# Progress in Montmorillonite Functionalized Artificial Bone Scaffolds: Intercalation and Interlocking, Nanoenhancement, and Controlled Drug Release

Authors: DongYing Li; Pin Li; Yong Xu; WenMing Guo; MengQi Li; MeiGui Chen; HaoYu Wang; HaiMei Lin
Journal: Journal of Nanomaterials
 (2022)

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## Abstract

Montmorillonite (MMT) has attracted widespread attention in the field of bone tissue engineering in recent years because of its interlayer domain structure. The progress of MMT application was reviewed in this article. Concretely, the application of MMT was mainly explained from the structural characteristics, mechanical strengthening mechanism, organic functionalization, and drug loading and release. Firstly, the polar polymer molecular chains are easily induced into the interlayer domain of MMT to form an interlock and achieve the mechanical strengthening of scaffold. Secondly, the “sandwich” sheet structure of MMT can be exfoliated into graphene-like MMT nanosheets, providing a nanostrengthening effect for polymer matrix. In addition, MMT’s interlayer domain provides a favorable environment for the loading and slow release of drugs, and it is an ideal platform for the functionalization of bone scaffolds. More importantly, MMT can be easily modified by cation exchange and chemical reaction to further improve the compatibility of composites: such as strengthening mechanical interlocking and nanostrengthening effects and achieving controllable loading and release of drugs. It is expected to provide a reference for improving the application of bone tissue engineering scaffolds.


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## Body

## 1. Introduction

With the increase in traffic accidents and the accelerated aging of the population, the demand for defective tissues, especially bone tissues, is increasing in recent years. Although autologous bone transplantation is still the gold standard for bone defect repair, its application is limited to a certain extent due to its limited materials, increased surgical trauma, and susceptibility to infection at the bone site [1, 2]. Allogeneic bone transplantation also has immune rejection, low survival rate, and risks of disease transmission and cross-infection [3, 4]. The three-dimensional porous artificial bone made of biodegradable materials has many unique advantages: (1) It has a shape and structure that matches the defected part and has sufficient mechanical properties to carry the stress transfer of the damaged part; (2) it has good osteogenic properties to induce cell growth and osteogenic differentiation, without rejection; (3) it has a suitable degradation rate, which makes room for new tissue until it is completely absorbed, and finally realizes the repair and functional reconstruction of the bone defect [5, 6].Commonly used bone scaffold materials include metals [7, 8], polymers [9, 10], bioceramics/glass [11, 12], and their composites [5, 13]. Metal materials have excellent mechanical properties and good biocompatibility, while their modulus is much higher than that of natural bone so that it is easy to produce a stress shielding effect, which seriously affects the stability of bone repair [14]. Polymer bone scaffolds generally have the defect of insufficient mechanical properties, and synthetic polymers even lack effective bioactivity and osteogenesis function [15]. Inorganic bioceramic materials exhibit good biological properties, but they have the defect of poor fracture toughness when used alone [16]. In brief, a single type of material is difficult to meet the requirements of bone scaffold materials.The strategy of composite materials is expected to be compatible with the advantages of each component material while avoiding its shortcomings, thereby providing a new way for the preparation of artificial bone for bone defect repair. Natural bone is mainly composed of organic part of type I collagen and inorganic part of hydroxyapatite crystals, as well as some cells, blood vessels and a small amount of growth factors, etc. [14, 17]. Among them, organics such as collagen not only acts as the growth framework of hydroxyapatite (HA) but also acts as a biological template to regulate its nucleation and growth, so that bone tissue has a very delicate structure and excellent mechanical properties [18]. Therefore, in order to prepare artificial bone that meets the needs of bone defect repair, more and more scholars hope to realize the controllable design of the mechanical properties, degradation performance, and osteogenesis function of bone scaffolds through the combination of materials with different properties and functions. A schematic diagram of a process for bone repair using a bone scaffold was presented, as shown in Figure 1.Figure 1
Schematic diagram of artificial bone scaffold used for bone repair.Montmorillonite (MMT), with a molecular formula of (Na, Ca) 0.33 (Al, Mg)2(Si4O10) (OH)2·nH2O [19], is a biodegradable monoclinic silicate mineral, which have a “sandwich” sheet structure composed of aluminum-oxygen octahedrons and silicon-oxygen tetrahedrons [20, 21]. As a typical clay mineral, it is widely used in the mechanical strengthening of bone scaffolds [22]. On the one hand, it can be transformed into graphene-like nanosheets with higher specific surface area through interlayer exfoliation behavior after undergoing chemical or physical action, so as to realize the nanostrengthening of the composite bone scaffold [23]. On the other hand, the rich cations between the layers of the MMT crystal structure have strong ion exchange capacity, which can induce the insertion of ionic polymer molecules into the interlayer domains of its “sandwich” structure through intercalation. In this way, molecular cross-linking bridges are used to achieve interlocking with the matrix, further enhancing the mechanical properties of the bone scaffold [24–27]. More importantly, through molecular intercalation and target-oriented functional modification, MMT is expected to build a drug or growth factor loading platform that realize the controlled and sustained release of loaded drugs or growth factors and the biofunctionalization of bone scaffolds [28].The latest research progress of montmorillonite in bone tissue engineering was briefly summarized in this article. The structural characteristics and properties of MMT as well as its application potential and advantages in bone tissue engineering were first explained. The focus was on the application of montmorillonite as a nanofiller in bone scaffolds and the latest research progress in organic modification to optimize its application. Besides, future prospects and development challenges for the application of MMT in bone tissue engineering are also prospected.


## 2. The Structure of Montmorillonite

MMT is a low-cost natural clay mineral and an important component of sedimentary rock and layered silicate-derived soil [29–31]. It is often used as an adsorbent to repair environmental pollution in the engineering field and as a drug carrier in the biomedicine field [32, 33]. The microstructure of MMT is a sandwich sheet structure constructed from nanocrystals with a thickness of about 1 nm. Specifically, its crystal lattice is composed of two O-Si-O tetrahedral flakes and one O-Al(Mg)-O octahedral flake, and O-Al(Mg)-O octahedrons and two O-Si-O tetrahedrons achieve bilateral fusion by sharing oxygen atoms, thus forming a sandwich-like interlayer [34, 35], as show in Figure 2.Figure 2
Schematic diagram of the montmorillonite crystal structure.The stability of the sandwich-like structure of MMT is mainly maintained by intermolecular forces such as van der Waals forces and electrostatic effects [23, 36]. At the same time, the intermolecular interaction force ensures the space of the interlayer domain in the steady state and provides a place for the loading of biomolecules such as anti-inflammatory drugs or the intercalation of modifiers such as polymer molecules [37]. More importantly, isomorphous substitution can occur in the MMT sandwich structure to generate charges. Specifically, in tetrahedral crystals, trivalent Al3+ can replace tetravalent Si4+, and Al3+ in octahedral crystals can be replaced by divalent Mg2+, so that a large number of negative charges are generated in the interlayer domain of MMT. Moreover, these negative charges can be absorbed hydrated cations (such as Na+) into the interlaminar space to achieve the effect of charge balance [38, 39]. Under certain conditions, these cations can be replaced by other cations; that is, cation exchange can be carried out. The phenomenon of cation exchange is expected to become the driving force for drug loading and molecular intercalation and to provide a guarantee for the functional modification of bone scaffolds.


## 3. Montmorillonite Improves the Mechanical Properties of the Scaffold

Scaffolds apply to bone tissue engineering should have strong mechanical strength as a physical support to resist internal and external pressure and at the same time provide a platform for cell adhesion, extension, proliferation, and differentiation. A single natural polymer or synthetic polymer often has insufficient mechanical properties. Therefore, it is necessary to add other materials to change this situation. In recent years, various studies have showed that the polymer matrix containing nanofillers not only has good biological properties but also has sufficient mechanical properties [40, 41]. As a natural clay nanomaterial, MMT not only has a large specific surface area, large aspect ratio, high rigidity, and good processibility but also can be well combined with polymeric matrix materials through physical or chemical effects [42, 43]. It has been reported that only a lower content of nanomontmorillonite filler was needed to significantly improve the mechanical properties of composite materials [38]. As shown in Table 1, some literature reported the improvement of unmodified MMT on the tensile/compressive strength and elastic strength of the polymer.Table 1
Montmorillonite enhances polymer mechanical properties.
MMT content (wt%)Polymer matrixMechanical performance enhancement resultsReferences0.2Polytetrafluoroethylene/glass fiberTensile strength achieved 23.1 MPa, increased by 41% and young’s modulus achieved 165 MPa, increased by 14%[43]2.0Poly (lactic acid)Elastic modulus achieved 4.3 GPa, increased by 34%. Tensile strength achieved 75.6 MPa, increased by 1.9%[38]Bacterial celluloseTensile strength was increased up to 210 MPa from 151.3 MPa[44]3.0Poly (vinyl alcohol)/chitosanTensile modulus and tensile strength increased by 35% and 33%, respectively[45]4.0Bacterial celluloseYoung’s modulus was increased up to 5.7 GPa from 4.2 GPa[44]4.5Poly-l-lactic acidTensile strength and modulus increased by 44.1% and 66.9%, respectively[46]5.0Poly (ε-caprolactone)Tensile strength and compressive strength increased by 83% and 78%, respectively[47]10Chitosan/hydroxyapatiteElastic modulus increased by 36%[48]Chitosan-gelatinYoung’s modulus was increased up to 20.18 MPa from 17.245 MPa. Compressive strength was increased up to 21.6 MPa from 4.48 MPa[49]In general, the ways that MMT nanofillers enhance the mechanical properties of the polymer matrix can be summarized as follows: (1) inducing polymer molecular chains to be embedded in the interlayer domain structure of MMT through molecular intercalation to achieve mechanical interlocking between the filler and the matrix; (2) by destroying the interlayer structure of MMT, it promotes the exfoliation of the lamellae to form graphene-like nanosheets, thereby achieving the enhancement of the mechanical properties of the polymer matrix through the nanoreinforcing effect.


### 3.1. Molecular Chain Intercalation Strengthening

It is well known that the interlayer domain of MMT has a swelling effect. In a liquid environment or in a molten state, its interlayer domain expands and increases, making polymer molecular chains easy to achieve intercalation behavior, as shown in Figure3. In detail, in the mixed solution of MMT and polymer, MMT absorbs water to cause the expansion effect of the interlayer domain, so that the polymer molecular chain is successfully embedded in the MMT interlayer domain, while the other end of the molecular chain is miscible with the polymer matrix. In short, under the synergistic effect of covalent or noncovalent intermolecular interactions and the mutual compatibility of organic materials, intercalated molecular chains are expected to build the link between MMT and polymers. As a result, a molecular cross-linking bridge is built between inorganic and organic substances; that is, an interlocking structure is formed in the composite material. The existence of this interlocking structure can significantly hinder the movement of polymer chains when the composite material system is subjected to external forces, thereby increasing the interaction between the polymer and MMT. In addition, the embedding of molecular chains increases the space between the MMT layers to a certain extent, that is, increases the contact area between the MMT nanofiller and the polymer matrix. In the composite material system, the formation of interlocking structure increases the molecular force between the two phases, and the expansion of the contact area between the two phases increases the number of interlocking structures in the system. Under this synergistic effect, it is expected to significantly improve the mechanical properties of composite materials. In other words, using MMT as the nanofiller phase is expected to achieve significant effects in enhancing the mechanical properties of polymer bone scaffolds.Figure 3
Schematic diagram of polymer molecular chain intercalate into MMT.The use of MMT as nanofiller to enhance the mechanical properties of polymer matrix has been widely accepted. Mauro et al. [52] added 1%, 3%, and 10 wt% MMT to hydrogels based on peptidomimetic polyamidoamine. When the degree of crosslinking of the hydrogel was constant, the shear storage modulus improved as the increase of MMT content. The shear storage modulus of hydrogel with MMT content of 10 wt% reached 200 KPa, which was 20 times higher than the original hydrogel. It might be attributed to the double action of the electrostatic force and the increase in contact area. This dual effect makes the hydrogel and MMT complete the intercalation structure, forming a complex and interconnected network. Paluszkiewicz et al. [53] prepared composite materials by adding 5 wt% MMT to chitosan (CS) solution in an acidic environment. FTIR results showed that the Si-O-Si group of MMT formed hydrogen bonds with the hydroxyl and amino groups of CS. This strong interfacial interaction promotes the effective transfer of interfacial stress. Therefore, compared with pure CS, the tensile strength of CS-MMT is increased by about 10-12 MPa, and the Young’s modulus is increased by 20%. A recent study [48] found that when the content of MMT was less than 4.5 wt%, the tensile strength of the PLLA/MMT composite scaffold increased with the increase of the content; the highest increase was 44.1% (reach 26.8 MPa). However, as the content of MMT continues to increase, the growth trend of the mechanical properties of the composite scaffold begins to reverse, resulting in a decrease in the mechanical properties. Meanwhile, the author further studied the cross-sectional morphology of the composite material to explore the mechanism of the incorporation of MMT to enhance the mechanical properties of the composite material and found that the PLLA/MMT scaffold has many folds and torn ligaments on the fracture surface compared to the pure PLLA scaffold. These studies showed that, as a nanofiller phase, MMT exhibited an amazing effect in enhancing the mechanical properties of a single polymer matrix.In addition, some studies have explored the influence of MMT as an incorporation on the mechanical properties of polymer composites. Bee et al. [54] added MMT to a composite matrix of polylactic acid (PLA) and naturally derived hydroxyapatite (HAp) in the molten state to form a ternary composite material, focusing on the mechanical properties of the composite material. They found that when the MMT content was 2%, the increase in tensile strength was most significant and believed that the improvement in the tensile properties of the composite material was mainly due to the following aspects: Firstly, the increase in the interfacial tension between PLA and HAp promoted PLA intercalation of molecules in the interlayer domain of MMT. Secondly, MMT nanoparticles restricted the migration of PLA molecules through their interlayer domains, thereby enhancing the PLA matrix. Thirdly, mutual repulsion at the interface between PO43- of nonpolar HAp and the polar site of MMT promoted the dispersion of MMT, thereby further releasing the potential of nanostrengthening effect.Olad et al. [51] studied the synergistic effect of graphene oxide (GO) and MMT on the performance of chitosan-gelatin (CS-Gel) scaffolds. XRD results showed that the characteristic peak of montmorillonite was located at 2θ=7.48°, while the characteristic peaks of MMT in CS-Gel/MMT and CS-Gel/GO/MMT were located at 2θ=4.98° and 2θ=4.78°, which proved that the polymer chain was inserted into the MMT interlayer domain. Compared with the CS-Gel scaffold, the mechanical properties of the CS-Gel/GO/MMT scaffold were significantly improved. Concretely, the Young’s modulus was increased from 17.56 MPa to 22.73 MPa, and the compressive strength was increased from 0.14 MPa to 0.2265 MPa. The author believed that this benefit from the ability of GO and MMT to improve the mechanical stability of the scaffold. Among them, GO has a significantly larger surface area and abundant oxygen-containing functional groups, which provided more landing sites for the attachment of polymer chains. It was similar to the reports of other researchers such as Ionita et al. [55]. The interlayer domain structure of MMT is conducive to the intercalation of polymer molecular chains, which provide a higher degree of geometric constraints for the mobility of polymer chains.As we all know, the pore shape, pore size, and porosity of the scaffold are important factors that affect its mechanical properties [56, 57]. Generally, the pore size and porosity have a negative correlation with the mechanical properties. That is, the larger the pore size and porosity, the worse the mechanical properties of the scaffold. Regarding the influence of the pore shape on the mechanical properties, researchers mainly design the pore shape of the bracket from the perspective of bionics to obtain more excellent mechanical properties. In the process of polymer molecular chain intercalation with MMT, in addition to the abovementioned increase in contact area and mechanical interlocking function, the thickness of the pore wall of the matrix can also be increased, and even the uniformly dispersed MMT can also obtain a uniformly distributed pore size. These behaviors are expected to further contribute to the improvement of the mechanical performance of the scaffold. Zheng et al. [58, 59] have done a lot of research on the enhancement of mechanical properties of gelatin and chitosan intercalated MMT. It was found that after adding MMT, the pore wall of the scaffold became thicker, and the tensile strength and Young’s modulus increased. Unfortunately, as the content of MMT continues to increase, the pore morphology becomes irregular, and Young’s modulus and tensile strength decrease instead. Lee et al. [60] added 5.79 vol% MMT to the poly-L-lactic acid (PLLA) solution and then fabricated a double-pore scaffold structure through cold compression molding and electrospinning. During the biodegradation process, the developed nanocomposite fiber scaffold exhibited significantly better strength and structural integrity than the original PLLA one. Ali et al. [61] studied the effect of MMT (1-5%) concentration on the performance of chitosan/xylan (1 : 1) bone scaffolds. The results showed that when the content of MMT was less than 3 wt%, the porosity of composite scaffold increases with the increased content. XRD results indicated that it may be attributed to the intercalation of polymer chains into the interlayer domain of MMT. In addition, the composite scaffold with 5 wt% MMT can produce needle-like apatite in simulated body fluid, which further improves the scaffold’s mechanical properties, as well as the biological activity.


### 3.2. Strengthening and Toughening of MMT Nanosheets

MMT is a layered minerals composed of extremely fine-grained hydrous aluminosilicates. H2O molecules and exchangeable cations are linked by weak hydrogen bonds to form a hydrated state and exist in the interlayer space. The number of water molecular layers is related to the cation valence, up to four layers. Studies have showed that under the action of high-energy ball milling, the impact and friction of the grinding balls will disrupt the molecular interaction between the layers, thereby destroying the stability of the MMT layered structure. It will cause the exfoliation of the single-layer MMT crystals, thereby obtaining graphene-like MMT nanosheets with a large specific surface area [62], as shown in Figure 4(a). As the ball milling progresses, the exfoliation effect of MMT becomes more obvious, and finally, complete exfoliation is realized, which leads to the disordered structure of MMT. Although the ball milling method can obtain MMT nanosheets, it is worth noting that it takes a long time to exfoliation of the MMT, and the final product faces the risk of contamination.Figure 4
The formation of MMT nanosheets and the mechanism of strengthening polymer composites: (a) the exfoliation mechanism of MMT; (b) the mechanism of strengthening polymer materials by MMT nanosheets; (c) the crack strengthening and toughening mechanism of polymer/MMT composites.
(a)(b)(c)In order to overcome the abovementioned problems, some new technologies have been studied, especially ultrasound-assisted methods, as shown in Figure4(a). Generally speaking, the exfoliation of MMT usually occurs in a liquid medium and is achieved under the assistance of mechanical stirring and ultrasound [63, 64]. In particular, the high-frequency vibration energy provided by ultrasound makes the solution in the action area quickly form high-energy microjets, and the energy generated is much greater than the molecular force between MMT layers, so as to disrupt the connection between MMT layers and realize exfoliating to obtain MMT nanosheets [65, 66].The obtained graphene-like MMT nanosheets not only have a higher aspect ratio and surface area but are more easily entangled with polymer chains to significantly increase the contact area, thereby strengthening the mechanical properties of the polymer matrix [67], as shown in Figure 4(b). Scholars [68] have studied the properties of polyvinyl alcohol (PVA)/MMT nanocomposites synthesized in aqueous solutions. It was found that when the content of montmorillonite is 4 wt%, the Young’s modulus of nanocomposites was increased by 2.5-3 times, compared with pure PVA. Their explanation for the sharp increase in the Young’s modulus of the polymer matrix includes the fact that MMT exfoliation produces a high aspect ratio and a large contact area with the polymer. Kosalczyk et al. [69] proved that the addition of exfoliated MMT nanosheets to polymer significantly increased the flexural modulus and strength. In addition, the exfoliated MMT nanosheets also help to improve the tensile strength and Young’s modulus of the polymer by restricting the movement of the polymer chains around it [70, 71]. Habibi et al. [72] added MMT to chitosan/gelatin composites. MMT functions as a compatibilizer, reducing the interfacial tension of chitosan and gelatin, and enhancing compatibility. Generally, an increase in the interfacial tension of a polymer will result in a decrease in mechanical properties [73, 74]. Therefore, the exfoliated MMT nanosheets significantly enhance the interfacial interaction to reduce the interfacial tension, which might be the essential reason for the enhanced tensile strength of the polymer composites containing MMT.The crack evolution process of composites contains a variety of toughening mechanisms, including crack deflection, interface delamination, crack bridging, and interface friction [75], as shown in Figure 4. The maximum stress of the initial polymer is concentrated at the crack tip, and when the exfoliated MMT is evenly distributed in the polymer matrix, it can not only increase the friction between the interface and increase the energy required for fracture but also limit the crack propagation; Secondly, it allows the main crack to change the trajectory of the expansion and extension, ensuring that it is difficult for the crack to penetrate the entire polymer matrix, as shown in Figure 4(c)-3. In addition, the exfoliated MMT nanosheets can bridge the crack branches, allowing them to form a closing force to achieve toughness, as shown in Figure 4(c)-4. Simply put, the exfoliated MMT nanosheets can carry a large number of external forces, reduce the stress concentration of cracks, and effectively improve the fracture toughness of the polymer matrix. For example, Jiang et al. [76] prepared PLA nanocomposites by direct melt extrusion. The results showed that the strength, modulus, and elongation at break of the obtained nanocomposites containing 2.5 wt% MMT were increased by 2%, 14%, and 288%, respectively, compared with pure PLA.Although the mechanical properties of the polymer matrix are enhanced by the interlocking of polymer chains and the exfoliating of MMT, but unembedded, unpeeled, excessive MMT will form aggregation in the polymer, causing stress concentration and formation of fracture strain, leading to failure [77, 78]. Recently, members of Zhang et al.’s [79] research team prepared polypropylene (PP)/MMT nanocomposites. When MMT content reached to 3 wt%, the tensile strength of polymer matrix was increased from 42.3 MPa to 49.6 MPa. However, when MMT content was exceeded 3 wt%, the tensile strength decreases instead. It is because the excessive MMT is unevenly distributed in the polymer matrix, leading to the formation of agglomeration. The agglomerated MMT particles were weak spots and the entry point for crack development and expansion, which ultimately reduced the mechanical properties of the polymer-MMT composites. Nawang et al. [80] studied the addition of montmorillonite to nanohydroxyapatite nanocomposites, and the results showed that the addition of 10% MMT increased the flexural strength and compressive strength of the composite by 18.9% and 107.9%, respectively. But 20% MMT increased the flexural strength and compressive strength of the composite by only 17.1% and 63.1%. This means that 20% of MMT has a stress concentration in the matrix. When the stress applied by these stress-concentrated parts exceeds a certain limit, cracks will occur, and the cracks will spontaneously propagate, leading to material failure. Therefore, compatibility of the MMT nanolayer with the polymer should be improved, and the montmorillonite is usually treated with organic modification.


## 3.1. Molecular Chain Intercalation Strengthening

It is well known that the interlayer domain of MMT has a swelling effect. In a liquid environment or in a molten state, its interlayer domain expands and increases, making polymer molecular chains easy to achieve intercalation behavior, as shown in Figure3. In detail, in the mixed solution of MMT and polymer, MMT absorbs water to cause the expansion effect of the interlayer domain, so that the polymer molecular chain is successfully embedded in the MMT interlayer domain, while the other end of the molecular chain is miscible with the polymer matrix. In short, under the synergistic effect of covalent or noncovalent intermolecular interactions and the mutual compatibility of organic materials, intercalated molecular chains are expected to build the link between MMT and polymers. As a result, a molecular cross-linking bridge is built between inorganic and organic substances; that is, an interlocking structure is formed in the composite material. The existence of this interlocking structure can significantly hinder the movement of polymer chains when the composite material system is subjected to external forces, thereby increasing the interaction between the polymer and MMT. In addition, the embedding of molecular chains increases the space between the MMT layers to a certain extent, that is, increases the contact area between the MMT nanofiller and the polymer matrix. In the composite material system, the formation of interlocking structure increases the molecular force between the two phases, and the expansion of the contact area between the two phases increases the number of interlocking structures in the system. Under this synergistic effect, it is expected to significantly improve the mechanical properties of composite materials. In other words, using MMT as the nanofiller phase is expected to achieve significant effects in enhancing the mechanical properties of polymer bone scaffolds.Figure 3
Schematic diagram of polymer molecular chain intercalate into MMT.The use of MMT as nanofiller to enhance the mechanical properties of polymer matrix has been widely accepted. Mauro et al. [52] added 1%, 3%, and 10 wt% MMT to hydrogels based on peptidomimetic polyamidoamine. When the degree of crosslinking of the hydrogel was constant, the shear storage modulus improved as the increase of MMT content. The shear storage modulus of hydrogel with MMT content of 10 wt% reached 200 KPa, which was 20 times higher than the original hydrogel. It might be attributed to the double action of the electrostatic force and the increase in contact area. This dual effect makes the hydrogel and MMT complete the intercalation structure, forming a complex and interconnected network. Paluszkiewicz et al. [53] prepared composite materials by adding 5 wt% MMT to chitosan (CS) solution in an acidic environment. FTIR results showed that the Si-O-Si group of MMT formed hydrogen bonds with the hydroxyl and amino groups of CS. This strong interfacial interaction promotes the effective transfer of interfacial stress. Therefore, compared with pure CS, the tensile strength of CS-MMT is increased by about 10-12 MPa, and the Young’s modulus is increased by 20%. A recent study [48] found that when the content of MMT was less than 4.5 wt%, the tensile strength of the PLLA/MMT composite scaffold increased with the increase of the content; the highest increase was 44.1% (reach 26.8 MPa). However, as the content of MMT continues to increase, the growth trend of the mechanical properties of the composite scaffold begins to reverse, resulting in a decrease in the mechanical properties. Meanwhile, the author further studied the cross-sectional morphology of the composite material to explore the mechanism of the incorporation of MMT to enhance the mechanical properties of the composite material and found that the PLLA/MMT scaffold has many folds and torn ligaments on the fracture surface compared to the pure PLLA scaffold. These studies showed that, as a nanofiller phase, MMT exhibited an amazing effect in enhancing the mechanical properties of a single polymer matrix.In addition, some studies have explored the influence of MMT as an incorporation on the mechanical properties of polymer composites. Bee et al. [54] added MMT to a composite matrix of polylactic acid (PLA) and naturally derived hydroxyapatite (HAp) in the molten state to form a ternary composite material, focusing on the mechanical properties of the composite material. They found that when the MMT content was 2%, the increase in tensile strength was most significant and believed that the improvement in the tensile properties of the composite material was mainly due to the following aspects: Firstly, the increase in the interfacial tension between PLA and HAp promoted PLA intercalation of molecules in the interlayer domain of MMT. Secondly, MMT nanoparticles restricted the migration of PLA molecules through their interlayer domains, thereby enhancing the PLA matrix. Thirdly, mutual repulsion at the interface between PO43- of nonpolar HAp and the polar site of MMT promoted the dispersion of MMT, thereby further releasing the potential of nanostrengthening effect.Olad et al. [51] studied the synergistic effect of graphene oxide (GO) and MMT on the performance of chitosan-gelatin (CS-Gel) scaffolds. XRD results showed that the characteristic peak of montmorillonite was located at 2θ=7.48°, while the characteristic peaks of MMT in CS-Gel/MMT and CS-Gel/GO/MMT were located at 2θ=4.98° and 2θ=4.78°, which proved that the polymer chain was inserted into the MMT interlayer domain. Compared with the CS-Gel scaffold, the mechanical properties of the CS-Gel/GO/MMT scaffold were significantly improved. Concretely, the Young’s modulus was increased from 17.56 MPa to 22.73 MPa, and the compressive strength was increased from 0.14 MPa to 0.2265 MPa. The author believed that this benefit from the ability of GO and MMT to improve the mechanical stability of the scaffold. Among them, GO has a significantly larger surface area and abundant oxygen-containing functional groups, which provided more landing sites for the attachment of polymer chains. It was similar to the reports of other researchers such as Ionita et al. [55]. The interlayer domain structure of MMT is conducive to the intercalation of polymer molecular chains, which provide a higher degree of geometric constraints for the mobility of polymer chains.As we all know, the pore shape, pore size, and porosity of the scaffold are important factors that affect its mechanical properties [56, 57]. Generally, the pore size and porosity have a negative correlation with the mechanical properties. That is, the larger the pore size and porosity, the worse the mechanical properties of the scaffold. Regarding the influence of the pore shape on the mechanical properties, researchers mainly design the pore shape of the bracket from the perspective of bionics to obtain more excellent mechanical properties. In the process of polymer molecular chain intercalation with MMT, in addition to the abovementioned increase in contact area and mechanical interlocking function, the thickness of the pore wall of the matrix can also be increased, and even the uniformly dispersed MMT can also obtain a uniformly distributed pore size. These behaviors are expected to further contribute to the improvement of the mechanical performance of the scaffold. Zheng et al. [58, 59] have done a lot of research on the enhancement of mechanical properties of gelatin and chitosan intercalated MMT. It was found that after adding MMT, the pore wall of the scaffold became thicker, and the tensile strength and Young’s modulus increased. Unfortunately, as the content of MMT continues to increase, the pore morphology becomes irregular, and Young’s modulus and tensile strength decrease instead. Lee et al. [60] added 5.79 vol% MMT to the poly-L-lactic acid (PLLA) solution and then fabricated a double-pore scaffold structure through cold compression molding and electrospinning. During the biodegradation process, the developed nanocomposite fiber scaffold exhibited significantly better strength and structural integrity than the original PLLA one. Ali et al. [61] studied the effect of MMT (1-5%) concentration on the performance of chitosan/xylan (1 : 1) bone scaffolds. The results showed that when the content of MMT was less than 3 wt%, the porosity of composite scaffold increases with the increased content. XRD results indicated that it may be attributed to the intercalation of polymer chains into the interlayer domain of MMT. In addition, the composite scaffold with 5 wt% MMT can produce needle-like apatite in simulated body fluid, which further improves the scaffold’s mechanical properties, as well as the biological activity.


## 3.2. Strengthening and Toughening of MMT Nanosheets

MMT is a layered minerals composed of extremely fine-grained hydrous aluminosilicates. H2O molecules and exchangeable cations are linked by weak hydrogen bonds to form a hydrated state and exist in the interlayer space. The number of water molecular layers is related to the cation valence, up to four layers. Studies have showed that under the action of high-energy ball milling, the impact and friction of the grinding balls will disrupt the molecular interaction between the layers, thereby destroying the stability of the MMT layered structure. It will cause the exfoliation of the single-layer MMT crystals, thereby obtaining graphene-like MMT nanosheets with a large specific surface area [62], as shown in Figure 4(a). As the ball milling progresses, the exfoliation effect of MMT becomes more obvious, and finally, complete exfoliation is realized, which leads to the disordered structure of MMT. Although the ball milling method can obtain MMT nanosheets, it is worth noting that it takes a long time to exfoliation of the MMT, and the final product faces the risk of contamination.Figure 4
The formation of MMT nanosheets and the mechanism of strengthening polymer composites: (a) the exfoliation mechanism of MMT; (b) the mechanism of strengthening polymer materials by MMT nanosheets; (c) the crack strengthening and toughening mechanism of polymer/MMT composites.
(a)(b)(c)In order to overcome the abovementioned problems, some new technologies have been studied, especially ultrasound-assisted methods, as shown in Figure4(a). Generally speaking, the exfoliation of MMT usually occurs in a liquid medium and is achieved under the assistance of mechanical stirring and ultrasound [63, 64]. In particular, the high-frequency vibration energy provided by ultrasound makes the solution in the action area quickly form high-energy microjets, and the energy generated is much greater than the molecular force between MMT layers, so as to disrupt the connection between MMT layers and realize exfoliating to obtain MMT nanosheets [65, 66].The obtained graphene-like MMT nanosheets not only have a higher aspect ratio and surface area but are more easily entangled with polymer chains to significantly increase the contact area, thereby strengthening the mechanical properties of the polymer matrix [67], as shown in Figure 4(b). Scholars [68] have studied the properties of polyvinyl alcohol (PVA)/MMT nanocomposites synthesized in aqueous solutions. It was found that when the content of montmorillonite is 4 wt%, the Young’s modulus of nanocomposites was increased by 2.5-3 times, compared with pure PVA. Their explanation for the sharp increase in the Young’s modulus of the polymer matrix includes the fact that MMT exfoliation produces a high aspect ratio and a large contact area with the polymer. Kosalczyk et al. [69] proved that the addition of exfoliated MMT nanosheets to polymer significantly increased the flexural modulus and strength. In addition, the exfoliated MMT nanosheets also help to improve the tensile strength and Young’s modulus of the polymer by restricting the movement of the polymer chains around it [70, 71]. Habibi et al. [72] added MMT to chitosan/gelatin composites. MMT functions as a compatibilizer, reducing the interfacial tension of chitosan and gelatin, and enhancing compatibility. Generally, an increase in the interfacial tension of a polymer will result in a decrease in mechanical properties [73, 74]. Therefore, the exfoliated MMT nanosheets significantly enhance the interfacial interaction to reduce the interfacial tension, which might be the essential reason for the enhanced tensile strength of the polymer composites containing MMT.The crack evolution process of composites contains a variety of toughening mechanisms, including crack deflection, interface delamination, crack bridging, and interface friction [75], as shown in Figure 4. The maximum stress of the initial polymer is concentrated at the crack tip, and when the exfoliated MMT is evenly distributed in the polymer matrix, it can not only increase the friction between the interface and increase the energy required for fracture but also limit the crack propagation; Secondly, it allows the main crack to change the trajectory of the expansion and extension, ensuring that it is difficult for the crack to penetrate the entire polymer matrix, as shown in Figure 4(c)-3. In addition, the exfoliated MMT nanosheets can bridge the crack branches, allowing them to form a closing force to achieve toughness, as shown in Figure 4(c)-4. Simply put, the exfoliated MMT nanosheets can carry a large number of external forces, reduce the stress concentration of cracks, and effectively improve the fracture toughness of the polymer matrix. For example, Jiang et al. [76] prepared PLA nanocomposites by direct melt extrusion. The results showed that the strength, modulus, and elongation at break of the obtained nanocomposites containing 2.5 wt% MMT were increased by 2%, 14%, and 288%, respectively, compared with pure PLA.Although the mechanical properties of the polymer matrix are enhanced by the interlocking of polymer chains and the exfoliating of MMT, but unembedded, unpeeled, excessive MMT will form aggregation in the polymer, causing stress concentration and formation of fracture strain, leading to failure [77, 78]. Recently, members of Zhang et al.’s [79] research team prepared polypropylene (PP)/MMT nanocomposites. When MMT content reached to 3 wt%, the tensile strength of polymer matrix was increased from 42.3 MPa to 49.6 MPa. However, when MMT content was exceeded 3 wt%, the tensile strength decreases instead. It is because the excessive MMT is unevenly distributed in the polymer matrix, leading to the formation of agglomeration. The agglomerated MMT particles were weak spots and the entry point for crack development and expansion, which ultimately reduced the mechanical properties of the polymer-MMT composites. Nawang et al. [80] studied the addition of montmorillonite to nanohydroxyapatite nanocomposites, and the results showed that the addition of 10% MMT increased the flexural strength and compressive strength of the composite by 18.9% and 107.9%, respectively. But 20% MMT increased the flexural strength and compressive strength of the composite by only 17.1% and 63.1%. This means that 20% of MMT has a stress concentration in the matrix. When the stress applied by these stress-concentrated parts exceeds a certain limit, cracks will occur, and the cracks will spontaneously propagate, leading to material failure. Therefore, compatibility of the MMT nanolayer with the polymer should be improved, and the montmorillonite is usually treated with organic modification.


## 4. Organic Modification of Montmorillonite

Polymer/montmorillonite is the most promising type of nanocomposite among many inorganic nanoparticles modified composites. Due to the unique layered one-dimensional nanostructure characteristics and morphological characteristics of montmorillonite, its interlayer space has designable reactivity, a large specific surface area (up to 750 m2/g), and a diameter/thickness ratio of more than 200. This nanostructure and morphological characteristics endow polymer materials with excellent mechanical properties, thermal properties, functional properties, and other physical properties. However, since MMT and polymer are inorganic and organic, respectively, the inherent differences in physical and chemical properties between them are likely to lead to poor compatibility, and defects such as uneven dispersion, aggregation, and stress concentration are likely to occur in composites. The existence of these defects weakens the effect of MMT nanofillers in enhancing the mechanical properties of the polymers. Recently, one of the effective methods to improve interface compatibility and promote uniform dispersion of fillers was organic modification. As shown in Table 2, modified MMT to improve the mechanical properties of polymers has been favored by many scholars, and the effect can be seen. The methods of organic modification of MMT mainly include surface graft modification and cation exchange modification. This section summarized the latest progress on organically modified MMT.Table 2
Mechanical properties of polymers increased by modified MMT.
MMT content (wt%)Polymer matrixOrganic modifierMechanical performance enhancement resultsReferences0.4Polytetrafluoroethylene/glass fiberQuaternary ammonium compoundsTensile strength achieved 25.5 MPa, increased by 48%[43]1.0Poly methyl methacrylateDimethyl dihydrogenated tallow ammoniumModulus increased by 76%[80]Methyl tallow bis-hydroxyethyl quaternary ammoniumModulus increased by 82% and toughness achieved 1256 J/m3Poly(ε-caprolactone)/poly (lactic acid)Methyl, bis-hydroxyethyl, and octadecyl ammoniumTensile strength and elongation at break increased by 48% and 78%, respectively[81]Polyethylene/polybutene-1Dimethyl dehydrogenated tallowOrganoclay reduced the interparticle distance and enhanced interface interaction[82]5.0Poly (ester amide)Organo-polyaniline nanofibrous chainsTensile strength achieved 14.3 MPa, increased by 98%[83]Poly(ε-caprolactone)/cellulose acetate butyrateMethyl dihydroxyl ethyl hydrogenated tallow ammoniumTensile modulus achieved 471 MPa, increased by 37%[84]Poly (hydroxybutyrate-co-hydroxyvalerate)Cloisite-30BYoung’s modulus achieved7.1±1.9 MPa, increased by 103%[85]10Chitosan/polygalacturonic acidAmino valeric acidCompressive modulus increased by 400%[86]Poly (lactic acid)Cloisite-30BYoung’s modulus was increased up to 5140(±210) MPa from 3800(±130) MPa[87]Poly(ε-caprolactone)(3-aminopropyl) triethoxysilaneTensile strength increased 3.46 times[47]Poly(ether ether ketone)Octadecyltrimethyl ammonium chlorideFlexural strength achieved 200 MPa. Yield strength achieved 105 MPa, increased by 10.5%[88]


### 4.1. Surface Graft Modification of MMT

Surface graft modification refers to the chemical reaction between nanoparticle surface functional groups and modifier molecules. Due to the covalent combination of organic and inorganic functional groups in the surface graft modification, it can not only change the surface structure and properties of nanoparticles but also make them have higher chemical stability [81]. Grafting is a feasible way to covalently immobilize organic functional groups on the surface of montmorillonite. During the grafting process, the surface hydroxyl groups of MMT provide a large number of anchor points for organic functionalization.The surface of MMT has a large number of hydroxyl groups, which is inherently hydrophilic, and has a weak affinity with most hydrophobic substrates [91]. However, surface-grafted modified MMT can better enhance the affinity between functional groups. Therefore, surface modification has become an important method to adjust material properties [92]. Grafting modification can occur on the outer surface of MMT without changing the basal plane spacing or on its interlayer space or “broken” edge, thereby expanding its interlayer space spacing [93–95]. Zulfiqar et al. [96] used aminobenzoic acid to modify MMT. They grafted amino (-NH2) functional groups on the hydroxyl groups on the outer surface of MMT. The results showed that the distance between the layers of MMT did not change. However, in the study of He et al. [93], they used the grafting reaction of the silylation reagent 3-aminopropyltriethoxysilane (APTES) with MMT. X-ray diffraction results showed that the interlayer spacing of MMT before grafting was 1.18 nm, and the interlayer spacing after grafting increased to 1.77 nm. Su et al. [97] also used APTES to silanize MMT. On the one hand, the interlayer spacing of montmorillonite increased up to 2.09 nm from 1.48 nm during silanization, as shown in Figure 5. At the same time, the modifiers react with each other to generate a siloxane bond, which causes an interlocking effect between the layers. This interlayer locking fixes the height of the MMT interlayer space, which is more convenient for the polymer to perform intercalation behavior. Besides, graft modification of MMT is also affected by factors such as the type of modifier and solvent and the degree of modification. For example, cetyltrimethylammonium modified MMT has poor dispersibility in alcohol with shorter carbon chains; on the contrary, it has better dispersibility in alcohol with longer carbon chains [98].Figure 5
Schematic diagram of the effect of APTES silanization on the interlayer space of montmorillonite.The surface modification of MMT has been proven to be an effective method to enhance the compatibility and uniform dispersion of nanofillers with polymers. Yang et al. [44] modified MMT (QACMMT) with quaternary ammonium compounds and used it to reinforce polytetrafluoroethylene/glass fiber (PTFE/GF) composites. They evaluated the dispersibility and dispersion stability of MMT and QACMMT in water. The results showed that pure MMT still aggregated after standing for 60 minutes. By comparison, QACMMT appears as more uniformly dispersed particles in the aqueous solution without stratification, indicating stable quality and good dispersion in water. In addition, QACMMT modified composites exhibit the highest tensile strength, modulus, fracture strain, and toughness. This is attributed to the good dispersibility of QACMMT in the polymer matrix, while enhancing the interface compatibility between glass fiber and polytetrafluoroethylene. Zhu et al. [83] studied the effects of surface-modified montmorillonite on the microphase dispersion and interfacial interaction in polycaprolactone PCL/polylactic acid PLA composites. The results showed that the modified montmorillonite not only significantly increased PCL/PLA interface interaction but also significantly improved the uniform dispersion of PLA. Meanwhile, the results also showed that the obtained PCL/PLA/MMT nanocomposite can be well balanced among Young’s modulus, tensile strength, and elongation. The reason was that the addition of MMT increases the interface interaction and more uniform phase dispersion, so that the polymer deforms simultaneously under stretching and has a higher elongation and tensile strength. Khosravi and Eslami-Farsani [99] used 3-glycidylpropyltrimethoxysilane (3-GPTS) to modify the mechanical properties of nanomontmorillonite (Na-MMT) on unidirectional basalt fiber (UDBF)/epoxy resin composites. The results showed that after adding 5 wt% of 3-GPTS/MMT, the flexural strength, tensile strength, and compressive strength increased by 28%, 11%, and 35%, respectively. The results showed that the adhesion between the modified MMT-enhanced composite samples was improved.Recently, the modification of polymers by incorporating two nanofillers at the same time has received great attention, and the existence of synergistic effects has been demonstrated [100–102]. For example, the combination of carbon nanotubes (CNT) and MMT has been successfully used to improve the mechanical properties of polymers, where CNTs are selectively distributed in the polymer matrix, while organic MMTs are distributed at the interface junction [103]. Zhu et al. [104] studied the modified MMT and carbon nanotubes (CNT) to synergistically improve the strength and toughness of the polymer. The modulus, tensile strength, and toughness of the polymer matrix had been greatly improved under the combined action of CNT and MMT. This was attributed to the good dispersion of the organically modified nanofillers; a large number of microcracks and an extremely stretched polymer matrix were generated during the stretching process, which dissipated a large amount of energy. Zhang et al. [105] prepared a CNT/MMT hybrid by growing CNT in situ on MMT. CNT/MMT hybrid material showed excellent reinforcement effect on polymer matrix such as nylon 6 [106].The above research provided a solid theoretical basis and sufficient reference for the surface grafting modified MMT to optimize the performance of the polymer matrix. The unique molecular structure and high specific surface area of MMT are not only conducive to graft functionalization but also become the best filler for the target polymer matrix, thereby forming high-quality composite materials.


### 4.2. Cation Exchange Modification

As we all know, the cations (mainly Na+) in the interlayer space of MMT are only loosely immobilized by the negative charges on the surface, commonly known as hydrated cations. Therefore, the cations between the layers remain exchangeable, as shown in Figure 6. In addition, the electrostatic interaction between the negative charges on the inner surface of the MMT and the organic cations or the hydrogen bonds formed on the inner surface of the organic chains drive the exchange of cations in the interlayer space [107, 108]. The cation exchange modification of MMT (OMMT) can occur in suspension, semisolid or solid state [109, 110]. In particular, it was currently the preferred method to prepare cationic modified MMT in water suspension [111]. In the water dispersion, with the aid of mechanical stirring or ultrasound, organic cations can also be quickly introduced into the intermediate layer of MMT at room temperature. In addition, cationic modification of MMT can be assisted by microwave-assisted heating. For example, Peng et al. [112] prepared OMMT by a mixed suspension of dioctadecyl tetrahydroxyethyl dibromopropane diammonium and MMT in a microwave-assisted heating system. Luo et al. [113] modified MMT with butane-1,4-bis (dodecyldimethylammonium bromide) through the synergistic effect of microwave and ultrasound.Figure 6
Schematic diagram of cation exchange of MMT.The cationic modification of montmorillonite is affected by the length of the organic cationic chain, the type of functional group, and the number of cations [79, 114, 115]. Cationic organic long carbon chain modifiers are more advantageous. Studies have clearly showed that when organic modifiers with longer carbon chains are inserted into the interlayer space of MMT, its intercalation/exfoliation was more likely to occur [116]. In contrast, organic modifiers with short chains (such as methacryloyl ethyl trimethyl ammonium chloride) are not closely arranged in the interlayer space of MMT, and do not effectively expand the interlayer space. MMT interlayer cation exchange capacity (CEC) can also affect its ion exchange performance [117, 118]. MMT with high CEC is generally easier to allow organic chains to enter the interlayer space and produce a large interlayer spacing, thereby making it easier to form an intercalation/exfoliation structure. With the increase of chain length, chain number, and cationic modifier loading, the base spacing of MMT also increases [119–121]. For example, when the cation exchange capacity of MMT was 4.0CEC; substrate spacing for adding cetyltrimethylammonium bromide and octyltrimethylammonium chloride was 3.9 nm and 4.1 nm [122]. When the modifier added to MMT is 4.0CEC, the basic spacing formed by cationic modifiers with different alkyl chain numbers (octadecyl trimethylammonium bromide and dioctadecyl-dimethylammonium bromide) was 4.1 nm and 5.5 nm, respectively. In addition, the alkyl chains of some organic cationic modifiers are arranged in parallel in the interlayer domain of MMT [123].Changing types of cations between MMT layers are also an important way and method to improve its uniform dispersion in the polymer matrix. The cation exchange of MMT reduces its surface energy and at the same time reduces the interface energy between it and the polymerization, making it easier to achieve intercalation and exfoliation, resulting in a more uniform dispersion of MMT in the polymer matrix to enhance mechanical properties. Cationic modified clay minerals had been adopted by more and more researchers to improve the mechanical properties of the matrix. In the study of Hamidabadi et al. [124], chitosan-modified MMT was used to enhance the mechanical properties of PVA.The results showed that the tensile properties, initial modulus, and toughness of the composite had been significantly improved. Shuai et al. [49] used (3-aminopropyl)-triethoxysilane (KH550) to organically modify MMT to enhance polycaprolactone (PCL). The positively charged end of KH550 entered the interlayer of montmorillonite through cation exchange, and the end with long alkyl carbon chain extended in the opposite direction. With the extension of long chains, the interlayer spacing of montmorillonite increased, making the intercalation of PCL molecular chains more convenient. This kind of ion exchange with MMT can often produce new bonds (such as hydrogen bonds) to prevent the movement of polymer molecular chains, thereby enhancing the interface bonding ability. The mechanical performance test results show that the modified MMT can significantly enhance the tensile and compressive properties of the polymer matrix. It was also found that the dispersion ability of modified MMT was significantly higher than that of unmodified MMT in the PCL matrix.In addition to improving the mechanical properties of polymer scaffolds, MMT is biocompatible and nontoxic in nature. Studies have shown that it can improve the ability of cell adhesion, migration, and proliferation on polymer scaffolds without causing positive immune responses and severe inflammatory reactions [125]. Relevant studies are summarized in Table 3. In general, MMT mainly affects the biocompatibility of scaffolds from the following aspects. Firstly, -OH, Si-O-Si, and other oxygen-containing groups of MMT can adjust the hydrophilicity of the surface of the scaffold to promote cell adhesion and growth. Secondly, the modified MMT can not only improve the hydrophobicity of scaffold surface but also can impart a positive charge to the surface scaffold, thereby further improving the biocompatibility of scaffolds. In addition, related studies have shown that modified MMT can not only enhance the mechanical properties of scaffolds but also improve its osteogenic properties [126, 127]. For example, Demir’s [128] research team prepared a porous scaffold of Zn-doped PCL-MMT. The results showed that the in vitro proliferation and osteogenic differentiation of human adipose stem cells (hASCs) were improved. Kundu et al. [129] found that the addition of MMT significantly increased the alkaline phosphatase (ALP) expression of mesenchymal stem cells (MSCs). Kim et al. [130] studied the effect and mechanism of nano-MMT on the differentiation of osteoblasts and osteoclasts in vivo and in vitro. On the one hand, nano-MMT improves the activity of ALP and the expression of related genes in osteoblasts, thereby stimulates the proliferation and differentiation of osteoblasts. On the other hand, nano-MMT repressed the expression of receptor activator for cathepsin K, nuclear factor kappa-B ligand, and tartrate-resistant acid phosphatase, which hinders the growth and reproduction of osteoclasts.Table 3
Montmorillonite enhances polymer biocompatibility.
MMT contentPolymer matrixBiocompatibility enhancement resultsReferences1.5 wt%ChitosanRecruit native cells and promote calvarial healing[37]3 wt%PLLA/PGAPromote MG-63 cell adhesion and proliferation[131]5 wt%Polyvinyl alcoholIncreased the water absorbency[132]10 wt%Poly(butylene adipate-co-terephthalate)Showed good biological safety, noncytotoxicity, and high hemocompatibility[133]22.2 wt%PLGAHighly biocompatible and nontoxic[134]1.67μg/mlChitosanShowed good biocompatibility and effectively stimulate cell proliferation[135]103ppmChitosanPromote proliferation of fibroblasts[136]6 g—Promote gene expression[137]Some researchers have used modified MMT to enhance the compatibility of two incompatible materials to enhance mechanical properties [138]. For example, in order to improve compatibility between PLLA and PGA, Yu et al. [131] modified composite materials by modifying MMT with octedacy trimethyl ammonium chloride. It is worth noting that the NH2 group in the modified MMT not only formed a hydrogen bond with the OH group of PLLA but also formed a hydrogen bond with the OH group of PGA, and the three formed an interlocking structure. Therefore, through the effect of cationic modification of MMT, the compatibility of PLLA and PGA was improved. In terms of mechanical properties, the tensile performance of the PLLA/PGA stent with cationic modified MMT was nearly doubled compared with the composite stent without addition. Bozolan’s research team [139] studied the effect of L-phenylalanine-modified montmorillonite on the mechanical properties of chitosan/carboxymethylcellulose/scleroglucan composite hydrogels. The results showed that the compressive stress values of hydrogel materials containing 0%, 1%, 3%, and 5% MMT were 126.45±8.83 kPa, 152.31±2.72 kPa, 210.94±2.36 kPa, and 266.99±6.60 kPa, respectively. The result was consistent with Liu [140] and Mohd’s [141]. In bone tissue engineering, some metal cations can not only promote the activity and proliferation of osteoblasts but also have anti-inflammatory and antibacterial effects [142, 143]. For example, the replaced silver ion undergoes an oxidation-reduction reaction in MMT, and formed metallic silver had antibacterial properties [144–146]. This showed that the organically modified nanoclay has abundant functional group combination; therefore, MMT was often used as a carrier for drug loading and sustained drug release.


## 4.1. Surface Graft Modification of MMT

Surface graft modification refers to the chemical reaction between nanoparticle surface functional groups and modifier molecules. Due to the covalent combination of organic and inorganic functional groups in the surface graft modification, it can not only change the surface structure and properties of nanoparticles but also make them have higher chemical stability [81]. Grafting is a feasible way to covalently immobilize organic functional groups on the surface of montmorillonite. During the grafting process, the surface hydroxyl groups of MMT provide a large number of anchor points for organic functionalization.The surface of MMT has a large number of hydroxyl groups, which is inherently hydrophilic, and has a weak affinity with most hydrophobic substrates [91]. However, surface-grafted modified MMT can better enhance the affinity between functional groups. Therefore, surface modification has become an important method to adjust material properties [92]. Grafting modification can occur on the outer surface of MMT without changing the basal plane spacing or on its interlayer space or “broken” edge, thereby expanding its interlayer space spacing [93–95]. Zulfiqar et al. [96] used aminobenzoic acid to modify MMT. They grafted amino (-NH2) functional groups on the hydroxyl groups on the outer surface of MMT. The results showed that the distance between the layers of MMT did not change. However, in the study of He et al. [93], they used the grafting reaction of the silylation reagent 3-aminopropyltriethoxysilane (APTES) with MMT. X-ray diffraction results showed that the interlayer spacing of MMT before grafting was 1.18 nm, and the interlayer spacing after grafting increased to 1.77 nm. Su et al. [97] also used APTES to silanize MMT. On the one hand, the interlayer spacing of montmorillonite increased up to 2.09 nm from 1.48 nm during silanization, as shown in Figure 5. At the same time, the modifiers react with each other to generate a siloxane bond, which causes an interlocking effect between the layers. This interlayer locking fixes the height of the MMT interlayer space, which is more convenient for the polymer to perform intercalation behavior. Besides, graft modification of MMT is also affected by factors such as the type of modifier and solvent and the degree of modification. For example, cetyltrimethylammonium modified MMT has poor dispersibility in alcohol with shorter carbon chains; on the contrary, it has better dispersibility in alcohol with longer carbon chains [98].Figure 5
Schematic diagram of the effect of APTES silanization on the interlayer space of montmorillonite.The surface modification of MMT has been proven to be an effective method to enhance the compatibility and uniform dispersion of nanofillers with polymers. Yang et al. [44] modified MMT (QACMMT) with quaternary ammonium compounds and used it to reinforce polytetrafluoroethylene/glass fiber (PTFE/GF) composites. They evaluated the dispersibility and dispersion stability of MMT and QACMMT in water. The results showed that pure MMT still aggregated after standing for 60 minutes. By comparison, QACMMT appears as more uniformly dispersed particles in the aqueous solution without stratification, indicating stable quality and good dispersion in water. In addition, QACMMT modified composites exhibit the highest tensile strength, modulus, fracture strain, and toughness. This is attributed to the good dispersibility of QACMMT in the polymer matrix, while enhancing the interface compatibility between glass fiber and polytetrafluoroethylene. Zhu et al. [83] studied the effects of surface-modified montmorillonite on the microphase dispersion and interfacial interaction in polycaprolactone PCL/polylactic acid PLA composites. The results showed that the modified montmorillonite not only significantly increased PCL/PLA interface interaction but also significantly improved the uniform dispersion of PLA. Meanwhile, the results also showed that the obtained PCL/PLA/MMT nanocomposite can be well balanced among Young’s modulus, tensile strength, and elongation. The reason was that the addition of MMT increases the interface interaction and more uniform phase dispersion, so that the polymer deforms simultaneously under stretching and has a higher elongation and tensile strength. Khosravi and Eslami-Farsani [99] used 3-glycidylpropyltrimethoxysilane (3-GPTS) to modify the mechanical properties of nanomontmorillonite (Na-MMT) on unidirectional basalt fiber (UDBF)/epoxy resin composites. The results showed that after adding 5 wt% of 3-GPTS/MMT, the flexural strength, tensile strength, and compressive strength increased by 28%, 11%, and 35%, respectively. The results showed that the adhesion between the modified MMT-enhanced composite samples was improved.Recently, the modification of polymers by incorporating two nanofillers at the same time has received great attention, and the existence of synergistic effects has been demonstrated [100–102]. For example, the combination of carbon nanotubes (CNT) and MMT has been successfully used to improve the mechanical properties of polymers, where CNTs are selectively distributed in the polymer matrix, while organic MMTs are distributed at the interface junction [103]. Zhu et al. [104] studied the modified MMT and carbon nanotubes (CNT) to synergistically improve the strength and toughness of the polymer. The modulus, tensile strength, and toughness of the polymer matrix had been greatly improved under the combined action of CNT and MMT. This was attributed to the good dispersion of the organically modified nanofillers; a large number of microcracks and an extremely stretched polymer matrix were generated during the stretching process, which dissipated a large amount of energy. Zhang et al. [105] prepared a CNT/MMT hybrid by growing CNT in situ on MMT. CNT/MMT hybrid material showed excellent reinforcement effect on polymer matrix such as nylon 6 [106].The above research provided a solid theoretical basis and sufficient reference for the surface grafting modified MMT to optimize the performance of the polymer matrix. The unique molecular structure and high specific surface area of MMT are not only conducive to graft functionalization but also become the best filler for the target polymer matrix, thereby forming high-quality composite materials.


## 4.2. Cation Exchange Modification

As we all know, the cations (mainly Na+) in the interlayer space of MMT are only loosely immobilized by the negative charges on the surface, commonly known as hydrated cations. Therefore, the cations between the layers remain exchangeable, as shown in Figure 6. In addition, the electrostatic interaction between the negative charges on the inner surface of the MMT and the organic cations or the hydrogen bonds formed on the inner surface of the organic chains drive the exchange of cations in the interlayer space [107, 108]. The cation exchange modification of MMT (OMMT) can occur in suspension, semisolid or solid state [109, 110]. In particular, it was currently the preferred method to prepare cationic modified MMT in water suspension [111]. In the water dispersion, with the aid of mechanical stirring or ultrasound, organic cations can also be quickly introduced into the intermediate layer of MMT at room temperature. In addition, cationic modification of MMT can be assisted by microwave-assisted heating. For example, Peng et al. [112] prepared OMMT by a mixed suspension of dioctadecyl tetrahydroxyethyl dibromopropane diammonium and MMT in a microwave-assisted heating system. Luo et al. [113] modified MMT with butane-1,4-bis (dodecyldimethylammonium bromide) through the synergistic effect of microwave and ultrasound.Figure 6
Schematic diagram of cation exchange of MMT.The cationic modification of montmorillonite is affected by the length of the organic cationic chain, the type of functional group, and the number of cations [79, 114, 115]. Cationic organic long carbon chain modifiers are more advantageous. Studies have clearly showed that when organic modifiers with longer carbon chains are inserted into the interlayer space of MMT, its intercalation/exfoliation was more likely to occur [116]. In contrast, organic modifiers with short chains (such as methacryloyl ethyl trimethyl ammonium chloride) are not closely arranged in the interlayer space of MMT, and do not effectively expand the interlayer space. MMT interlayer cation exchange capacity (CEC) can also affect its ion exchange performance [117, 118]. MMT with high CEC is generally easier to allow organic chains to enter the interlayer space and produce a large interlayer spacing, thereby making it easier to form an intercalation/exfoliation structure. With the increase of chain length, chain number, and cationic modifier loading, the base spacing of MMT also increases [119–121]. For example, when the cation exchange capacity of MMT was 4.0CEC; substrate spacing for adding cetyltrimethylammonium bromide and octyltrimethylammonium chloride was 3.9 nm and 4.1 nm [122]. When the modifier added to MMT is 4.0CEC, the basic spacing formed by cationic modifiers with different alkyl chain numbers (octadecyl trimethylammonium bromide and dioctadecyl-dimethylammonium bromide) was 4.1 nm and 5.5 nm, respectively. In addition, the alkyl chains of some organic cationic modifiers are arranged in parallel in the interlayer domain of MMT [123].Changing types of cations between MMT layers are also an important way and method to improve its uniform dispersion in the polymer matrix. The cation exchange of MMT reduces its surface energy and at the same time reduces the interface energy between it and the polymerization, making it easier to achieve intercalation and exfoliation, resulting in a more uniform dispersion of MMT in the polymer matrix to enhance mechanical properties. Cationic modified clay minerals had been adopted by more and more researchers to improve the mechanical properties of the matrix. In the study of Hamidabadi et al. [124], chitosan-modified MMT was used to enhance the mechanical properties of PVA.The results showed that the tensile properties, initial modulus, and toughness of the composite had been significantly improved. Shuai et al. [49] used (3-aminopropyl)-triethoxysilane (KH550) to organically modify MMT to enhance polycaprolactone (PCL). The positively charged end of KH550 entered the interlayer of montmorillonite through cation exchange, and the end with long alkyl carbon chain extended in the opposite direction. With the extension of long chains, the interlayer spacing of montmorillonite increased, making the intercalation of PCL molecular chains more convenient. This kind of ion exchange with MMT can often produce new bonds (such as hydrogen bonds) to prevent the movement of polymer molecular chains, thereby enhancing the interface bonding ability. The mechanical performance test results show that the modified MMT can significantly enhance the tensile and compressive properties of the polymer matrix. It was also found that the dispersion ability of modified MMT was significantly higher than that of unmodified MMT in the PCL matrix.In addition to improving the mechanical properties of polymer scaffolds, MMT is biocompatible and nontoxic in nature. Studies have shown that it can improve the ability of cell adhesion, migration, and proliferation on polymer scaffolds without causing positive immune responses and severe inflammatory reactions [125]. Relevant studies are summarized in Table 3. In general, MMT mainly affects the biocompatibility of scaffolds from the following aspects. Firstly, -OH, Si-O-Si, and other oxygen-containing groups of MMT can adjust the hydrophilicity of the surface of the scaffold to promote cell adhesion and growth. Secondly, the modified MMT can not only improve the hydrophobicity of scaffold surface but also can impart a positive charge to the surface scaffold, thereby further improving the biocompatibility of scaffolds. In addition, related studies have shown that modified MMT can not only enhance the mechanical properties of scaffolds but also improve its osteogenic properties [126, 127]. For example, Demir’s [128] research team prepared a porous scaffold of Zn-doped PCL-MMT. The results showed that the in vitro proliferation and osteogenic differentiation of human adipose stem cells (hASCs) were improved. Kundu et al. [129] found that the addition of MMT significantly increased the alkaline phosphatase (ALP) expression of mesenchymal stem cells (MSCs). Kim et al. [130] studied the effect and mechanism of nano-MMT on the differentiation of osteoblasts and osteoclasts in vivo and in vitro. On the one hand, nano-MMT improves the activity of ALP and the expression of related genes in osteoblasts, thereby stimulates the proliferation and differentiation of osteoblasts. On the other hand, nano-MMT repressed the expression of receptor activator for cathepsin K, nuclear factor kappa-B ligand, and tartrate-resistant acid phosphatase, which hinders the growth and reproduction of osteoclasts.Table 3
Montmorillonite enhances polymer biocompatibility.
MMT contentPolymer matrixBiocompatibility enhancement resultsReferences1.5 wt%ChitosanRecruit native cells and promote calvarial healing[37]3 wt%PLLA/PGAPromote MG-63 cell adhesion and proliferation[131]5 wt%Polyvinyl alcoholIncreased the water absorbency[132]10 wt%Poly(butylene adipate-co-terephthalate)Showed good biological safety, noncytotoxicity, and high hemocompatibility[133]22.2 wt%PLGAHighly biocompatible and nontoxic[134]1.67μg/mlChitosanShowed good biocompatibility and effectively stimulate cell proliferation[135]103ppmChitosanPromote proliferation of fibroblasts[136]6 g—Promote gene expression[137]Some researchers have used modified MMT to enhance the compatibility of two incompatible materials to enhance mechanical properties [138]. For example, in order to improve compatibility between PLLA and PGA, Yu et al. [131] modified composite materials by modifying MMT with octedacy trimethyl ammonium chloride. It is worth noting that the NH2 group in the modified MMT not only formed a hydrogen bond with the OH group of PLLA but also formed a hydrogen bond with the OH group of PGA, and the three formed an interlocking structure. Therefore, through the effect of cationic modification of MMT, the compatibility of PLLA and PGA was improved. In terms of mechanical properties, the tensile performance of the PLLA/PGA stent with cationic modified MMT was nearly doubled compared with the composite stent without addition. Bozolan’s research team [139] studied the effect of L-phenylalanine-modified montmorillonite on the mechanical properties of chitosan/carboxymethylcellulose/scleroglucan composite hydrogels. The results showed that the compressive stress values of hydrogel materials containing 0%, 1%, 3%, and 5% MMT were 126.45±8.83 kPa, 152.31±2.72 kPa, 210.94±2.36 kPa, and 266.99±6.60 kPa, respectively. The result was consistent with Liu [140] and Mohd’s [141]. In bone tissue engineering, some metal cations can not only promote the activity and proliferation of osteoblasts but also have anti-inflammatory and antibacterial effects [142, 143]. For example, the replaced silver ion undergoes an oxidation-reduction reaction in MMT, and formed metallic silver had antibacterial properties [144–146]. This showed that the organically modified nanoclay has abundant functional group combination; therefore, MMT was often used as a carrier for drug loading and sustained drug release.


## 5. Drug Loading-Release of MMT

Common problems such as bacterial infections in bone tissue engineering greatly increase the risk of bone repair [147]. Drug delivery is one of the most important applications of organic functionalized montmorillonite. After proper modification, MMT has received more and more attention as a drug carrier and drug sustained release agent. It can deliver drugs continuously, controllable and targeted, thereby improving its therapeutic effect.The sustained release of drugs in montmorillonite usually requires drug molecules to be inserted into the interlayer space of montmorillonite, as shown in Figure7. Otherwise, simple surface adsorption will result in a faster release rate. It may be attributed to the fact that the layered structure of MMT not only hinders the external erosion of drug but also inhibits the diffusion of drug from the interlayer domain to outside. Sadeghianmaryan et al. [148] studied a spinning scaffold made of polycaprolactone/montmorillonite nanocomposites loaded with curcumin (Cur). The results showed that by introducing MMT into the polymer matrix, the sudden release level was reduced, and PCL/20% MMT had the lowest curcumin release. Silver nanoparticles are an effective antibacterial agent, with strong bactericidal activity and a wide range of bactericidal types and no bacteria resistance [149, 150]. However, Ag-based antibacterial materials face some problems. Ag nanoparticles are easy to aggregate in the polymer matrix and lead to rapid release of Ag+. Guo et al. [151] loaded Ag+ into the interlayer space of MMT through ion exchange and was further chemically reduced to metal Ag with higher stability. When the MMT loaded with Ag was added to the PGA stent manufactured by 3D printing, the Ag exhibited uniform dispersion, and the composite scaffold exhibited antibacterial performance of sustained release of Ag.Figure 7
Drug loading/release of MMT.In order to further evaluate the release mechanism of the drug in MMT, people usually fit the drug release data to the kinetic formula, such as Korsmeyer-Peppas model, Higuchi model, and zero-order and first-order, as shown in Table4 [152, 153]. Among them, Higuchi model [154] (Equation (1)) and Korsmeyer-Peppas model [155] (Equation (2)) were the most commonly used in the research of MMT drug loading and release. According to the research of Panahi et al. [156] and Li et al. [157], under simulated gastric juice and simulated intestinal juice, the release kinetics of clarithromycin and dexibuprofen in MMT were confirmed to be consistent with the Higuchi model. In addition, some studies have concluded that the diffusion mechanism of drugs in MMT can be explained by the Korsmeyer-Peppas model. Among them, 0.45≤n corresponds to Fickian diffusion mechanism, 0.45<n<0.89 corresponds to non-Fickian transport, n=0.89 corresponds to case II transport, and n>0.89 corresponds to super case II transport [158, 159].
(1)ft=Q=AD2C−CsCst,Table 4
Kinetic models and mechanisms of montmorillonite release drugs.
DrugsKinetic modelsDiffusional release mechanismsReferences5-FluorouracilKorsmeyer-PeppasFickian diffusion[164]ChlorpromazineNon-Fickian diffusion[165]Hydrophobic ibuprofenNon-Fickian diffusion[166]Sodium diclofenacCase II transport[167]CiprofloxacinSuper case II transport[168]DexibuprofenHiguchiNon-Fickian diffusion[157]Tanshinone IIANon-Fickian diffusion[169]ClarithromycinFickian diffusion[156]Sodium diclofenacZero-orderSuper case II transport[170]5-FluorouracilFirst-order—[171]CurcuminPower lawNon-Fickian diffusion[148]Paracetamol PaRitger–PeppasNon-Fickian diffusion[172]whereQ is the amount of drug released in time t per unit area A. C is the drug initial concentration. Cs is the drug solubility in the matrix media, and D is the diffusivity of the drug molecules (diffusion coefficient) in the matrix substance.
(2)MtM∞=Ktn,whereMt/M∞ is a fraction of drug released at time t. k is the release rate constant, and n is the release exponent.The release of drugs in MMT is mainly affected by the following factors. Firstly, when the drug-carrying system is exposed to the dissolving medium, the concentration gradient of the drug is the main driving force, which makes it exhibit explosive release behavior in the early period [160]. Secondly, the interlayer structure of MMT creates interconnected and tortuous barriers, which hinders the dissolution and diffusion of drug molecules and reduces the release rate of drugs [161]. Furthermore, there are intermolecular forces such as hydrogen bonds [156], covalent bonds [162], and ionic bonds [163] between drug molecules and MMT, which further hinder the release of drugs.The sustained release of drugs in MMT depends on its content, the increase of interlayer spacing, and its dispersion in the matrix [160]. The research results of Bakre et al. [173] showed that in polycaprolactone/montmorillonite composite materials, as the concentration of MMT increases, the sustained release rate of curcumin slows down. The increased interlayer spacing after modification of MMT can optimize the sustained release performance. Yu et al. [174] used cetyltrimethylammonium bromide (CTAB) modified natural sodium montmorillonite (Na-MMT) as a carrier to carry the drug-amoxicillin (AMX) for tissue engineering. The results showed that the cumulative release rate of AMX with modified MMT at 75 h was only 45%, while the cumulative release rate of AMX scaffold without modified MMT at 75 h reaches 80%. Bounabi et al. [172] developed a drug delivery carrier for paracetamol, which was composed of a hydrogel composed of poly (2-hydroxyethyl methacrylate) and MMT. Among them, MMT was used as a polymer crosslinking agent, which prolongs the release time of paracetamol, delays the clearance of the drug, and reduces the need for multiple administrations.


## 6. Conclusions and Prospects

The unique “sandwich” structure of montmorillonite can not only interlock with polymer molecular chains to enhance the mechanical properties of the polymer but also can be exfoliated into graphene-like nanosheets to uniformly disperse and enhance the mechanical properties of the polymer. For the interlayer space of polymer molecular chain intercalation MMT, firstly, under the complex action of MMT and water molecules, the basic distance between the interlayer space increases. Then, one end of the polymer molecular chain forms a bond with the MMT under the action of electrostatic force or van der Waals force, while the other end forms a common compatibility with the polymer and finally forms an interlocking structure. Ball milling and ultrasound-assisted methods usually destroy the stability of the MMT structure and exfoliate it into a graphene-like nanosheet structure. The peeled MMT sheet not only has a higher aspect ratio but is also easier to be entangled with the polymer chain, so that the contact area of the polymer-MMT composite is increased, and the fracture consumes more energy. In addition, the exfoliated graphene-like montmorillonite nanosheets have a good effect on resisting crack propagation.The organic modification of MMT includes surface graft modification and cation exchange modification. The modified MMT can not only achieve a surprising dispersion effect in the polymer matrix but also act as a compatibilizer to improve interfacial compatibility, providing an effective method for improving the performance of the polymer matrix. In addition, the organically modified MMT has abundant functional groups, providing a natural platform for drug loading and sustained drug release. The combination of these two aspects is of great significance to the development of ultra-high-performance and low-cost bone tissue engineering scaffolds.Undoubtedly, montmorillonite has a positive effect on improving the performance of bone tissue engineering scaffolds. However, for polymer-MMT nanomaterials, researches on MMT functionalized bone tissue artificial scaffolds are in the ascendant. Firstly, more new assemblies of MMT nanomaterials and polymer materials should be discovered to provide more advanced functional materials for bone scaffolds. Secondly, more nontoxic and harmless modifiers should be developed, thereby paving the way for the preparation of polymer/MMT composite scaffolds with adjustable mechanical properties. Besides, we should deeply explore the potential of MMT for drug delivery in bone tissue engineering and build systematic drug-loading and drug release systems to provide support for the preparation of drug-loaded scaffolds with controllable release performance.



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*Source: 7900382-2022-01-30.xml*