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Injectable and 3D Bioprinted Polysaccharide Hydrogels: From Cartilage to Osteochondral Tissue Engineering Janani Radhakrishnan, Anuradha Subramanian, Uma Maheswari Krishnan, and Swaminathan Sethuraman Biomacromolecules, Just Accepted Manuscript • DOI: 10.1021/acs.biomac.6b01619 • Publication Date (Web): 30 Nov 2016 Downloaded from http://pubs.acs.org on November 30, 2016
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Injectable and 3D Bioprinted Polysaccharide
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Hydrogels: From Cartilage to Osteochondral
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Tissue Engineering
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Janani Radhakrishnan, Anuradha Subramanian, Uma Maheswari Krishnan and
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Swaminathan Sethuraman*
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School of Chemical & Biotechnology, SASTRA University
Centre for Nanotechnology & Advanced Biomaterials
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Thanjavur-613401, India
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ABSTRACT: Biomechanical performance of functional cartilage is executed by the exclusive
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anisotropic composition and spatially varying intricate architecture in articulating ends of
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diarthrodial joint. Osteochondral tissue constituting the articulating ends comprise superfical soft
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cartilage over hard subchondral bone sandwiching interfacial soft-hard tissue.
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absorbent, lubricating property of cartilage and mechanical stability of subchondral bone regions
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are rendered by extended chemical structure of glycosaminoglycans and mineral deposition
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respectively.
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class of hydrogels investigated for restoration of functional cartilage.
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hydrogels have gained momentum as it offers patient compliance, tunable mechanical properties,
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cell deliverability and facile administration at physiological condition with long term
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functionality and hyaline cartilage construction.
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groups in carbohydrate polymers impart tailorability of desired physicochemical properties and
The shock-
Extracellular matrix glycosaminoglycans analogous polysaccharides are major Recently, injectable
Interestingly, facile modifiable functional
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versatile injectable chemistry for the development of highly potent biomimetic in situ forming
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scaffold. The scaffold design strategies have also evolved from single component to bi- or multi-
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layered and graded constructs with osteogenic properties for deep subchondral regeneration. This
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review highlights the significance of polysaccharide structure-based-functions in engineering
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cartilage tissue, injectable chemistries, strategies for combining analogous matrices with
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cells/stem cells and biomolecules and multi-component approaches for osteochondral mimetic
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constructs. Further, the rheology and precise spatio-temporal positioning of cells in hydrogel
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bioink for rapid prototyping of complex three-dimensional anisotropic cartilage have also been
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discussed.
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KEYWORDS: injectable, osteochondral, bioinks, bioprinting, polysaccharides
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Arthritis, an age-associated degeneration in articular cartilage is the leading cause for disability
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affecting daily activities in about 23% of older adults.1 Around 9.6% men and 18% women
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worldwide over 60 years of age have been diagnosed with symptomatic arthritis.2,3 The disease
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causes joint tenderness, varying degrees of inflammation, joint pain, occasional effusion and
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degeneration of the joint. These sequential events in the disease progression limits the movement
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and function, leading to poor quality of life thereby causing significant societal and economic
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burden equivalent to cardiovascular disease.2,4,5
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imbalance between synthesis and degradation of matrix materials leads to osteoarthritis.2,4
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Although the injury in articular cartilage initiates wound healing response, drawbacks such as
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limited proliferation of terminally differentiated chondrocytes, their catabolic response to
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pathological mediators and avascularity restricting immigration of regenerative cells prevents
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complete regeneration and restoration of native ECM structure and composition.6,7 Current
1. Introduction
Destabilization in tissue homeostasis causing
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treatment modalities such as microfracture, mosaicplasty, autologous chondrocyte transplantation,
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and osteochondral allograft transplantation relieves pain and improves joint function.5,8 However,
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the cartilage produced by these approaches lack integration with neighbouring host tissue and
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most often constitute collagen I, which is inferior both chemically and mechanically to hyaline
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articular cartilage that has collagen II.1,8,9 Hence, once distorted, neither reversion nor retardation
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of degeneration occurs in the native joint, thereby leaving the field wideopen for regenerative
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strategies like tissue engineering and cell based therapies to address the existing lacuna.5
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Articular cartilage, a low friction articulation diarthrodial joints at knees, hips, fingers, and lower
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spine region is an unusual biphasic tissue. Solid matrix of this tissue is composed of collagen II
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and proteoglycans as primary extracellular matrix (ECM) components and fluid phase is
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synovium.10,11 Cartilage is avascular, aneural and alymphatic in nature and unlike other tissues,
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homogenous population of sparse chondrocytes (2–5%) residing in porous matrix contribute to
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the ECM maintenance, which in turn nourishes the chondrocytes.10,12 The components of a few
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millimeters thick cartilage provides required mechanical properties enabling biomechanical
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functions such as dissipation of compressive loads, shock absorption and allows frictionless pain-
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free movement.4,10,13,14 Complementary to the cartilage compressive strength, the underlying
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harder subchondral bone contributes due to its large area and provides anchorage for collagen
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fibrils of articular cartilage.15 However, in the case of arthritic joint evidenced by extensive
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cartilage tissue damage, an imbalance occurs between the matrix synthesis and degradation of the
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major matrix glycocomponents due to loss of chondrocytes and adverse inflammatory
22
responses.16 The pathogenesis of severe full-thickness defects involves degenerative changes in
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cartilage and subchondral bone with notable remodelling changes in response to the applied
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stress.15 Polysaccharides possess several characteristics that can be harnessed to address the 3 ACS Paragon Plus Environment
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problems encountered at an osteoarthritic joint.
The sol-gel transition phenomenon of
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polysaccharides and tailorability offers designing of spatially varying multi-layered anisotropic
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constructs that mimic the anisotropic composition and micro-macro structural organization
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thereby providing a viable therapeutic strategy to combat osteoarthritis and related joint disorders.
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The use of polysaccharides for a variety of material science applications with special emphasis on
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biomedical applications has been increasing since it resembles the glycan constituent of native
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extracellular matrix (ECM).17 Owing to their beneficial inherent physicochemical properties
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such as biocompatibility, biodegradability, tailorable functional groups and role in cell signaling,
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research on structural and functional polysaccharide biomaterials have gained pace in the recent
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decades. They find diverse utility ranging from extracorporeal devices to intricate implants
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addressing highly specialized medical requirements.18 Hence, many novel synthetic routes and
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newer sources are being explored recently for polysaccharide polymers though most of the
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currently available polysaccharides have been sourced from natural origin.19,20 Polysaccharides
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have been used to design tissue engineered constructs for blood vessels, myocardium, heart
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valves, bone, articular and tracheal cartilage, intervertebral discs, menisci, skin, liver, skeletal
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muscle, neural tissue, urinary bladder and for transplantation of Islets and ovarian follicles.21,22
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In particular, different types of polysaccharides have been explored as cartilage substitutes for the
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functional restoration of load bearing tissue. It has been found extremely useful for articular
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cartilage whose degeneration causes major disabilities in elders.9,23 Three-dimensional Hyaff 11®,
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Hyalograft–C® implants and intra-articular injections of hyaluronic acid as viscosupplement have
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proven effective for the management of patients suffering from osteoarthritis.9,23
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Tissue engineering integrates the principles of engineering and life sciences towards designing
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various scaffolds as biological substitutes either with or without cells and biomolecules to repair,
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regenerate and restore functional tissue at the lesion.24–27 Success of tissue engineering relies
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largely on the extent to which a scaffold mimics the native extracellular matrix (ECM) of the
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respective tissues in terms of structure and composition.28,29
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biomaterials are being explored for the fabrication of matrices, polymers have gained prime
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significance towards the development of ECM analogues.
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biodegradable properties, and ease of functionalization exhibited by polymers favors tailoring of
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the desired composition and intricate architecture as per requirement.30
Although various classes of
Wide range of mechanical and
Natural polymers
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intrinsically possess diverse functions. This category includes proteins that serve as structural
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materials and catalysts while the polysaccharides play vital role in storage, cell recognition and
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intracellular communication.31 Designing an ideal tissue engineered substitute for the restoration
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of articulation and load-bearing function requires constructs with highly mimetic multi-layered
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osteochondral features. The integration of superficial chondral and mineralized subchondral
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bone regions to establish smooth transitional soft-hard tissue interface in the scaffold is
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pivotal.32,33 Recently, 3D printing and patterning of cellular and matrix materials promises to
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replicate the components of anisotropic osteochondral tissue.
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bioprinting enables reconstitution of intrinsic architectural organization and functional
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performance of the tissue. However, the choice of hydrogel material with cytocompatibility, cell
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dispensability and required viscoelastic properties that ensures stability of printed construct is
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indispensible for bioprinting.34 This review compiles the state-of-the-art in polysaccharide based
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injectable scaffolds, multi-component constructs and printable bioinks for functional restoration
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of cartilage that helps in comprehending the relevance of chemistry on physical and biological
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properties of the cartilage tissue mimics. 5 ACS Paragon Plus Environment
This exciting prospect of
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2.
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Polysaccharides are carbohydrate macromolecules built by repeating monosaccharide units linked
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by glycosidic bonds that play vital roles in living systems.21,35 These abundant polysaccharides
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intrinsically possess desirable properties such as biocompatibility, biodegradability and
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functional groups that facilitate facile chemical modifications for tailorability, cytocompatibility
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and organized macro-structural features making them more promising as biomaterials.21,35
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Complementary to the chemical and structural features, biologically the saccharide units play a
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remarkable role in cell signaling thereby mediating cellular processes at molecular level.21
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Polysaccharides majorly explored for tissue engineering include starch, cellulose, chitosan,
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pectins, alginate, agar, dextran, pullulan, gellan, xanthan and glycosaminoglycans.21 Based on
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the chemistry, these repeating units of saccharide possess both beneficial and detrimental
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properties towards tissue engineering applications, which are tabulated in table 1 and 2. These
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polysaccharides have been used alone or in combinations with other natural and synthetic
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polymers for the fabrication of robust matrices with multiple cues to achieve tissue
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morphogenesis. Polysaccharides can be categorized based on the chemical composition (homo-
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and heteropolysaccharides), structure (linear and branched), function in the organism (structural,
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storage and secreted polysaccharides), charge (cationic, anionic and neutral) or source (animal
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origin, plants, algal and microbial).21,35 Figures 1 and 2 depict the structures of linear and
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branched polysaccharides explored for tissue engineering applications. As the physicochemical
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characteristics and functions depends on the structure of polysaccharides, classification based on
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the structure (linear and branched) with the properties has been discussed in the following
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sections (figure 1, 2 & 3).
Polysaccharides as functional biomaterials
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Figure 1. Chemical structures of linear polysaccharide polymers with disaccharide repeating
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units [A] Hyaluronic Acid; [B] Chitin; [C] Chitosan; [D] Heparin; [E] Chondroitin Sulfate; [F]
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Carrageenans; and [F] Pectins. 7 ACS Paragon Plus Environment
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2.1
Linear polysaccharides
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2.1.1
Hyaluronic acid (HA) is a linear polysaccharide consisting of alternate N-acetyl-D
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glucosamine and D-glucuronic acid linked by β(1→3) and β(1→4) glycoside bonds (figure
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1A).36–38 It is a unique linear non-sulfated proteoglycan in the native ECM of connective tissues,
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particularly cartilage and the synovial fluid, with high water adsorption and retention
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capability.23,36,38 The highly viscous and viscoelastic synovial fluid containing HA as the major
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component exhibits protective physicochemical properties as lubricant and shock absorber in
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articular joint cavities.23,36,39 HA is also known to interact with CD44 glycoprotein receptors on
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the chondrocyte surface and enhance the cellular functions through the chondrocyte-specific
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CD44/HA signaling pathway.36,39,40 It stimulates chondrocyte metabolism and exhibits multiple
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chondroprotective roles such as production of collagen II and proteoglycans. This is achieved
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through associations of HA core with keratin sulfate and chondroitin sulfate, scavenging of
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reactive oxygen species, regulation of immune complex interaction and fibroblast
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proliferation.38,39 Though it has many desirable properties such as lubricating and cushioning
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effects for restoring the viscosity and elasticity of the synovial fluid, poor mechanical strength
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and faster degradation of HA restricts its potential in visco-supplementation therapy for treatment
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of early osteoarthritis.4,39,41,42
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hydrophobic poly(caprolactone) units or suitable chemical modifications such as esterification,
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methacrylation and divinyl sulfone/dialdehyde crosslinking at the carboxyl and hydroxyl
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functional groups of HA.38,43,44 Heris et al., have reported a smart injectable hybrid HA / gelatin
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hydrogel particles with Young’s modulus of 22±2.5 kPa by indentation test and shear modulus of
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75±15 Pa at 1 Hz, embedded in HA network as substrate for viable cell adhesion.45
However, these demerits can be overcome by including
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2.1.2 Chitin or poly(β(1→4) N-acetyl-D-glucosamine) is an aminopolysaccharide sourced
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majorly from the crustaceans (shrimps and crabs) and is the second most abundant natural
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polymer (figure 1B).46,47
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viscosity, polyelectrolyte tendency, polyoxy salt formation and metal chelation.48 Additionally,
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the inherent biocompatibility, biodegradability and low immunogenicity of chitin are
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advantageous for biomedical applications such as wound dressing, immobilization of enzymes
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and cells.46,47,49,50 The microfibrillar structure of chitin films and fibers has promoted its use as a
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bioabsorbable suture material.47 Scaffolds of β-chitin has supported chondrocyte morphology
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and synthesis of specific ECM similar to hyaline cartilage.51 A ternary polyethylene oxide /
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chitin / chitosan scaffold showed cartilaginous regenerative potential as the N-acetyl glucosamine
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of chitin favors chondrogenic expression.52 However, its hydrophobicity and insolubility in
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various media limits its applications and hence chitosan, its deacetylated dissolvable form finds
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wide utility as a biomaterial. 48,53
The properties of this highly basic polysaccharide include high
14 15
Table 1. Various linear polysaccharide polymers, their repeating units along with their
16
advantages and disadvantages towards tissue engineering applications
17 Polymer
Repeating units
Properties Beneficial
Disadvantages
Hyaluronic acid (HA)
N-acetyl-D- glucosamine and D-glucuronic acid linked by (1→3) and (1→4) glycoside bonds
High viscoelasticity, chondro-protective, high water adsorption and retention
Poor mechanical strength, faster degradation
Chitin
(1→4) linked N-acetyl glucosamine
High viscosity, polyelectrolyte tendency, metal chelation, enzymatic degradation
Hydrophobicity and insolubility in various media
Chitosan
(1→4) linked D-glucosamine and N-acetyl glucosamine
Process-ability, microbicidal, structural
Brittleness
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similarity to glycosaminoglycans Chondroitin Sulfates
D-glucuronic acid and Nacetyl galatosamine sulfated at either 4- or 6- positions
Water and nutrient absorption, chondroprotective, antiinflammatory
Highly hydrophilic, solvability
Heparin
(1→4)-linked uronic acids (mainly D-glucuronic, Liduronic or L-2-sulfated iduronic) and glucosamine (mainly D-N-acetyl glucosamine and D-di-N-6sulfate glucosamine)
Protein binding, anticoagulation, complement activation, anti-angiogenic, anti-cancer and antiinflammatory
Provokes immunogenic responses
Cellulose
D-glucopyranose linked by β(1→4) glycosidic bonds.
Varied mechanical properties to suit soft and hard tissues, machinable, bioadhesives
Requires intense processing to obtain pure form
Pectins
α (1→4)-galacturonic acids with varying degrees of methylation in carboxylic acid groups
Ionic gels
Highly hydrophilic
Alginate
β-D-mannuronic acid and αL-guluronic acid
Ionic gels, poor protein binding affinity, promotes cell spheroids
Poor mechanical properties, difficult to handle
Carrageenans
(1→3)-linked β-D-galactose and (1→4)-linked α-Dgalactose
Thermo and ionic gelling, structural similarity with glycosaminoglycans, soft to firm gels
Inflammatory
Gellan gum
(1→4)-L-rhamnose-α(1→3)D-glucose-β(1→4)-Dglucuronic acid-β(1→4)-Dglucose
Thermo and ionic gelling, structural similarity with glycosaminoglycans, varying mechanical properties based on acetylation
Slow degradation
Pullulan
α(1→6) linked maltotriose
Flexible with elastic and compressible properties
High hydrophilicity
1 2
2.1.3 Chitosan is a linear aminopolysaccharide consisting of β(1→4) linked D-glucosamine
3
residues and N-acetyl-glucosamine groups (figure 1C). It is a semi-synthetic polymer derived by 10 ACS Paragon Plus Environment
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the partial deacetylation of the poorly soluble chitin.7,54 The cationic chitosan possess bioactive
2
properties such as biocompatibility, biodegradability and microbicidal property that renders
3
versatile applications for this polysaccharide.55
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electrostatically with negatively charged ECM molecules like glycosaminoglycans, which in turn
5
links with cytokines and growth factors to regulate the cellular fate processes.55 Additionally,
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nontoxicity, processability, ease of chemical modification via hydroxyl and amine functional
7
groups, mechanical properties and unique engineered structures of chitosan are advantageous for
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cell and tissue engineering applications.56–58 Moreover, due to its chemical structural similarity
9
with diverse glycosaminoglycans (GAGs), the predominant ECM molecules in cartilage and
10
meniscus, chitosan mimics the native micro-environment for chondrocytes and meniscus cells,
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and promotes chondrogenic activity and cartilage-specific protein expression.36,54,59 Chitosan-
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polycaprolactone copolymers blended with varying ratios of collagen II to construct layer by
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layer mimetic porous microstructured scaffolds crosslinked with sodium tripolyphosphate for
14
articular cartilage repair. The graded architecture and zonal variation in mechanical properties
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exhibited positive impact on the cellular behavior of chondrocytes cultured.60
16 17
2.1.4 Heparin is another linear highly sulfated glycosaminoglycan with alternating units of
18
β(1→4) linked uronic acids (mainly D-glucuronic, L-iduronic or L-2-sulfated iduronic) and
19
glucosamine residues (mainly D-N-acetyl glucosamine and O- and N-sulfated glucosamine).61–63
20
Existence of reactive groups such carboxyl and sulfate groups (figure 1D) provides charge
21
specificity that enables chemical modification as well as electrostatic interactions. The highly
22
negative charge of heparin interacts electrostatically with proteins including growth factors,
23
proteases and chemokines and regulates various cellular signaling.64 Its affinity towards growth
24
factors and cell surface receptors could facilitate stabilization of trophic molecules against
The positive charge of chitosan interacts
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degradation and enable extended localization with enhanced binding.64,65 In addition to cellular
2
interactions, heparin exhibits anti-coagulation, complement activation, anti-angiogenic, anti-
3
cancer and anti-inflammatory effects in biological system.61,64,66 An enzymatically crosslinked
4
injectable heparin and dextran based hydrogel displayed higher storage modulus (~48kPa),
5
chondro-compatibilty and cartilage matrix secretion.63
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2.1.5 Chondroitin sulfate (CS) is an anionic polyelectrolyte glycosaminoglycan consisting of
8
repeating disaccharide units of β-(1→4) D-glucuronic acid and β-(1→3) N-acetyl galactosamine
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with sulfate groups (figure 1E). CS is the major matrix component of cartilage,67–69 associated
10
with proteoglycans to form aggrecan and syndecan that function as structural component and
11
receptor respectively.70 Chondroitin sulfate not alone mediates the osmotic swelling pressure for
12
matrix expansion and collagen network tension, but also possesses high affinity towards the
13
growth factors via electrostatic interaction thereby exhibiting both structural and biological role
14
in articular cartilage.70
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activity, water and nutrient retention, potent wound healing for functional cartilage restoration are
16
the other major beneficial properties of CS.68,69 Since the presence of CS influences compressive
17
strength of the scaffold by promoting proteoglycan secretion, many attempts has been made to
18
integrate CS with other polymers like poly(ethylene glycol) through its reactive hydroxyl and
19
carboxyl functional groups.68,69
Host tissue integration, chondro-protectiveness, anti-inflammatory
20 21
2.1.6 Carrageenan is a linear carbohydrate polymer with (1→3)-linked β-D-galactose and
22
(1→4)-linked α-D-galactose units, which vary in the degree of substitution and are modified into
23
the 3, 6-anhydro derivative depending on the source and extraction conditions.
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disaccharides in the backbone of hydrophilic carrageenans have been in special focus, as its 12 ACS Paragon Plus Environment
Sulfated
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structure resembles glycosaminoglycans. Based on the sulfation of disaccharides, three classes of
2
carrageenans are available. They are the least sulfated κ-carrageenan (kappa), highly sulfated λ
3
and the ι-type with intermittent sulfation.
4
Kappaphycus cottonii forms strong rigid gels, while the highly sulfated lambda (λ) form does not
5
self-gel. On the other hand, elastic, dry, soft gels are produced by the iota (ι-type) in the presence
6
of calcium ions.
7
potassium concentration.71 Instead of ionic gelation, carrgeenans even form thermoreversible
8
hydrogels since it can undergo upper critical solution temperature mediated sol-gel
9
transformation.71
The κ-carrageenan (kappa) extracted from
Firmness of the κ-carrageenan hydrogels has been tailored by changing
10 11
2.1.7 Pectins are natural polysacchardies found in most primary cell walls and non-woody parts
12
of terrestrial plants.21 Repeats of largely methoxylated 1,4-linked α-D-galactosyluronic acid
13
constitutes pectin (figure 1G).21,72
14
combined with many hydrophobic polymers to improve wetablility in polymeric blends.21,72 In
15
addition to biodegradability and cytocompatibility, the facile gelling ability of pectin on exposure
16
to divalent or multivalent cations to form physical hydrogels has gained much attention for tissue
17
engineering applications as injectables.21,73,74 Further, the diols of pectins can be oxidized to
18
aldehyde groups that form Schiff‘s base by reacting with amino groups to form chemical
19
hydrogels.74
The excellent hydrophilic property of pectin could be
20 21
2.1.8 Cellulose constitutes the primary component of plant cell wall structure and is well known
22
as renewable and sustainable biopolymer.75,76 It is a linear homopolymer with D-glucopyranose
23
repeating units linked by β-(1→4) glycosidic bonds (figure 2A).76 Apart from its hydrophilicity,
24
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the chair structure, minimizes flexibility and confers exceptional mechanical strength due to
2
hydrogen bonding and high cohesiveness.75,77
3 4
2.1.9 Alginate is an anionic block polysaccharide with repeating disaccharide units of β-D-
5
mannuronic acid and α-L-guluronic acid residues linked through 1→4 glycosidic linkages (figure
6
2B). It is derived from brown sea weed family which includes Laminaria japonica, Laminaria
7
hyperborea, Ascophyllum nodosum, Eclonia maixma, Lesonia negrescens, Macrocystis pyrifera,
8
and Sargassum.78–81
9
mucoadhesive.80,82,83 The carboxylate groups in guluronic acid residues have been used to form
10
ionic hydrogels through their interaction with divalent cations such as calcium, zinc and
11
strontium.80,83,84
12
crosslinks.85 However, poor binding affinity of alginate to serum proteins restricts adhesion of
13
certain mammalian cell types on alginate scaffolds. However, alginate scaffolds have been found
14
to aid non-adherent cells such as chondrocytes to maintain their native spherical morphology.84
15
Apart from the most commonly evaluated ionically crosslinked physical hydrogels, chemical
16
hydrogels have also been reported by modifying alginic acid groups to aldehyde via acid
17
hydrolysis. This step is followed by oxidation to form injectable poly(aldehyde guluronate)
18
(PAG) hydrogel stabilized by exposure to adipic acid dihydrazide for crosslinking molecule.86 A
19
calcium crosslinked alginate hydrogel with rapid curing and homogeneous mechanical stability
20
showed near-elastic property with Young’s modulus of
21
periosteum-derived chondrogenesis.87
This biocompatible carbohydrate is reported to be biodegradable and
The block structures of alginate determines the structure of the ionic
0.17±0.01 MPa and enhanced
22 23
2.1.10
Gellan gum is a high molecular weight anionic exopolysaccharide secreted by the
24
bacteria Sphingomonas paucimobilis (Sphingomonas elodea) during aerobic fermentation.88 The 14 ACS Paragon Plus Environment
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linear tetrasaccharide consists of (1→4)-L-rhamnose-α(1→3)-D-glucose-β(1→4)-D-glucuronic
2
acid-β(1→4)-D-glucose as repeating unit, with one carboxylic side group (figure 2C).89 Gellan
3
gum properties such as non-toxicity, easy processability, injectability, pseudoplasticity and
4
structural similarity of glucuronic acid residue with glycosaminoglycans are advantageous.89 The
5
mechanical properties of gellan gum varies from soft, elastic to hard, brittle in the acetylated and
6
deacetylated forms respectively.89 Gellan gum forms non-toxic, ionic gels and thermo-gels close
7
to body temperature and has been used for various drug delivery and tissue engineering
8
applications.88,90–92 Though this polysaccharide tends to form ordered double helix structure on
9
cooling, true gel network formation has been initiated upon aggregation and ionic crosslinking.88
10
Oliveira JT et al., 2010, reported that the valency of the ions determines the strength of ionic
11
hydrogel. Presence of monovalent cations induces gelation in gellan gum via screening of
12
electrostatic repulsion amongst the carboxylate groups whereas divalent cations connects two
13
carboxylate groups of glucuronic acid molecules in addition to the screening effect.89 Thus
14
divalent cations form stonger gels than monovalent cations and hence influence the viscoelastic
15
behavior too. This gellan gum also forms photocrosslinkable hydrogels via methacrylation.
16 17
2.1.11 Pullulan occurs as a part of the cell wall in the yeast-like fungus Aureobasidium
18
pullulans.5,93 It comprises linear maltotriose residues linked by α(1→4) glycosidic bonds with
19
consecutive α(1→6) glycosidic bonds (figure 2D).5,93–95 Its molecular weight ranges between
20
1,000,000-2,000,000 daltons depending on the culture conditions of fungi.93 This non-ionic
21
polysaccharide is biocompatible (non-immunogenic), non-toxic, blood compatible, non-
22
mutagenic and non-carcinogenic, impermeable to oxygen, non-hygroscopic and non-
23
reducing.93,94 Presence of a unique linkage in its structure imparts structural flexibility with
24
elastic and compressible properties.93 The highly water soluble pullulan has been modified via 15 ACS Paragon Plus Environment
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chemical functionalization or blending with other organic or inorganic materials that improve the
2
stability.93,94 Carboxymethylated pullulan conjugated with heparin and hydroxyapatite / pullulan
3
/ dextran composite have been developed as tissue regenerative constructs.93,94
4 5
Figure 2. Chemical structures of linear polysaccharide polymers [A] Cellulose; [B] Alginic acid;
6
[C] Gellan gum and [D] Pullulan.
7 8
2.2.
9
2.2.1. Starch is an abundant, renewable, hydrophilic storage polysaccharide in plants comprising
10
of two polymers – linear α-amylose (20-30%) and highly branched amylopectin (70-80%)
11
consisting of α(1→4) glucan and α(1→4)-glucan with α(1→6) linkages at branches (figure
12
3A).21,24,31,96–98
13
contributes to its inferior mechanical properties with brittleness.77
Branched polysaccharides
It has high molecular mobility and weak interaction between chains that
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However, due to its
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biocompatibility, biodegradability and versatile processability, starch has been used in
2
combination with other polymers and as composites in various tissue regenerative strategies
3
especially orthopaedics and in drug delivery.77,97–99 In a stimuli responsive injectable chitosan-
4
starch combined hydrogel, the increase starch content enhanced the elastic modulus evaluated by
5
dynamic mechanical analysis, induced chondrogenesis of encapsulated adipose derived stromal
6
cells and enhanced carilage matrix secretion.98
7 8
2.2.2. Dextran derived from bacteria consists primarily of repeating α(1→6) linked D-
9
glucopyranose residues with less percent of α(1→2), α(1→3), or α(1→4) linked side chains
10
(figure 3B).100,101 Dextran known for its high hydrophilicity shows low protein adsorption,
11
biodegradability, biocompatibility and has been widely used for development of cell
12
microcarriers, drug delivery vehicles and tissue engineering constructs.100–102 It bears three
13
hydroxyl groups per glucose unit, which facilitates the formation of both physically and
14
chemically cross-linked hydrogels.100,102,103 Dextran derivatized with lactic acid oligomers form
15
physical crosslinks while dextran modified with bifunctional isocyanates, glutaraldehyde, or by
16
partial oxidation of hydroxyl groups to aldehydes that were then crosslinked with gelatin forms
17
chemically crosslinked gels.100 In addition, liquid-liquid phase separation mediated scaffolds has
18
been fabricated from dextran for tissue engineering applications.100 Dextran-tyramine conjugated
19
HA enzymatic injectable biomimetic hydrogel demonstrated high moduli of 370 to 18,000 Pa,
20
enhanced bovine chondrocyte viability, proliferation and matrix secretion.104
21 22
2.2.3. Agar sourced from marine red seaweeds is a complex polysaccharide mixture of linear
23
agarose gelling element and branched agaropectin.
24
comprises of 1→3-linked-β-D-galactose (G) and 1→4-linked 3,6-anhydro-α-L-galactose whereas
The linear agarose polymer skeleton
17 ACS Paragon Plus Environment
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branched agaropectin comprises agarose with presence of several substituent groups (eg. sulfate
2
esters, methyl esters, pyruvate acid ketals) at 4-linked α-L-galactose residues (figure 3C).84,105
3
The self-gelling property of agar restricts its processing ability for use as a biomaterial, although
4
it possesses exciting properties and has gained recent attention in the form of nanofibers.105
5
Linear agarose forms hydrogels by hydrogen bonding and has been employed for three-
6
dimensional chondrocytes encapsulation.84
7
synthesis of matrix components on exposure to dynamic deformational loading as well as
8
maintained chondrocyte phenotype upto four days.12
9
tendency to resist invasion of blood vessel that results in low oxygen tension and thereby
10
Chondrocyte-seeded agarose hydrogels induced
In addition, agarose possess intrinsic
supports the formation of avascular cartilage tissue.84
11 12
Table 2. Branched polysaccharide polymers, their repeating units along with their advantages
13
and disadvantages towards tissue engineering applications Polymer
Repeating units
Properties Beneficial
Disadvantages
Starch
20-30% of α-amylose (glucose linked by α(1→4) bonds) and 70-80% of amylopectin α(1→4)linked glucose, branched with α(1→6) linked for every 24 to 30 glucose residues
Versatile processability, imparted with thermoplasticity
Inferior mechanical properties with brittleness
Agar
Agarose [(1→3)-β-Dgalactopyranose-(1→4)-3,6anhydro-α-L-galactopyranose units] and agaropectin [(1→3)-βD-galactopyranose-(1→4)-3,6anhydro-α-L-galactopyranose and 4-linked α-L-galactose]
Self-gelling, agarose resists blood vessel invasion supporting avascularity
Difficult to process, quick gelation, limited degradation of agarose
Dextran
α(1→6) linked D-glucopyranose with less percent of α(1→2),
Swellability and rheology
Very high hydrophilicity
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α(1→3), or α(1→4) linked side chains Xanthan
Two D-glucose, two D-mannose and one D-glucuronic acid. Mannose-glucuronic acidmannose trisaccharide is attached to every second glucose by (1→3) linkage
Pseudo-plastic, rheology similar to HA
Difficult to handle high viscosity
1 2 3
2.2.4. Xanthan gum is an extracellular heteropolysaccharide produced by the bacterium
4
Xanthomonas campestris. Its molecular weight ranges from 2 X 106 to 20 X 106 Da.4,106,107
5
Xanthan consists of repeating units of five monosaccharides comprising two D-glucose, two D-
6
mannose and one D-glucuronic acid units (figure 3D).4,108 The glucose residues forms the
7
primary structure similar to the cellulose backbone. The alternating glucose units are substituted
8
with trisaccharide units of glucuronic acid flanked by mannose units.106,107
9
around half of the terminal mannose units are pyruvated while the inner mannose linked to the
10
backbone are acetylated.106,107 In the fivefold helical secondary structure, the backbone remains
11
protected from the outside environment by wrapping of the side chains via hydrogen bonding.4,106
12
This complexity in structure contributes to its high viscosity at lower concentration, stability at
13
various pH, temperature, ion concentrations and pseudo-plasticity similar in rheology to
14
hyaluronic acid.4 The non-toxic, biocompatible, bioadhesive and biodegradable xanthan has been
15
reported for various medical applications such as wound healing, implantation, drug carriers and
16
as hydrogels components.106,108 The administration of intra-articular xanthan injection has been
17
found to be protective on the articular cartilage in papain-induced osteoarthritic rabbit models.4
18
Further, carboxymethyl derivatization at the glucose residues of xanthan has been reported for
19
microcapsule encapsulation of chondrocytes and act as artificial matrix.107
20 19 ACS Paragon Plus Environment
Approximately
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1 2
Figure 3. Chemical structures of branched polysaccharide polymers [A] Starch; [B] Dextran; [C]
3
Agar and [D] Xanthan.
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3. Articular cartilage: Structure, function, injuries, healing responses and therapeutics
2
A comprehensive understanding of the structure, function and injuries of articular cartilage with
3
intrinsic responses ensures progress in the design of appropriate therapeutic modalities.109 The
4
hyaline articular cartilage functions to provide gliding low-friction surface, shock absorption and
5
protects the underlying supporting subchondral bone from pressure which in turn complement the
6
mechanical strength.15,110–112 The biomechanical role especially the compressive properties of
7
cartilage is based on the interstitial fluid swelling pressure of proteoglycans, mostly associated in
8
aggregates comprising complexes of aggrecans.
9
attachment of brush-like chondroitin sulfate or keratin sulfate with the protein core (aggrecan)
10
with hyaluronic acid and a link protein for stabilization.11,110–112 Additionally, hydrated collagen
11
II stabilized by collagen IX and XI, non-collagenous proteins, lesser amounts of small
12
proteoglycans such as biglycan, fibromodulin and decorin contributes to the organization and
13
thereby regulates functions of the cartilage matrix.4,10,110,111 The additional functions of matrix
14
include protection for chondrocytes, mediation of nutrient transfer, serve as store for cytokines
15
and growth factors for chondrocyte homeostasis.110
16
sparsely distributed cells that synthesize and maintain their resident matrix by regulating matrix
17
metabolism.
18
collagenous proteins differentially at varying depths to highly specialized tissue that enable their
19
functions. The compositional and morphological variations in the depth of cartilage matrix is
20
divided into four zones – superficial, transitional, middle (radial) or deep zone and calcified
21
cartilage zone.110 The superficial zone consists of thinnest collagen fibres at highest density to
22
form oriented lamina splendens covering the joint.
23
permeability and contributes to the tensile strength.111 The transitional zone contains highest
24
proteoglycan content, with collagen orientation varying from tangential to random in the
Aggrecan aggregates are generated by the
Chondrocytes are highly specialized,
The chondrocytes precisely organize the collagen, proteoglycans and non-
This resists shearing, monitors fluid
21 ACS Paragon Plus Environment
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underlying regions. The collagen fibrils orient perpendicularly in the deeper zone, and delineates
2
at the tidemark thereby differentiating the deep zone from calcified zone.111 This arrangement of
3
collagen improves the integration of soft and hard tissues at the cartilage-bone interface. In
4
contrast, calcified zone comprises collagen X for mineralization and structural integrity. The
5
proper functioning of the cartilage depends on the contributions from each zone.111
6
maintenance of required levels and distribution of proteoglycan and collagen fibers is crucially
7
significant for imparting the compressive and tensile strength respectively of articular
8
cartilage.113 The deepest or lattermost region of diarthrodial bone end is subchondral bone that
9
lies between calcified cartilage and trabecular bone.15 Unlike the cartilage region, subchondral
10
bone is highly vascularized which nourishes itself and the overlying cartilage.15 In addition to
11
providing anchorage for collagen fibrils of adjacent cartilage, the subchondral bone plays critical
12
role in absorption and maintenance of joint shape.15 The mechanical strength of bone is rendered
13
by extracellular matrix composed collagen and other organic components reinforced with
14
inorganic calcium phosphate nanocrystallites for superior rigidity.114
The
15 16
Trauma or degeneration that causes imbalance between the matrix biosynthesis and degradation
17
leads to destruction of articular cartilage tissue with subsequent extensive damage at various
18
zones.16 Such defects or injuries are categorized based on the depth into partial-thickness and
19
full-thickness, with the subchondral bone affected in the latter (figure 4). The avascular nature of
20
cartilage has no access to progenitor cells and blood cells, hindering the ability to initiate normal
21
wound healing process in partial thickness defects, thus exhibiting it poor intrinsic
22
reparability.110,111
23
chondrocyte proliferation with subsequent synthesis of ECM in an attempt to heal the lesion. In
24
most cases, the reparative efforts fail as the chondrocyte number does not increase to meet the
However, metabolic and enzymatic activities at the lesion site promote
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1
demands at the injured site that ultimately compromises functioning.111
2
thickness wounds penetrating the calcified and subchondral bone witness the migration of bone
3
marrow cells followed by spontaneous immune response and subsequent healing process to
4
restore the tissue. The subchondral bone plays integral role in the osteoarthritis pathogenesis as it
5
undergoes bone remodeling induced by the applied stress.15 The characteristic remodeling in
6
osteoarthritis is increased sclerotic subchondral bone, osteophytes spikes at the joint margins,
7
subchondral bone cyst formation and remodeling of trabeculae.115
8
osteoarthritis evidences 20 fold higher turn over of subchondral bone with associated higher
9
secretion of bone markers such as alkaline phosphatase, osteocalcin and osteopontin than normal
10
bone.115 Subsequently, these changes enhances anabolic activity of subchondral bone osteoblasts
11
and contributes to osteophytic bone formation.115 Such immature osteoids in subchondral bone
12
are unmineralized that lacks in the properties of native bone. This eventually leads to bone-bone
13
articulation of abnormal subchondral bone and the presence of unmyelinated free nerve endings
14
causes severe pain experienced in patients.15
15
components including contrasting layers of osteochondral tissue comprising both soft superficial
16
cartilage and hard subchondral bone with irregular defects.
On the contrary, full
The progression of
Thus, the distortion affects the entire joint
17 18
Management of clinical osteoarthritis includes pharmacological, non-pharmacological and
19
surgical options.116
20
administration of simple analgesia, opioid analgesia, non-steroidal anti-inflammatory drugs
21
(NSAIDs), calcitonin, topical applications, nutraceuticals such as glucosamine and chondroitin
22
sulfate, intra-articular injections of hyaluronic acid and corticosteroids.116,117 Management of the
23
condition by awareness, exercise and weight reduction approach are categorized as non-
24
pharmacological approaches.116
Pharmacological therapies mostly aim at alleviating the pain by
In patients refractory to both pharmacological and non23 ACS Paragon Plus Environment
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Page 24 of 74
1
pharmacological therapy, surgical total joint replacement option remains definitive for substantial
2
improvement in pain and function, but has finite life expectancy.109,116 Regenerative modalities
3
such as microfracture, mosaicplasty, allograft transplantation and autologous chondrocytes
4
transplantation reduce pain, restore tissue and improve joint function but lead to the formation of
5
inferior fibrocartilage consisting of collagen I.1,5,8,9 The high proportion of fibrocartilage may fail
6
in lateral integration with neighboring host tissue of the defect site.118 To overcome the existing
7
demerits, tissue engineering based interventional regenerative strategies aim at augmenting the
8
cartilage repair with subchondral bone.
9
substituting the damaged matrix and / or cells, biomolecules in combinations to restore the
10
superficial hyaline cartilage composed of collagen II and the mineralized subchondral bone
11
region. Emergence of injectable glycosaminoglycan mimetic polysaccharide scaffold addresses
12
major unresolved clinical complications due to its facile administration, patient compliance,
13
spatio-temporal distribution of cells and biomolecules, structural and composition mimetic design.
14
Furthermore, the fabrication of multi-layered matrices that are analogous to superficial resilient
15
cartilage and mineralized deeper subchondral zones are most relevant recent strategies in tissue
16
engineering.
These strategies employ biomaterial scaffolds for
17 18
4. Sol-gel transition chemistry of polysaccharide materials
19
Hydrogels are three dimensional, hydrophilic, water-insoluble networks of physical or chemically
20
crosslinked homopolymer or copolymer chains that retain water and possess properties similar to
21
native cartilage.37,83,104,119,120
22
transition from sol state at ambient conditions to gel phase on exposure to physiological
23
conditions are collectively termed as injectable or in situ forming hydrogels.83 An ideal in situ
24
forming injectable hydrogel should be formed in compatible aqueous medium without releasing
Such polymeric hydrogels formed on the rationale of phase
24 ACS Paragon Plus Environment
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1
harmful by-products.
Further, gelation rate should ideally ensure homogeneous mixing of
2
clinically meaningful number of cells with the functional efficacy.84 Such in situ gelling systems
3
permit premixing of cells and bioactive agents thereby facilitating facile spatiotemporal
4
incorporation into the hydrogel depot at the target site.37,121 These hydrogels are superior to
5
preformed scaffolds in terms of improved patient compliance and overcome the risk of implant
6
migration. Other advantages include simple cell encapsulation and ease of clinical
7
implementation via minimally invasive route for the treatment of geometrically irregular, larger
8
and deeper lesions.8,37,83,122,123
9
crosslinking, chemical crosslinking, enzymatic crosslinking, pH-induced gelation, temperature-
10
induced gelation, electric field, magnetic field, ionic interaction, hydrophobic interactions,
11
antigen mediation and their combinations.8,83,101,124–129 The crosslinking chemistry requires the
12
presence of specific functional groups inherently present in the polymer structure or introduced
13
through various chemical modifications. In the case of polysaccharides, such cross-links are
14
established at the carboxyl, amino and hydroxyl groups (Table 3).
15
injectability, simultaneous tailoring of physical properties such as mechanical strength to meet
16
the requirements of scaffolds could be dictated through appropriate functionalization. In addition,
17
injectable hydrogels derived from two or more polymers results in the integration of properties
18
from parent polymers.
19
interpenetrating, semi-interpenetrating or double network hydrogels.130,131 Based on the types of
20
bonds formed or associative interactions that leads to gelation, in situ forming hydrogels are
21
categorized as chemical hydrogels or physical hydrogels.124
The gelation strategies for these systems include photo-
Apart from imparting
Sol-gel transformation strategies facilitate the formation of
22
25 ACS Paragon Plus Environment
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1 2
Figure 4. Functional groups involved in crosslinking chemistries and the resultant bonds formed
3
by [A] Michael addition reaction; [B] Schiff’s base formation; [C] Click reactions and [D]
4
Enzymatic coupling mediated injectable hydrogels. 26 ACS Paragon Plus Environment
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1 2
Table 3. Functional groups of polysaccharides, chemical modifications to desired reactive
3
groups and their respective gelation mechanism
4 Polysaccharide Hyaluronic acid
Functional groups Carboxyl (-COOH) Carboxyl (-COOH) Carboxyl (-COOH) Carboxyl (-COOH) Carboxyl (-COOH) Carboxyl (-COOH) Hydroxyl (-OH) Vicinal diols
Chitosan
Amino (-NH2) Amino (-NH2) Amino (-NH2) Amino (-NH2) Amine (-NH2) Amine (-NH2) Amine (-NH2)
Chemical Modifications Thiolation by carbodiimide mediated Dithiothreitol (DTT) coupling Maleimide functionalization Modification with furylamine Tyramine conjugation Carbodiimide activated 1,3diaminopropane crosslinking Coupling with amineterminated dextran– tyramine conjugates Methacrylation Periodate oxidation to form aldehydes
Gelation mechanism Michael addition
Reference Censi et al., 2010; Jin et al., 2010 104,122
Michael addition Diels-Alder click Enzymatic Thixotropic inter-penetrating networks Enzymatic
Photo crosslinking Schiff’s base formation
Jin et al., 2010 132 Yu et al., 2013 133 Lee et al., 2009 134 Barbucci et al., 2010 22 Jin et al., 2010 132
Levett et al., 2014 135
Monocarboxylated pluronics glycolic acid/phloretic acid
Thermal
Millane et al., 2015; Sheu et al., 2013; Su et al., 2013; Tan et al., 2009 123,136–139 Park et al., 2009 54
Enzymatic
Jin et al., 2009 140
Methacrylic acid and lactic acid grafted via carbodiimide Succinyl modified
Thermal
N-Acetyl-L-Cysteine conjugation Grafted with poly (Nisopropylacrylamide) Methacrylated glucol derivative
Hong et al., 2008 141
Schiff’s base formation Schiff’s base formation Thermal Photo crosslinking
27 ACS Paragon Plus Environment
Tan et al., 2009 136 Zhang et al., 2011 130
Chen and Cheng, 2009 36 Park et al., 2013 142
Biomacromolecules
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Amine (-NH2) Amine (-NH2) Hydroxyl (-OH) Chondroitin Sulfates
Carboxyl (-COOH)
Hydroxyl (-OH) Vicinal diols
N-Acetyl-L-Cysteine conjugation Carbamate Carbodiimide bond activated 1,3Ester bond diaminopropa ne crosslinking Acrylation Derivatized with tri(2carboxyethyl)phosphine (TCEP) Methacrylation Periodate oxidation to form aldehydes
Photo crosslinking Schiff’s base formation
Barbucci et al., 2010 22
Jo et al., 2010 70
Levett et al., 2014 135
Dawlee et al., 2005; Millan et al., 2015 137,144
Kim et al., 2011 145
Periodate oxidation to form aldehydes
Michael addition Schiff’s base formation
Carbodiimide activated 1,3diaminopropane crosslinking Oxidation to form aldehydes
Thixotropic inter-penetrating networks Schiff’s base formation
Barbucci et al., 2010 22
Carboxyl (-COOH) Carboxyl (-COOH)
---
Ionic crosslinking Ionic crosslinking
Vicinal diols
Periodate oxidation to form aldehydes
Schiff’s base formation
Sulfate (-SO4),
---
Ionic crosslinking
Cellulose
Carboxy methyl cellulose
Carboxyl (-COOH)
Pectins
Vicinal diols
Carrageenans
Michael addition
Teng et al., 2010 143
Jin et al., 2011 63
Tyramine coupled using carbodiimide activation Thiolation
Alginate
Michael addition Thixotropic inter-penetrating networks
Enzymatic
Carboxyl (-COOH) Carboxyl (-COOH) Vicinal diols
Heparin
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28 ACS Paragon Plus Environment
Balakrishnan et al., 2013 146
Takei et al., 2010 74
Mishra et al., 2012 73
Cho et al., 2009; Popa et al., 2011; Stevens et al., 2004 71,87,147 Balakrishnan et al., 2014; Millan et al., 2015 124,137 Popa et al., 2011 71
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Dextran
Carboxyl (-COOH) Hydroxyl (-OH) Hydroxyl (-OH) Hydroxyl (-OH)
Hydroxyl (-OH) Vicinal diols
Xanthan
Hydroxyl (-OH)
Thiol
Tyramine conjugation
Michael addition Michael addition Enzymatic
Hydroxyethyl methacrylate conjugation Periodate oxidation to form aldehydes
Photocrosslinking Schiff’s base formation
Carboxymethylation
Ionic crosslinking
Vinylsulfone
Hiemstra et al., 2007 148 Hiemstra et al., 2007 148 Jin et al., 2007; Jin et al., 2010; Teixeira et al., 2012 104,149,150 Pescosolido et al., 2011 151 Zhang et al., 2011 130
Mendes et al., 2012 107
1 2
4.1.
3
This category comprises hydrogels that are formed by covalent interactions between the
4
functional groups in the polymer chains. The commonly encountered covalent coupling reactions
5
are discussed in the following sections.
Chemical hydrogels
6 7
4.1.1. Michael addition: Michael addition reaction is based on the addition of nucleophiles
8
(Michael donor) across the carbon-carbon multiple bonds of activated electrophilic olefins or
9
alkynes (Michael acceptor) to form a ‘Michael adduct’ (figure 4A).152 Polysaccharides have been
10
chemically modified to carry both electrophilic (acrylates, vinyl sulfone, maleimide) and
11
nucleophilic groups (thiol, phosphine).70,102,104,122,132,148,153 Michael addition reactions have been
12
widely employed to tailor the properties of polysaccharide hydrogels for tissue engineering
13
applications. The water-solubility of chondroitin sulfate (CS) was reduced by formation of a
14
hydrogel through phosphine-mediated Michael addition involving the crosslinking of CS-acrylate 29 ACS Paragon Plus Environment
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1
and CS-tri(2-carboxyethyl)phosphine (TCEP).
2
incorporated by grafting acrylic acid and TCEP to the adipic acid dihydrazide linked CS 70. The
3
hydroxyl groups of dextran were substituted to varying degrees with thiol (Dex-SH) and vinyl
4
sulfones as nucleophile and electrophile respectively for Michael reaction. The rheology and
5
degradation behavior of the resultant hydrogel was compared with similar hydrogel formed using
6
tetra-acrylated poly(ethylene glycol) as synthetic polymer electrophile.
7
exhibited high elasticity while delayed degradation was exhibited by hydrogel formed from the
8
synthetic polymer.148 Similarly, other polysaccharides such as hyaluronic acid and chitosan have
9
been modified to carry maleimide and thiol groups that exhibits in situ gelling ability via Michael
10
Both the electrophile and nucleophile were
Both the hydrogels
addition reaction.104,132,143
11 12
4.1.2. Click reaction: Click reactions are those that can be used for a wide range of functional
13
groups and result in high yields. The by-products can be easity separated from the reaction
14
mixture and are performed in easilty removable solvents. Click reaction cross-linked hydrogels
15
are advantageous over traditional physical and chemical crosslinking as they have higher yield
16
with rapid reactivity at mild conditions, superior chemo-selectivity and specificity with non-toxic
17
byproducts.133,154,155 The common click chemistries include azide-alkyne (3+2) cycloaddition,
18
thiol-alkene addition catalyzed by Cu(I) and furan-maleimide (4+2) Diels–Alder (DA)
19
cycloaddition (figure 4C).133,154,156 Since, the micro-molar toxicity of Cu(I) catalyst restricts
20
cellular applications, the catalyst-free Diels-Alder cycloaddition catalyst has gained attention in
21
recent years for cartilage tissue engineering applications.133 In addition to facile encapsulation of
22
cells by pre-mixing, the unreacted azide or acetylene groups can be further functionalized
23
thereby aiding modification of the hydrogel properties.155 An interpenetrating hydrogel network
24
of gelatin, HA and chondroitin sulfate (CS) was developed by the crosslinking of HA modified 30 ACS Paragon Plus Environment
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1
with furylamine at the carboxyl group and furan carboxylic acid modified gelatin via Diels-Alder
2
click reaction.133
3
network hydrogels holds promise for their use as potent matrices for cartilage tissue
4
engineering.133 Meng et al., have recently reviewed the click reactions based polysaccharide
5
modifications for various biomedical applications.157
The tunable mechanical properties and rheology of these interpenetrating
6 7
4.1.3. Schiff’s base formation: The reaction between amines and aldehydes to form Schiff base
8
without any chemical crosslinking agents has been the basis for the formation of injectable
9
hydrogels (figure 4B).155 The gelation kinetics and physical properties of these hydrogels can be
10
tailored by varying the ratio of amine and aldehyde groups, while the remaining functional
11
groups can be conjugated with therapeutic moieties.155
12
hyaluronic acid, chitosan, dextran, cellulose and chondroitin sulfates have been derivatized to
13
form the precursors of Schiff base hydrogels.130,139,144,146,155
14
have been oxidized by periodate that cleaves the vicinal diols in monosaccharide residues and
15
forms aldehydes.74,123 These aldehydes forms hydrogels by crosslinking with amine or hydrazide
16
groups via imine or hydrazine bonds respectively.74,123
17
enhances the degradability of the polysaccharide backbone.74
18
introduction of Schiff’s base crosslinking to an existing disulfide crosslinked in situ forming
19
thiolated chitosan and oxidized dextran hydrogel system enhanced the mechanical properties and
20
reduced the gelation duration.130 Recently, oxidized hyaluronic acid, alginate and chondroitin
21
sulfate based Schiff‘s base crosslinked microtissues have been engineered by cell aggregation to
22
rejuvenate and restore chondrogenic potential of aged mesenchymal stem cells and synthesize
23
collagen II. 137
Various polysaccharides such as
Polysaccharides with vicinal diols
Further, such periodate oxidation
24 31 ACS Paragon Plus Environment
Zhang et al., reported that
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1
4.1.4. Photo-crosslinking: Photo-crosslinked hydrogels are formed by in situ polymerization of
2
acrylate and methacrylate groups carrying monomers or polymers mediated by the free radicals
3
generated by photoinitiators on exposure to light of specific wavelengths.83
4
polymerization occurs at physiological pH and temperature, this in situ forming strategy enables
5
the spatio-temporal distribution of functional bioactive factors and viable cells.83,119,158 Factors
6
such as polymer concentration, photoinitiators and light exposure time can be modified to control
7
the spatio-temporal gelation.159
8
biocompatibility and usage of low viscous monomer or macromer precursor solutions for
9
gelation.119 A variety of chemistries could be achieved with unsaturated C=C carrying groups in
10
polymers such as chitosan, alginate, chondroitin sulfate and hyaluronic acid thereby imparting
11
photo-crosslinking potential.83,160
As the
Photo-crosslinking hydrogels are advantageous for their
12 13
4.1.5. Enzyme-mediated cross-linking: Injectable, biodegradable and biocompatible hydrogels
14
formed upon the coupling of tyramine phenol moieties catalyzed by oxidation reaction of
15
horseradish peroxidase (HRP) enzyme and hydrogen peroxide (H2O2) finds applications as
16
artificial ECM and cargo delivery systems.134,140,161
17
conditions, exhibits tailorable mechanical strength and gelation time.140,162
18
conjugates synthesized by the grafting of glycolic acid / phloretic acid and tyramine at the amine
19
groups of chitosan and hydroxyl groups of dextran respectively formed cross-linked hydrogels
20
catalyzed by HRP and H2O2 (figure 4D).140,149,162
21
hydrogels supported the viability, phenotype retention and ECM production of the encapsulated
22
chondrocytes.140,149,162 HRP-mediated co-crosslinking of tyramine conjugated dextran (Dex-TA)
23
and heparin (Hep-TA) developed enzymatic hydrogels to achieved chondrogenesis by
24
glycosaminoglycan and collagen synthesis. The carboxyl groups of heparin was substituted by
These hydrogels formed at optimal Polysaccharide
Both the enzyme-mediated cross-linked
32 ACS Paragon Plus Environment
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1
tyramine and the increase in ratio of Dex-TA/Hep-TA resulted in faster gelation and increased
2
storage modulus.63
3 4
4.2.
5
Physical hydrogels are stabilized through secondary forces of associations such as electrostatic
6
interactions, hydrogen bonding, hydrophobic forces, etc,. The following sections describe the
7
salient features of physical hydrogels stabilized by each type of secondary associative forces.
Physical hydrogels
8 9
4.2.1. Electrostatic interaction: Oppositely charged ionic polyelectrolytes exhibit electrostatic
10
interactions leading to the formation of hydrogels in situ.83 For example, addition of divalent
11
ions such as strontium (Sr2+), calcium (Ca2+) and barium (Ba2+) cross-links anionic alginate
12
quickly via G-blocks (comprising L-guluronate residues) stacking thereby leading to ionotropic
13
hydrogel network formation with an egg-box structure.147 The egg-box model represents a
14
structure with corrugated surface with interstices that host cations for electrostatic association
15
with their counter ions on the polymer chain.147 Similarly, anionic gellan gum forms hydrogel in
16
the presence of cations such as Ca2+ and Mg2+.88 These ionotropic hydrogels are formed by
17
crosslinking low millimolar concentrations of cations thereby exhibiting low toxicity towards the
18
encapsulated cells.88 The degradation and mechanical properties of these hydrogels relies on the
19
length of the polymer chain and charge density. The electrostatic interactions between two
20
oppositely charged macromolecules form cytocompatible polyelectrolyte hydrogels with rapid
21
gelation.129 For instance, cationic polysaccharide chitosan formed resorbable polyelectrolyte
22
hydrogel on interaction with a natural anionic polypeptide polyglutamate that was found to
23
support the adhesion and proliferationof cells.129 Photocrosslinkable chitosan was reported to
24
exhibit electrostatic interactions with the extracellular matrix components chondroitin sulfate and 33 ACS Paragon Plus Environment
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1
collagen II to form an injectable hydrogel formulation. This composite hydrogel was found to
2
support proliferation and matrix protein synthesis by the encapsulated chondrocytes.163
3 4
4.2.2. Temperature or pH induced gelation: The intrinsic behavior of certain polymers to exhibit
5
sol-gel transition at critical solution temperature (CST) and above critical gel concentration
6
(CGC) has been used for developing injectable hydrogels.126,164 Polymers exhibiting sol-gel
7
transition above CST due to the hydrophobic interactions are collectively termed as lower critical
8
solution temperature (LCST) polymers. On the contrary, upper critical solution temperature
9
(UCST) polymers form gel at temperatures below their CST.164 This reversible transition occurs
10
rapidly to the energetically most favored state which is either a sol or gel.165 For instance, the
11
LCST of poly(N-isopropylacrylamide) (PNIPAM) and its copolymers is around 32°C, above
12
which inter and intra-molecular hydrophobic interactions facilitate the release of water from the
13
vicinity of isopropyl group thereby forming hydrogel.126,160,164 This temperature induced gelation
14
has been combined with other existing strategies to improve the structural integrity of the
15
hydrogel network. Censi et al., developed an in situ forming hydrogel based on the simultaneous
16
Michael addition and thermo-responsiveness of a triblock copolymer poly(ethylene glycol)
17
flanked by N-isopropylacrylamide (PNIPAM)/N-(2-hydroxypropyl) methacrylamide dilactate
18
and thiolated hyaluronic acid which was found to have enhanced structural and mechanical
19
stability.122 Thermosensitive chitosan-pluronic hydrogel was developed by grafting a synthetic
20
monocarboxylated
21
poly(ethylene oxide), to the amino group of chitosan thereby imparting thermoresponsive phase
22
transition at room temperature.54
23
solubilizes in aqueous medium at pH lower than 6.2 and as the basicity increases, the cationic
24
amine groups are neutralized due to deprotonation that is evidenced by gel-like precipitation.
pluronic
polymer,
poly(ethyleneoxide)-b-poly(propylene
Changes in pH mediates gelation in chitosan.
34 ACS Paragon Plus Environment
oxide)-b-
Chitosan
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1
This responsiveness to pH can also be achieved on addition of polyols such as β-
2
glycerophosphate (GP) salt.82,83
3 4
4.2.3. Supramolecular assembly: Supramolecular assemblies stabilized by secondary forces of
5
attraction can be employed as injectables due to their ability to undergo sol-gel transition in
6
response to the environmental stimuli.
7
assembly of polymeric chains into cyclodextrins (CD). Cyclodextrins are water-soluble cyclic
8
oligosaccharides with hydrophobic internal cavities consisting of six to eight α-D-glucopyranose
9
units designated as α-CD, β-CD and γ-CD.166 Self-assembly occurs as the guest linear polymer
10
chains penetrates and threads into the hydrophobic cavities of cyclic host molecules to form
11
inclusion complexes with unique supramolecular architectures.166
12
complex formation includes combination of geometric compatibility and secondary forces such
13
as hydrophobic interactions, hydrogen bonding of hydroxyl groups along the rims of neighboring
14
CDs and van der Waals forces.166 The properties of these supramolecular hydrogels can be
15
tailored by modifying the host and guest moieties. A bivalent β-CD/adamantane has been
16
synthesized and coupled with the HA derivatives as guest polymer that assembles as
17
hydrogels.167
Supramolecular hydrogels are formed by molecular
The forces driving the
18 19
4.2.4. Shear thinning hydrogel: Certain proteins, colloidal systems, peptides and polymeric
20
blends possess phenomenal self-assembling behavior that causes gelation on withdrawal of
21
shear.168,169 Based on this property, highly viscous polymer solution or minimally cross-linked
22
gel deforms or flows under shearing force while being injected and forms hydrogel in situ.169
23
The polymeric chains of HA form random coils and molecular entanglements leading to gelation,
24
while on application of shear stress, it deforms and flows due to the alignment of molecules along 35 ACS Paragon Plus Environment
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1
the direction of stress.170 This unique rheology of HA has been blended with thermal gelling
2
methyl cellulose to develop an injectable hydrogel. The gelation was found to be rapid in this
3
system due to the integration of two gelation strategies.170 Barbucci et al., have studied a class of
4
thixotropic injectable inter-penetrating hydrogels by combining polysaccharides.
5
combinations of negatively charged carboxymethyl cellulose with positively charged chitosan,
6
neutral guar gum and negatively charged hyaluronic acid exhibited interesting mechanical and
7
biological properties as interpenetrating hydrogel networks.22 Among the various polysaccharide
8
combinations, the hydrogel formed by combining oppositely charged polysaccharides exhibited
9
better mechanical properties.22 In a recent approach, Ding et al., have integrated associations
10
based on electrostatic interactions and supramolecular assembly using heparin and α-cyclodextrin.
11
The dual physically bonded hydrogel exhibited shear thinning property, cell compatibility and
12
sustained growth factor delivery.171
The
13 14
5.
15
Ideally, biomaterials to be used as scaffolds for cartilage regeneration should possess properties
16
such as biocompatibility, biodegradability, high porosity, compressibility, non-cytotoxicity and
17
non-antigenicity.172
18
cartilage, provide mechanical support, biochemical cues, and promote cell-matrix interactions for
19
initiating tissue reparative process.14,150 A typical tissue engineering triad comprises of cells and
20
biomolecules as biological components apart from scaffolds as structural support.83,173,174 This
21
section discusses the progress of polysaccharide based injectable scaffolds and its combinations
22
with biological components towards the fabrication of ideal constructs that repair and regenerate
23
the complex functional articular hyaline cartilage. The emergence of hydrogels as in situ forming
24
scaffolds and bioprinted constructs for cartilage tissue has been depicted in figure 5.
Injectable strategies for cartilage tissue engineering
The scaffolds should mimic the native ECM architecture of articular
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1 2 3
Figure 5. Schematic representation of progressive degeneration of articular cartilage. The white
4
patches in superficial cartilage tissue represents partial-thickness defect of articulating surface
5
and red patches denotes the full-thickness defect of articular surface with subchondral bone. In
6
situ forming hydrogel that fills the irregular osteochondral defect serves as depot for cells and
7
growth factors encapsulated. The fabrication of 3D printed irregular construct and administration
8
has also been depicted.
9 10
5.1.
11
Articular cartilage tissue majorly consists of the extracellular matrix with very low cell-to-matrix
12
ratio.14 Regeneration of this structure based functional tissue could be augmented by scaffolds as
13
temporary
14
morphogenesis.109,175 Scaffold biomaterials should preferably be porous to facilitate infiltration
15
of cells. It should be biodegradable to permit neo-tissue formation, permeable to facilitate
16
nutrient and gas exchange and possess appropriate mechanical strength, apart from being
17
chondro-inductive and chondro-conductive.109
18
properties play a vital role in regenerating the injured cartilage tissue.14,135 Various injectable 37
Polysaccharides as structural substitutes
biocompatible
matrices
that
provide
form,
shape
and
direct
tissue
Thus, development of scaffolds with ideal
ACS Paragon Plus Environment
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1
acellular scaffolds based on polysaccharides have been developed and evaluated for their
2
potential to infiltrate and support the cell fate processes of mammalian chondrocytes. Biji et al.,
3
have developed Schiff’s reaction based injectable hydrogels formed by self-crosslinking of the
4
oxidized polysaccharides namely carboxymethyl cellulose or alginate with gelatin in the presence
5
of borate for cartilage tissue engineering.124,146 Such self-crosslinks result in porous hydrogels
6
with biocompatibility, biodegradability, tunable gelation kinetics and host tissue integration.124,146
7
In addition, infiltration, proliferation, phenotype maintenance and GAG deposition of murine
8
chondrocytes was promoted by these scaffolds.124,146 The aldehyde of oxidized hyaluronic acid
9
reacts with the amino group of water-soluble N-succinyl-chitosan to form Schiff‘s base
10
crosslinked hydrogel within 4 minutes.136 The resultant hydrogels exhibit microporous structure,
11
desired swelling, adequate compressive modulus, in vitro degradation, and support viability and
12
spherioid formation of bovine chondrocytes that were introduced either by surface seeding or
13
encapsulation.136 Though the scaffold acts as a structural substitute and recruits cells to the
14
injured site, this approach would not be potent in the case of larger, deeper defects that suffer
15
severe loss of chondrocytes. Hence, combinational strategies involving scaffolds with cells and
16
trophic factors have emerged as promising alternates for accomplishing cartilage tissue
17
regeneration. These alternates have been discussed in the following sections.
18 19
5.2.
20
The lack of neurovascular supply in the articular cartilage restricts the migration of regenerative
21
cells while poor proliferation of differentiated chondrocytes decreases the intrinsic regenerative
22
potential of the tissue.176–178 Some of the current treatment modalities such as mosaicplasty,
23
autologous chondrocytes transplantion (ACT) and microfracture are based on recruiting
24
chondrocytes and mesenchymal stem cells from bone marrow at the injured site to enhance
Cell / Stem cell carriers
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1
regeneration.
The ACT procedure involves harvesting chondrocytes from non-load bearing
2
unaffected cartilage surfaces and expansion in vitro prior to transplantation at the injured site.176
3
Besides chondrocytes, stem cells are being sourced to overcome the limitation of chondrocyte
4
availability especially in cases of severely degenerated articular cartilage. The microfracture
5
treatment aims at recruiting bone marrow mesenchymal stem cells by infiltration through the
6
fracture into the defect site.179 Embryonic stem cells, bone marrow stromal cells, synovium
7
derived stem cells and human induced pluripotent stem cells (hiPSCs) have been extensively
8
investigated to attain functional cartilage regeneration and restoration.176,180–184 The role and
9
efficiency of different cells in the scenario of cartilage tissue repair and regeneration have been
10
comprehensively reviewed by several groups.185–188 The features of regenerated tissue largely
11
rely on the cell types involved in the synthesis of matrix components. For instance, autologous
12
chondrocytes therapy generates better hyaline-like cartilage compared to microfracture based
13
mesenchymal cell therapy.179 Success of cell-based therapeutics requires efficient localization
14
and retention of transplanted cells at the injured site, thus necessitating the need for integration of
15
biomaterial matrices with cells.189,190 In comparison with the preformed scaffolds, in situ forming
16
hydrogels augment the uniformity in distribution of cells throughout the scaffolds. The following
17
section discusses on the various polysaccharide based injectable strategies evaluated for its
18
potential as carriers of viable differentiated cells and stem cells. Table 4 summarizes some of the
19
scaffolds evaluated in vitro for chondrogenesis.
20 21
Table 4. Polysaccharide polymer based injectable hydrogel scaffolds that have been evaluated in
22
vitro for cartilage regenerative potential Polymer
Gelation Mechanism
Cell/Stem cells
Study Inference Duration
39 ACS Paragon Plus Environment
Reference
Biomacromolecules
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60
Oxidized Alginate / Gelatin
Schiff’s base formation
Murine chondrocytes
Carboxymethylated Enzymatic crosslinking Pullulan / Chondroitin Sulfate
Porcine chondrocytes
Chitosan / Alginate / Fibrin with Strontium Ranelate nanoparticles
Human Mesenchymal stem cells
21 days
14 days
14 days
Page 40 of 74
Infiltration of chondrocytes, phenotype retention and functionality
Balakrishnan et al., 2014
Proliferation of chondrocytes and cartilage specific protein expression
Chen et al., 2016 191
Chondrogenic differentiation with enhanced ECM synthesis
Deepthi et al., 2016 192
Infiltration of chondrocytes, phenotype retention and functionality
Balakrishnan et al., 2013
124
Oxidized Carboxymethyl cellulose / Gelatin
Schiff’s base formation
Murine chondrocytes
Alginate / Polyvinyl alcohol
Ionic crosslinking
Chondrocytes 28 days (immortalized human costal chondrocyte cell line) C28/I2 cultured
Chondrocytes increased and deposition of glycosaminoglycans
Cho et al., 2009 147
RGD-Alginate / Hyaluronate
Crosslinking due to Specific interactions between cell and polymer
Primary chondrocytes
6 weeks
In vivo subcutaneous injection into the dorsum of SPF/VAF immunedeficient mice
Park and Lee, 2011 193
Oligo (poly(ethylene glycol) fumarate) (OPF) / PEG diacrylate
Ionic crosslinking
Rabbit marrow mesenchymal stem cells (MSCs)
2 weeks
Chondrogenic differentiation
Park et al., 2007 194
Methacrylated glycol chitosan and hyaluronic acid
Photocrosslinking
Chondrocytes
28 days
Proliferation and ECM deposition
Park et al., 2013 142
Chitosan / Hyaluronic acid
Schiff’s base formation
Bovine articular chondrocytes encapsulation and adhesion
24 hours
Promoted cell survival and retention of chondrocytes phenotype
Tan et al., 2009 139
40 ACS Paragon Plus Environment
146
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onto the scaffold Hyaluronic acid / PEG
Michael addition
Primary Bovine chondrocytes
3 weeks
Production of glycosaminoglycans and collagen II
Jin et al., 2010 132
Chitosan / HA / PNIPAM
Thermoreversible
Articular chondrocytes from New Zealand rabbits
5 weeks
Cell proliferation, differentiation and morphology retention, Increased ECM deposition and mechanical properties
Chen and Cheng, 2009 36
Oxidized gellan gum
Temperature Porcine dependent chondrocytes
150 days
Cell proliferation and specific matrix formation
Gong et al., 2009 195
Dextran-Tyramine
Enzymatic crosslinking
Primary Bovine chondrocytes
28 days
Sustain chondrocyte viability, phenotype retention and de novo ECM synthesis in vitro
Jin et al., 2010 104
Chitosan- beta glycerophosphatehydroxyethyl cellulose
Temperature Human/rat mesenchymal stem cells
28 days
Chondrogenic differentiation
NaderiMeshkin et al., 2014 196
1 2
5.2.1. Chondrocytes: Injectable polysaccharide hydrogels extensively characterized for their
3
physicochemical properties, have been evaluated further using chondrocytes sourced from rabbit,
4
bovine and other mammals for their potential to establish cell-matrix interaction, phenotype
5
maintenance, proliferation and synthesis of hyaline cartilage ECM.132,136,197,198
6
developed by various injectable strategies, the cell sources, duration of the in vitro study and the
7
potential exhibited in cartilage regeneration have been consolidated in table 4.
8
chondrocytes were well dispersed in poly(ethylene glycol) (PEG) - vinyl sulfone precursor
9
solution prior to hydrogel formation in association with thiolated HA through Michael 41 ACS Paragon Plus Environment
Hydrogels
Bovine
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1
addition.199 In addition to tunable gelation time, storage modulus and degradation behavior can
2
be tailored by varying the molecular weight of HA and degree of substitution in the functional
3
groups. The HA-PEG hydrogel exhibited good compatibility with over 95% viable chondrocytes
4
and enhanced production of GAGs and hyaline-specific collagen II for three weeks.199 HA based
5
H2O2 and HRP mediated enzymatic hydrogels (HA-g-dextran-tyramine conjugates) have been
6
demonstrated to form biomimetic hydrogel within two minutes in situ. This hydrogel resembled
7
the macromolecular structure of proteoglycans and exhibited chondrocyte compatibility,
8
increased proliferation and matrix deposition for three weeks.197
9
glycosaminoglycan of the cartilage namely hyaluronic acid and chondroitin sulfate were
10
methacrylated and included with photocurable gelatin-methacrylamide based biomimetic
11
hydrogel to investigate the role of compositional cue on the chondrocytes behavior.135 Presence
12
of glycosaminoglycans in the in situ forming gel positively influenced the chondrogenic
13
redifferentiation of expanded chondrocytes, chondrogenesis, matrix distribution and mechanical
14
properties.135
The most abundant
15 16
Autologous chondrocytes seeded alginate was injected subcutaneously in mice and the construct
17
remained localized and promoted cartilage formation.80 Injectable Michael addition mediated
18
heparin-thiol and PEG-diacryl hydrogel has demonstrated in vitro spontaneous re-differentiation
19
of de-differentiated chondrocytes without growth factor or chondrogenic moieties after one week
20
of culture.200 The cell-hydrogel construct showed re-differentiation and cartilage formation in
21
vivo following subcutaneous implantation in nude mice.200 In a typical partial-thickness rabbit
22
knee defect model, the heparin based cell bearing demonstrated re-differentiation, endogenous
23
TGF-β1 retention, excellent regeneration and integration with the host tissue in 4 months when
24
compared with the cell-free hydrogel.200 42 ACS Paragon Plus Environment
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1
Despite its regenerative potential, the limited availability of differentiated chondrocytes,
2
morbidity of source site, intrinsic low expansion potential and trans-differentiation in vitro
3
restricts the clinical utility of well differentiated chondrocytes from autologous sources.201,202 On
4
the other hand, other allogeneic or xenogeneic sources can induce unfavorable immune responses
5
and transmit diseases.111 Hence, interest towards the investigation of undifferentiated stem cells
6
of various origin for their chondrogenic potential thereby enabling repair and regeneration of the
7
articular cartilage tissue are being explored.
8 9
5.2.2. Stem cells
10
The undifferentiated stem / progenitor cells possess high proliferative potential, differentiate to
11
specialized cells, have good accessibility and availability thereby proving to be a promising
12
alternate source to meet the clinical demands of chondrocytes.111,202,203 Stem cells from various
13
sources such as embryonic, fetal or adult tissues are allowed for expansion and differentiation in
14
vitro or by microenvironment of the transplanted area in vivo.202 The chondrogenic stem cells
15
perform the cellular processes to restore the extracellular matrix and thereby re-establish the
16
articular cartilage.
17
turnaround time for allogeneic therapy and eliminates the requirement for harvesting patients’
18
cells. 201
In addition, increasing numbers of stem cell banks ensures reduced
19 20
Embryonic stem cells (ESCs)
21
ESCs derived from the inner cell mass of blastocysts are highly proliferative, undifferentiated
22
cells with pluripotent ability.203,204 The intrinsic potential of differentiation to several somatic
23
cell lineages challenges the efficient direction of differentiation to specific chondrogenic lineages
24
203
. Several approaches using biomaterials, co-culturing with chondrocytes, 3D culture combined 43 ACS Paragon Plus Environment
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with growth factors, biophysical stimuli, hypoxia and mechanical stimulations have been
2
attempted to achieve chondrogenic differentiation of ESCs.203,204 Development of efficient mode
3
for enhanced differentiation is crucial as undifferentiated ESCs enhances the risk of teratoma
4
formation and can turn tumorigenic in vivo.204 Toh et al., demonstrated the direct differentiation
5
of human ESCs (hESCs) in high-dense microenvironment combined with TGFβ-1 and expanded
6
as hESC-derived chondrogenic cells for 16 population doublings in selective growth factor
7
combination (TGFβ-1, FGF2 and PDGF-bb). Further, the chondrogenic cells cultured in the
8
hyaluronic acid based hydrogel (GlycosilTM) were implanted in rat osteochondral defect model
9
and exhibited chondro-inductive potential without teratoma formation.203
Human iPSCs
10
reprogrammed to possess embryonic stem cell-like epigenetic status have been induced to
11
undergo chondrogenic differentiation in vitro using alginate hydrogel constructs prior to
12
implantation at a rat osteochondral defect site.183 The chondro-induced hiPSCs exhibited repair
13
and regeneration of cartilage with enhanced quality.183
14 15
Adult stem cells
16
Multipotent adult mesenchymal stem cells (MSCs) isolated from different sources like bone
17
marrow, adipose tissue (ASCs), umbilical cord matrix, skin, dental pulp and synovial tissue have
18
been evaluated for their cartilage regenerative potential.44,188,201,205
19
isolated from bone marrow of healthy adult donors’ posterior iliac crest were expanded and
20
encapsulated in thermosensitive hydrogel made from water soluble chitosan and poly(N-
21
(isopropyl acrylamide)) copolymer.
22
chondrogenic differentiation of hMSCs in vitro and showed increased expression of
23
chondrogenic markers such as aggrecan and collagen II.206 Similarly, thermoresponsive chitosan-
24
beta glycerophosphate-hydroxyethyl cellulose exhibited chondrogenic property apart from
Human MSCs (hMSCs)
This injectable cell-polymer complex promoted
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promoting the survival and proliferation of encapsulated bone marrow derived human/rat
2
MSCs.196 In order to enhance the engraftment of bone marrow hMSCs at the injured cartilage,
3
cyto-adhesive pullulan exopolysaccharide was employed. This carbohydrate favored maintenance
4
of viability, enhanced proliferation and chondrogenesis of MSCs in a cartilage explant in vitro
5
model.5 Portron et al., demonstrated the role of hypoxia on chondrogenesis in a polysacchraide
6
scaffold by simulating the native avascular cartilage hypoxic environment via preconditioning of
7
adult stem cells with low oxygen tension in vitro. The chondrogenic pre-disposition of adipose
8
derived stromal cells (ASCs) by hypoxia were further assessed for in vivo regenerative potential
9
using rabbit cartilage defect and nude mice subcutis models.202 Polysaccharide based silanized
10
hydroxypropyl methylcellulose (Si-HPMC) pre-mixed with chondrogenic rabbit and human
11
ASCs were reticulated to form hydrogels prior to administration in rabbit and mice
12
respectively.202 The study concluded that chondrogenic differentiation using specific medium in
13
vitro promoted optimal cartilage regeneration irrespective of oxygen preconditioning.202
14
composite injectable hydrogel reinforced using two-dimensional nanomaterial has been reported
15
for hMSCs encapsulation.
16
hydrogel exhibited high cell viability, physiological stability and shear thinning phenomenon
17
ideal for cell delivery in cartilage tissue engineering and bioprinting.207 While the stem cells
18
encapsulated in polysaccharide constructs were induced for chondrogenic differentiation using
19
growth factors in culture media, other approaches of simultaneous loading of specific growth
20
factors with stem cells have also been examined.113,194,206
A
This nanosilicate reinforced methacrylated kappa–carrageenan
21 22
5.3.
23
Growth factors are a class of bioactive polypeptides that mediate cell signaling and influence
24
cellular fate processes thereby regulating homeostasis and involving in reparative processes.208
Bio-molecular signal delivery
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There are several such signaling growth factors involved in the development of articular cartilage
2
that serve as therapeutics for defects in articular cartilage.208 Growth factors designated for
3
cartilage therapeutics should essentially be anti-catabolic and pro-anabolic by decreasing the
4
inflammatory cytokines and stimulating chondrocyte ECM deposition respectively.208 Tropic
5
factors such as transforming growth factor-β (TGF-β) superfamily, bone morphogenetic protein-2
6
(BMP-2), BMP-7, insulin-like growth factor-1 (IGF-1), fibroblast growth factor (FGF), platelet-
7
derived growth factor (PDGF), etc., augments cartilage repair by displaying variety of vital roles
8
in regeneration.113,208–210 These growth factors have been explored alone or in combinations for
9
cartilage regeneration. For instance, co-encapsulation of TGF-β3 and bFGF in an injectable
10
thermo-responsive hydrogel, promoted chondrogenic differentiation of rabbit MSCs by mediating
11
molecular and cellular processes that resemble in vivo chondrogenesis.211
12 13
TGF-β1 is a 25 kDa protein that potentially promotes chondrogenic differentiation, proliferation
14
and cartilage ECM synthesis.87,212,213
15
degradable injectable hydrogel demonstrated proliferation of chondrocytes for over 21 days while
16
minimizing de-differentiation.212 On culturing periosteum in polysaccharide gels such as agarose
17
and alginate with TGF-β1, proliferation and differentiation of chondrogenic precursor cells in the
18
cambium layer promoted chondrogenesis.87 Such autologous periosteal-derived cartilage tissue
19
formation is advantageous as template for directional tissue evolution and act as a source for
20
chondrogenic growth factors with minimal morbidity at donor site.87
21
chondrogenic commitment in stem cells prior to implantation was demonstrated by the
22
administration of autologous adipose stem cells (ASC) in the treatment of rabbit articular
23
cartilage defect.
24
chondrogenic ASC enhanced collagen II and aggrecan expression while down regulating collagen
Co-encapsulation of chondrocytes with TGF-β1 in a
The efficiency of
Injectable gellan gum combined with TGF-β1 and BMP-2 preconditioned
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I, thereby emerging as a promising construct for cartilage repair.92 Thus, stem cells predisposed
2
to chondrogenic differentiation in vitro leads to improved tissue repair compared to
3
undifferentiated stem cells.92,202
4 5
IGF-1 also known as somatomedin C, primarily acts in an anabolic fashion to increase the
6
synthesis of proteoglycan and collagen II.113
7
localized and sustained delivery to mediate vital cellular processes including differentiation and
8
proliferation of progenitor cells and ECM synthesis.113 The self-crosslinked alginate / gelatin
9
scaffold that gelates within two seconds had demonstrated cell attraction, adhesion and enhanced
10
chondrocyte functionality by the incorporation of bioactive platelet derived growth factor
11
(PDGF-BB), dexamethasone, chondroitin sulfate, and its combinations.124
12
dexamethasone and chondroitin sulfate improved glycosaminoglycan deposition within the
13
matrix, and PDGF-BB increased the chondrocyte proliferation, thus promoting neo-cartilage
14
formation for treating osteoarthritis.124
Growth factors loaded in scaffolds achieve
The presence of
15 16
Other signaling sources such as platelet lysate rich in growth factors and anti-inflammatory
17
cytokines could serve as potent chemoattractants and signaling mediators for cellular processes.
18
Autologous blood derived platelet rich plasma has demonstrated enhanced biological activity
19
such as proliferation and chondrogenic differentiation of mesenchymal stromal cells in dextran-
20
tyramine hydrogel. The injectable dexran-tyramine system with desired mechanical strength and
21
biological cues had integrated into the osteoarthritic site and opens up new vistas towards cell-
22
free approach in cartilage regeneration.150
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5.4.
2
Research on pharmaceutical therapeutics for osteoarthritis focus primarily on developing disease-
3
modifying osteoarthritis drugs (DMOADs) and connective tissue structure-modifying agents
4
(CTSMAs) such as doxycycline (Dx).117 Polysaccharide hydrogel scaffolds that deliver drugs
5
such as doxycycline and resveratrol have been reported to integrate the effects of
6
pharmacological and regenerative strategies.117,138 Doxycycline (Dx) indirectly protects collagen
7
II and aggrecan by inhibiting synthesis of inducible nitric oxide synthase, which suppresses
8
chondrocyte secretion of remodeling proteins, matrix metalloproteinases (MMPs). In addition,
9
Dx had been demonstrated to improve subchondral bone structure, minimize joint-space
10
narrowing and also exhibited anti-inflammatory effects by suppressing inflammatory cytokines
11
(interleukin-1 (α/β) and interleukin-6).117 Electrostatic interactions between Dx and HA based
12
injectable hydrogel crosslinked through zinc chelation have been reported. This hydrogel when
13
used to treat osteoarthritis in rabbit model, exhibited additive effects of both the polysaccharide
14
and chondroprotective drug.
15
property that were useful for treating osteoarthritis.117 Resveratrol (Res), an anti-inflammatory
16
drug modified to carry amino group was allowed to react with oxidized HA to form Schiff‘s base
17
crosslinked hydrogel and was used for chondrocyte delivery.138 The HA/Res hydrogel was found
18
to be chondrocyte compatible. The chondrocytes synthesized ECM through upregulation of the
19
cartilage specific genes collagen II, aggrecan and sox-9 while downregulating genes encoding for
20
MMPs, interleukin-1b. Further, the scaffold also reduced LPS-induced inflammation that is
21
associated with chondrocyte damage which augers well for cartilage regeneration.138
Pharmaceutical strategies
This included viscoelasticity, anti-inflammatory and analgesic
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6. Combinational multi-component design strategies
2
Progress in the design of biomimetic scaffolds has promoted the exploration of combination
3
strategies for engineering complex articular cartilage tissue.214 The lacuna existing in different
4
single component approaches can be overcome by integrating strategies with properties
5
complementing each other.214
6
poly(lactide-co-glycolide) (PLGA) with defined shape and size have been piled to form three-
7
dimensional porous scaffold that demonstrated chondrocyte and stem cell compatibility.141
8
However, mechanical strength and uncontrolled movement of microcarriers in vivo, restrict its
9
utility as scaffolding material. To overcome this limitation, microcarriers have been coated with
10
collagen and incorporated into crosslinkable chitosan grafted with methacrylic acid and lactic
11
acid. This composite scaffold that integrated hydrogel with cell microcarriers exhibited improved
12
elastic modulus and chondrocyte viability with phenotype retention for 12 days.141 Recently, a
13
simple combination of heparin and self-assembling peptide RAD16-I enhanced the chondrogenic
14
commitment of adipose-derived stem cells (ADSC) due to the existence of growth factor binding
15
domain in heparin.65 Further, integration of nanofibrous peptide amphiphiles (RAD16-I) and
16
gellable heparin increased the expression of mature cartilage markers like collagen II and
17
proteoglycans.65
18
variation to mimic the anisotropic architecture of native tissue.
Injectable cell microcarriers of polylactide (PLA) and
The multi-component strategies have been further designed with spatial
19 20
6.1.
21
Typically, the degenerative changes in osteoarthritis are associated with both articular cartilage
22
and underlying subchondral bone.215
23
mediated reparative cartilage tissue tends to delaminate that remains a threat in clinical scenario
24
of arthritis. 216 Therefore, designing of constructs that integrates soft cartilaginous phase and hard
Osteochondral mimetic approaches
The spontaneous response or surgical interventions
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osseous phase with soft-hard interface plays crucial role in regenerative process and integrate
2
with neighbouring host tissue.
3 4
6.1.1. Bioceramics and Bioglass
5
Calcium phosphates such as hydroxyapatite (HA) and tricalcium phosphates are bioceramic that
6
potentially enhance biomineralization and are indispensible biomaterials for bone tissue
7
engineering.217 Bioceramic based implants induce the deposition apatite layer on the surface
8
similar to native bone and thereby promotes integration with adjacent host bone.217 Though
9
bioceramics are brittle, the mechanical stiffness, bioactive osteoconductive properties and
10
tailorable biodegradability are inevitable for bone scaffolds.217,218 Bioactive glasses are a class of
11
bioceramics which include 45S5 Bioglass® composed of 45 wt % SiO2, network modifiers of
12
24.5 wt % Na2O and 24.5 wt % CaO, and 6 wt % P2O5 that simulate Ca/P composition of
13
HA.217,219 Bioactive glasses have been reported with rapid bonding to bone compared to
14
bioceramics.217,219
15
mechanical properties of bone layer and interfacial properties has become a promising strategy in
16
the fabrication of multi-layered osteochondral scaffold.
17
osteochondral constructs with bioceramics in calcified layer have been tabulated in table 5.
Incorporation of inorganic bioceramics or bioactive glasses to enhance the
The various multi-component
18 19
Table 5: Bioceramics based subchondral mimetics in various bi- or multi-layered osteochondral
20
strategies for regeneration of functional articuar cartilage Phasicity
Phase
Biphasic
Chondral phase
Biomaterials Bovine decellularized articular cartilage extracellular
Fabrication Modified temperature gradientguided thermalinduced phase separation (TIPS)
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Inference Superior biomechanical properties in vitro. Biphasic scaffold with
Reference Da et al., 2013 220
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Bony phase with compact bone
Multilayered
Gradient
Bilayered
matrix
technique
PLGA / βTricalcium phosphate
Rapid prototyping technique
Cartilaginous Collagen – I layer
chondrogenic and osteogenic induced BMSCs enhanced integration and functional regeneration of osteochondral tissue in vivo.
Intermediate layer
Collagen – I (40%), hydroxyapatite (60%)
Standardized Good early Kon et al., physical processing stability of 2010 221 biomaterial in Standardized pilot clinical study physical processing of 6 months. and neoossification
Subchondral bone layer
Collagen – I (30%), hydroxyapatite (70%)
Standardized physical processing and neoossification
Superficial zone
Chitosan with gelatin
Thermoresponsive hydrogel
Radial zone
Chitosan with hyaluronic acid
Thermoresponsive hydrogel
Calcified zone
Chitosan with β- Thermoresponsive Tricalcium hydrogel phosphate
Cartilage layer
Chitosan
Freeze-dried at 80ºC
Bone layer
Hydroxyapatite in polyurethane sponge
Sintered at 1300ºC
Depth dependent variation in mechanical, rheological and structural properties.
Walker and Madihally, 2014 222
Adhesion, proliferation and differentiation of goat bone marrow stromal cells to osteoblasts and chondrocytes in respective layers.
Oliveira et al., 2006 223
1 2 3
6.1.2. Bi- or Multi-layered osteochondral constructs
4
In view of replicating the contrasting soft cartilage and hard bone tissues, bilayered scaffolds
5
have been designed with β-tricalcium phosphate, hydroxyapatite and bioactive glass constituted 51 ACS Paragon Plus Environment
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in the deep layer.219,220,223 Enrichment of the subchondral bone layer with such bioceramics have
2
improved the mechanical and physical characteristics, as well as enhanced the osteogenic
3
potential of the bilayered construct.219,220,223
4
between the cartilage-like and bone-like layers in the multi-phasic constructs have been designed
5
to mimic soft-hard transitional interface of the osteochondral tissue.221,222 In another approach,
6
the anisotropic architecture of cartilage matrix has been mimicked by varying the depth-based
7
composition of chitosan as continuous phase in the scaffold. The polysaccharide chitosan based
8
temperature sensitive injectable hydrogel consisted of gelatin, hyaluronic acid and β-tricalcium
9
phosphate in the superficial, radial, and calcified zones respectively. The anisotropic hydrogel
10
demonstrated depth dependent variations in mechanical, rheological and structural properties in
11
addition to its gelling nature both in vitro and in in vivo BALB/c mouse subcutaneous model.222
12
Thus, developing integrated constructs could emerge as superior strategy to closely mimic the
13
complex articular cartilage tissue with potential for repair and restoration.
Furthermore, sandwiching an interfacial layer
14 15
7. 3D rapid prototype using polysaccharides bioinks
16
The advent of 3D bioprinting is set to revolutionize the field of regenerative medicine and
17
conventional tissue engineering approaches towards complex tissues such as osteochondral,
18
kidney, brain, blood vessel, etc,.224–226 Bioprinting aims at building intricate heterogeneous 3D
19
architecture of tissue by spatio-temporal positioning of cells and biomolecules with micrometer
20
precision using bioinks that exactly mimic the native tissue.224,225,227 Figure 6A & B shows the
21
various patterns printed using thermoresponsive pluronic hydrogel as bioink. The homogeneity
22
of dispensed print and layer by layer precise deposition of prints have been shown in figure 6C
23
and 6D-E respectively using curcumin (green) and sulforhodamine (red) as fluorophores under
24
confocal laser scanning microscopy (CLSM). Unlike traditional scaffold-based strategies, this 52 ACS Paragon Plus Environment
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bottom-up technology offers high cell seeding density with strong cell-cell communication,
2
which in turn facilitates cell-ECM interactions that aid regeneration of the tissue. Success of 3D
3
construction of tissues largely depends on the bioink properties as it should possess both
4
biological features to pattern cells as well as the physical requirements for printability which are
5
contrasting to each other.151,224,228 The feasibility of printing viable cells (green stained by
6
calcein-AM) using alginate has been shown in figure 7B with absence of dead cells (no red cells
7
– ethidium bromide staining). Figure 7A shows the CLSM micrographs of alginate bioprints
8
loaded with dansylated bovine serum albumin as model protein. The versatile properties of
9
polysaccharides such as injectability, flowability, cell deliverability and cytocompatibility in
10
addition to chondrogenesis have led to their exploration as bioinks for 3D bioprinting of tissues.
11 12
Among the various cartilages in the human system, aesthetic cartilage like ear and articular
13
cartilage have been bioprinted using polysaccharides like alginate, hyaluronic acid, cellulose,
14
gellan gum and dextran that have been investigated for their printability parameters.151,224,225,228–
15
234
16
was used to print 3D cartilage structures such as human ear and sheep meniscus using blueprints
17
of magnetic resonance images and computed tomography images. The shape fidelity of alginate
18
was achieved by nanofibrillated cellulose and it was successfully used to print the human
19
nasoseptal chondrocytes thereby demonstrating the potential of the blend bioink for printing 3D
20
tissues.224 Pescosolido et al., have developed a semi-interpenetrating network (semi-IPN) based
21
on two polysaccharides namely hyaluronic acid and hydroxyethyl-methacrylate-derivatized
22
dextran (dex-HEMA). The bioink dispensed by the print head of bioprinter exhibited shear-
23
thinning rheology and the photopolymerized construct showed appreciable mechanical strength.
A bioink formulated with shear thinning nanofibrillated cellulose and quick gelling alginate
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The HA/dex-HEMA printed scaffolds showed high porosity with well-defined strand spacing and
2
supported the viability of encapsulated chondrocytes.151
3 4 5
Figure 6. Spatio-temporal patterning of thermoresponsive Pluronic hydrogels by 3D bioprinting:
6
[A] Macroscopic views of various patterns; [B] Optical micrographs (4X); Confocal laser
7
scanning micrographs (CLSM) (10X) of [C] curcumin stained bioprint, [D] layer by layer
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printing (Green: curcumin, Red: sulforhodamine) 2D micrograph, and [E] 3D reconstructed
2
image.
3
4 5
Figure 7. Bioprinting of calcium crosslinked alginate hydrogel: confocal laser scanning
6
micrographs (10X) showing [A] different regions of the printed construct loaded with dansylated
7
bovine serum albumin (blue), and [B] merged images of 3D printed hydrogel (grey) loaded with
8
viable cells (green).
9 10
Biofabrication of two compartments consisting of chondrocyte encapsulated hyaluronic acid and
11
osteoblast encapsulated collagen-I have been printed as osteochondral tissue-mimetic
12
structures.225
13
chondrocytes in Dulbecco’s Modified Eagle Medium (DMEM) for chondral section, while
14
collagen-I in DMEM constituted the osteo section. The cell survival and functioning were
15
appreciable for 14 days, thus validating the choice of bioinks.225 An attempt to mimic the various
The polysaccharide bioink was composed of hyaluronic acid, alginate and
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zones of osteochondral tissue by deposition of multiple bioinks to develop clinically relevant
2
sized constructs was reported by Levato et al.
3
expanded in polylactic acid microcarriers and encapsulated in gelatin methacrylamide-gellan gum
4
bioink for printing viable constructs. The osteogenic and chondrogenic differentiation of stromal
5
cells has been established by gellan gum bioink with and without microcarriers respectively.233
6
Though the layer-by-layer assembly of 3D printed constructs mimics the complex structural
7
organization of the native tissue, establishing desirable bio-mechanical properties still remains a
8
challenge in this strategy. Hence, attempts have been made to enhance the mechanical properties
9
of 3D dual cell-laden printed scaffold for osteochondral tissue engineering by sequential
10
dispensing of cell-alginate bioink over a thermoplastic polycaprolactone (PCL) framework.235
11
The fabricated construct retained the viable osteoblasts and chondrocytes in their respective
12
layers.235
Mesenchymal stromal cells (MSCs) were
13 14
8. Translation of cartilage tissue engineering to clinics
15
The ultimate goal of cartilage tissue engineering is to translate a promising therapeutic strategy to
16
the clinics for regeneration and restoration of functional articular cartilage. Polysaccharide based
17
scaffolds have attained a remarkable place among the biomaterials evaluated for clinical
18
regeneration of cartilage tissue in osteoarthritic patients. The FLEXX trial assessed the efficacy
19
and safety of 1% sodium hyaluronate (EUFLEXXA®) for painful knee osteoarthritis therapy at 26
20
weeks. It was observed that the EUFLEXXA® therapy demonstrated significant pain relief in
21
patients with improved joint functioning, treatment satisfaction and health-related quality of life
22
compared to saline-control.236
23
hyaluronate (Hyalgan or Supartz), Hylan G-F 20 (Synvisc) and Orthovisc, a high molecular
24
weight HA are available in the United States.23
A variety of other hyaluronan preparations such as sodium
Though HA intra-articular injections are
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administered to compensate the loss of endogenous HA, the fate or exact role of exogenous HA
2
remains ambiguous. As an effort to decipher the effect of exogenous HA in arthritis, the wear
3
particles released by degradation was monitored in the synovial fluid human knees before
4
EUFLEXXA® injections (four times) using bio-ferrography.
5
magnetically labeled osseous and carilagenous particles in addition to a questionnaire assessment.
6
The study concluded that HA potentially reduced the joint degradation rate.237 A pilot clinical
7
study has evaluated the short-term stability of osteochondral biomimetic multi-layered
8
scaffold.221
9
hydroxyapatite nanoparticles for subchondral bone. The high resolution MRI evaluation of early
10
6 months follow up in thirteen patients (15 defects) confirmed the stability of this acellular
11
gradient scaffold. 221
The study analyzed the
The gradient scaffold has been based on collagen-I and nucleated with
12 13
Autologous chondrocyte implantation (ACI) is the most relied treatment for symptomatic
14
chondral or osteochondral defects.238
15
differentiation, inhomogenous distribution and leakage of cells from the site necessitate
16
integration of scaffolding with cellular therapeutics for patients.238
17
available tissue engineered graft is Hyalograft® C (Fidia Advanced Biopolymers Laboratories,
18
Abano Terme, Italy) consisting of autologous chondrocytes cultured on Hyaff-11®, an esterified
19
form of hyaluronan with controlled degradation and complete resorption in 3 months.239 Two to
20
five year follow-up reports of Hyalograft® C administration for arthritis proved improvement in
21
the repair of cartilage with hyaline-like appearance.38 A monolayer-expanded cartilage cell
22
product, CARTIPATCH (Tissue Bank of France, Lyon, France)
23
dimensional agarose-alginate hydrogel was reported to improve cell phenotype retention.238 In
24
the phase-II clinical study, symptomatic arthritis patients were implanted with the cellular
However, limitations such as chondrocyte de-
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combined with three-
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agarose-alginate hydrogel and investigated for
clinical, radiological, arthroscopic and
2
histological outcomes after a minimum of two years follow-up.238 The arthritic condition was
3
significantly improved in all patients including patients with larger and deeper lesions.238 Such
4
clinical trials at different phases increase the translation of promising tissue engineering
5
approaches from bench-to-bedside of patients for potentially regenerating the injured tissue.
6 7
Though there are numerous preclinical studies in the development of tissue engineered medical
8
products, clinical trials are very limited due to the regulatory issues, differences in
9
patients‘ healing response, large scale fabrication and skilled expertise for handling in production
10
as well as implantation constraints. The introduction of advancements such as tissue engineered
11
products into clinics faces regulatory restrictions as it creates ethical dilemmas for physicians,
12
patients and wider public.240 In contrast to the well-defined defects of preclinical studies, clinical
13
osteoarthritic defect is irregular with varied size and severity based on the patient‘s history.
14
Personalization of osteochondral constructs to integrate with both soft and hard tissue based on
15
the specific requirement of patient remains challenging. Fabrication of large tissue engineered
16
constructs with intricate 3D structures, strictly defined mechanical, chemical and biological
17
characteristics limits the clinical translation.241
18
defined 3D structures comprising of soft and hard zones with interface in between. However,
19
expansion of clinically meaningful number of cells and complex tissue analogous matrices at
20
rapid rate to meet the clinical requirement has not been realized practically.
21
commercialization and industrialization of tissue engineered medical products is nascent, the
22
unmet prime requirements include massive fabrication, sterilization, stringent quality control of
23
3D scaffolds for human trials and affordability of medical expenditure restricts translation of this
24
technology.241–243
Bioprinting technology offeres to fabricate
As
Finally, well trained expertise in handling the complex osteochondral 58 ACS Paragon Plus Environment
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1
constructs at various stages from production to implantation in patients is also bottleneck that
2
holds effective translation of this technology.241
3 4
9.
5
The advent of tissue engineering principles has undoubtedly changed the scenario of
6
osteoarthritis therapeutics to improve the patient’s quality of life. Polysaccharide biomaterials
7
have been extensively studied for fabrication of cartilage engineering scaffolds due to their
8
desirable
9
processability, viscoelasticity and biological activities such as cytocompatibility and
10
chondrogenic potential. Further, the facile modifications of accessible functional groups in
11
polysaccharides have overcome the inferior mechanical properties and faster degradation apart
12
from imparting injectability to these biomaterials. The most challenging aspects of cartilage
13
regeneration include homogeneous distribution of functional chondrocytes, retention of
14
chondrocyte phenotype, restoration of lubricative ECM with collagen-II rich hyaline-like
15
cartilage and long term durability for pain free locomotion. In vitro and in vivo evaluation of
16
injectable polysaccharide based in situ forming hydrogels has demonstrated the ability to deposit
17
collagen-II and aggrecan, which are the key markers of hyaline cartilage. The emergence of
18
injectable and printing technologies for the construction of osteochondral mimetics represents a
19
significant milestone in the repair and regeneration of articular cartilage at complexly injured
20
osteoarthritic sites. In addition, 3D bioprinting strategy could potentially contribute to the next
21
generation personalized therapeutics that addresses each patient‘s requirement as it is an
22
automated, computerized, rapid technology to establish cell-cell communication with complex
23
architecture of native tissues. However, translation to clinics requires meticulous choice of the
Conclusions and Future Perspectives
properties
such
as
tailorable
chemistry,
biodegradability,
59 ACS Paragon Plus Environment
biocompatibility,
Biomacromolecules
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1
appropriate polysaccharide with robust features such as mechanical properties, compatibility,
2
degradability and regenerative potential assessed in suitable pre-clinical trials.
3 4
Corresponding Author
5
*Dr. Swaminathan Sethuraman
6
Director, Centre for Nanotechnology & Advanced Biomaterials
7
Orchid Chemicals & Pharmaceuticals Chair Professor
8
School of Chemical & Biotechnology,
9
SASTRA University,
10
Tamil Nadu, India
11
E-mail:
[email protected] 12 13
Author Contributions
14
The manuscript was written through contributions of all authors. All authors have given approval
15
to the final version of the manuscript.
16 17
Acknowledgements
18
The authors wish to acknowledge Nano Mission (SR/NM/PG-16/2007) and the FIST program
19
(SR/FST/ST/LSI-453/2010) of the Department of Science & Technology (DST), Government of
20
India for their financial support. The joint financial support from the Drugs & Pharmaceuticals
21
Research Programme, DST, India and SASTRA University is also acknowledged. First author is
22
thankful to Innovation in Science Pursuit for Inspired Research (INSPIRE), DST, India for Senior
23
Research Fellowship (IF120692).
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Table of Contents
7 8 Crosslinked Gel Osteochondral defect
Z motion X motion
Sol
Y motion
Polymeric Bioink
Printed hydrogel construct
9
3D Bioprinted Gel
74 ACS Paragon Plus Environment
