template, the nano-HA with class spindle is prepared. When the concentration of CS is 0.5%, the width is approximately 7-8 nm, and the length is between 30 and 40 nm. The CS template changes the appearance of nano-HA [49, 50].The nano-HA/ChS complex was prepared using chondroitin sulfate (ChS) with high density of negatively charged groups, and the nano-HA, which was a length of 12 nm and a width of 6 nm, was produced after joining ChS. The long axis of ChS was parallel to thec axis of nano-HA crystals. Nano-HA nucleation and growth depended on the chemical interaction between nano-HA and ChS. The periodic arrangement of the carboxylic acid group on the ChS can chelate Ca2+ format Ca–ChS complex. This complex formed the vug-critical size by absorbing Ca2+, PO43-, CO32-, and OH− [51, 52]. The nano-HA crystals were subject to the regulation of the group on the ChS template and the spatial hindrance of nano-HA crystals; thus, a certain size of particles was formed. When no ChS template was present, nano-HA grew naturally without limits [53].


## 3.1. Chemosynthesis

Many methods exist for preparing HA powder, such as wet and dry methods. Wet methods include precipitation, hydrothermal synthesis, sol-gel, ultrasonic synthesis, and microemulsion [18, 19].


### 3.1.1. Chemical Precipitation Method

Chemical precipitation is mixed aqueous solution of certain concentration of calcium salt and phosphate salt, by controlling the pH value, produces chemical reaction, generates colloidal hydroxyapatite sediment, and gets its crystalline powder by calcining grinding. This method is simple and easy to prepare, and the powder has high purity, fine particle, and low cost [20]. It is a widely used method for preparing medical HA powder [21, 22].


### 3.1.2. Hydrothermal Synthesis

The hydrothermal synthesis method is in a closed pressure vessel, using aqueous solution as the reaction medium, heating the reaction vessel, so that the dissolved or insoluble substance is dissolved and recrystallized in normal conditions [23, 24]. The main advantage of this method is that the product has high crystallinity and no need for secondary crystallization treatment, thus reducing the chance of HA particle agglomeration during recrystallization [25]. However, the method also has a complex process, and it is difficult to obtain the HA powder of quantitative Ca/P, and the quality is unstable.


### 3.1.3. Sol-Gel Method

Sol-gel method is to dissolve alcohol salt in organic solvents and to dissolve, polymerize, and form sol by adding distilled water. Then, with the addition of water, the sol turns to gel, and the gel is processed by low temperature drying and high temperature calcining through vacuum state, which can obtain nanometer powder ceramics. The advantage of this method is that the purity is high, which is beneficial to the preparation of high purity biological ceramics, and the particle size distribution is narrow. However, the application of this method is restricted by the high raw material price, the toxicity of organic solvents, and the easy and rapid aggregation of particles during heat treatment. Sodium- (Na-) doped hydroxyapatite HA powder with crystallite sizes varying from 35 nm to 65 nm and pore sizes that vary between 100 and 300μm can be prepared by sol-gel method [26].


### 3.1.4. Dry Method

The dry method is the solid-state reaction method. The process involved mixing solid calcium phosphate and other compounds; when the temperature is higher than 1000°C, reaction will occur with the presence of water vapor and resulted in hydroxyapatite powders. Though dry method prepared hydroxyapatite powder exhibited no lattice contraction and good crystallization performance, impurity phase often exists. Phase pure highly crystalline HA derived from eggshell was reported recently. A flower-like morphology HA (Figure1) can be obtained via a direct solid-state sintering method after preheat treatment of calcined eggshell and dicalcium phosphate dihydrate at 800°C [27].Figure 1
SEM images of the flower-like morphology HA powder derived from eggshell [27].


## 3.1.1. Chemical Precipitation Method

Chemical precipitation is mixed aqueous solution of certain concentration of calcium salt and phosphate salt, by controlling the pH value, produces chemical reaction, generates colloidal hydroxyapatite sediment, and gets its crystalline powder by calcining grinding. This method is simple and easy to prepare, and the powder has high purity, fine particle, and low cost [20]. It is a widely used method for preparing medical HA powder [21, 22].


## 3.1.2. Hydrothermal Synthesis

The hydrothermal synthesis method is in a closed pressure vessel, using aqueous solution as the reaction medium, heating the reaction vessel, so that the dissolved or insoluble substance is dissolved and recrystallized in normal conditions [23, 24]. The main advantage of this method is that the product has high crystallinity and no need for secondary crystallization treatment, thus reducing the chance of HA particle agglomeration during recrystallization [25]. However, the method also has a complex process, and it is difficult to obtain the HA powder of quantitative Ca/P, and the quality is unstable.


## 3.1.3. Sol-Gel Method

Sol-gel method is to dissolve alcohol salt in organic solvents and to dissolve, polymerize, and form sol by adding distilled water. Then, with the addition of water, the sol turns to gel, and the gel is processed by low temperature drying and high temperature calcining through vacuum state, which can obtain nanometer powder ceramics. The advantage of this method is that the purity is high, which is beneficial to the preparation of high purity biological ceramics, and the particle size distribution is narrow. However, the application of this method is restricted by the high raw material price, the toxicity of organic solvents, and the easy and rapid aggregation of particles during heat treatment. Sodium- (Na-) doped hydroxyapatite HA powder with crystallite sizes varying from 35 nm to 65 nm and pore sizes that vary between 100 and 300μm can be prepared by sol-gel method [26].


## 3.1.4. Dry Method

The dry method is the solid-state reaction method. The process involved mixing solid calcium phosphate and other compounds; when the temperature is higher than 1000°C, reaction will occur with the presence of water vapor and resulted in hydroxyapatite powders. Though dry method prepared hydroxyapatite powder exhibited no lattice contraction and good crystallization performance, impurity phase often exists. Phase pure highly crystalline HA derived from eggshell was reported recently. A flower-like morphology HA (Figure1) can be obtained via a direct solid-state sintering method after preheat treatment of calcined eggshell and dicalcium phosphate dihydrate at 800°C [27].Figure 1
SEM images of the flower-like morphology HA powder derived from eggshell [27].


## 3.2. Template Synthesis

Nanometer apatite with crystal structures, orientations, and morphology that are similar to that of human bone tissue is difficult to prepare by traditional chemical methods because crystal morphology and orientation are affected by numerous factors, such as solution pH, temperature, ionic strength, solvents, anionic, ratio of reactants, and crystallization behavior of the material [30–33]. In human bones, apatite is formed with nanometer needle or columnar structure, and theC axis along the long axis of the collagen fibers is arranged (Table 1), thus gaining better mechanical properties than single crystals or the chemical synthesis of nano-HA. In the biological activity, the chemical synthesis of nano-HA has certain bone conduction performance, but its bone induction performance is low, out of the standard requirement to replace the function of the bone tissue [34, 35]. Therefore, measures must be taken for improvement. At present, differences exist among the morphology, size, calcium and phosphorus ratio, and crystallinity of chemically synthesized nanoapatite granules and apatite crystals in natural bone tissue. Therefore, reduction of the calcium and phosphorus ratio is important. Template synthesis occurs under conditions similar to the physical environment, thereby avoiding cumbersome steps and the use of special equipment during chemical synthesis methods. In recent years, the principle and method of synthesis have been introduced into the synthesis of nanohydroxyapatite. An important method of biomimetic synthesis is using a template to control the growth direction of the crystal and to obtain nanoapatite that has a similar structure and components as that in the natural bone. The current popular research template has two kinds: organic polymer and biological templates. Template synthesis yields nanoapatite with relatively low degree of crystallinity and low calcium phosphate ratio [36–38].Table 1
Difference between natural bone and synthetic nanoapatite.
Morphology Dimension Calcium phosphate ratio Crystallinity Representative method Natural bone Needle 5–10 nm 1.50 low Flaky 20–50 nm  Chemical synthesis Needle 130–170 nm 1.640–1.643 high Hydrothermal 15–25 nm Coprecipitation  Organic polymer template synthesis Needle 70–90 nm 1.35 low Polyamide 10–20 nm Polyacrylic acid 2–5 nm Polyethylene glycol Spherical 30–50 nm Polyethyleneimine 1–5µm  Biological template synthesis Flaky 7–8 nm 1.5–1.66 low Collagen Fusiform 30–40 nm Chitosan Flaky 6 nm Chondroitin sulfate 12 nm


### 3.2.1. Organic Polymer Templates

Polyacrylic acid (PAA) is added to the nanohydroxyapatite synthesis to prepare a HA/PAA composite material; and through molecular thermodynamic simulation of the performance of nano-HA-PA interface, the nHA grows along the c axis of nano-HA under the guidance of polyacrylic acid [39, 40]. Polyethylene glycol (PEG) is an organic matter that is avirulent, without excitants, and has good water solubility. A spherical nanohydroxyapatite with a size of 30 to 50 nm can be obtained by adding 2%–6% PEG to spherical nanometer hydroxyapatite. The nano-HA crystallinity increases with the increase of PEG concentration. The different concentrations of PEG and the interaction between calcium greatly affect the nanometer apatite morphology and growth (Figure 2) [28, 41]. Cross-linked polyethylene imine (PEI) is a template to preparing nHA/PEI in composite materials. The unlinked PEI cannot guide the nucleation and growth of calcium phosphate, and the cross-linked PEI can form a spongy ball with a diameter of 1–5 μm. The inorganic membrane on the ball is made up of tiny, slightly spherical particles that further form larger spheres. The main contents include calcium phosphate and nanoscale hydroxyapatite, and the inorganic phase content accounts for only 10% (Ca/P ratio is 1.35) [42, 43].Figure 2
TEM pictures of nanometer apatite morphology using PEG as template. (a) Control, (b) 8% PEG6000, and (c) 16% PEG6000 [28].
(a) (b) (c)


### 3.2.2. Biological Templates

Inspired by oysters, which have strong adsorption ability, dopamine was investigated by polymerization. Polydopamine easily adheres to almost any surface in alkaline solutions without requiring advanced treatment processes for the other surface. The amino and sulfonium groups of polydopamine can further take Schiffer’s alkali or Michael addition reactions with other substances to graft other biological molecules with functional activity, thus modifying the surface of biomaterials. Polydopamine can promote the adhesion of cells to multiple surfaces. Given its structure, polydopamine-o-phenol diphenol can chelate calcium ions, adsorb phosphate ions, and activate nanoapatite crystal nucleation as a template for nanoapatite synthesis. On the other hand, hydroxyl of phthalates can react to functional groups of polypeptides or proteins, as the intermediate means of grafting biological molecules. Recently, scholars reported the use of polydopamine-coated nanoapatite, titanium materials, and polyglycolic acid materials for enhancing bone implant material surfaces that improved bone tissue regeneration. Moreover, polydopamine promotes the adhesion function of cells on multiple surfaces. The structure of polydopamine catechol can chelate calcium; thus, further adsorption of phosphate ions is used for the template synthesis of nanoapatite to start the nanoapatite crystal. Catechol hydroxyl can react with peptides or protein amino functional groups, such as in grafting biologically functional molecules [44–46].Gelatin has protein-rich functional groups, such as carboxyl and hydroxyl groups. It is the local hydrolysis product of collagen, which is the main organic composition of the tendon and bone tissue and has a typical three-dimensional spiral structure. It contains numerous specific spiral biometric sites in the molecular chain. Control experimental conditions can cause the nano-HA to nucleate directly on the gelatin fibers, and the c axis is along the direction of the collagen helix. Self-assembly method confirms that the gelatin can regulate nano-HA so that it can be directed to grow along a certain direction [47, 48].Chitosan (CS) is a rare and positively charged alkaline polysaccharide. It is a product of deethylation (1–4), and its chemical name is 2-amino-2-deoxy-beta-d-glucose. By using chitosan as the template, the nano-HA with class spindle is prepared. When the concentration of CS is 0.5%, the width is approximately 7-8 nm, and the length is between 30 and 40 nm. The CS template changes the appearance of nano-HA [49, 50].The nano-HA/ChS complex was prepared using chondroitin sulfate (ChS) with high density of negatively charged groups, and the nano-HA, which was a length of 12 nm and a width of 6 nm, was produced after joining ChS. The long axis of ChS was parallel to thec axis of nano-HA crystals. Nano-HA nucleation and growth depended on the chemical interaction between nano-HA and ChS. The periodic arrangement of the carboxylic acid group on the ChS can chelate Ca2+ format Ca–ChS complex. This complex formed the vug-critical size by absorbing Ca2+, PO43-, CO32-, and OH− [51, 52]. The nano-HA crystals were subject to the regulation of the group on the ChS template and the spatial hindrance of nano-HA crystals; thus, a certain size of particles was formed. When no ChS template was present, nano-HA grew naturally without limits [53].


## 3.2.1. Organic Polymer Templates

Polyacrylic acid (PAA) is added to the nanohydroxyapatite synthesis to prepare a HA/PAA composite material; and through molecular thermodynamic simulation of the performance of nano-HA-PA interface, the nHA grows along the c axis of nano-HA under the guidance of polyacrylic acid [39, 40]. Polyethylene glycol (PEG) is an organic matter that is avirulent, without excitants, and has good water solubility. A spherical nanohydroxyapatite with a size of 30 to 50 nm can be obtained by adding 2%–6% PEG to spherical nanometer hydroxyapatite. The nano-HA crystallinity increases with the increase of PEG concentration. The different concentrations of PEG and the interaction between calcium greatly affect the nanometer apatite morphology and growth (Figure 2) [28, 41]. Cross-linked polyethylene imine (PEI) is a template to preparing nHA/PEI in composite materials. The unlinked PEI cannot guide the nucleation and growth of calcium phosphate, and the cross-linked PEI can form a spongy ball with a diameter of 1–5 μm. The inorganic membrane on the ball is made up of tiny, slightly spherical particles that further form larger spheres. The main contents include calcium phosphate and nanoscale hydroxyapatite, and the inorganic phase content accounts for only 10% (Ca/P ratio is 1.35) [42, 43].Figure 2
TEM pictures of nanometer apatite morphology using PEG as template. (a) Control, (b) 8% PEG6000, and (c) 16% PEG6000 [28].
(a) (b) (c)


## 3.2.2. Biological Templates

Inspired by oysters, which have strong adsorption ability, dopamine was investigated by polymerization. Polydopamine easily adheres to almost any surface in alkaline solutions without requiring advanced treatment processes for the other surface. The amino and sulfonium groups of polydopamine can further take Schiffer’s alkali or Michael addition reactions with other substances to graft other biological molecules with functional activity, thus modifying the surface of biomaterials. Polydopamine can promote the adhesion of cells to multiple surfaces. Given its structure, polydopamine-o-phenol diphenol can chelate calcium ions, adsorb phosphate ions, and activate nanoapatite crystal nucleation as a template for nanoapatite synthesis. On the other hand, hydroxyl of phthalates can react to functional groups of polypeptides or proteins, as the intermediate means of grafting biological molecules. Recently, scholars reported the use of polydopamine-coated nanoapatite, titanium materials, and polyglycolic acid materials for enhancing bone implant material surfaces that improved bone tissue regeneration. Moreover, polydopamine promotes the adhesion function of cells on multiple surfaces. The structure of polydopamine catechol can chelate calcium; thus, further adsorption of phosphate ions is used for the template synthesis of nanoapatite to start the nanoapatite crystal. Catechol hydroxyl can react with peptides or protein amino functional groups, such as in grafting biologically functional molecules [44–46].Gelatin has protein-rich functional groups, such as carboxyl and hydroxyl groups. It is the local hydrolysis product of collagen, which is the main organic composition of the tendon and bone tissue and has a typical three-dimensional spiral structure. It contains numerous specific spiral biometric sites in the molecular chain. Control experimental conditions can cause the nano-HA to nucleate directly on the gelatin fibers, and the c axis is along the direction of the collagen helix. Self-assembly method confirms that the gelatin can regulate nano-HA so that it can be directed to grow along a certain direction [47, 48].Chitosan (CS) is a rare and positively charged alkaline polysaccharide. It is a product of deethylation (1–4), and its chemical name is 2-amino-2-deoxy-beta-d-glucose. By using chitosan as the template, the nano-HA with class spindle is prepared. When the concentration of CS is 0.5%, the width is approximately 7-8 nm, and the length is between 30 and 40 nm. The CS template changes the appearance of nano-HA [49, 50].The nano-HA/ChS complex was prepared using chondroitin sulfate (ChS) with high density of negatively charged groups, and the nano-HA, which was a length of 12 nm and a width of 6 nm, was produced after joining ChS. The long axis of ChS was parallel to thec axis of nano-HA crystals. Nano-HA nucleation and growth depended on the chemical interaction between nano-HA and ChS. The periodic arrangement of the carboxylic acid group on the ChS can chelate Ca2+ format Ca–ChS complex. This complex formed the vug-critical size by absorbing Ca2+, PO43-, CO32-, and OH− [51, 52]. The nano-HA crystals were subject to the regulation of the group on the ChS template and the spatial hindrance of nano-HA crystals; thus, a certain size of particles was formed. When no ChS template was present, nano-HA grew naturally without limits [53].


## 4. Nanoapatite Functional Modification

Combination of growth factors is an important approach for nano-HA functional transformation. According to different applications and application requirements, different kinds of growth factors are loaded on the nano-HA to exert different effects, such as the loading on BMP-2 with nano-HA for bone repair that can effectively induce osteogenesis of mesenchymal cells [54]. Loading of vascular endothelial growth factor nano-HA can effectively promote vascularization when used as a bone implant [55, 56]. The loading of fibroblast growth factor can promote the healing of bone defect and acceleration of bone growth [57, 58]. However, because growth factor is an active protein, it is expensive and its preservation over a prolonged period is difficult, thus greatly restricting its clinical application. Therefore, chemical synthesis method was adopted in vitro according to the active nucleotide sequence of growth factor. The active peptide with the function of growth factor was prepared. This short peptide synthesis is affordable, and its preservation and transportation conditions are not as demanding as those of the growth factor. It is a biological medical material with potential clinical applications. According to the core function sequence of BMP-2, 24 amino acid active peptides were designed and synthesized, further carrying out the activity of short peptide nanoreorganization of HA/collagen/PLA composite artificial bone. In vivo experiments on rats show that the scaffold materials can promote new bone formation and have good biomedical application [59, 60]. Arginine-glycine-aspartate (RGD) peptides modified HA for the promotion of bone integration function. The results showed that HA with RGD peptides has good adsorption capacity for fibronectin, vitronectin, and fibrinogen, which play decisive roles in post-echo and survival of bone mesenchymal stem cells (BMSC) (Figure 3). The newborn bone is directly bound to the HA implant of RGD, but the RGD has a significant inhibitory effect on bone formation [29, 61]. Kasugai et al. [62] grafted six aspartic acid (Asp) molecules to nano-HA by experimental research in mice and demonstrated that the compound material can effectively deliver drugs to the bone tissue. Kim et al. [63] reported for the first time that the bone formation polypeptide is derived from the polypeptide sequence of the BMP-7 nonmature region, which is composed of 15 amino acids. The study confirmed that this peptide can promote BMSCs to differentiate into osteoblasts, and its osteogenic activity is better than that of BMP-7.Figure 3
Cell morphology of BMSCs on PLGA/NHA-RGD ((a) and (b)) and PLGA/NHA ((c) and (d)) 3D scaffold surfaces depicted by ESEM after 1- and 4-day cell incubation [29].


## 5. Conclusion

At present, the physical and chemical properties of the nanometer apatite (crystallinity, calcium phosphate scale, etc.) and induced osteogenesis performance need further improvements. No systematic research has been conducted on the reduction of the nanocalcium phosphorus ratio and crystallinity of apatite and on the improvement of the osteogenic induction activity of the synthesized nanoapatite, which are significant for the regeneration of clinical bone tissue. Therefore, further investigations must be conducted.



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*Source: 3106214-2018-04-01.xml*