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Mina Mekhail and Maryam Tabrizian* was not until the 1970s that interest in chitosan was rekindled, mainly due to the desire of the fishery industry to utilize the shell wastes produced.[10] Nowadays, chitosan is being investigated in a wide range of applications in agriculture, pharmaceuticals, food additives, and biomedical engineering.[10–13] The ability to solubilize chitosan in slightly acidic conditions made it feasible to fabricate injectable chitosan-based scaffolds (ICS) for applications in regenerative medicine. In this Progress Report, ICS refers to chitosan-based solutions that undergo gelation in response to physical, chemical, or biological stimuli. Injecting a scaffold into the target tissue using minimally invasive surgery is much more desirable in a clinical setting than standard surgical implantation of the scaffold. Minimally invasive surgery reduces hospitalization time and eliminates the risk of post-surgical wound infections. Moreover, in order to enhance the biocompatibility of ICS, various chemical modifications have been implemented to solubilize chitosan in neutral rather than acidic conditions.[14–16] The number of publications on ICS has been growing exponentially in the last decade, reaching up to 27% of all the publications on injectable scaffolds (IS) (Figure 1A). Further analysis of ICS publications demonstrated that 41% of the publications did not mention a specific biomedical application; 36% investigated ICS for bone and cartilage regeneration; 6% investigated ICS for cardiovascular regeneration; and the remaining 17% were for other applications such as cancer treatments, neural regeneration, wound healing, corneal repair, kidney repair, periodontal healing, adipose tissue regeneration, peripheral nerve regeneration and, as embolization agents (Figure 1B). It is thus evident from the growing interest in the literature that ICS provide promising therapeutic modalities for a multitude of applications in regenerative medicine. Regenerative medicine is the field concerned with the replacement of diseased or injured tissues with regenerated, healthy, and functional tissue. Biomaterials (both synthetic and natural) play a pivotal role in regenerative medicine. They are used in the fabrication of scaffolds that provide cellular attachment, migration, proliferation, and in some cases differentiation. In the past decade, IS have been extensively explored due to their many desirable properties, such as: 1) ease of administration in vivo using minimally invasive surgery; 2) high

Injectable scaffolds (IS) are polymeric solutions that are injected in vivo and undergo gelation in response to physiological or non-physiological stimuli. Interest in using IS in regenerative medicine has been increasing this past decade. IS are administered in vivo using minimally invasive surgery, which reduces hospitalization time and risk of surgical wound infection. Here, chitosan is explored as an excellent candidate for developing IS. A literature search reveals that 27% of IS publications in the past decade investigated injectable chitosan scaffolds (ICS). This increasing interest in chitosan stems from its many desirable physicochemical properties. The first section of this Progress Report is a comprehensive study of all physical, chemical, and biological stimuli that have been explored to induce ICS gelation in vivo. Second, the use of ICS is investigated in four major regenerative medicine applications, namely bone, cartilage, cardiovascular, and neural regeneration. Finally, an overall critique of the ICS literature in light of clinical translatability is presented. Even though ICS have been widely explored in the literature, very few have progressed to clinical trials. The authors discuss the current barriers to moving ICS into the clinic and provide suggestions regarding what is needed to overcome those challenges.

1. Introduction Chitosan, a linear polysaccharide, is one of the most explored naturally derived biomaterials in the field of regenerative medicine.[1] Its extensive use is attributed to its many desirable physicochemical characteristics such as biocompatibility,[2,3] biodegradability,[4,5] anti-microbial properties,[5,6] mucoadhesive properties,[7] and versatility in fabrication/modification.[8,9] Moreover, it is readily available and cheap. Chitosan is obtained through the deacetylation of chitin, a polysaccharide found in the shells of crustaceans such as shrimp and crabs. Deacetylation of chitin produces D-glucosamine units that are randomly distributed within the polymeric chain containing N-acetyl-D-glucosamine monomers. The presence of cationic amine groups makes chitosan (unlike chitin) soluble in acidic solutions. Even though chitosan was first synthesized more than a century and a half ago (in 1859) by Dr. C. Rouget, it M. Mekhail, Prof. M. Tabrizian Biomedical Engineering Duff Medical Building Room 313, McGill Montreal H3A 2B4, Canada E-mail: [email protected]

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Injectable Chitosan-Based Scaffolds in Regenerative Medicine and their Clinical Translatability

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water retention and excellent porosity that can allow cellular infiltration; 3) ability to provide controlled release of entrapped therapeutic molecules through diffusion and/or degradation; 4) biodegradability at a rate similar to rate of tissue remodeling; and 5) versatility in fabrication that allow manipulation of specific physicochemical properties. In this Progress Report, a comprehensive analysis of the different ICS formulations developed for regenerative medicine applications was conducted. Moreover, the four most widely investigated applications were further discussed, namely bone, cartilage, cardiovascular, and neural regeneration. These four applications are also the areas that can greatly benefit from ICS technologies based on the current clinical demand. Unfortunately, from all the developed ICS compositions, only one thermosensitive ICS made it to clinical trials and is now commercially available for articular cartilage regeneration. The barriers to moving ICS from lab to clinic are discussed, and suggestions are proposed by the authors regarding the development of clinically feasible ICS.

2. Modes of Gelation “Modes of gelation” refer to physical, chemical, or biological stimuli that trigger chitosan gelation in vivo. The various modes of gelation discussed in this Progress Report have been organized under the following categories: self-triggered gelation in response to physiological conditions (temperature and pH); physically/chemically-triggered gelation (photocrosslinking, chemical crosslinking, ionic crosslinking, and polymer–polymer interactions); and biologically-triggered gelation (enzymatic crosslinking) (Figure 2).

Mina Mekhail is a Ph.D. candidate in the department of Biomedical Engineering at McGill University. He has over 9 years of experience in the field of biomaterials and has developed novel scaffolds during his graduate studies. For his Ph.D. thesis, he developed an injectable, rapidly gelling, chitosan sponge for applications in tissue engineering and drug delivery. He is currently interested in applying his injectable chitosan sponge in vivo for spinal cord injury repair. Maryam Tabrizian is a Professor in the department of Biomedical Engineering at McGill University. Her expertise lies is in the development of advanced materials and interfaces for applications in nanomedicine, regenerative medicine, and biorecognition systems. Some ongoing projects in her lab are using layer-by-layer-coated liposomes for targeted drug delivery, design of microfluidic devices for biorecognition applications, using surface plasmon resonance for pathogen detection, and development of novel injectable biomaterials.

2.1. Self-Triggered Gelation in Response to Physiological Conditions In this section, a discussion of ICS formulations designed to undergo gelation at body temperature (37 °C) or at different pH environments found in the body (acidic pH of the stomach, neutral pH of most tissues, or slightly alkaline pH of the intestines) is presented.

2.1.1. Temperature-Responsive ICS ICS that undergo gelation in response to temperature elevation, usually from room temperature to 37 °C, are referred to

Figure 1. A) Graph demonstrating the total number of publications on IS and ICS over the past decade; B) Pie chart illustrating the percentage of publications that utilized ICS in different regenerative medicine applications.

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as thermosensitive ICS. This mode of gelation has been the most widely explored (75% of total ICS publications) after the publication of the landmark study by Chenite et al. that demonstrated the use of β-glycerophosphate (GP) for thermal gelation of neutral chitosan solutions.[17] Other molecules such as ammonium hydrogen phosphate and sodium bicarbonate have also been explored, but not as widely as GP. GP is a weak base that was used in the 1990s as an essential medium supplement to differentiate human mesenchymal stem cells (MSCs) into the osteoblast lineage.[18,19] In 2000, Chenite et al. reported that the addition of GP to an acidic chitosan solution on ice increased its pH up to a neutral pH while keeping the chitosan soluble. However, increasing the temperature prompted chitosan gelation. This was the first physically crosslinked, thermosensitive chitosan hydrogel reported in the literature.[17] Multiple mechanisms were proposed to explain the gelation process. First it was suggested that strong hydrogen bonds between chitosan and water molecules at low temperatures and in the presence of GP kept the chitosan soluble. However, with increasing temperature, these hydrogen bonds weakened due to the increased vibration of energized water molecules. This led to the displacement of water molecules surrounding chitosan with GP, which in turn provided a screening effect that favored hydrophobic interactions between chitosan chains, ultimately leading to gelation (Figure 3A). Other secondary mechanisms that contributed to gelation were ionic attractions between anionic phosphate groups in GP and cationic amine groups in chitosan, and the weakening of repulsive forces between amine groups in chitosan due to their deprotonation in the basic environment created by GP.[17] Moreover, an inverse correlation between the degree of deacetylation (DOD) of chitosan and the gelation temperature was discovered; the higher the DOD, the lower the transition temperature. Therefore, the ideal DOD for tissue engineering applications was found to be 91%, which provided a gelation temperature close to 37 °C.[17] Further investigation of the CS/GP gelation mechanism demonstrated that the main driving force for gelation was the hydrophobic interactions between chitosan chains.[20] Ionic crosslinking was deemed to contribute weakly to the gelation process since the ratio of protonated amine groups to anionic phosphate groups decreased with increasing temperature.[20]

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Figure 2. A pie chart demonstrating the percentages of publications using the various modes of gelation explored.

Ammonium hydrogen phosphate was also explored for the fabrication of thermosensitive ICS.[21] Ammonium hydrogen phosphate added to a chitosan solution at a low temperature was able to raise the pH of the solution to 7–7.2 without precipitating the chitosan. It was therefore suggested that ammonium hydrogen phosphate does not cause chitosan gelation through only ionic interactions. Similar to CS/GP, gelation occurred mainly due to the hydrophobic interactions between chitosan chains with increasing temperature.[21] Sodium bicarbonate, a weak base, was found to induce chitosan gelation at concentrations between 0.08 and 0.12 M.[22] The addition of sodium bicarbonate to an acidic chitosan solution prompted the formation of carbon dioxide and water due to the reaction between hydrogen and carbonate ions. The production of carbon dioxide raised the pH of the solution and triggered gelation through formation of intramolecular hydrogen bonds between chitosan chains.[22] In another investigation, gelatin was added to the solution to avoid irregular precipitation of chitosan during pH increase and provided more homogeneous gel formation with increasing temperature.[23] In addition to introducing components (GP, ammonium hydrogen phosphate, and sodium bicarbonate) to the chitosan solution to create thermosensitive ICS, chemical modifications of chitosan have also been explored, to render the modified chitosan polymer thermosensitive. The most widely explored chemical modification is grafting poly(ethylene glycol) (PEG) to chitosan and fabricating PEG-chitosan block copolymers. PEG is a water-soluble polyether that has been widely investigated in regenerative medicine and drug delivery applications.[24,25] Coating proteins, genes, or drug delivery vehicles such as micro- and nanoparticles with PEG has been shown to prolong their blood circulation life by camouflaging them from immune cells and shielding them from proteolytic enzymes.[26,27] In regenerative medicine, PEG has been used to reduce both scaffold degradation and immunogenicity.[28] At a low temperature, PEG-grafted chitosan was soluble at neutral pH due to strong hydrogen bonds between the chains and the surrounding water molecules.[29] As temperature increased the hydrophobic interactions between the polymer chains became more pronounced, and gelation occurred.[29] This gelation mechanism is similar to CS/GP and CS/ammonium hydrogen phosphate. However, the amount of grafted PEG was critical for the formation of a thermosensitive hydrogel. Grafting less than 40% (w/w) of PEG on the chitosan backbone did not render chitosan soluble at a neutral pH. While grafting PEG at weight ratios higher than 55% w/w, increased hydrophilicity and weakened the hydrophobic interactions between chitosan chains, thus preventing gelation.[29] It is important to note that these PEG-grafting values were optimized for PEG with a molecular weight (MW) of 2000; the use of different MWs will yield different grafting values.[29] In another study, Kang et al. fabricated PEG-poly(L-alanine-co-Lphenyl)-grafted chitosan, which was shown to form micelles at low temperatures (10 °C), but increased in size through aggregation at a higher temperature (35 °C).[30] Thermal gelation was attributed to two major factors, namely the deprotonation of amine groups of chitosan and the dehydration of PEG with increasing temperature.[30]

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Figure 3. Schematic diagrams illustrating one example from each of the seven modes of gelation. A) Thermosensitive CS/GP gelation mechanism; B) pH-responsive N-palmitoyl chitosan solution; C) Photopolymerization reaction of methacrylated glycol chitosan using visible blue light; D) Chemical crosslinking using genipin; E) Ionic crosslinking using Guanosine 5′-diphosphate (GDP); F) Polymeric blending of N-succinyl chitosan and aldehyde hyaluronic acid; G) Enzymatically-mediated crosslinking of chitosan using the enzyme urease.

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2.1.2. pH-Responsive ICS Like thermosensitive hydrogels, pH-responsive hydrogels take advantage of the physiological environment to trigger gelation. Chitosan precipitates at a pH greater than 6.5; therefore, some modifications were used to provide a homogeneous hydrogel formation during the transition to a neutral pH. One method was to conjugate the hydrophobic group, palmitoyl, to the free amine groups of chitosan.[38] The polymeric solution had shear-thinning properties, which made it possible to inject, and as the pH increased from 6.5 to 7 in vivo, the solution underwent gelation.[38] Gelation occurred due to the increased hydrophobic interactions between the palmitoyl groups with increasing pH and the reduction in repulsive forces between deprotonated amine groups.[38] In another study, N, O-carboxymethyl chitosan was blended with alginate and genipin to produce a semi-interpenetrating polymeric network.[39] Genipin crosslinked the free amine groups in N, O-carboxymethyl chitosan, turning the hydrogel

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dark blue. The hydrogel was responsive to pH and swelled more in a neutral pH than in an acidic environment. In an acidic solution, strong hydrogen bonding between N, O-carboxymethyl chitosan and alginate limited the swelling; however, at a neutral pH, the carboxylic groups were ionized, leading to more swelling due to electrostatic repulsion.[39] Similarly, another study investigated the preparation of chitosan/poly(ethylene oxide) that swelled more under basic conditions as compared to neutral or acidic conditions.[40] Designing smart, pH-responsive ICS that can swell in different pH environments is critical for the future of targeted drug and cell delivery.

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In addition to PEG-grafting, PEG-chitosan block copolymers (PEG–CS) were synthesized to fabricate thermosensitive hydrogels.[31] The solubility of PEG–CS in neutral PBS increased with increasing the molar ratio of PEG since PEG disrupted the crystalline structure of chitosan and weakened the chitosan intrachain hydrogen bonding.[31] Similar to polyol salts and PEG-grafted chitosan, the major driving force for gelation was attributed to the increased hydrophobic interactions between PEG–CS chains as temperature increased.[31] In another study, thiolated glycol chitosan was crosslinked using oligo(acryloyl carbonate)-b-poly(ethylene glycol)-b-oligo(acryloyl carbonate) triblock copolymer via the Michael-type addition reaction.[32] The acryloyl groups provided sites for functionalizing thiolcontaining bioactive moieties prior to reaction with thiolated glycol chitosan, which is a desirable feature for fabricating bioactive scaffolds. Gelation occurred rapidly when thiolated glycol chitosan and oligo(acryloyl carbonate)-b-poly(ethylene glycol)-boligo(acryloyl carbonate) were mixed under physiological conditions (pH 7.4 and 37 °C) due to the presence of thiolate anions, which are a reactive species of the Michael-type addition.[32] Pluronic acid, the trade name for a widely used poloxamer, poly(ethylene oxide)-b-poly(propylene oxide)-b-poly(ethylene oxide), is thermosensitive in nature and has been incorporated with chitosan in order to produce thermosensitive ICS. Pluronic acid grafted on chitosan using EDC/NHS chemistry was shown to undergo gelation at 25 °C.[33] Increasing the temperature increased hydrophobic interactions between the pluronic chains and increased their aggregation into a more crystalline structure. In addition, chitosan dehydration and hydrophobic interactions between chitosan chains contributed to gelation.[33] Poly(N-isopropylacrylamide) or PNIPAm is a another thermosensitive polymer that has been widely explored in tissue engineering and drug delivery applications.[34,35] NIPAm was grafted onto chitosan using cerium ammonium nitrate (CAM) as an initiator. N-isopropylacrylamide-grafted chitosan was soluble at pH 7, and underwent gelation above 32 °C due to the formation of inter- and intrachain hydrophobic interactions between the N-isopropylacrylamide-grafted chitosan chains.[36,37]

2.2. Physically/Chemically Triggered Gelation 2.2.1. Photoinitiated Polymerization Unlike temperature and pH that are physiological stimuli, photoinitiated polymerization uses light to induce gelation. The ability to use specific wavelengths to control spatiotemporal hydrogel properties makes photocrosslinking highly desirable.[41] The period of exposure can be used to control the degree of polymerization and thus the mechanical properties of the hydrogel and its rate of degradation.[42] However, photoinitiators used in the literature have been shown to be cytotoxic to a variety of cell types through the formation of free radicals, which increase oxidative stress.[43,44] Chitosan modified with methacrylic acid and lactic acid was photopolymerized using 365 nm UV light and the photoinitiator Iragacure2959.[45] Iragacure 2959 was chosen since it is considered to be the least cytotoxic.[44] It dissociates into two primary radicals (benzoyl and 2-hydroxypropyl) upon absorption of UV, which in turn react with vinyl groups (H2C CH2) to initiate polymerization. Gelation took place in approximately 9 min.[45] In another study, chitosan was modified by incorporating glycol acrylate methacrylate through the Michael addition reaction to produce methacryloyloxy ethyl carboxyethyl chitosan.[46] Methacryloyloxy ethyl carboxyethyl chitosan hydrogels were fabricated by blending with a photoinitiator, D-2959, and exposing the solution to UV (320–480 nm) for 15 min.[46] In order to avoid the harmful effects of UV, such as protein denaturation and cell damage, photoinitiators activated by wavelengths in the visible spectrum have been explored. Riboflavin (vitamin B2) was investigated as a photoinitiator to induce the gelation of methacrylated glycol chitosan using blue light (400–500 nm) (Figure 3C).[47] Even though the blue light was in the visible spectrum, cell exposure for more than 600 s significantly reduced cell viability.[47] The most rapid gelation (12 s) took place using 24 × 10−6 M of riboflavin. Riboflavin is converted to the leuco form in the presence of trace amounts of oxygen, and the free radicals formed initiate polymerization of methacrylated glycol chitosan.[47] In order to improve the mechanical properties, hyaluronic acid was added to form a semi-interpenetrating polymeric network with methacrylated glycol chitosan.[48] 2.2.2. Chemical Crosslinking Chemical crosslinking of ICS provides some advantages over physical crosslinking, such as enhanced mechanical properties,

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in vivo stability, better control over the degree of crosslinking, and the ability to fine tune the rate of degradation. However, chemical crosslinking also has disadvantages, such as cytotoxicity of crosslinking agents to cells, slower rate of gelation, and difficulty injecting pre-gelled hydrogels. Illustrated in this section are the studies that were able to overcome these challenges and fabricate effective chemically crosslinked ICS. Fiejdasz et al. used a blend of chitosan and collagen crosslinked with genipin as an ICS (Figure 3D).[49] Genipin is a natural crosslinking molecule derived from the fruits of Gardenia jasminoides Ellis that has been shown to be more biocompatible compared to glutaraldehyde (a synthetic crosslinking agent).[50] Genipin molecules crosslink primary amine groups found in both chitosan and collagen and induce a color change to dark blue.[51] Interestingly, it was found that the gelation time of genipin-crosslinked chitosan/collagen blends was within the range of 3–7 min, which is more rapid than some physically crosslinked ICS.[49] In another investigation, Deng et al. used EDC (1-ethyl3-(3-dimethylaminopropyl) carbodimiide) chemistry to crosslink chitosan and collagen blends.[52] In contrast to the previous study, Deng et al. crosslinked the chitosan/collagen blend for 8 min prior to injection and demonstrated that the blend was still injectable. EDC is a zero-length crosslinker, which means it mediates the crosslinking of two functional groups without being incorporated in the link. EDC mediates the formation of amide bonds between carboxylic acid groups and primary amine groups. In this case, collagen contains both carboxylic acid and amine groups, while chitosan only contains amine groups. Gelation took place within 4 min, which is again more rapid than other physical crosslinking methods.[52] Finally, water-soluble chitosan hydrogels were fabricated via a radical polymerization reaction using ammonium persulfate as an oxidant and N,N,N′,N′-tetramethylethylenediamine (TMEDA) as a reducer.[53] The redox reaction that took place caused the crosslinking of C C that results in alkyl linkages, and the reaction took place in less than 6 min.[53] In addition, Boesel et al. developed a new, “greener” approach to fabricate methacrylated oligomers of chitosan that were polymerized in the presence of sodium persulfate and ascorbic acid.[54] The gelation time was adjustable from 1.5 to 60 min by changing the methacrylation reaction conditions such as time, temperature, and concentration of oligomers.[54]

area. Ionotropic gelation provides a simple way for fabricating ICS, induces rapid gelation, and is highly reproducible. Hsieh et al. were first to investigate the combination of poly(glutamate) and chitosan to produce porous scaffolds.[56] Poly(glutamate) is a natural polyanionic polymer produced by Bacillus subtilis; it is hydrophilic, biocompatible, and biodegradable.[56,57] Mixing poly(glutamate) and chitosan solutions led to rapid hydrogel formation (

Injectable chitosan-based scaffolds in regenerative medicine and their clinical translatability.

Injectable scaffolds (IS) are polymeric solutions that are injected in vivo and undergo gelation in response to physiological or non-physiological sti...
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