Smart Polymeric Hydrogels for Cartilage Tissue Engineering: A

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Review

Smart Polymeric Hydrogels for Cartilage Tissue Engineering: A Review on the Chemistry and Biological Functions Niloofar Eslahi, Marjan Abdorahim, and Abdolreza Arash Simchi Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01235 • Publication Date (Web): 24 Oct 2016 Downloaded from http://pubs.acs.org on October 26, 2016

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Smart Polymeric Hydrogels for Cartilage Tissue Engineering: A Review on the Chemistry and Biological Functions Niloofar Eslahi,†, ‡ Marjan Abdorahim,‡ Abdolreza Simchi‡,§,* †

Department of Textile Engineering, Science and Research Branch, Islamic Azad University,

P.O. Box 14515/775, Tehran, Iran ‡

Department of Materials Science and Engineering, Sharif University of Technology, Azadi

Avenue, P.O. Box 11365/8639, Tehran, Iran §

Institute for Nanoscience and Nanotechnology, Sharif University of Technology, Azadi

Avenue, P.O. Box 11365/8639, Tehran, Iran

ACS Paragon Plus Environment

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KEYWORDS: Smart hydrogel; Stimuli responsive; Nanostructure; Cartilage tissue engineering

ABSTRACT: Stimuli responsive hydrogels (SRHs) are attractive bio-scaffolds for tissue engineering. The structural similarity of SRHs to the extracellular matrix (ECM) of many tissues offers great advantages for a minimally invasive tissue repair. Among various potential applications of SRHs, cartilage regeneration has attracted significant attention. The repair of cartilage damage is challenging in orthopedics owing to its low repair capacity. Recent advances include development of injectable hydrogels to minimize invasive surgery with nanostructured features and rapid stimuli-responsive characteristics. Nanostructured SRHs with more structural similarity to natural ECM up-regulate cell-material interactions for faster tissue repair and more controlled stimuli-response to environmental changes. This review highlights most recent advances in the development of nanostructured or smart hydrogels for cartilage tissue engineering. Different types of stimuli-responsive hydrogels are introduced and their fabrication processes through physico-chemical procedures are reported. The applications and characteristics of natural and synthetic polymers used in SRHs are also reviewed with an outline on clinical considerations and challenges.


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1. Introduction Tissue engineering (TE) has evolved as a promising technology by combining cells, engineering and materials methods to provide a more functionally biological tissue.1, 2 Biomimetic scaffolds have been developed with controlled structures and on-demand properties to regulate specific cellular behaviors.3 The basic principle of cartilage tissue engineering requires isolation of appropriate cells which are proliferated on a 3D matrix and subsequently implanted into the site of injury.4, 5 Among different biomaterials being developed and utilized for TE, hydrogels have attracted considerable attention owing to their capability to mimic the physiochemical and biological properties of the natural cartilage ECM.6 Hydrogels are 3D networks of hydrophilic polymers with a high degree of water uptake while maintaining structural integrity because of their crosslinked structure.7 Chemically crosslinked networks have permanent covalent bonds, whereas physical networks have temporary joints resulting from molecular entanglements or physical interactions.8, 9 Low interfacial tension, biomimetic microstructure, and high permeability to undersized molecules have made hydrogels a promising material for various biomedical applications including tissue regeneration, drug delivery devices, and wound dressing.10-13 The porous structure of hydrogels enables the transfer of low molecular weight nutrients and cellular waste, which is vital for cellular viability.4 In addition, biocompatible and biodegradable hydrogels can direct the migration, growth and arrangement of cells during cartilage regeneration.14 Recently, particular interest has been attracted in smart or stimuli-responsive hydrogels (SRHs) in which a distinct transition is caused by small alterations in the environment.15 These hydrogels


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can undergo reversible volume phase transitions (in chemically crosslinked hydrogels) or sol-gel transitions (in physically crosslinked hydrogels) in response to various external physiochemical factors such as changes in temperature16-18, pH19-21, ionic strength22, solvent composition23, wavelength or intensity of light24, 25, electric and magnetic fields26-28, or chemical triggers like glucose.29, 30 To achieve swift and significant response to environmental stimuli, it is important to use smart hydrogels in diverse applications including actuators, sensors, scaffolds, and drug delivery.31-33 Because of the attractive properties of SRHs, there is now a great attention on the design of SRH scaffolds which satisfy all the essential requirements of cartilage regeneration. Recent advances include development of injectable SRHs with nanostructured features (i.e. nanogels, nanofibers and nanocomposite hydrogels) and rapid stimuli-responsive characteristics.34 In-situ forming hydrogels in conjunction with stimulatory growth factors can act as an ideal match with cartilage defects. The main advantage of injectable hydrogels for cartilage repair is their application using minimally invasive surgery.35-37 With rapidly emerging science of nanotechnology over the last decade, nanostructured biomaterials have received significant attention since they can mimic surface properties of natural tissues.2, 38 Besides dimensional resemblance to natural cartilage ECM, nanomaterials also possess unique characteristics including superior cytocompatibility, mechanical, optical, electrical, and magnetic properties in comparison with conventional materials.39 Thus, novel nanostructured hydrogels have been regarded as promising candidates for cartilage repair. Previous studies have overviewed various polymeric hydrogels for cartilage repair 4, 36, 40, stimuli responsive hydrogels for different biomedical applications16, 41, 42, and nanocomposite hydrogels used in medicine.43, 44 This review paper summarizes the recent progresses on the development of nanostructured, smart, or in-situ forming hydrogels with rapid stimuli-responsive


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characteristics for cartilage TE applications. It aims to give an overview of various biomaterials currently under investigation for cartilage regeneration and highlights some of the most recent advances in the field with a focus on nanostructured materials and their chemical and biological functions. Besides, requirements of the tissue engineering hydrogels are discussed with an outline on future directions and challenges. 2. Cartilage tissue engineering Articular cartilage is a flexible tissue where chondrocytes are barely distributed into a highly organized ECM containing glycosaminoglycans (GAGs), proteoglycans, and collagen fibers.14 Chondrocytes (CHONs), as the main metabolically active cells within cartilage, are responsible for the production, organization, and maintenance of the articular cartilage ECM.45 The treatment of articular cartilage defects, which can occur by injury, trauma, disease, and tumors, is one of the most challenging clinical issues in orthopedics due to its low capacity for repair.3 The limited regenerative characteristics of cartilage arises from reduced availability of CHONs in the dense nanostructured ECM, limited proliferating potential of CHONs and absence of vascular networks required for effective cartilage regeneration.4, 46 Various surgical procedures have already been attempted to treat cartilage damage, but each has its own shortcomings.47-49 Autografts, as the gold standard for cartilage repair, suffer from donor site morbidity and restrained availability. Besides, allografts have some limitations including delayed vascular penetration, high incidence of non-union, and disease transmission.50 Tissue engineering approaches are potential alternatives for cartilage reconstruction to provide a more functionally biological scaffold mimicking native tissue.1 Specifically, cartilage tissue engineering aims to evolve biomimetic tissues that recapitulate the structural, biological, and functional features of native cartilage, prevent the progression of cartilage degeneration, and enhance long-term functional outcomes


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for patients.3 Different parameters including structural environment, mechanical stimuli, cellbiomaterial interactions, and biological signals affect the development of cartilage in a tissueengineered scaffold.4 To fulfill the criteria for tissue-engineered articular cartilage, the scaffolds should exhibit biocompatibility, easy handling, and mechanical properties similar to native tissue.38 Among different types of scaffolds, hydrogels can act as promising scaffolds for cartilage regeneration because their viscoelastic properties, biomimetic structure, and high water content can support cell functions.50 Tissue engineering hydrogels should be biocompatible, biodegradable, non-toxic, non-immunogenic, and non-inflammatory. To date, a variety of hydrogels from different biomaterials have been attempted for articular cartilage regeneration, but only a few could closely mimic the environment and ECM network with temporal and spatial complexity.3, 38, 51 Tissue engineering of an ideal articular cartilage requires three components: (i) a cell population with the ability to proliferate and differentiate into CHONs; (ii) a biocompatible and biodegradable scaffold enabling regulation of cellular functions and possessing enough mechanical strength for withstanding physiologic loads during joint movement; (iii) biological factors capable of stimulating cellular response and ECM synthesis.1 Incorporation of bioactive components such as cells, growth factors, genes, peptides and proteins into the hydrogels has recently gained much attention to enhance biomimetic scaffolds with bioactive functions.36, 37 Among these biological agents, stem cells are prominent due to their availability and lack of donor morbidity.3, 52 Different types of stem cells have been used for cartilage tissue engineering such as embryonic stem cells, adipose-derived stem cells (ADSCs) and bone marrow-derived mesenchymal stem cells (BMSCs).53, 54 Several factors including chemical compositions, degradation rate, biological properties and mechanical performance of hydrogels play an important role in cartilage tissue engineering.55, 56


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For instance, cell metabolism and tissue formation can be regulated by manipulating scaffold properties (pore sizes and their distribution, surface topography, etc.) and incorporating of ECM motifs and bioactive substances in the scaffolds.51 However, matching mechanical strength of native tissue is challenging. In the case of cartilage, a compressive modulus of 0.7 to 0.8 MPa, a shear modulus of about 0.7 MPa and a tensile modulus of 0.3–10 MPa are required.38 Besides, compressive strain values vary within cartilage tissue owing to its anisotropic structure.57 With recent advances, researchers have attempted to boost the mechanical properties of hydrogels by a number of solutions such as addition of covalent crosslinks, increasing crosslinking density, and addition of nanofillers (i.e. nanoparticles, nanofibers, nanotubes).58 Novel biomaterials include SRHs which are regarded as highly suitable scaffolds mimicking the dynamic nature of ECM to accommodate cultured cells for growth.6, 59 With the rapidly emerging science of nanotechnology, there has been interest in the potential application of nanostructured hydrogels in TE.60, 61 Application of nanotechnology for regenerative medicine becomes significant by observing nature. The biomimetic features (owing to nanometer dimension of natural tissues) and excellent physiochemical properties of nanomaterials are significant in stimulating cell growth and guiding tissue regeneration. Therefore, the development of nanomaterials with excellent mechanical properties has been of great interest to improve cellular functions and facilitate tissue regeneration.2, 39 3. Stimuli responsive hydrogels Stimuli responsive or smart hydrogels can undergo reversible volume phase or sol-gel phase transitions upon slight changes in the environment.7, 59 SRHs can be implanted in vivo through minimally invasive procedures, providing therapeutic treatments without invasive surgical


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methods for biomedical applications including cartilage TE.62 The stimuli-responsive properties in hydrogels are principally adjusted by polymer–polymer and polymer–solvent interactions.42 Various physical and chemical stimuli have been used to induce responses in smart hydrogels. The physical stimuli consist of temperature, electric fields, light, sound and magnetic fields, while the chemical or biochemical stimuli include pH, ions and specific molecular recognitions.9, 41

Different types of smart hydrogels and their operating mechanisms are summarized in Table 1.

Table 1 Different types of smart hydrogels and the governing mechanisms for their stimuli responsive behavior.

Stimuli

Mechanism

Temperature

Schematic representation

Materials

References

Changes in hydrophilichydrophobic balance

NIPAAm, Pluronic, PEG copolymers

41,63,64,65

pH

Association/disassociation of pendant acidic/basic groups with hydrogen ions

Polyelectrolytes, PAAc, Chitosan, poly(Nvinylcaprolactam)

66,67,68

Light

Incorporation of photosensitive functional groups into the hydrogel networks

Azobenzenes, Poly(cinnamic acid), Triphenylmethane

69,70,42

Electric field

Generation of electrical polarization in polyelectrolytes

Chitosan/PAN, Hyaluronic acid/PVA, Alginate/ PMAA

71,72,73,74

Mechanical stress

Induced conformational changes

Peptides, Acrylatebased block copolymers

75-77

Biomolecules

Dissolution–precipitation dynamics

Glucose-sensitive, DNA-responsive, Enzyme-sensitive hydrogels

78,79,80,41


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3.1 Temperature responsive hydrogels Amongst SRHs, temperature-responsive hydrogels are the most widely studied one. The temperature-dependent transformation of these hydrogels has been ascribed to the hydrophilic/hydrophobic balance in the network.42, 81 These hydrogels are categorized into positive or negative thermoresponsive systems. Positive temperature responsive hydrogels shrink by cooling below the upper critical solution temperature (UCST). On the other hand, negative thermo-reversible hydrogel shrink by heating above the lower critical solution temperature (LCST). Because changes in the environmental temperature induce gelation without any need for chemical or physical treatment, polymers with LCST less than human body temperature could be potentially used in injectable applications.9, 56 Thermosensitive polymers undergo sol-gel transition upon temperature alteration, causing changes in solubility, configuration, and hydrophilic-hydrophobic balance.82 This behavior has been assigned to the distinct interactions between polymer and solvent.42 Under the LCST, water molecules between the polymeric chains can form hydrogen bonds with polar groups within the hydrogel making it soluble. However, at a critical temperature, polymer–polymer and water– water interactions dominate. In fact, above the LCST, polymer chains shrink and become hydrophobic and insoluble, resulting in gelation.9, 83 N-isopropyl-acrylamides (NIPAAm), Poloxamers and different PEG-based polymers are common examples of temperature-responsive hydrogels.63 Park et al.84 introduced thermosensitive chitosan–Pluronic hydrogels as a potential candidate for cartilage TE. Sa-Lima et al.85 designed a biocompatible poly(N isopropylacrylamide)-g-methylcellulose thermoreversible hydrogel which could support cell encapsulation and GAGs production required for cartilage regeneration. Recently, Abbadessa et al.86 synthesized thermoresponsive hydrogels based on PEG copolymers for cartilage bioprinting.


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It was found that incorporation of polysaccharides improved the mechanical properties and stability of the hydrogels. In our previous work, we also developed a novel thermosensitive nanocomposite hydrogel based on fibrous protein/block copolymers. The synthesized hydrogels underwent in situ crosslinking and rapid gelation under physiological conditions and had the potential as injectable SRHs for cartilage TE application.87 The advantage of temperaturetriggered hydrogels is the ease of fabrication with no need for external crosslinking agents using minimally invasive implantations.81 However, the gelation time, temperature and pH of the polymer solution should be suitable for clinical applications having no harmful effect on cell viability.4 3.2 pH responsive hydrogels The pH-responsive hydrogels are composed of polymers with acidic or basic groups that can exchange protons depending on pH.19, 66 While the sensitivity of thermoresponsive hydrogels is limited by thermal diffusion, pH-responsive hydrogels can be also restricted by hydrogen ion diffusion.9 The mechanism of pH-responsive hydrogels involves dissociation and association with hydrogen ions in response to the environmental pH.42 This type of hydrogel is studied extensively for drug delivery applications since the pH profile of pathological tissues, e.g. in case of inflammation, infection, and cancer, differentiates from that of normal tissue.88 Polyelectrolytes with a large number of ionizable groups are the frequently used polymers for pH-sensitive hydrogels as they show different swelling properties depending on the surrounding pH.66 Poly(acrylic acid) (PAAc) is the sample of weak poly acids that is protonated at acidic pH and deprotonated at neutral and basic pH. Meanwhile, poly(4-vinylpyriden) is a poly base that accepts proton at basic pH.67, 89 Block copolymers can also be used for preparation of pH-


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responsive hydrogels. With decreasing pH below the pKa, the ionization of the polymer increases electrostatic repulsions of the polymer molecules, resulting in destabilization.82 For instance, poly(ethylene oxide)-block-poly(N,N-diethylaminoethylmethacrylate) diblock copolymer (PEOPDEAMA) is soluble at acidic pH; however, the hydrophilic block (PDEAMA, pKa=7.3) is deprotonated at alkaline pH and turns hydrophobic.90 Bonina et al.91 prepared pH-sensitive hydrogels using chitosan and polyacrylamide. According to the obtained results, the swelling ratio of the hydrogels was dependent on the pH/ionic strength of the medium and the degree of crosslinking. Strehin et al.92 developed pH-responsive chondroitin sulfate-PEG adhesive hydrogels with potential application in regenerative medicine including cartilage repair. It was found that the hydrogel stiffness, swelling properties, and kinetics of gelation could be controlled by varying the initial pH of the precursor solutions. Halacheva et al.93 designed pH-sensitive hydrogels from poly(meth)acrylic acid-containing crosslinked particles with high porosity, elasticity and ductility. The enhanced mechanical properties of the produced hydrogels made them a good candidate for regenerative medicine. Injectable SRHs based on chitosan-β-glycerophosphate-starch were also introduced by Sa-Lima et al.94, which could induce chondrogenic differentiation of ADSCs for cartilage TE using minimal invasive strategies. Although pH-responsive hydrogels have been studied for cartilage regeneration, it is difficult to predict the pH at the diseased site clinically which may cause undesired tissue response.4 3.3 Photo-responsive hydrogels Light is another interesting stimulus that can be imposed instantly and managed easily to modulate hydrogel properties.24 Photo-responsive hydrogels are usually produced by


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incorporation of photo-sensitive functional groups. Typical photoreactive agents, usually photochromic chromophores, can be incorporated in polymer matrices to create photo-triggered hydrogels.9 There are three classes of light-induced reactions including photoisomerization, photocleavage and photodimerization.24 For example, photo-sensitive chromophores such as azobenzenes (and its derivatives) exhibit cis–trans photoisomerization upon UV irradiation inducing reversible volume changes in the hydrogel network.95 Photodimerization processes based on coumarin96, nitrocinnamate97, anthracene98 and poly(cinnamic acid)70 have been applied for the formation of photo-responsive hydrogels. Moreover, photocleavable groups, such as triphenylmethane leuco derivatives, can dissociate into ion pairs by UV light leading to lightinduced swelling of the hydrogels.42 For instance, Kloxin and coworkers69 synthesized a photodegradable PEG-based hydrogel using 2-nitrobenzyl crosslinker. Under UV irradiation, the hydrogel was eroded depending on light intensity and wavelength. The observed photodegradation properties were attributed to the cleavage of crosslinks inside the hydrogel network. Light-sensitive hydrogels can be used for cartilage TE. Anseth et al.99 introduced hydrogel networks based on PEG and PVA which underwent transdermal photopolymerization after subcutaneous injection. Levett et al.100 designed photocrosslinkable hydrogels based on gelatinmethacrylamide with enhanced chondrogenic differentiation and improved mechanical properties for cartilage repair. Recently, Giammanco et al. 101 developed photoresponsive hydrogels by coordination of alginate–acrylamide hybrid gels with ferric ions. The physicochemical properties of the hydrogels could be tuned by visible light irradiation as scaffolds for cartilage TE. While photo-crosslinking has better spatial and temporal control over gelation, their practical use is


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limited due to the requirement of a photoinitiator, which may cause toxicity problems via a local temperature increase and prolonged irradiation time.4 It is worth mentioning that the strong scattering properties of tissues in the UV region lead to low penetration depth of the photo-sensitive hydrogels. However, by employing photosensitive groups which are responsive to higher wavelengths, the UV light could be replaced by a NIR laser (700–1000 nm range) with lower scattering properties and deeper penetration depth. The NIR-absorbing materials can convert the photon energy into heat for targeted release of chemotherapeutics.102 For instance, Wang et al.103 designed an injectable hydrogel consisting of α-cyclodextrin and PEG-modified dendrimer-encapsulated platinum nanoparticles which underwent photothermo-sensitive degradation to release the entrapped therapeutic agents upon NIR irradiation. 3.4 Electro-sensitive hydrogels Electro-sensitive hydrogels are generally composed of polyelectrolytes similar to pH-sensitive hydrogels.9 These hydrogels exhibit swelling, shrinking or bending in the presence of an applied electric field depending on their position towards the electrodes. For instance, bending and deswelling occur when the hydrogel is parallel and perpendicular to the electrodes, respectively.104, 105 By applying an electrical field in a polyelectrolyte hydrogel, mobile counter ions in the solution bind to the polymer network inducing the generation of an opposite potential difference across the gel.42 Thus, the hydrogel bends and swells locally depending on the strength, direction and duration of the electrical stimulus.106 Other factors including solution pH and ionic strength also influence the electromechanical behavior of the hydrogels.104, 105 In addition, crosslinking density also affects the bending direction. Hydrogels having high


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crosslinking density show unidirectional bending while those having low crosslinking density exhibit bi-directional bending.107 Polycation hydrogels such as CH/PAN72 and CH/PDADMAC108 bend toward the anode whereas polyanion ones such as hyaluronic acid /PVA109 and alginate/PMMA73 bend toward the cathode under electric stimulus. Electricsensitive hydrogels can be widely used as sensors, actuators, artificial muscles, membrane separation devices and drug delivery systems. Osada et al.110 reported an electrically responsive artificial muscle system with the ability to convert chemical energy to mechanical energy. Rahimi et al.106 designed an electro-responsive hydrogel composed of PAAc and fibrin. They found that under electrical field the produced hydrogel can direct muscle cell alignment and facilitate distribution of cells within the structure. Takahashi et al.74 synthesized an electroconductive hydrogel based on pyrrole being polymerized electrochemically into PAAc hydrogel. The zero-order safranin release profile was shown to be responsive to alterations in the electrical potential, ionic strength, and pH. According to Murdan et al.104, electro-responsive drug release from hydrogels originates from the electro-induced changes such as swelling or deswelling in response to an electric field. Jensen et al.111 evaluated the electro-stimulated release of peptides from chondroitin 4-sulphate hydrogels which was shown to be dependent on the molecular size and shape of guest molecules. It should be noted that the efficacy of electroresponsive hydrogels may be limited by biocompatibility issues as well as hydrolysis in the presence of electrical fields particularly at high voltage.112 3.5 Mechanical responsive hydrogels Mechano-sensitive hydrogels could change conformation over a range of mechanical forces. In order to design such dynamic SRHs, one should apply specific molecular interactions in conjunction with well–defined polymer chemistry and engineering methods to mimic the


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modular conformations of functional proteins in natural ECM.75 For instance, mechanical responsiveness can be induced by self-assembly. Schneider et al.76 produced responsive hydrogels by linking the intramolecular folding of small designed peptides due to their ability to self-assembly. Mechanical forces can stimulate polymer matrices easily to release drug/growth factors in mechanically stressed environments.113, 114 Xiao et al.75 created mechano-responsive hydrogels using self-assembled acrylate-based block copolymer micelles. They showed strain– dependent reversible deformation of the elastomeric hydrogels which could be effectively used for controlled drug release in response to externally applied mechanical forces. Wang et al.115 designed a superhydrophobic polymeric composite capable of strain-dependent delivery of anticancer agents. Jaspers et al.77 introduced strain-stiffening hydrogels with a universal mechanical response at sufficient stress. They investigated the effect of controllable variables (i.e. concentration, polymer length and temperature) on the mechanical properties of semiflexible polymer hydrogels and observed a transition from a low-viscous liquid to an elastic gel upon applying minute stresses at the critical point. Recently, Kia et al.116 developed lignin supramolecular hydrogels with mechanically responsive and self-healing properties. The obtained results confirmed the potential use of the synthesized hyperbranched copolymer as smart biomaterial. Since musculoskeletal tissues are constantly under mechanical forces within their microenvironment, this type of stimulus ultimately affect cellular behaviors in biological entities.117, 118 Matrix stiffness and elasticity regulate cell signaling and significantly affect cells adhesion, growth, and differentiation.119 Given the mechanically demanding environment of articular cartilage, mechanical forces play a key role in regulating cartilage development and chondrogenic gene expression.120 According to Mauck et al.121, dynamic compression enhanced


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matrix distribution and the mechanical properties of CHONs-seeded hydrogels for cartilage TE. Similarly, Huang et al.122 studied the influence of mechanical loading on chondrocyte metabolism. They reported an increase in the amount of chondrogenesis in 3D cultures using MSCs upon exposure to mechanical stimulations. Lim et al.107 developed dynamic electromechanical hydrogel matrices which could support proliferation and differentiation of stem cells for potential TE applications. Trumbull et al.54 also investigated the effect of mechanical loading on ADSCs differentiation to create mechanical loading platforms for musculoskeletal tissue engineering. 3.6 Biomolecular-sensitive hydrogels Hydrogels with molecular recognition capabilities have been developed from polymers with responsive dissolution–precipitation dynamics. The biomolecular-responsive hydrogels can be used for regulating homeostasis in the body by detecting different analytes.42 For instance, glucose-sensitive hydrogels have been utilized as modulated insulin delivery systems.78 Controlled delivery of insulin is important in diabetes because insulin must be delivered meticulously at the exact time.66 One of the most commonly used enzymes for this purpose is glucose oxidase which catalyzes the oxidation of glucose. The anionic charge in the reduced state induces osmotic pressure within the hydrogel and results in hydrogel swelling.123 So far, different hydrogels having glucose oxidase have been developed as glucose-responsive hydrogels.78, 124, 125 Concanavalin A (Con A), a glucose-binding protein, has also been applied for self-regulated insulin delivery. Insulin molecules are usually functionalized in this system to introduce glucose.9 For reversible sol–gel phase transition, glucose-responsive crosslinking is required. In other words, there should be a specific interaction between glucose and Con A for the formation of crosslinks between glucose-containing polymer chains.66 Matsumoto et al.126


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designed a modulated insulin delivery system using phenylborate derivatives which could form covalent complexes with polyol compounds. The hydrogel swelling kinetics could be manipulated by polyvalent phenylboronate–glucose interactions.42 Enzyme-responsive materials, which usually have enzyme-reactive groups either along the polymeric backbone or as a side group, are also designed to undergo macroscopic transition through catalytic activities of selective enzymes. Specific enzymatic reactions have been employed for network formation, network degradation and release of loaded biomolecules.59 Skaalure et al.79 designed an enzyme-sensitive hydrogel based on photoclickable thiol-ene PEG crosslinked with a peptide derived from aggrecanase-cleavable site in aggrecan. The introduced hydrogel had the potential for cartilage repair due to its degradability by CHONs. Jin et al.127 developed chitosan-based hydrogels through enzymatic crosslinking with horseradish peroxidase (HRP) and hydrogen peroxide (H2O2). They also synthesized injectable hydrogels using hyaluronic acid-dextran-tyramine conjugates with potential application for cartilage TE.128 The hydrogels were formed via enzymatic crosslinking of the tyramine residues in the presence of HRP. It should be noted that the high concentration of H2O2 during injection could induce cytotoxicity in these hydrogels.129 Attempts have been made to develop hydrogels responsive to specific proteins. Recently, Parmar et al.130 have developed a biodegradable hydrogel using a collagen-like protein modified with hyaluronic acid (HA) or chondroitin sulfate binding peptides and cross-linked with metalloproteinase 7 (MMP7)-sensitive peptide. The viability and chondrogenic differentiation of human mesenchymal stem cells (hMSCs) was improved in functionalized hydrogel in comparison with control. DNA-responsive hydrogels also undergo swelling and shrinking in response to specific DNAs. These types of hydrogels show high specificity for detecting DNA


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concentrations and as a result they have high potential in diagnostics and genomic applications.80, 131-133

4. In-situ forming hydrogels The implantation of scaffolds through surgical procedures may cause problems such as tissue damage and pain. To confront the limitations of such conventional methods, injectable hydrogels that are formed in situ after injection have been developed.134 The development of in-situ forming hydrogels has received a lot of attention in recent years.4, 50 The merits of using these hydrogels are their high flexibility, capability of cell encapsulation, and controlled delivery at the defect site using minimally invasive procedures. In fact, injectable hydrogels provide biological and mechanical cues for tissue repair after formation in-situ.135-137 The requirements of low viscosity and high fluidity upon injection, mild gelation conditions, biodegradability, biocompatibility and nontoxicity should be fulfilled for practical biomedical applications.36 Different strategies have been employed to make injectable hydrogels from polymers. In this type of system, the liquid solution of the polymer is injected to fill the tissue defect and then it is polymerized in situ to transform into a solid gel shortly.4 In-situ forming hydrogels are classified into chemical and physical hydrogels based on the gelation mechanism. Chemical hydrogels are produced by covalent crosslinking of polymers whereas physical hydrogels are prepared by noncovalent intermolecular interactions of polymers with finite lifetimes, e.g. stereocomplexation, hydrogen bonding, hydrophobic and electrostatic interactions, and Van der Waals forces.55, 134 In recent years, hybrid-type hydrogels have been developed by combined physical and chemical crosslinking metods to overcome the drawbacks of each type.138 Figure 1


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schematically shows the mechanisms and engineering design of polymers for injectable hydrogels. More specific details of different in-situ forming hydrogels are explained below.

Figure 1 Schematic representation of different methods for production of physically and chemically crosslinked hydrogels. 4.1 Physical crosslinking Physically crosslinked gels are of great attention for biomedical applications because toxic crosslinking reagents are not used in the preparation of these materials. However, they are generally unstable and mechanically weak.9, 55 Commonly, ionic interactions have been used to produce physically crosslinked hydrogels. For instance, alginate hydrogels are formed by calcium ions.139, 140 Hydrogen bonding occurs in poly(methacrylic acid-g-ethylene glycol) [P(MMc-g-EG)] hydrogel.141 Physically crosslinked hydrogels can also be prepared from selfassembly of multi-block copolymers or graft copolymers through hydrophobic interactions.142 Similarly, polypeptide-conjugated polymers are used for hydrogel formation via self-assembly of peptide sequences.143, 144 Kisiday et al.145 developed a self-assembling peptide hydrogel for


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cartilage regeneration. The designed scaffold supported CHONs growth and promoted ECM synthesis in 3-D cell cultures after 4 weeks. The formation of stereocomplex by poly(lactide) (PLA) was first reported by Tsuji.146 The stereocomplexation can occur in the blends of PLA homopolymers as well as PLA and poly(ethylene glycol) (PEG) block copolymers.147 Although hydrogels can be easily formed upon mixing each polymer solution using stereocomplexation, there exists only a limited number of compositions suitable for this type of crosslinking.9 4.2 Chemical crosslinking In chemical crosslinking, permanent covalent bonds are involved which result in more stable hydrogels with better mechanical strength as compared with physical crosslinking method.50 However, incorporation of chemical crosslinkers may cause toxicity problems. Covalent crosslinking of polymers occur by a variety of chemical reactions including radical polymerization, click chemistry, Michael addition, and enzyme-mediated polymerization.62 In radical polymerization, as one of the most popular reactions, radicals are produced from initiator molecules and then propagated via unreacted double bonds during polymerization.148 The crosslinked polymeric networks will be formed finally after reacting polymer chains with each other. However, full control over the final product is difficult owing to various termination mechanisms.134 Of particular interest, PEG-based monomers have successfully been polymerized of photon irradiation and utilized as cell delivery vehicles for tissue regeneration.37 Chemical crosslinking can also be performed by mixing polymers having nucleophilic and electrophilic groups through Michael-type reactions. But biological compounds can pose the risk of side reactions through competing with nucleophiles.134, 149 Lee et al.64 have developed injectable HA/Pluronic F127 hydrogels. The composites were prepared by conjugation of HA


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with dopamine and mixed with thiol end-capped Pluronic F127 copolymer. As a result of Michael-type catechol-thiol addition reactions, lightly cross-linked hydrogels were obtained. The superior tissue-adhesion properties and in vivo stability of hydrogels make them practical for drug delivery applications. As for click chemistry, the polymer chains should be functionalized with azide and alkyne groups to form a triazole compound. The main disadvantage of this method is the application of potentially toxic copper as catalyst.150 Recently, different kinds of enzymes have been implemented for hydrogel formation.151-153 Merits of enzymatic crosslinking are the high level of substrate specificity preventing unwanted reactions as well as the mild gelation conditions favorable for tissue regeneration.55 Different kinds of polymers have been functionalized with tyramine, tyrosine or aminophenol side groups to fabricate in-situ forming hydrogels using hydrogen peroxide as the oxidizing agent catalyzed by horseradish peroxidase (HRP).128, 154-156 4.3 Hybrid crosslinking Hybrid hydrogels employ both physical and chemical crosslinking methods to overcome their respective limitations while preserving their merits.138, 157 Therefore, hybrid hydrogels have attracted interest for biomedical applications recently. Polysaccharide crosslinked hydrogels for sustained protein delivery systems was developed by Shimoda et al.158 They utilized acryloyl group-modified cholesterol-bearing pullulan as a building block and multiarmed poly(ethylene glycol) with thiol groups as a cross-linker. Besides, an injectable hybrid crosslinkable gellan hydrogel was produced via gellan thiolation by Du et al.159 The thiolated gellan hydrogel exhibited rapid gelation, low gelling temperature, stable structure and nontoxicity and therefore it could be used as injectable system. Shachaf et al.160 designed a thermoresponsive hydrogel from


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conjugation of fibrinogen to Pluronic F127 by free-radical polymerization using light-induced chemical crosslinking. Mura et al.161 also introduced a novel injectable chitosan hydrogel by joint ionic and covalent cross-linking. They found that the pH barrier of the chitosan solution could be overcome not only to preserve its thermosensitivity, but also to increase crosslinking degree in the hydrogel network. Results showed that the gel was formed rapidly at the injection site and remained for at least 1 week. Boere et al.162 also combined the temperature-induced physical crosslinking with native chemical ligation (NCL) to produce mechanically strong hydrogels for tissue engineering applications. A novel monomer of N-(2-hydroxypropyl)methacrylamide-cysteine (HPMA-Cys) was copolymerized with N-isopropylacrylamide (NIPAAm) to achieve thermo-responsive polymers functionalized with cysteine. Besides, thioester cross-linkers were produced using PEG and hyaluronic acid. It was found that a rapid gelation occurred with increasing temperature up to 37°C owing to the self-assembly of the pNIPAAm chains. In order to enhance the stability of the formulations, covalent cross-linking between thioester and cysteine functionalities was formed which led to a stronger hydrogel. 5. Nanostructured hydrogels With the advances of nanotechnology, material design can be exploited into the nanometer scale.163 Using nanotechnology, novel materials have been developed to mimic the complex and hierarchical structure of the native cartilage tissue. Cells can interact with the ECM due to the nanoscale dimension of natural tissues. The biomimetic properties and superior physiochemical features of nanomaterials (i.e. increased surface area, roughness and area to volume ratios) stimulate cell growth and tissue regeneration in comparison with conventional materials.39 Therefore, nanostructured biomaterials are becoming increasingly important particularly in tissue engineering.


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As mentioned, hydrogels have high potential to replicate the native hydrated cartilage ECM. Swift and significant response to a particular stimuli and high elasticity are of great importance to use smart hydrogels in various applications. To increase the response of gel dynamics, several strategies have been explored, among which, reducing gel size down to nanoscale is one of the most promising techniques known to achieve rapid kinetics.9, 34 By tailoring mechanical and physical properties of hydrogels at nanometer scale, 3D nanostructured hydrogel scaffolds could be designed for TE applications with the ability of controlling cellular fate and tissue regeneration via cell-matrix interactions.164 However, only a limited number of nanostructuring techniques can be practically utilized for the development of novel hydrogel networks. For instance, it is seemingly impossible to apply lithographic methods owing to the high water content of hydrogels. On the contrary, nanostructured hydrogels can be simply achieved via selfassembly of hydrophobic segments (nanodomains) of polymers in aqueous environment.165 As an example, Frisman et al.164 presented a method to provide nanostructure into hydrogels through micellar self-assembly with the aim of mimicking natural ECM for TE applications. Various strategies to take the advantages of nanotechnology in the preparation of smart hydrogels are outlined below and depicted in Figure 2.


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Figure 2 Different types of nanostructured hydrogels.

5.1 Nanoparticles hydrogels (Nanogels) Nanogels are nanoscale hydrogel nanoparticles (

Smart Polymeric Hydrogels for Cartilage Tissue Engineering: A

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