Nanocomposite hydrogels for cartilage tissue engineering: a review

Abstract

The loss of cartilaginous tissues is an important challenge to orthopaedic surgeons. Injury to cartilage tissue due to its properties is along with movement difficulties. Tissue engineering is a developing field that can be used for regeneration or replacement of damaged tissues. In this field, an appropriate scaffold that support the recruitment, adhesion, proliferation and differentiation of cells is necessary. Hydrogels recently considered as materials that resemble the extracellular matrix (ECM) and efficiently replace defective tissues, but they have limited mechanical strength. So nanomaterials are embedded in the hydrogel’s matrix to improve their properties. Nanoparticles, such as organic/polymeric and inorganic (hydroxyapatite, clay, graphene and metallic nanoparticles), can be used as fillers to reinforce the hydrogel matrix. Utilizing those nanocomposites could help in better performance of hydrogels applicable in cartilage regeneration practices. This review presents some of nanocomposite hydrogel (NCH) systems that used in cartilage tissue engineering.

Keywords:

• Hydrogels
• cartilage tissue engineering
• nanocomposite
• scaffold

Introduction

Cartilage tissue is mainly composed of chondrocytes that produce ECM proteins, mainly type II collagen and aggrecan. These proteins have roles in the tissue resistance to tensile and strength. Degradation of ECM units is a leading cause of cartilage dysfunction [1].

The loss of cartilaginous tissues because of pathological conditions such as osteoarthritis (OA), rheumatoid arthritis (RA), traumatic conditions including intra-articular fracture and ligament injury is an important challenge to orthopaedic surgeons [2]. Injury to cartilage tissue due to its properties is not easy problem. For example, this tissue is avascular, has a complex structure, very few cells, high amount of heterogeneity and functions under an intensely strenuous environment [3]. The injury can be partially self-regeneratable when the diameter of the damage is less than 3 mm, but when the diameter of the damage is more than 4 mm, self-repair ability would be apparently limited [4]. Recent treatments, include mosaicplasty, autologous chondrocytes injection or micro-fracture, have some success rates. But common disadvantages of these treatments are that the newly made articular cartilage does not have the structural organization of native cartilage and the biomechanical properties in comparison to native tissue are poor [5]. So it is important to develop new strategies to efficiently return cartilage function and avoid surgery [1].

Tissue engineering is a developing field that can be a permanent remedy to tissue damage that implicates millions of people each year [6]. Its aim is to replace damaged or diseased tissues with bioengineered implants. In this field, there are three major blocks: isolated cells, the bioactive molecules such as drugs or growth factors and the three-dimensional (3-D) scaffolds [7–9].

Chondrocytes, fibroblasts, stem cells and genetically modified cells have all been considered cell source for cartilage repair [6]. The supply of autologous chondrocytes is very limited [10]. Instead, mesenchymal stem (MSCs) because of their properties such as easy-accessibility in a minimally invasive procedure, collecting in large quantities from different tissues in the human body, abilities to self-renew and to differentiate into several tissue lineages, such as cartilage, bone and adipose tissues, are an important source in the field of regenerative medicine since1970s [2,11].

The most commonly used bioactive agents in the culturing medium or on the biomaterials scaffold are the growth/differentiation factors (GFs). They are proteins that have fundamental roles in cell proliferation, migration, differentiation and maturation of functional tissues precursors [12,13]. A number of growth factors like transforming growth factor β(TGF-β), fibroblast growth factor (FGF), insulin-like growth factor (IGF) and bone morphogenetic proteins (BMP) have been studied for cartilage tissue engineering [6]. TGF-β that is more important in the growth and differentiation of cells into articular cartilage [14].

Tissue regeneration needs an appropriate microenvironment for the adhesion, proliferation differentiation and recruitment of cells, and this can be facilitated by the use of biomimetic materials [15]. Tissue engineering for cartilage needs a simulated ECM that stimulates the cells to proliferate and differentiate towards new tissue generation [2,16].

Some characteristics are necessary for scaffold. For example: three-dimensional (3-D) and highly porous structures with interconnected pore networks in order to allow transportation of nutrients and metabolic waste, appropriate surface for cell attachment, support cell growth and differentiation, biocompatible with a controllable degradation and resorption rate to match cell/tissue growth in vitro and/or in vivo, mechanical properties to match those of the tissues at the site of implantation, must perfectly fit with the surrounding natural healthy tissue at the injury site and no potential for immunological reactions [17–20].

Both natural and synthetic materials have been considered as scaffold for cartilage tissue engineering. Specially polymeric materials in the forms of hydrogels, fibrous meshes and sponges are attractive [6]. In particular, hydrogel composites have been intensively studied [4,21–23]. In this review paper, we have collected and discussed some studies about nanocomposite hydrogels for cartilage tissue engineering.

Scaffolds for cartilage

In cartilage tissue, chondrocytes are surrounded by their pericellular matrix (PCM) composed of type-VI collagen and proteoglycans. The PCM and ECM together form the cartilage matrix, which is mainly consisting of water, type-II collagen and proteoglycans [24]. The proteoglycans in cartilage matrix are highly negative charged so attract water and resulting in a high pressure tissue [19].

To better mimic the nanostructure in natural ECM, scaffolds constructed from nanofibres, nanoparticles, nanotubes and hydrogels. Hydrogels recently have considered as scaffolds that resemble the ECM and efficiently replace damaged tissues [25].

Hydrogel applications for biomedical are attractive because of their highly hydrated nature that resemblances the ECM [26]. Cells can be combined into a hydrogel’s 3D network which provide structural support while allowing the transport of oxygen, nutrients and metabolites [27].

Hydrogel as scaffold for cartilage tissue engineering

Hydrogels are 3D cross-linked polymeric networks that absorb and hold large amounts of water. The “network” indicates, crosslinks hinder dissolution of the hydrophilic polymer chains into the aqueous phase [28]. They are smart materials that respond to environmental stimuli (temperature pH, ionic strength, electric field, the presence of enzyme, etc.) and swell or shrink accordingly [29]. When the networks are held together by molecular entanglements or secondary forces such as ionic, H-bonding or hydrophobic forces, they are called “reversible” or “physical” gels. Hydrogels are called “permanent” or “chemical” gels when they are cross-linked together covalently [30].

Porosity have an important role on the diffusion of nutrients and oxygen, particularly in the lack of a functional vascular system [31]. Natural tissues have pores in the range of nanometer. So there are more studies which have demonstrated that hydrogels with smaller pore size have better effects. But few studies have recommended that bigger interconnected pores are suitable for cell proliferation and ECM production [32,33].

In a scaffold to ensure adequate cell–cell interactions, cell seeding method and density of cells should be considered. Hydrogels provide uniform cell distributions if cells are sufficiently suspended through gelation [6].

1–3 Classification of hydrogel

The hydrogel can be classified on different bases as in Table 1 [30,34,35].

Table 1. The hydrogel classifications.

NO	Classification basis	Types	Ref
1	Based on source of hydrogels	Natural Synthetic	[35]
2	According to polymeric composition	Homopolymeric Copolymeric Multipolymer interpenetrating polymeric hydrogel(IPN)	[34]
3	Based on type of cross linking	chemically cross linked physically cross linked	[30]
4	Based on physical appearance	Matrix Film Microsphere	[34]
5	Based on network electrical charge	Neutral Ionic Amphoteric electrolyte Zwitterionic	[34]

Injectable hydrogel

In order to transplant preformed tissue-engineered constructs into defects, invasive surgery may be necessary. Injectable scaffolds have advantages such as fill irregular-shaped defects, simple incorporation of bioactive factors and limited surgical invasion [36]. Hydrogels can be used as injectable scaffolds because they simply fill defects with any shape and size, implanted in a minimally invasive way, their tissue-like properties and controllability of release behaviour [6,37]. Injectable hydrogels have two forms: in situ-forming and preformed hydrogels. In situ-forming hydrogels formation is occurred in physiological conditions after injection. Before administration they have the form of clear polymer solutions that change into a gel in response to alterations in external stimuli such as temperature, pH or by chemical crosslinking with enzymes¸ Schiff base¸ Michael addition¸ photopolymerization, etc. at the site of administration. Also preformed gels, which is identified as shear-thinning hydrogel, can also be applied as injectable materials [38,39]. Temperature sensitive hydrogels have advantages in comparison to injectable materials with chemical gelation mechanism. For example, sol–gel transition of them does not need any of toxic chemical reagents (such as crosslinkers, organic solvents, catalysts) [40]. Despite their advantages, there is a risk of leakage of the sol phase to unwanted sites before the beginning of the sol–gel phase transition [41].

Challenge of hydrogels

Hydrogels due to their high water content have limited mechanical strength that may fail the construct of the hydrogel under mechanically challenging conditions, as found in the load-bearing bone and cartilage tissues. In order to solve this problem, one approach is increasing polymer concentration and crosslinking density. But this impacts the diffusion rates of nutrients, bioactive factors and cell metabolites through the hydrogel [42].

Materials for scaffolds

Polymeric materials are the dominant material for most tissue engineering, because of their excellent biological and mechanical properties, flexible processing capabilities and low cost [43]. Cartilage ECM is an appropriate material for cartilage scaffold. Also, it has been suggested that cartilage ECM-derived scaffolds protect the main constituents of native cartilage, good biocompatibility and provide a natural microenvironment for the cell attachment, proliferation and differentiation into chondrocytes [10]. Natural and synthetic materials has been considered as scaffold for cartilage tissue engineering. Hydrogels based on both synthetic and natural polymers have advantages for the encapsulation of cells [30].

Natural polymers have more considered in tissue engineering because they are either components of or have macromolecular properties similar to the natural ECM [44]. Natural hydrogels have abundant adhesive proteins such as laminin and fibronectin for the support of cell adhesion and migration [37]. But this polymers have disadvantages such as the processability of them is difficult, their mechanical properties are not optimal, limited degradation profiles, batch-to-batch difference from animal and plant sources and may have pathogens or cause immune responses [43,45,46].

Synthetic polymers are more controllable. Chemical and physical properties of a polymer can be improved to alter mechanical and degradation features of them [3]. But they have some drawback for example: direct cell-scaffold interactions are low, the limited in situ biodegradability and the use of toxic chemicals for manufacturing [6,19].

Improvement of hydrogel properties with nanoparticles

The highly hydrated condition of hydrogels and their poor mechanical properties are challenges in vitro and in vivo. To overcome these limitations, combinations of both materials are a highly attractive approach [47]. The application of hydrogel may be better if a nanomaterials is inserted in the hydrogel’s matrix. Therefore, current studies in hydrogel have led to the development of NCHs [48]. Nanoparticles, containing organic/polymeric and inorganic (such as hydroxyapatite, clay, graphene and metallic nanoparticles) can be used as fillers to reinforce the hydrogel matrix [49]. Indeed the nanoscale dimensions have the more surface area to volume ratios, which increase the surface reactivity, the release of loaded bioactive agents, bioavailability and superior mechanical properties. Also due to their ability to penetrate into tissues through capillaries and epithelial lining, they improve transport properties and a more efficient delivery of therapeutic agents to target cells [50–52]. Nanoparticles in comparison to microparticles have many advantages. For example, when particles are embedded into the polymeric matrix during the crosslinking of scaffold, they have more homogeneous distribution and much more particles are available for the same equivalent weight of carriers [53].

The role of nano in cartilage tissue engineering

Natural tissues and organs have nanometer dimensions and cells directly interact with nanostructured ECM. So the biomimetic properties of nanomaterials is important in cell growth as well as tissue regeneration [54]. The structure of the scaffold at the nano and microscale is also essential for cartilage tissue engineering. Nanomaterials such as nanofibers and materials with nanostructured surfaces have been investigated to mimic the ECM of cartilage [55]. Cartilage tissue contains a small percentage of chondrocytes but dense nanostructured ECM rich in proteoglycans, collagen and elastin fibres. So the applying of nanomaterials have excellent mechanical and biomimetic in terms of their nanostructure properties [54].

Bioactive factor delivery in hydrogels

The aim of controlled bioactive factor delivery systems in hydrogels is releasing bioactive molecules to target places in the body with pre-programmed rates over a period of time [45]. Direct encapsulation of bioactive molecules in hydrogel have limitations such as poor control of release profiles. Often, there will a burst release profile, which may lead to adverse tissue reactions [37]. In comparison to other proteins, growth factors have short half-lives, act locally and have short diffusion distances through the ECMs. Usually, growth factors are degraded enzymatically or chemically or deactivated in physiological conditions in a very limited time frame [50,56]. One attractive method to overcome this problem is incorporation of micro or nanoparticle delivery system into the hydrogels [37]. There are some advantages for them such as safe delivery, protection of bioactive agents, reduction of side effects and the ability to deliver the bioactive agents locally [50].

Polymeric NCH

Thermally responsive hydrogels are interesting because their gelation and swelling behaviour can be triggered by temperature change [26]. These copolymers at temperatures below their lower critical solution temperature (LCST) are completely soluble in aqueous and they form a gel at temperatures above their LCST [47].

Chitin and chitosan due to their properties, such as high biocompatibility, antibacterial activity, biodegradability, non-antigenicity and high adsorption, are excellent materials for tissue engineering [57]. Various glycosaminoglycans (GAGs) that exist in articular cartilage and chitosan are similar in structure, which makes it a suitable matrix for cartilage repair [58].

Chitosan/glycerophosphate (CS/GP) hydrogel, a thermosensitive hydrogel due to its sol–gel-phase transition in body temperature is an important applicable biological substance. Chitosan in complex with other materials such as silk have been studied for biomedical applications. Silk fibroin is a natural fibrous protein and due to its excellent biological compatibility and mechanical strength has been used as a biomaterial for some biomedical applications such as tissue engineering. Fereshteh Mirahmadi and coworkers used degummed chopped silk fibres and electrospun silk fibres to the thermosensitive chitosan/glycerophosphate hydrogels. Their aim was to reinforce the hydrogel constructs for cartilage tissue engineering. They showed that hybrid of hydrogel with two layers of electrospun silk fibres improvesignificantlymechanical properties of them. The expression of GAG and collagen type II indicated the chondrogenic phenotype for chondrocytes with a significant increase in degummed silk fibre-hydrogel composite for GAG content and in two-layer electrospun fibre-hydrogel composite for Col II [23].

PVA hydrogel, is a non-degradable synthetic polymer, has high potential for cartilage tissue engineering due to its structure and material properties similarities with natural cartilage. But the PVA hydrogel has some problems, such as its insufficient mechanical property and porosity, small pore size and poor cell adhesion ability. Min Zeng et al applied nano-hydroxyapatite (nano-HA), as a bone repair material, in order to improve the mechanical property of scaffolds and poly (lactic-co-glyclic acid) (PLGA), a degradable polymer to improve the biocompatibility. The results of cell culture indicated that human MSCs were able to attach, grow and proliferate well in the composites. In addition, the composites could stimulate the proliferation and differentiation toward chondrocytes in vitro [4].

The fundamental building blocks of ECM are similar and include a fibrous protein network embedded within a gelatinous polysaccharide ground substance [21]. The building of nanofibres is morphologically similar to the fibrillar constituents of the native ECM [59]. Jeannine Coburn et al fabricated an injectable fibre–hydrogel composite for cartilage tissue engineering. They prepared polyɛ-caprolactone (PCL) fibre and mixed with the hydrogel macromer(Poly(ethylene glycol)-diacrylate) [21]. PCL is a hydrophobic, semicrystalline, biodegradable polyester polymer and has extended resorption time that makes it a suitable scaffold for cartilage tissue engineering [2]. The resulting integrated fibre–hydrogel composite has significantly greater mechanical properties and induces enhanced biological responses from adult stem cells to produce more tissue [21].

Alginate is one of the natural biomaterials that have some properties such as forming hydrogels or sponges, excellent biocompatibility, low immunological motivation, degradability and flexibility to process into ideal geometries [35]. Its hydrogel gelation procedure does not need any toxic chemicals that may prevent the activity of growth factors, good binding affinity with growth factors and relatively slow release of them widely used for delivery of the growth factors.

In the study by Sung Mook Lim et al, they used alginate solution and included BMP-7 and polyion complex nanoparticles (containing TGF-b2) to differentiate MSCs into cartilage tissue. The dual growth factors (BMP-7/TGF-b2)-loaded nanoparticle/hydrogel system released both of the growth factors in controlled way: BMP-7 was released faster and a slower release for TGF-b2 was observed, approximately 80% and 30% release at the end of an incubation period (21 days), respectively. Each growth factor release from the dual growth factors-loaded hydrogel (without the nanoparticles) had much slower than that of the nanoparticle/hydrogel system. This is due to the aggregation between growth factors during the hydrogel fabrication stage [60].

Fibrin is the polymer of fibrinogen molecules. Fibrin hydrogels have been usually used as scaffolds for tissue engineering [35]. In a study, the effects of growth factor loaded in nanoparticles combined in fibrin scaffold for chondrogenic differentiation were studied. Heparin have electrostatic nature and specific affinity for growth factors so delivers growth factors in a controlled manner. TGF-b3 was loaded in heparin nanoparticles and embedded in fibrin scaffolds. They evaluated specific cartilage extracellular matrix components in vitro and in vivo by bone marrow–derived stromal cells (BMSCs). Histological and immunohistochemical assays showed that great amounts of type II and proteoglycan were produced from BMSCs embedded in fibrin constructs, while collagen type I were decreased in constructs that include nanoparticles that were loaded with TGF-b both in vitro and in vivo. The results showed that TGF-b3-loaded nanoparticles induced BMSCs differentiation to cartilage in a greater amount [61].

Poly(lactide-co-caprolactone) (PLCL) scaffolds can degraded very slowly and have elastic mechanical properties similar to articular cartilage so compensate the weakness of hydrogels. In a study, PLCL scaffolds were synthesized by gel-pressing method with 85% porosity and 300–500 mm pore size range. Heparin-functionalized nanoparticles, composed of poly(lactide-co-glycolide) (PLGA), Pluronic F-127, and heparin, and then, TGF-b1 was loaded to the nanoparticles. Then, adipose-derived stem cells (hASCs), fibrin gels and TGF-b1-loaded nanoparticles was embedded into the PLCL scaffolds. The results of studies both in vivo and in vitro showed that hASCs differentiate to the cartilage and sustained release for TGF-b1 from the heparin-functionalized nanoparticles were observed [62].

Figure 1. Hydrogels can be used as injectable scaffolds because they simply fill defects with any shape and size.

Silica NCH

Synthetic silicates are a class of nanomaterials have properties such as high degree of anisotropy, functionality and due to surface to volume ratio interact with biological entities in a different way. Further interaction with polymeric networks, they form physically crosslinked networks and significantly increasing the mechanical stiffness of polymer [63]. The addition of highly exfoliated silicate nanoparticles to the PEG chains significantly increase the rheological behaviour of the injectable precursor. They can lead to scaffold be able to tolerate the normal loads and stresses of native cartilage (type-II collagen), before the cells begin constructing functional ECM. Amirali Nojoomi et al designed injectable PEG-based hydrogels containing laponite particles for cartilage tissue engineering. Tensile testing and oscillatory shear stress assay also indicated that by the addition of the laponite nanoparticles into PEG-based hydrogels, elastic modulus of scaffold enhance. Cell viability tests by hMSC showed biocompatibility of the PEG hydrogels. The addition of laponite particles slightly decreased the cell viability, but the nanocomposite hydrogels shown reasonable biocompatibility (>90%) [41].

Mesoporous silica nanofibres because of their properties, such as anisotropy, diameter around 50 nm, their mesoporosity, pore volume and high specific surface, can be used as reservoirs for bioactive molecules (cell growth and differentiation factors). Nela Buchtova and coworkers designed nanocomposite hydrogels based on siloxane-derived hydroxypropylmethylcellulose (Si-HPMC) interlinked with mesoporous silica nanofibres for cartilage tissue engineering. The covalent binding between biopolymer and silica fibers, checking simultaneously cell viability in such a material. This biomaterial made of siloxane derivative of HPMC interlinked with silica nanofibres is shown to be mechanically tunable and cytocompatible [64].

Conclusions

Tissue engineering for cartilage need an appropriate microenvironment that act as ECM in which the cells can proliferate and differentiate following by new tissue generation. Hydrogels are appropriate for this purpose because of their highly hydrated nature that resemblances the ECM. Specially, injectable hydrogels due to their limited surgical invasion are attractive. But they have some disadvantages such as limited mechanical strength. So recent studies have developed NCHs nanoparticles, such as polymeric and silica, were used in composites. Also direct encapsulation of bioactive molecules in hydrogel have poor control of release profiles, so incorporation of them in nanoparticle can solve this problem.

Acknowledgements

This work is funded by Faculty of Advanced Medical Sciences (Grant number: 95/2–2/3) Tabriz University of Medical Sciences, Tabriz, Iran.

Disclosure statement

No potential conflict of interest was reported by the authors.

Additional information

Funding

This work is funded by Faculty of Advanced Medical Sciences (Grant number: 95/2–2/3) Tabriz University of Medical Sciences, Tabriz, Iran.